AMP-activated protein kinase (AMPK) activation increases inflammation-related fibrosis in the lungs.

Metformin reverses established lung fibrosis in a bleomycin model

Fibrosis is a pathological result of a dysfunctional repair response to tissue injury and occurs in a number of organs, including the lungs1. Cellular metabolism regulates tissue repair and remodelling responses to injury2-4. AMPK is a critical sensor of cellular bioenergetics and controls the switch from anabolic to catabolic metabolism5. However, the role of AMPK in fibrosis is not well understood. Here, we demonstrate that in humans with idiopathic pulmonary fibrosis (IPF) and in an experimental mouse model of lung fibrosis, AMPK activity is lower in fibrotic regions associated with metabolically active and apoptosis-resistant myofibroblasts. Pharmacological activation of AMPK in myofibroblasts from lungs of humans with IPF display lower fibrotic activity, along with enhanced mitochondrial biogenesis and normalization of sensitivity to apoptosis. In a bleomycin model of lung fibrosis in mice, metformin therapeutically accelerates the resolution of well-established fibrosis in an AMPK-dependent manner. These studies implicate deficient AMPK activation in non-resolving, pathologic fibrotic processes, and support a role for metformin (or other AMPK activators) to reverse established fibrosis by facilitating deactivation and apoptosis of myofibroblasts.

Role of AMP-activated protein kinase in mechanism of metformin action.

Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. Its glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization. Metformin's beneficial effects on circulating lipids have been linked to reduced fatty liver. AMP-activated protein kinase (AMPK) is a major cellular regulator of lipid and glucose metabolism. Here we report that metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformin's inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders.

Activated AMPK boosts the Nrf2/HO-1 signaling axis—A role for the unfolded protein response

In light of the emerging interplay between redox and metabolic signaling pathways we investigated the potential cross talk between nuclear factor E2-related factor 2 (Nrf2) and AMP-activated kinase (AMPK), central regulators of the cellular redox and energy balance, respectively. Making use of xanthohumol (XN) as an activator of both the AMPK and the Nrf2 signaling pathway we show that AMPK exerts a positive influence on Nrf2/heme oxygenase (HO)-1 signaling in mouse embryonic fibroblasts. Genetic ablation and pharmacological inhibition of AMPK blunts Nrf2-dependent HO-1 expression by XN already at the mRNA level. XN leads to AMPK activation via interference with mitochondrial function and activation of liver kinase B1 as upstream AMPK kinase. The subsequent AMPK-mediated enhancement of the Nrf2/HO-1 response does not depend on inhibition of the mammalian target of rapamycin, inhibition of glycogen synthase kinase 3β, or altered abundance of Nrf2 (total and nuclear). However, reduced endoplasmic reticulum stress was identified and elaborated as a step in the AMPK-augmented Nrf2/HO-1 response. Overall, we shed more light on the hitherto incompletely understood cross talk between the LKB1/AMPK and the Nrf2/HO-1 axis revealing for the first time involvement of the unfolded protein response as an additional player and suggesting tight cooperation between signaling pathways controlling cellular redox, energy, or protein homeostasis.

Phosphorylation of Janus kinase 1 (JAK1) by AMP-activated protein kinase (AMPK) links energy sensing to anti-inflammatory signaling

Adenosine 5′-monophosphate–activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Rα/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.

Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function

Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.

Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling

Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK–ERK–MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo

Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. However, the mechanisms behind these effects are unclear. Here we show that nicotine, a major constituent of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which in turn phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating its proteasome-dependent degradation. The nicotine-dependent reduction of MKP1 induces the aberrant activation of both p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. Phosphorylation of IRS1 leads to its degradation, protein kinase B inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results establish AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity.

A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4

S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

Morin Attenuates Ovalbumin-Induced Airway Inflammation by Modulating Oxidative Stress-Responsive MAPK Signaling.

Asthma is one of the most common inflammatory diseases characterized by airway hyperresponsiveness, inflammation, and remodeling. Morin, an active ingredient obtained from Moraceae plants, has been demonstrated to have promising anti-inflammatory activities in a range of disorders. However, its impacts on pulmonary diseases, particularly on asthma, have not been clarified. This study was designed to investigate whether morin alleviates airway inflammation in chronic asthma with an emphasis on oxidative stress modulation. In vivo, ovalbumin- (OVA-) sensitized mice were administered with morin or dexamethasone before challenge. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained to perform cell counts, histological analysis, and enzyme-linked immunosorbent assay. In vitro, human bronchial epithelial cells (BECs) were challenged by tumor necrosis factor alpha (TNF-α). The supernatant was collected for the detection of the proinflammatory proteins, and the cells were collected for reactive oxygen species (ROS)/mitogen-activated protein kinase (MAPK) evaluations. Severe inflammatory responses and remodeling were observed in the airways of the OVA-sensitized mice. Treatment with morin dramatically attenuated the extensive trafficking of inflammatory cells into the BALF and inhibited their infiltration around the respiratory tracts and vessels. Morin administration also significantly suppressed goblet cell hyperplasia and collagen deposition/fibrosis and dose-dependently inhibited the OVA-induced increases in IgE, TNF-α, interleukin- (IL-) 4, IL-13, matrix metalloproteinase-9, and malondialdehyde. In human BECs challenged by TNF-α, the levels of proteins such as eotaxin-1, monocyte chemoattractant protein-1, IL-8 and intercellular adhesion molecule-1, were consistently significantly decreased by morin. Western blotting and the 2',7'-dichlorofluorescein assay revealed that the increases in intracellular ROS and MAPK phosphorylation were abolished by morin, implying that ROS/MAPK signaling contributes to the relief of airway inflammation. Our findings indicate for the first time that morin alleviates airway inflammation in chronic asthma, which probably occurs via the oxidative stress-responsive MAPK pathway, highlighting a novel profile of morin as a potent agent for asthma management.

Activin–like kinase–3 activity is important for kidney regeneration and reversal of fibrosis

Molecules associated with the transforming growth factor β (TGF-β) superfamily, such as bone morphogenic proteins (BMPs) and TGF-β, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-β1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.

Induction of AMPK activity corrects early pathophysiological alterations in the subtotal nephrectomy model of chronic kidney disease.

The rat kidney ablation and infarction (A/I) model of subtotal or 5/6th nephrectomy is the most commonly studied model of nondiabetic chronic kidney disease (CKD). The A/I kidney at 1 wk exhibits reductions in kidney function, as determined by glomerular filtration rate, and diminished metabolic efficiency as determined by oxygen consumption per sodium transport (QO2/TNa). As renoprotective AMPK activity is affected by metabolic changes and cellular stress, we evaluated AMPK activity in this model system. We show that these early pathophysiological changes are accompanied by a paradoxical decrease in AMPK activity. Over time, these kidney parameters progressively worsen with extensive kidney structural, functional, metabolic, and fibrotic changes observed at 4 wk after A/I. We show that induction of AMPK activity with either metformin or 5-aminoimidazole-4-carboxamide ribonucleotide increases AMPK activity in this model and also corrects kidney metabolic inefficiency, improves kidney function, and ameliorates kidney fibrosis and structural alterations. We conclude that AMPK activity is reduced in the subtotal nephrectomy model of nondiabetic CKD, that altered regulation of AMPK is coincident with the progression of disease parameters, and that restoration of AMPK activity can suppress the progressive loss of function characteristic of this model. We propose that induction of AMPK activity may prove an effective therapeutic target for the treatment of nondiabetic CKD.

Histone acetyltransferase PCAF regulates inflammatory molecules in the development of renal injury.

Kidney diseases, including chronic kidney disease (CKD) and acute kidney injury (AKI), are associated with inflammation. The mechanism that regulates inflammation in these renal injuries remains unclear. Here, we demonstrated that p300/CBP-associated factor (PCAF), a histone acetyltransferase, was overexpressed in the kidneys of db/db mice and lipopolysaccharide (LPS)-injected mice. Moreover, elevated histone acetylation, such as H3K18ac, and up-regulation of some inflammatory genes, such as ICAM-1, VCAM-1, and MCP-1, were found upon these renal injuries. Furthermore, increased H3K18ac was recruited to the promoters of ICAM-1, VCAM-1, and MCP-1 in the kidneys of LPS-injected mice. In vitro studies demonstrated that PCAF knockdown in human renal proximal tubule epithelial cells (HK-2) led to downregulation of inflammatory molecules, including VCAM-1, ICAM-1, p50 subunit of NF-κB (p50), and MCP-1 mRNA and protein levels, together with significantly decreased H3K18ac level. Consistent with these, overexpression of PCAF enhanced the expression of inflammatory molecules. Furthermore, PCAF deficiency reduced palmitate-induced recruitment of H3K18ac on the promoters of ICAM-1 and MCP-1, as well as inhibited palmitate-induced upregulation of these inflammatory molecules. In summary, the present work demonstrates that PCAF plays an essential role in the regulation of inflammatory molecules through H3K18ac, which provides a potential therapeutic target for inflammation-related renal diseases.

NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.

The early events leading to the development of rheumatoid arthritis (RA) remain unclear, but formation of autoantibodies to citrullinated protein antigens (ACPAs) is considered a key pathogenic event. Neutrophils isolated from patients with various autoimmune diseases display enhanced neutrophil extracellular trap (NET) formation, a phenomenon that exposes autoantigens in the context of immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers, and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and RA synovial fluid neutrophils compared to neutrophils from healthy controls and from patients with osteoarthritis (OA). Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules, and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. Indeed, during NETosis, neutrophils externalized the citrullinated autoantigens implicated in RA pathogenesis, and anti-citrullinated vimentin antibodies potently induced NET formation. Moreover, the inflammatory cytokines interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines, and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease.

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression.

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

Activation of AMP-activated protein kinase suppresses oxidized low-density lipoprotein-induced macrophage proliferation.

Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [(3)H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKalpha1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27(kip) suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21(cip) and p27(kip) expression via AMPK activation, and small interfering RNA (siRNA) of p21(cip) and p27(kip) restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.

Dual role of interleukin-10 in the regulation of respiratory syncitial virus (RSV)-induced lung inflammation.

RSV lower respiratory tract infections (LRTI) are among the most common diseases necessitating hospital admission in children. In addition to causing acute respiratory failure, RSV infections are associated with sequelae such as secondary bacterial infections and reactive airway disease. One characteristic host response observed in severe RSV-induced LRTI and/or subsequent development of asthma is increased expression of interleukin (IL)-10. However, contradictory results have been reported regarding whether IL-10 inhibits asthmatic responses or intensifies the disease. We aimed to reconcile these discordant observations by elucidating the role of IL-10 in regulating the host response to RSV LRTI. In this study, we used a lung-specific, inducible IL-10 over-expression (OE) transgenic mouse model to address this question. Our results showed that the presence of IL-10 at the time of RSV infection not only attenuated acute inflammatory process (i.e. 24 h post-infection), but also late inflammatory changes [characterized by T helper type 2 (Th2) cytokine and chemokine expression]. While this result appears contradictory to some clinical observations where elevated IL-10 levels are observed in asthmatic patients, we also found that delaying IL-10 OE until the late immune response to RSV infection, additive effects rather than inhibitory effects were observed. Importantly, in non-infected, IL-10 OE mice, IL-10 OE alone induced up-regulation of Th2 cytokine (IL-13 and IL-5) and Th2-related chemokine [monocyte chemoattractant protein 1 (MCP-1), chemokine (C-C motif) ligand 3 (CCL3) and regulated upon activation normal T cell expressed and secreted (RANTES)] expression. We identified a subset of CD11b(+)CD11c(+)CD49b(+)F4/80(-)Gr-1(-) myeloid cells as a prinicipal source of IL-10-induced IL-13 production. Therefore, the augmented pathological responses observed in our 'delayed' IL-10 over-expression model could be attributed to IL-10 OE alone. Taken together, our study indicated dual roles of IL-10 on RSV-induced lung inflammation which appear to depend upon the timing of when elevated IL-10 is expressed in the lung.

Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis.

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice.

Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction.

AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy

The SNF1/AMP-activated protein kinase (AMPK) family maintains the balance between ATP production and consumption in all eukaryotic cells. The kinases are heterotrimers that comprise a catalytic subunit and regulatory subunits that sense cellular energy levels. When energy status is compromised, the system activates catabolic pathways and switches off protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation. Surprisingly, recent results indicate that the AMPK system is also important in functions that go beyond the regulation of energy homeostasis, such as the maintenance of cell polarity in epithelial cells.

CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein–coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase–independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

Protective effects of exercise and phosphoinositide 3-kinase(p110alpha) signaling in dilated and hypertrophic cardiomyopathy.

Physical activity protects against cardiovascular disease, and physiological cardiac hypertrophy associated with regular exercise is usually beneficial, in marked contrast to pathological hypertrophy associated with disease. The p110alpha isoform of phosphoinositide 3-kinase (PI3K) plays a critical role in the induction of exercise-induced hypertrophy. Whether it or other genes activated in the athlete's heart might have an impact on cardiac function and survival in a setting of heart failure is unknown. To examine whether progressive exercise training and PI3K(p110alpha) activity affect survival and/or cardiac function in two models of heart disease, we subjected a transgenic mouse model of dilated cardiomyopathy (DCM) to swim training, genetically crossed cardiac-specific transgenic mice with increased or decreased PI3K(p110alpha) activity to the DCM model, and subjected PI3K(p110alpha) transgenics to acute pressure overload (ascending aortic constriction). Life-span, cardiac function, and molecular markers of pathological hypertrophy were examined. Exercise training and increased cardiac PI3K(p110alpha) activity prolonged survival in the DCM model by 15-20%. In contrast, reduced PI3K(p110alpha) activity drastically shortened lifespan by approximately 50%. Increased PI3K(p110alpha) activity had a favorable effect on cardiac function and fibrosis in the pressure-overload model and attenuated pathological growth. PI3K(p110alpha) signaling negatively regulated G protein-coupled receptor stimulated extracellular responsive kinase and Akt (via PI3K, p110gamma) activation in isolated cardiomyocytes. These findings suggest that exercise and enhanced PI3K(p110alpha) activity delay or prevent progression of heart disease, and that supraphysiologic activity can be beneficial. Identification of genes important for hypertrophy in the athlete's heart could offer new strategies for treating heart failure.

6-phosphogluconate dehydrogenase links oxidative PPP, lipogenesis and tumor growth by inhibiting LKB1-AMPK signaling

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB.

We show that NF-kappaB and transcriptional targets are activated in liver by obesity and high-fat diet (HFD). We have matched this state of chronic, subacute 'inflammation' by low-level activation of NF-kappaB in the liver of transgenic mice, designated LIKK, by selectively expressing constitutively active IKK-b in hepatocytes. These mice exhibit a type 2 diabetes phenotype, characterized by hyperglycemia, profound hepatic insulin resistance, and moderate systemic insulin resistance, including effects in muscle. The hepatic production of proinflammatory cytokines, including IL-6, IL-1beta and TNF-alpha, was increased in LIKK mice to a similar extent as induced by HFD in in wild-type mice. Parallel increases were observed in cytokine signaling in liver and mucscle of LIKK mice. Insulin resistance was improved by systemic neutralization of IL-6 or salicylate inhibition of IKK-beta. Hepatic expression of the IkappaBalpha superrepressor (LISR) reversed the phenotype of both LIKK mice and wild-type mice fed an HFD. These findings indicate that lipid accumulation in the liver leads to subacute hepatic 'inflammation' through NF-kappaB activation and downstream cytokine production. This causes insulin resistance both locally in liver and systemically.

WK175, a novel antitumor agent, decreases the intracellular nicotinamide adenine dinucleotide concentration and induces the apoptotic cascade in human leukemia cells.

We recently developed a class of novel antitumor agents that elicit a potent growth-inhibitory response in many tumor cells cultured in vitro. WK175, a member of this class, was chosen as a model compound that showed strong in vitro efficacy. WK175 interferes with the intracellular steady-state level of NAD(+), resulting in a decreased cellular NAD(+) concentration. We found that WK175 induces apoptotic cell death without any DNA-damaging effect. The apoptotic death signaling pathway initiated by WK175 was examined in detail: mitochondrial membrane potential, cytochrome c release, caspase 3 activation, caspase 3 and poly(ADP-ribose) polymerase cleavage, and the appearance of a sub-G(1) cell cycle population were determined in time course studies in THP-1 (a human monocytic leukemia cell line) cells. We found activation of this cascade after 24 h of treatment with 10 nM WK175. Induction of apoptosis was prevented by bongkrekic acid, Z-Asp-Glu-Val-Asp-fluoromethylketone, and Z-Leu-Glu-His-Asp-fluoromethylketone, inhibitors of the mitochondrial permeability transition and of caspase 3 and 9, respectively, but not by Ac-Tyr-Val-Ala-Asp-CHO, a specific caspase 1 inhibitor, suggesting the involvement of the permeability transition pore, caspase 3, and caspase 9 in the WK175-induced apoptotic cascade. These results imply that decreased NAD(+) concentration initiates the apoptotic cascade, resulting in the antitumor effect of WK175.

Resolution of acute inflammation in the lung.

Acute inflammation in the lung is essential to health. So too is its resolution. In response to invading microbes, noxious stimuli, or tissue injury, an acute inflammatory response is mounted to protect the host. To limit inflammation and prevent collateral injury of healthy, uninvolved tissue, the lung orchestrates the formation of specialized proresolving mediators, specifically lipoxins, resolvins, protectins, and maresins. These immunoresolvents are agonists for resolution that interact with specific receptors on leukocytes and structural cells to blunt further inflammation and promote catabasis. This process appears to be defective in several common lung diseases that are characterized by excess or chronic inflammation. Here, we review the molecular and cellular effectors of resolution of acute inflammation in the lung.

Integrin-linked kinase expression is elevated in human cardiac hypertrophy and induces hypertrophy in transgenic mice.

BACKGROUND Although numerous signaling pathways are known to be activated in experimental cardiac hypertrophy, the molecular basis of the hypertrophic response inherent in human heart diseases remains largely unknown. Integrin-linked kinase (ILK) is a multifunctional protein kinase that physically links beta-integrins with the actin cytoskeleton, suggesting a potential mechanoreceptor role. METHODS AND RESULTS Here, we show a marked increase in ILK protein levels in hypertrophic ventricles of patients with congenital and acquired outflow tract obstruction. This increase in ILK was associated with activation of the Rho family guanine triphosphatases, Rac1 and Cdc42, and known hypertrophic signaling kinases, including extracellular signal-related kinases (ERK1/2) and p70 S6 kinase. Transgenic mice with cardiac-specific expression of a constitutively active ILK (ILK(S343D)) or wild-type ILK (ILK(WT)) exhibited a compensated ventricular hypertrophic phenotype and displayed an activation profile of guanine triphosphatases and downstream protein kinases concordant with that seen in human hypertrophy. In contrast, transgenic mice with cardiomyocyte-restricted expression of a kinase-inactive ILK (ILK(R211A)) were unable to mount a compensatory hypertrophic response to angiotensin II in vivo. CONCLUSIONS Taken together, these results identify ILK-regulated signaling as a broadly adaptive hypertrophic response mechanism relevant to a wide range of clinical heart disease.

Redox-relevant aspects of the extracellular matrix and its cellular contacts via integrins.

SIGNIFICANCE The extracellular matrix (ECM) fulfills essential functions in multicellular organisms. It provides the mechanical scaffold and environmental cues to cells. Upon cell attachment, the ECM signals into the cells. In this process, reactive oxygen species (ROS) are physiologically used as signalizing molecules. RECENT ADVANCES ECM attachment influences the ROS-production of cells. In turn, ROS affect the production, assembly and turnover of the ECM during wound healing and matrix remodeling. Pathological changes of ROS levels lead to excess ECM production and increased tissue contraction in fibrotic disorders and desmoplastic tumors. Integrins are cell adhesion molecules which mediate cell adhesion and force transmission between cells and the ECM. They have been identified as a target of redox-regulation by ROS. Cysteine-based redox-modifications, together with structural data, highlighted particular regions within integrin heterodimers that may be subject to redox-dependent conformational changes along with an alteration of integrin binding activity. CRITICAL ISSUES In a molecular model, a long-range disulfide-bridge within the integrin β-subunit and disulfide bridges within the genu and calf-2 domains of the integrin α-subunit may control the transition between the bent/inactive and upright/active conformation of the integrin ectodomain. These thiol-based intramolecular cross-linkages occur in the stalk domain of both integrin subunits, whereas the ligand-binding integrin headpiece is apparently unaffected by redox-regulation. FUTURE DIRECTIONS Redox-regulation of the integrin activation state may explain the effect of ROS in physiological processes. A deeper understanding of the underlying mechanism may open new prospects for the treatment of fibrotic disorders.

Effective Use of PI3K and MEK Inhibitors to Treat Mutant K-Ras G12D and PIK3CA H1047R Murine Lung Cancers

Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-alpha catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-alpha mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-alpha H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.

Notch promotes survival of pre–T cells at the β-selection checkpoint by regulating cellular metabolism

Notch signals are necessary for the functional outcomes of T cell receptor β-selection, including differentiation, proliferation and rescue from apoptosis. The mechanism underlying this requirement for T cell development is unknown. Here we show that Notch receptor and Delta-like 1 ligand interactions promoted the survival of CD4−CD8− pre–T cells through the maintenance of cell size, glucose uptake and metabolism. Furthermore, the trophic effects of Notch signaling were mediated by the pathway of phosphatidylinositol-3-OH kinase and the kinase Akt, such that expression of active Atk overcame the requirement for Notch in β-selection. Collectively, our results demonstrate involvement of Notch receptor–ligand interactions in the regulation of cellular metabolism, thus enabling the autonomous signaling capacity of the pre–T cell receptor complex.*Note: In the version of this article initially published online, in the fourth sentence of the abstract, the term "Atk" was a misspelling; this should be "Akt. " In the fourth sentence of the second paragraph of the introduction, the name of the second kinase mentioned, "PI(3)K-dependent kinase 1," was incorrect; this should read "phosphoinositide-dependent kinase 1. " These errors have been corrected for the HTML and print versions of the article.

Cyclic AMP-dependent phosphorylation of thromboxane A(2) receptor-associated Galpha(13).

Although it is well established that cAMP inhibits platelet activation induced by all agonists, the thromboxane A(2) signal transduction pathway was found to be particularly sensitive to such inhibition. Therefore, we examined whether cAMP-dependent kinase mediates phosphorylation of the thromboxane A(2) receptor-G-protein complex. It was found that cAMP induces protein kinase A-dependent [gamma-(32)P]ATP labeling of solubilized membrane proteins in the region of Galpha subunits, i.e. 38-45 kDa. Moreover, ligand affinity chromatography purification of thromboxane A(2) receptor-G-protein complexes from these membranes revealed that 38-45-kDa phosphoproteins co-purify with thromboxane A(2) receptors. Immunoprecipitation of the affinity column eluate with a Galpha(13) antibody demonstrated that 8-Br-cAMP increased phosphorylation of thromboxane A(2) receptor-associated Galpha(13) by 87 +/- 27%. In separate experiments, immunopurification of Galpha(13) on microtiter wells coated with a different Galpha(13) antibody revealed that 8-Br-cAMP increased Galpha(13) phosphorylation by 53 +/- 19%. Finally, treatment of (32)P-labeled whole platelets with prostacyclin resulted in a 90 +/- 14% increase in phosphorylated Galpha(13) that was abolished by pretreatment with the adenylate cyclase inhibitor MDL-12. These results provide the first evidence that protein kinase A mediates phosphorylation of Galpha(13) both in vitro and in vivo and provides a basis for the preferential inhibition of thromboxane A(2)-mediated signaling in platelets by cAMP.

Acetic Acid Activates the AMP-Activated Protein Kinase Signaling Pathway to Regulate Lipid Metabolism in Bovine Hepatocytes

The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid) and BML-275 (an AMPKα inhibitor). Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

AMP-Activated Protein Kinase: An Ubiquitous Signaling Pathway With Key Roles in the Cardiovascular System

The AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis, which acts to restore energy homoeostasis whenever cellular energy charge is depleted. Over the last 2 decades, it has become apparent that AMPK regulates several other cellular functions and has specific roles in cardiovascular tissues, acting to regulate cardiac metabolism and contractile function, as well as promoting anticontractile, anti-inflammatory, and antiatherogenic actions in blood vessels. In this review, we discuss the role of AMPK in the cardiovascular system, including the molecular basis of mutations in AMPK that alter cardiac physiology and the proposed mechanisms by which AMPK regulates vascular function under physiological and pathophysiological conditions.

Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer

Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/β-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.

Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome

The ectopic expression of transcription factors can reprogram cell fate, yet it is unknown how the initial binding of factors to the genome relates functionally to the binding seen in the minority of cells that become reprogrammed. We report a map of Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) on the human genome during the first 48 hr of reprogramming fibroblasts to pluripotency. Three striking aspects of the initial chromatin binding events include an unexpected role for c-Myc in facilitating OSK chromatin engagement, the primacy of O, S, and K as pioneer factors at enhancers of genes that promote reprogramming, and megabase-scale chromatin domains spanned by H3K9me3, including many genes required for pluripotency, that prevent initial OSKM binding and impede the efficiency of reprogramming. We find diverse aspects of initial factor binding that must be overcome in the minority of cells that become reprogrammed.

Wortmannin, a phosphoinositide 3-kinase inhibitor, selectively enhances cytotoxicity of receptor-directed-toxin chimeras in vitro and in vivo.

BACKGROUND Generalized resistance of some neoplastic cell lines to treatment with ligand-toxin chimeras has been attributed to an increased rate of lysosomal uptake and degradation following endocytosis of the chimera-receptor complex. Because phosphoinositide 3-kinase (Pl 3-kinase) activity is known to play a role in intracellular trafficking, particularly from endosomes to lysosomes, we hypothesized that co-exposing cells to the Pl 3-kinase inhibitor, wortmannin, might enhance cytotoxicity of ligand-toxin chimeras. METHODS In vitro, cytotoxicity of five receptor directed-toxin chimeras (bFGF-SAP, bFGF-PE, aFGF-PE, HBEGF-SAP, bFGF-gelonin) and an immunotoxin (11A8-SAP) was examined in the presence or absence of this Pl 3-kinase inhibitor against a panel of human neoplastic cell lines: SK-MEL-5 (melanoma), PA-1 (ovarian teratocarcinoma), DU145 (prostatic carcinoma) and MCF-7 (breast carcinoma). In vivo, antitumor activity of a treatment regimen combining wortmannin (1 or 2 mg/kg i.p.) and bFGF-SAP (10 micrograms/kg i.v.) once a week for 4 weeks was evaluated compared to administration of each agent alone in C3H/HeN mice implanted with the FSallC murine fibrosarcoma. RESULTS At concentrations greater than the reported Ki for Pl 3-kinase inhibition (1-10 microM), wortmannin enhanced cytotoxicity when combined with saporin or gelonin chimeras, but produced subadditive cytotoxicity when combined with Pseudomonas exotoxin chimeras. When low nanomolar concentrations selective for Pl 3-kinase inhibition (5-100 nM) were examined for effects on one receptor directed-toxin chimera, wortmannin dramatically enhanced bFGF-SAP cytotoxicity in three of the four cell lines. A different Pl 3-kinase inhibitor, LY294002 (Ki approximately 1 microM), however, failed to potentiate bFGF-SAP. When administered to mice, wortmannin combined with bFGF-SAP resulted in a significant decrease in tumor volumes compared to vehicle-treated controls that was not observed in mice treated with either agent alone. CONCLUSIONS Taken together, these results suggest that although wortmannin increases the cytotoxic efficacy of some receptor-directed chimeras, potentiation may occur through an alternative pathway not involving Pl 3-kinase inhibition.

Matrix Remodeling Promotes Pulmonary Hypertension through Feedback Mechanoactivation of the YAP/TAZ-miR-130/301 Circuit.

Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPAR?-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.

Ly6Chi monocytes direct alternatively activated profibrotic macrophage regulation of lung fibrosis.

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a devastating disease. Antiinflammatory therapies, including corticosteroids, are of no benefit. The role of monocytes and macrophages is therefore controversial. OBJECTIVES To define the role of monocytes and macrophages during lung fibrogenesis and resolution, and explore the phenotype of the cells involved. METHODS We used multiple in vivo depletional strategies, backed up by adoptive transfer techniques. Further studies were performed on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS Depletion of lung macrophages during fibrogenesis reduced pulmonary fibrosis as measured by lung collagen (P = 0.0079); fibrosis score (P = 0.0051); and quantitative polymerase chain reaction for surrogate markers of fibrosis Col1 (P = 0.0083) and a-smooth muscle actin (P = 0.0349). There was an associated reduction in markers of the profibrotic alternative macrophage activation phenotype, Ym1 (P = 0.0179), and Arginase 1. The alternative macrophage marker CD163 was expressed on lung macrophages from patients with IPF. Depletion of Ly6Chi circulating monocytes reduced pulmonary fibrosis (P = 0.0052) and the number of Ym1- positive alternatively activated lung macrophages (P = 0.0310). Their adoptive transfer during fibrogenesis exacerbated fibrosis (P = 0.0304); however, adoptively transferred CD45.1 Ly6Chi cells were not found in the lungs of recipient CD45.2 mice. CONCLUSIONS We demonstrate the importance of circulating monocytes and lung macrophages during pulmonary fibrosis, and emphasize the importance of the alternatively activated macrophage phenotype. We show that Ly6Chi monocytes facilitate the progression of pulmonary fibrosis, but are not obviously engrafted into lungs thereafter. Finally, we provide empirical data to suggest that macrophages may have a resolution-promoting role during the reversible phase of bleomycin-induced pulmonary fibrosis.

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells.

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser(485/491) of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser(485/491) phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser(485/491) phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser(485/491) phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser(491) in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser(491) that plays a negative role in insulin signaling in muscle cells.

BRAF mutation predicts for MEK-dependence in non-small cell lung cancer (NSCLC).

5247 Constitutive ERK signaling is common in human cancer and is often the result of activating mutations of BRAF, RAS and upstream receptor tyrosine kinases. Missense BRAF kinase domain mutations are frequently observed in melanoma, colon and thyroid cancers and less frequently in lung and other cancer types. The vast majority (>90%) involve a glutamic acid for valine substitution at codon 600 (V600E), which results in elevated BRAF kinase activity. BRAF kinase domain mutations with intermediate and impaired kinase activity have also been identified, most frequently in NSCLC. We have previously reported that tumors with V600E BRAF mutation are selectively sensitive to MEK inhibition. Using the potent and selective MEK1/2 inhibitor PD0325901 (Pfizer), we examined a panel of NSCLC cell lines with mutant EGFR, KRAS, and/or low, intermediate and high-activity BRAF kinase domain mutations for MEK dependence. In all but one case, EGFR, KRAS and BRAF mutations were mutually exclusive with the exception being a cell line with concurrent NRAS and intermediate activity BRAF mutations. Consistent with our prior results, NSCLC cells with V600E BRAF mutation were exquisitely sensitive to MEK inhibition (PD0325901 IC50 of 2nM). The proliferation of cells with non-V600E mutations, including those with high (G469A), intermediate (L597V) and impaired (G466V) kinase activities, was also MEK dependent with IC50’s ranging between 2.7 and 80 nM. Inhibition of MEK in these cells resulted in downregulation of cyclin D1 and G1 growth arrest, with variable induction of apoptosis. Despite high basal ERK activity, NSCLC tumor cells with EGFR mutation were uniformly resistant to MEK inhibition (at doses of up to 500nM), despite effective and prolonged inhibition of ERK phosphorylation. Tumor cells with RAS mutation had a more variable response, with some cell lines demonstrating sensitivity, while others were completely resistant. There was no correlation between basal ERK activity and sensitivity to MEK inhibition. A strong inverse correlation between Akt activity and PD0325901 sensitivity was observed. These results suggest that MEK inhibition may be useful therapeutically in tumors with V600E and non-V600E BRAF kinase domain mutations. The results also suggest that inhibition of both MEK and Akt signaling may be required in NSCLC tumors with high basal AKT activity.

CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4(+) T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.

Myeloid-specific Krüppel-like factor 2 inactivation increases macrophage and neutrophil adhesion and promotes atherosclerosis.

RATIONALE Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. METHODS AND RESULTS Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2(-/-)) mice were similar to myeKlf2(+/+) macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2(+/+) macrophages, myeKlf2(-/-) macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2(-/-) mice also adhered more robustly to endothelial cells, and fewer myeKlf2(-/-) neutrophils survived in culture over a 24-hour period in comparison with myeKlf2(+/+) neutrophils. When myeKlf2(-/-) mice were mated to Ldlr(-/-) mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2(-/-)Ldlr(-/-) mice compared with myeKlf2(+/+)Ldlr(-/-) littermates. The increased atherosclerosis in myeKlf2(-/-)Ldlr(-/-) mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2(+/+)Ldlr(-/-) mice. CONCLUSIONS This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.

Nuclear Factor-�B Affects Tumor Progression in a Mouse Model of Malignant Pleural Effusion

We developed a novel mouse model of malignant pleural effusion (MPE) by injecting Lewis lung cancer (LLC) cells directly into the pleural space of syngeneic C57B/6 mice. The pleural effusions in this model share common cellular and biochemical features with human MPEs. Implantation and growth of pleural tumors triggers a host inflammatory response characterized by a mixed inflammatory cell influx into the pleural fluid. LLC cells exhibited high basal nuclear factor (NF)-κB activity in vitro and in vivo, which we used to drive expression of a NF-κB–dependent green fluorescent protein-firefly luciferase fusion reporter construct. NF-κB–dependent reporter expression allowed intravital tracing of pleural tumors. Inhibition of NF-κB in LLC cells did not affect cell viability in culture; however, injection of LLC cells expressing a dominant NF-κB inhibitor resulted in decreased tumor burden, decreased pleural effusion volume, and decreased pleural effusion TNF-α levels. These studies indicate that tumor NF-κB a...

Targeted blockade of TGF-β and IL-6/JAK2/STAT3 pathways inhibits lung cancer growth promoted by bone marrow-derived myofibroblasts

To investigate the role of TGF-β and IL-6 in myofibroblasts (MFs) - lung cancer cell interactions, lung cancer cells (Lewis and CTM-167 cell lines) were stimulated by IL-6, MF-conditioned medium (MF-CM) or MFs, with or without TGF-β signaling inhibitor - SB431542 and/or JAK2/STAT3 inhibitor - JSI-124. MFs were stimulated by TGF-β, cancer cell-CM or cancer cells, with or without SB431542 and JSI-124. Cell proliferation, the levels of cytokines, expression of mRNA and protein were determined. Mice bearing xenograft tumors were intraperitoneally treated with SB431542 or JSI-124 and monitored for up to 45 days. In co-culture systems, MFs secreted high levels of IL-6, while cancer cells produced high levels of TGF-β. Recombinant IL-6 and MF-CM activated STAT3 and upregulated TGF-β in cancer cells. In contrast, cancer cell-CM or TGF-β stimulated MFs to produce IL-6. Blockade of JAK2/STAT3 and TGF-β signaling by specific inhibitors significantly inhibited cell proliferation in vitro and tumor growth in vivo of lung cancer cells. Our study demontrated that the TGF-β and IL-6/JAK2/STAT3 signaling pathways form a positive feedback signaling loop that mediated the interactions between MFs and lung cancer cells. Targeted inhibiton of this signaling loop could be a new approach for lung cancer prevention and therapy.

Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo.

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4(-/-) fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4(+/+) cells in competitive repopulation assays. However, hematopoietic "KLF4(-/-) chimeras" generated by transplantation of KLF4(-/-) fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115(+)Gr1(+)) monocytes, and had reduced numbers of resident (CD115(+)Gr1(-)) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4(-/-) chimeras, bone marrow monocytic cells from KLF4(-/-) chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding.

The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.

Disruption of the Hepcidin/Ferroportin Regulatory System Causes Pulmonary Iron Overload and Restrictive Lung Disease

Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.

Neutrophil elastase-mediated lung disease.

Elastases of both the neutrophil and macrophage have been implicated in lung disease initiation and progression. Although it is unlikely that these proteases evolved for the purpose of injuring lung tissue, the elastin-rich connective tissue framework of the lungs appears to be particularly susceptible to the action of elastolytic proteases. Assuming that neutrophil elastase most likely plays a role in the migration of neutrophils toward a site of inflammation and degradation of proteins from invading organisms or other products of the inflammatory response, it is the role of inhibitors of this protease to protect normal tissues from its effects. In alpha-1 antitrypsin deficiency we find an experiment of nature that disrupts this protease-anti-protease balance, resulting in an increased risk of destructive lung disease.

Identification of secreted proteins that mediate cell-cell interactions in an in vitro model of the lung cancer microenvironment.

Non-small cell lung cancer (NSCLC) cells with somatic mutations in K-ras recruit to the tumor a variety of cell types (hereafter collectively termed "stromal cells") that can promote or inhibit tumorigenesis by mechanisms that have not been fully elucidated. Here, we postulated that stromal cells in the tumor microenvironment alter the tumor cell secretome, including those proteins required for tumor growth and dissemination, and we developed an in vitro model to test this hypothesis. Coculturing a murine K-ras mutant lung adenocarcinoma cell line (LKR-13) with a murine lung stromal cell (macrophage, endothelial cell, or fibroblast) enhanced stromal cell migration, induced endothelial tube formation, increased LKR-13 cell proliferation, and regulated the secretion of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial-to-mesenchymal transition. Among these proteins, CXCL1 has been reported to promote NSCLC development, whereas interleukin-18 (IL-18) has an undefined role. Genetic and pharmacologic strategies to inhibit CXCL1 and IL-18 revealed that stromal cell migration, LKR-13 cell proliferation, and LKR-13 cell tumorigenicity required one or both of these proteins. We conclude that stromal cells enhanced LKR-13 cell tumorigenicity partly through their effects on the secretome of LKR-13 cells. Strategies to inhibit tumor/stromal cell interactions may be useful as therapeutic approaches in NSCLC patients.

High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling.

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung.

The mechanisms by which a primary tumor affects a selected distant organ before tumor cell arrival remain to be elucidated. This report shows that Gr-1+CD11b+ cells are significantly increased in lungs of mice bearing mammary adenocarcinomas before tumor cell arrival. In the premetastatic lungs, these immature myeloid cells significantly decrease IFN-gamma production and increase proinflammatory cytokines. In addition, they produce large quantities of matrix metalloproteinase 9 (MMP9) and promote vascular remodeling. Deletion of MMP9 normalizes aberrant vasculature in the premetastatic lung and diminishes lung metastasis. The production and activity of MMP9 is selectively restricted to lungs and organs with a large number of Gr-1+CD11b+ cells. Our work reveals a novel protumor mechanism for Gr-1+CD11b+ cells that changes the premetastatic lung into an inflammatory and proliferative environment, diminishes immune protection, and promotes metastasis through aberrant vasculature formation. Thus, inhibition of Gr-1+CD11b+ cells could normalize the premetastatic lung environment, improve host immunosurveillance, and inhibit tumor metastasis.

Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells.

Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence.

Cellular senescence is considered a major tumour-suppressor mechanism in mammals, and many oncogenic insults, such as the activation of the ras proto-oncogene, trigger initiation of the senescence programme. Although it was shown that activation of the senescence programme involves the up-regulation of cell-cycle regulators such as the inhibitors of cyclin-dependent kinases p16INK4A and p21CIP-1, the mechanisms underlying the senescence response remain to be resolved. In the case of stress-induced premature senescence, reactive oxygen species are considered important intermediates contributing to the phenotype. Moreover, distinct alterations of the cellular carbohydrate metabolism are known to contribute to oncogenic transformation, as is best documented for the phenomenon of aerobic glycolysis. These findings suggest that metabolic alterations are involved in tumourigenesis and tumour suppression; however, little is known about the metabolic pathways that contribute to these processes. Using the human fibroblast model of in vitro senescence, we analysed age-dependent changes in the cellular carbohydrate metabolism. Here we show that senescent fibroblasts enter into a metabolic imbalance, associated with a strong reduction in the levels of ribonucleotide triphosphates, including ATP, which are required for nucleotide biosynthesis and hence proliferation. ATP depletion in senescent fibroblasts is due to dysregulation of glycolytic enzymes, and finally leads to a drastic increase in cellular AMP, which is shown here to induce premature senescence. These results suggest that metabolic regulation plays an important role during cellular senescence and hence tumour suppression.

Coupling cAMP signaling to transcription in the liver: pivotal role of CREB and CREM.

Transcriptional factors binding to cAMP-responsive elements (CREs) in the promoters of various genes belong to the basic domain-leucine zipper superfamily and are composed of three genes in mammals, CREB, CREM, and ATF-1. A large number of CREB, CREM, and ATF-1 proteins are generated by posttranscriptional events, mostly alternative splicing, and regulate gene expression by acting as activators or repressors. Activation is classically brought about by signaling-dependent phosphorylation of a key acceptor site (Ser133 in CREB) by a number of possible kinases, including PKA, CamKIV, and Rsk-2. Phosphorylation is the prerequisite for the interaction of CBP (CREB-binding protein), a co-activator that has also histone acetyltransferase activity. Repression may involve dynamic dephosphorylation of the activators and thus decreased association with CBP. Another pathway of transcriptional repression on CRE sites implicates the inducible repressor ICER (inducible cAMP early repressor), a product of the CREM gene. Being an inducible repressor, ICER is involved in autoregulatory feedback loops of transcription that govern the down-regulation of early response genes, such as the proto-oncogene c-fos. The liver represents a remarkable physiological setting where cAMP-responsive signaling plays a major role. Indeed, a finely tuned program of gene expression is triggered by partial hepatectomy, so that through specific checkpoints a coordinated regeneration of the tissue is obtained. Temporal kinetics of transcriptional activation after hepatectomy reveals a pattern of early induction for several genes, some of them controlled by the CREB/CREM transcription factors. An important role of CREM in liver physiology was suggested by the robust induction of ICER after partial hepatectomy. The delay in tissue regeneration in CREM-deficient mice confirmed the important function of this factor in regulating hepatocyte proliferation. As gene induction is accompanied by critical changes in chromatin organization, the deciphering of the specific modification codes that histones display during liver regeneration and physiology will provide exciting new insights into the dynamics of chromatin architecture.

Targeted nanoparticles containing the proresolving peptide Ac2-26 protect against advanced atherosclerosis in hypercholesterolemic mice

Chronic, nonresolving inflammation is a critical factor in the clinical progression of advanced atherosclerotic lesions. In the normal inflammatory response, resolution is mediated by several agonists, among which is the glucocorticoid-regulated protein called annexin A1. The proresolving actions of annexin A1, which are mediated through its receptor N-formyl peptide receptor 2 (FPR2/ALX), can be mimicked by an amino-terminal peptide encompassing amino acids 2–26 (Ac2-26). Collagen IV (Col IV)–targeted nanoparticles (NPs) containing Ac2-26 were evaluated for their therapeutic effect on chronic, advanced atherosclerosis in fat-fed Ldlr−/− mice. When administered to mice with preexisting lesions, Col IV–Ac2-26 NPs were targeted to lesions and led to a marked improvement in key advanced plaque properties, including an increase in the protective collagen layer overlying lesions (which was associated with a decrease in lesional collagenase activity), suppression of oxidative stress, and a decrease in plaque necrosis. In mice lacking FPR2/ALX in myeloid cells, these improvements were not seen. Thus, administration of a resolution-mediating peptide in a targeted NP activates its receptor on myeloid cells to stabilize advanced atherosclerotic lesions. These findings support the concept that defective inflammation resolution plays a role in advanced atherosclerosis, and suggest a new form of therapy.

Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts.

Activated fibroblasts are associated with many different tumors. Myofibroblasts, activated fibroblasts, and perivascular mesenchymal cells such as pericytes play a role in cancer progression. Many studies suggest that myofibroblasts facilitate tumor growth and cancer progression. The source for myofibroblasts and other activated fibroblasts within the tumors is still debated. Although de novo activation of quiescent fibroblasts into alpha-smooth muscle actin (alpha SMA)-positive myofibroblasts is one likely source, epithelial to mesenchymal transition and bone marrow recruitment are also evolving as possible mechanisms for the emergence of a heterogeneous population of carcinoma-associated fibroblasts. Here, we show that transforming growth factor-beta1 could induce proliferating endothelial cells to undergo a phenotypic conversion into fibroblast-like cells. Such endothelial to mesenchymal transition (EndMT) is associated with the emergence of mesenchymal marker fibroblast-specific protein-1 (FSP1) and down-regulation of CD31/PECAM. Additionally, we show EndMT in tumors using the B16F10 melanoma model and the Rip-Tag2 spontaneous pancreatic carcinoma model. Crossing Tie2-Cre mice with R26Rosa-lox-Stop-lox-LacZ mice allows for irreversible tagging of endothelial cells. We provide unequivocal evidence for EndMT at the invasive front of the tumors in these transgenic mice. Collectively, our results show that EndMT is a unique mechanism for the accumulation of carcinoma-associated fibroblasts and suggest that antiangiogenic treatment of tumors may have a direct effect in decreasing activated fibroblasts that likely facilitate cancer progression.

Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer.

Activating mutations in the EGF receptor (EGFR) are associated with clinical responsiveness to EGFR tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib. However, resistance eventually arises, often due to a second EGFR mutation, most commonly T790M. Through a genome-wide siRNA screen in a human lung cancer cell line and analyses of murine mutant EGFR-driven lung adenocarcinomas, we found that erlotinib resistance was associated with reduced expression of neurofibromin, the RAS GTPase-activating protein encoded by the NF1 gene. Erlotinib failed to fully inhibit RAS-ERK signaling when neurofibromin levels were reduced. Treatment of neurofibromin-deficient lung cancers with a MAP-ERK kinase (MEK) inhibitor restored sensitivity to erlotinib. Low levels of NF1 expression were associated with primary and acquired resistance of lung adenocarcinomas to EGFR TKIs in patients. These findings identify a subgroup of patients with EGFR-mutant lung adenocarcinoma who might benefit from combination therapy with EGFR and MEK inhibitors.

Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway

Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

miR-205 hinders the malignant interplay between prostate cancer cells and associated fibroblasts.

AIMS Tumor microenvironment is a strong determinant for the acquisition of metastatic potential of cancer cells. We have recently demonstrated that cancer-associated fibroblasts (CAFs) elicit a redox-dependent epithelial-mesenchymal transition (EMT) in prostate cancer (PCa) cells, driven by cycloxygenase-2/hypoxia-inducible factor-1 (HIF-1)/nuclear factor-κB pathway and enhancing tumor aggressiveness. Here, we investigated the involvement of microRNAs (miRNAs) in tumor-stroma interplay to identify possible tools to counteract oxidative stress and metastasis dissemination. RESULTS We found that miR-205 is the most downmodulated miRNA in PCa cells upon CAF stimulation, due to direct transcriptional repression by HIF-1, a known redox-sensitive transcription factor. Rescue experiments demonstrated that ectopic miR-205 overexpression in PCa cells counteracts CAF-induced EMT, thus impairing enhancement of cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. In addition, miR-205 blocks tumor-driven activation of surrounding fibroblasts by reducing pro-inflammatory cytokine secretion. INNOVATION Overall, such findings suggest miR-205 as a brake against PCa metastasis by blocking both the afferent and efferent arms of the circuit between tumor cells and associated fibroblasts, thus interrupting the pro-oxidant and pro-inflammatory circuitries engaged by reactive stroma. CONCLUSION The evidence that miR-205 replacement in PCa cells is able not only to prevent but also to revert the oxidative/pro-inflammatory axis leading to EMT induced by CAFs sets the rationale for developing miRNA-based approaches to prevent and treat metastatic disease.

Alteration of the Antitumor Immune Response by Cancer-Associated Fibroblasts

Among cells present in the tumor microenvironment, activated fibroblasts termed cancer-associated fibroblasts (CAFs), play a critical role in the complex process of tumor-stroma interaction. CAFs, one of the prominent stromal cell populations in most types of human carcinomas, have been involved in tumor growth, angiogenesis, cancer stemness, extracellular matrix remodeling, tissue invasion, metastasis, and even chemoresistance. During the past decade, these activated tumor-associated fibroblasts have also been involved in the modulation of the anti-tumor immune response on various levels. In this review, we describe our current understanding of how CAFs accomplish this task as well as their potential therapeutic implications.

Deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice

Although mast cell functions have classically been related to allergic responses, recent studies indicate that these cells contribute to other common diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, aortic aneurysm and cancer. This study presents evidence that mast cells also contribute to diet-induced obesity and diabetes. For example, white adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Furthermore, in the context of mice on a Western diet, genetically induced deficiency of mast cells, or their pharmacological stabilization, reduces body weight gain and levels of inflammatory cytokines, chemokines and proteases in serum and WAT, in concert with improved glucose homeostasis and energy expenditure. Mechanistic studies reveal that mast cells contribute to WAT and muscle angiogenesis and associated cell apoptosis and cathepsin activity. Adoptive transfer experiments of cytokine-deficient mast cells show that these cells, by producing interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), contribute to mouse adipose tissue cysteine protease cathepsin expression, apoptosis and angiogenesis, thereby promoting diet-induced obesity and glucose intolerance. Our results showing reduced obesity and diabetes in mice treated with clinically available mast cell-stabilizing agents suggest the potential of developing new therapies for these common human metabolic disorders.

An integrin β3–KRAS–RalB complex drives tumour stemness and resistance to EGFR inhibition

Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, αvβ3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, αvβ3 expression and the resulting KRAS–RalB–NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains.

Early region 3 (E3) of group C human adenoviruses (Ad) encodes several inhibitors of tumor necrosis factor alpha (TNF-alpha) cytolysis, including an E3 14.7-kDa protein (E3-14.7K) and a heterodimer containing two polypeptides of 10.4 and 14.5 kDa. To understand the mechanism by which the viral proteins inhibit TNF-alpha functions, the E3-14.7K protein was used to screen a HeLa cell cDNA library to search for interacting proteins in the yeast two-hybrid system. A novel protein containing multiple leucine zipper domains without any significant homology with any known protein was identified and has been named FIP-2 (for 14.7K-interacting protein). FIP-2 interacted with E3-14.7K both in vitro and in vivo. It colocalized with Ad E3-14.7K in the cytoplasm, especially near the nuclear membrane, and caused redistribution of the viral protein. FIP-2 by itself does not cause cell death; however, it can reverse the protective effect of E3-14.7K on cell killing induced by overexpression of the intracellular domain of the 55-kDa TNF receptor or by RIP, a death protein involved in the TNF-alpha and Fas apoptosis pathways. Deletion analysis indicates that the reversal effect of FIP-2 depends on its interaction with E3-14.7K. Three major mRNA forms of FIP-2 have been detected in multiple human tissues, and expression of the transcripts was induced by TNF-alpha treatment in a time-dependent manner in two different cell lines. FIP-2 has consensus sequences for several potential posttranslational modifications. These data suggest that FIP-2 is one of the cellular targets for Ad E3-14.7K and that its mechanism of affecting cell death involves the TNF receptor, RIP, or a downstream molecule affected by either of these two molecules.

TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.

Dendritic Cell KLF2 Expression Regulates T Cell Activation and Proatherogenic Immune Responses.

Dendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including atherosclerosis. However, the molecular mechanisms by which DCs mitigate or promote inflammatory pathogenesis are only partially understood. Previous studies have shown an important anti-inflammatory role for the transcription factor Krüppel-like factor 2 (KLF2) in regulating activation of various cell types that participate in atherosclerotic lesion development, including endothelial cells, macrophages, and T cells. We used a pan-DC, CD11c-specific cre-lox gene knockout mouse model to assess the role of KLF2 in DC activation, function, and control of inflammation in the context of hypercholesterolemia and atherosclerosis. We found that KLF2 deficiency enhanced surface expression of costimulatory molecules CD40 and CD86 in DCs and promoted increased T cell proliferation and apoptosis. Transplant of bone marrow from mice with KLF2-deficient DCs into Ldlr-/- mice aggravated atherosclerosis compared with control mice, most likely due to heightened vascular inflammation evidenced by increased DC presence within lesions, enhanced T cell activation and cytokine production, and increased cell death in atherosclerotic lesions. Taken together, these data indicate that KLF2 governs the degree of DC activation and hence the intensity of proatherogenic T cell responses.

Post-translational modifications of the beta-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization.

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic alpha subunit and regulatory beta and gamma subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the alpha subunit. The beta subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the beta-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the beta subunit from a particulate extranuclear distribution to a more homogenous cell distribution. Mutation of the serine-108 phosphorylation site to alanine is associated with enzyme inhibition, but no change in cell localization. In contrast, the phosphorylation site mutations, SS24, 25AA and S182A, while having no effects on enzyme activity, are associated with nuclear redistribution of the subunit. Taken together, these results indicate that both myristoylation and phosphorylation of the beta subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.

LIN28B enhanced tumorigenesis in an autochthonous KRASG12V-driven lung carcinoma mouse model

LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.

Pharmacological correction of a defect in PPARγ signaling ameliorates disease severity in Cftr-deficient mice

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (encoded by Cftr) that impair its role as an apical chloride channel that supports bicarbonate transport. Individuals with cystic fibrosis show retained, thickened mucus that plugs airways and obstructs luminal organs as well as numerous other abnormalities that include inflammation of affected organs, alterations in lipid metabolism and insulin resistance. Here we show that colonic epithelial cells and whole lung tissue from Cftr-deficient mice show a defect in peroxisome proliferator-activated receptor-gamma (PPAR-gamma, encoded by Pparg) function that contributes to a pathological program of gene expression. Lipidomic analysis of colonic epithelial cells suggests that this defect results in part from reduced amounts of the endogenous PPAR-gamma ligand 15-keto-prostaglandin E(2) (15-keto-PGE(2)). Treatment of Cftr-deficient mice with the synthetic PPAR-gamma ligand rosiglitazone partially normalizes the altered gene expression pattern associated with Cftr deficiency and reduces disease severity. Rosiglitazone has no effect on chloride secretion in the colon, but it increases expression of the genes encoding carbonic anhydrases 4 and 2 (Car4 and Car2), increases bicarbonate secretion and reduces mucus retention. These studies reveal a reversible defect in PPAR-gamma signaling in Cftr-deficient cells that can be pharmacologically corrected to ameliorate the severity of the cystic fibrosis phenotype in mice.

Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration.

Acute skeletal muscle injury triggers an expansion of fibro/adipogenic progenitors (FAPs) and a transient stage of fibrogenesis characterized by extracellular matrix deposition. While the perpetuation of such phase can lead to permanent tissue scarring, the consequences of its suppression remain to be studied. Using a model of acute muscle damage we were able to determine that pharmacological inhibition of FAP expansion by Nilotinib, a tyrosine kinase inhibitor with potent antifibrotic activity, exerts a detrimental effect on myogenesis during regeneration. We found that Nilotinib inhibits the damage-induced expansion of satellite cells in vivo, but it does not affect in vitro proliferation, suggesting a non cell-autonomous effect. Nilotinib impairs regenerative fibrogenesis by preventing the injury-triggered expansion and differentiation of resident CD45(-):CD31(-):α7integrin(-):Sca1(+) mesenchymal FAPs. Our data support the notion that the expansion of FAPs and transient fibrogenesis observed during regeneration play an important trophic role toward tissue-specific stem cells.

Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells.

We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis.

The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

EMSY overexpression disrupts the BRCA2/RAD51 pathway in the DNA-damage response: implications for chromosomal instability/recombination syndromes as checkpoint diseases

EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention.

p38MAPK controls expression of multiple cell cycle inhibitors and islet proliferation with advancing age.

Aging is a complex organismal process that is controlled by genetic, environmental, and behavioral factors. Accumulating evidence supports a role for different cell cycle inhibitors in mammalian aging. Little is known, however, about the upstream signals that induce their expression. Here, we explore the role of p38MAPK by generating a dominant-negative allele (p38(AF)) in which activating phosphorylation sites Thr180 and Tyr182 are mutated. Heterozygous p38(AF) mice show a marked attenuation of p38-dependent signaling and age-induced expression of multiple cell cycle inhibitors in different organs, including pancreatic islets. As a result, aged p38(AF/+) mice show enhanced proliferation and regeneration of islets when compared to wild-type littermates. We further find an age-related reduction in expression of the p38-specific phosphatase Wip1. Wip1-deficient mice demonstrate decreased islet proliferation, while Wip1 overexpression rescues aging-related decline in proliferation and regenerative capacity. We propose that modulation of p38MAPK activity may provide new avenues for treating certain age-related degenerative diseases.

Cancer-related inflammation

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumours. In some types of cancer, inflammatory conditions are present before a malignant change occurs. Conversely, in other types of cancer, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumours. Regardless of its origin, 'smouldering' inflammation in the tumour microenvironment has many tumour-promoting effects. It aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, subverts adaptive immune responses, and alters responses to hormones and chemotherapeutic agents. The molecular pathways of this cancer-related inflammation are now being unravelled, resulting in the identification of new target molecules that could lead to improved diagnosis and treatment.

LIN28 Regulates Stem Cell Metabolism and Conversion to Primed Pluripotency.

The RNA-binding proteins LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency, but their exact functions are poorly understood. Here, we show that, like LIN28A, LIN28B can function effectively with NANOG, OCT4, and SOX2 in reprogramming to pluripotency and that reactivation of both endogenous LIN28A and LIN28B loci are required for maximal reprogramming efficiency. In human fibroblasts, LIN28B is activated early during reprogramming, while LIN28A is activated later during the transition to bona fide induced pluripotent stem cells (iPSCs). In murine cells, LIN28A and LIN28B facilitate conversion from naive to primed pluripotency. Proteomic and metabolomic analysis highlighted roles for LIN28 in maintaining the low mitochondrial function associated with primed pluripotency and in regulating one-carbon metabolism, nucleotide metabolism, and histone methylation. LIN28 binds to mRNAs of proteins important for oxidative phosphorylation and modulates protein abundance. Thus, LIN28A and LIN28B play cooperative roles in regulating reprogramming, naive/primed pluripotency, and stem cell metabolism.

The precursor form of IL-1alpha is an intracrine proinflammatory activator of transcription.

Although most cytokines are studied for biological effects after engagement of their specific cell surface membrane receptors, increasing evidence suggests that some function in the nucleus. In the present study, the precursor form of IL-1alpha was overexpressed in various cells and assessed for activity in the presence of saturating concentrations of IL-1 receptor antagonist to prevent receptor signaling. Initially diffusely present in the cytoplasm of resting cells, IL-1alpha translocated to the to nucleus after activation by endotoxin, a Toll-like receptor ligand. The IL-1alpha precursor, but not the C-terminal mature form, activated the transcriptional machinery in the GAL4 system by 90-fold; a 50-fold increase was observed using only the IL-1alpha propiece, suggesting that transcriptional activation was localized to the N terminus where the nuclear localization sequence resides. Under conditions of IL-1 receptor blockade, intracellular overexpression of the precursor and propiece forms of IL-1alpha were sufficient to activate NF-kappaB and AP-1. Stable transfectants overproducing precursor IL-1alpha released the cytokines IL-8 and IL-6 but also exhibited a significantly lower threshold of activation to subpicomolar concentrations of tumor necrosis factor alpha or IFN-gamma. Thus, intracellular functions of IL-1alpha might play an unforeseen role in the genesis of inflammation. During disease-driven events, the cytosolic precursor moves to the nucleus, where it augments transcription of proinflammatory genes. Because this mechanism of action is not affected by extracellular inhibitors, reducing intracellular functions of IL-1alpha might prove beneficial in some inflammatory conditions.

CD11b+ monocytes abrogate Th17 CD4+ T cell-mediated experimental autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) represents a Th17 T cell-mediated mouse model of postinflammatory heart disease. In BALB/c wild-type mice, EAM is a self-limiting disease, peaking 21 days after alpha-myosin H chain peptide (MyHC-alpha)/CFA immunization and largely resolving thereafter. In IFN-gammaR(-/-) mice, however, EAM is exacerbated and shows a chronic progressive disease course. We found that this progressive disease course paralleled persistently elevated IL-17 release from T cells infiltrating the hearts of IFN-gammaR(-/-) mice 30 days after immunization. In fact, IL-17 promoted the recruitment of CD11b(+) monocytes, the major heart-infiltrating cells in EAM. In turn, CD11b(+) monocytes suppressed MyHC-alpha-specific Th17 T cell responses IFN-gamma-dependently in vitro. In vivo, injection of IFN-gammaR(+/+)CD11b(+), but not IFN-gammaR(-/-)CD11b(+), monocytes, suppressed MyHC-alpha-specific T cells, and abrogated the progressive disease course in IFN-gammaR(-/-) mice. Finally, coinjection of MyHC-alpha-specific, but not OVA-transgenic, IFN-gamma-releasing CD4(+) Th1 T cell lines, together with MyHC-alpha-specific Th17 T cells protected RAG2(-/-) mice from EAM. In conclusion, CD11b(+) monocytes play a dual role in EAM: as a major cellular substrate of IL-17-induced inflammation and as mediators of an IFN-gamma-dependent negative feedback loop confining disease progression.

Non-receptor tyrosine kinase Etk is involved in the apoptosis of small cell lung cancer cells.

Epithelial and endothelial tyrosine kinase (Etk), also known as Bmx (bone marrow X kinase) plays an important role in apoptosis of epithelial cells. The goal of this study was to investigate whether Etk is involved in apoptosis of small cell lung cancer (SCLC) cells and correlated with the expression levels of apoptosis-associated proteins such as Bcl-2, Bcl-X(L) and p53. One hundred and seventy-one cases of lung cancer specimens including seventy-one SCLCs and one hundred NSCLCs were immunostained for Etk, Bcl-2, Bcl-X(L) and p53. Parental SCLC H446 cell line, and its subline (H446-Etk) that overexpresses Etk, were used to study the role of Etk in apoptosis induced by doxorubicin. It was found that high expression of Etk occurs in 74.6% of SCLC cases, but only in 40% of NSCLC cases, and there is marked difference in the expression levels of Bcl-2, Bcl-X(L) and p53 between Etk-positive and Etk-negative SCLC cases. Furthermore, the levels of Bcl-2 and Bcl-X(L) significantly increased in H446-Etk cells than that in H446 cells after doxorubicin treatment, and were positively associated with Etk expression. However, p53 did not correspond with Etk expression although its expression decreased greatly with apoptosis both in H446-Etk and H446 cells. After doxorubicin treatment, the cell viability was significantly higher in H446-Etk cells than in parental H446 cells. Downregulation of Etk by Etk siRNA sensitized H446 cells to doxorubicin. Our results indicate that upregulation of tyrosine kinase Etk may be a new mechanism involved in protection of SCLC cells from apoptosis. Bcl-2 and Bcl-X(L) but not p53 may contribute to doxorubicin-induced apoptosis through Etk pathway.

Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis.

Myelin-directed autoimmunity is considered to play a key role in the pathogenesis of multiple sclerosis (MS). Increased production of both pro- and anti-inflammatory cytokines is a common finding in MS. Interleukin-17 (IL-17) is a recently described cytokine produced in humans almost exclusively by activated memory T cells, which can induce the production of proinflammatory cytokines and chemokines from parenchymal cells and macrophages. In situ hybridisation with synthetic oligonucleotide probes was adopted to detect and enumerate IL-17 mRNA expressing mononuclear cells (MNC) in blood and cerebrospinal fluid (CSF) from patients with MS and control individuals. Numbers of IL-17 mRNA expressing blood MNC were higher in patients with MS and acute aseptic meningoencephalitis (AM) compared to healthy individuals. Higher numbers of IL-17 mRNA expressing blood MNC were detected in MS patients examined during clinical exacerbation compared to remission. Patients with MS had higher numbers of IL-17 mRNA expressing MNC in CSF compared to blood. This increase in numbers of IL-17 mRNA expressing MNC in CSF was not observed in patients with AM. Our results thus demonstrate increased numbers of IL-17 mRNA expressing MNC in MS with higher numbers in CSF than blood, and with the highest numbers in blood during clinical exacerbations.

[Neutrophils and macrophages related to the pathogenesis and disease development of chronic obstructive pulmonary disease by the inflammatory reaction].

Chronic obstructive pulmonary disease (COPD) is a chronic airway disorder characterized by obstructive airflow limitation which is not completely reversible with treatment. Inflammatory changes in the peripheral airways, especially those with the diameter less than 2mm (so-called small airway disease) have been speculated to be initial steps of COPD. And so it must be quite clear that neutrophils and macrophages play an essential role in the pathogenesis of these lesions. Studies with bronchoalveolar lavage demonstrated an increase in neutrophil numbers and the neutrophil chemoattractant interleukin-8. Recent studies demonstrated that neutrophils and macrophages are increased and contain a variety of proteases, which are involved in cell infiltration and activation. Studies with gene-engineered animals and anti-cytokine treatment will facilitate better understanding the role of neutrophils and macrophages, and eventual novel therapy.

Kruppel-like factor 2 (KLF2) regulates monocyte differentiation and functions in mBSA and IL-1β-induced arthritis.

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b(+)F4/80(+)Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion.

Early local tumor invasion in breast cancer results in a likely encounter between cancer cells and mature adipocytes, but the role of these fat cells in tumor progression remains unclear. We show that murine and human tumor cells cocultivated with mature adipocytes exhibit increased invasive capacities in vitro and in vivo, using an original two-dimensional coculture system. Likewise, adipocytes cultivated with cancer cells also exhibit an altered phenotype in terms of delipidation and decreased adipocyte markers associated with the occurrence of an activated state characterized by overexpression of proteases, including matrix metalloproteinase-11, and proinflammatory cytokines [interleukin (IL)-6, IL-1β]. In the case of IL-6, we show that it plays a key role in the acquired proinvasive effect by tumor cells. Equally important, we confirm the presence of these modified adipocytes in human breast tumors by immunohistochemistry and quantitative PCR. Interestingly, the tumors of larger size and/or with lymph nodes involvement exhibit the higher levels of IL-6 in tumor surrounding adipocytes. Collectively, all our data provide in vitro and in vivo evidence that (i) invasive cancer cells dramatically impact surrounding adipocytes; (ii) peritumoral adipocytes exhibit a modified phenotype and specific biological features sufficient to be named cancer-associated adipocytes (CAA); and (iii) CAAs modify the cancer cell characteristics/phenotype leading to a more aggressive behavior. Our results strongly support the innovative concept that adipocytes participate in a highly complex vicious cycle orchestrated by cancer cells to promote tumor progression that might be amplified in obese patients.

Integrins promote cytokinesis through the RSK signaling axis.

Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.

DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress

The stress-activated kinase JNK mediates key cellular responses to oxidative stress. Here we show that DAP kinase (DAPk), a cell death promoting Ser/Thr protein kinase, plays a main role in oxidative stress-induced JNK signaling. We identify protein kinase D (PKD) as a novel substrate of DAPk and demonstrate that DAPk physically interacts with PKD in response to oxidative stress. We further show that DAPk activates PKD in cells and that induction of JNK phosphorylation by ectopically expressed DAPk can be attenuated by knocking down PKD expression or by inhibiting its catalytic activity. Moreover, knockdown of DAPk expression caused a marked reduction in JNK activation under oxidative stress, indicating that DAPk is indispensable for the activation of JNK signaling under these conditions. Finally, DAPk is shown to be required for cell death under oxidative stress in a process that displays the characteristics of caspase-independent necrotic cell death. Taken together, these findings establish a major role for DAPk and its specific interaction with PKD in regulating the JNK signaling network under oxidative stress.

Phosphoinositide 3-kinase signalling--which way to target?

Abstract Phosphoinositide 3-kinases (PI3Ks) are central to the control of cell growth, proliferation and survival, and drive the progression of tumours by activating phosphoinositide-dependent kinase, protein kinase B and the target of rapamycin. Other downstream effectors link PI3K to cell motility and the control of cardiovascular parameters. Current knowledge indicates that PI3Ks might qualify as drug targets for the treatment of cancer, chronic inflammation, allergy and cardiovascular failure. However, PI3Ks also modulate vital processes such as metabolic control and nutrient uptake. Here, mechanistic data and mouse phenotypic analyses are summarised, and the possible success of therapeutic inhibition of distinct PI3K isoforms is discussed.

Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation.

Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion

BACKGROUND Metformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms. METHODS Ninety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways. RESULTS Infarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect. CONCLUSIONS Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

mir-1-mediated paracrine effect of cancer-associated fibroblasts on lung cancer cell proliferation and chemoresistance.

Lung cancer is the leading cause of cancer-related mortality in humans worldwide. Moreover, the overall 5-year survival rate is only 15%. Pathologically almost 80% of all lung cancer cases are non-small cell lung cancer (NSCLC). Cancer-associated fibroblasts (CAFs) have been found to exist in a large number of NSCLCs. CAFs have been proven to promote tumor progression, metastasis and resistance to therapy through paracrine effects in most solid tumors. In the present study, firstly we isolated CAFs from patient tissues and demonstrated that they promoted cell proliferation and chemoresistance to cisplatin in the lung cancer cell lines A549 and 95D in a paracrine manner. Secondly, using ELISA and quantative PCR, we found that a higher amount of stromal cell-derived factor 1 (SDF-1) existed in the CAFs rather than that observed in the normal fibroblasts (NFs). Thirdly, we detected that SDF-1 facilitated lung cancer cell proliferation and drug resistance via the CXCR4-mediated signaling pathway which involved NF-κB and Bcl-xL. Moreover, we also confirmed that the expression level of SDF-1 in the CAFs was negatively regulated by microRNA mir-1 through microRNA overexpression and quantitative PCR. Overall, our data provide one explanation for the effects of CAFs on lung cancer cells. Meanwhile, our results also suggest CAFs as a potential therapeutic target in tumor treatment.

Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication.

TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetic modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests that TLR3 can act as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects.

Naringenin inhibits allergen-induced airway inflammation and airway responsiveness and inhibits NF-kappaB activity in a murine model of asthma.

Naringenin, a flavonoid, has antiinflammatory and immunomodulatory properties. We investigated whether naringenin could attenuate allergen-induced airway inflammation and its possible mechanism in a murine model of asthma. Mice were sensitized and challenged with ovalbumin. Some mice were administered with naringenin before ovalbumin challenge. We evaluated the development of airway inflammation and airway reactivity. Interleukin (IL)4, IL13, chemokine (C-C motif) ligand (CCL)5, and CCL11 in bronchoalveolar lavage fluid and serum total IgE were detected by ELISA. IkappaBalpha degradation and inducible nitric oxide synthase (iNOS) in lungs were measured by Western blot. We also tested NF-kappaB binding activity by electrophoretic mobility shift assay. The mRNA levels of iNOS, CCL5, and CCL11 were detected by real-time PCR. Naringenin attenuated ovalbumin-induced airway inflammation and airway reactivity in experimental mice. The naringenin-treated mice had lower levels of IL4 and IL13 in the bronchoalveolar lavage fluid and lower serum total IgE. Furthermore, naringenin inhibited pulmonary IkappaBalpha degradation and NF-kappaB DNA-binding activity. The levels of CCL5, CCL11, and iNOS were also significantly reduced. The results indicated that naringenin may play protective roles in the asthma process. The inhibition of NF-kappaB and the decreased expression of its target genes may account for this phenomenon.

Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.

Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification.

The role of neutrophils in the pathogenesis of systemic lupus erythematosus.

PURPOSE OF REVIEW Recent discoveries implicate neutrophils as important regulators of innate and adaptive immunity and in the development of organ damage in systemic autoimmune diseases, including systemic lupus erythematosus (SLE). RECENT FINDINGS Various putative SLE biomarkers are neutrophil-related, including neutrophil granular proteins and histones undergoing post-translational modifications during neutrophil extracellular trap (NET) formation. In the bone marrow, lupus neutrophils can drive B and T cell abnormalities, at least in part, by their enhanced production of type-I interferons, tumor necrosis factor-alpha (TNFα) and the B-cell stimulating factors B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Lupus neutrophils and, in particular, lupus low-density granulocytes (a distinct pathogenic subset) display epigenetic modifications and genomic alterations that may be relevant to their deleterious roles in SLE. Proteins and enzymes externalized by lupus NETs can affect vascular health by inducing endothelial apoptosis and oxidizing lipoproteins. Hampering NET formation through peptidylarginine deiminase inhibitors abrogates lupus phenotype and atherosclerosis in murine studies. SUMMARY Recent discoveries support the notion that neutrophils, low-density granulocytes and aberrant NET formation and clearance play important roles in lupus pathogenesis. Future studies should focus on how to selectively target these immunostimulatory pathways in this disease.

Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms. Interruption of IL18 action reduces atherosclerosis in mice. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E–deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or from Apoe−/− mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe−/− mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

Kruppel-like factor 2 is a transcriptional regulator of chronic and acute inflammation.

Although myeloid cell activation is requisite for an optimal innate immune response, this process must be tightly controlled to prevent collateral host tissue damage. Kruppel-like factor 2 (KLF2) is a potent regulator of myeloid cell proinflammatory activation. As an approximately 30% to 50% reduction in KLF2 levels has been observed in human subjects with acute or chronic inflammatory disorders, we studied the biological response to inflammation in KLF2(+/-) mice. Herein, we show that partial deficiency of KLF2 modulates the in vivo response to acute (sepsis) and subacute (skin) inflammatory challenge. Mechanistically, we link the anti-inflammatory effects of KLF2 to the inhibition of NF-κB transcriptional activity. Collectively, the observations provide biologically relevant insights into KLF2-mediated modulation of these inflammatory processes that could potentially be manipulated for therapeutic gain.

Recruitment of Nck by CD3ϵ Reveals a Ligand-Induced Conformational Change Essential for T Cell Receptor Signaling and Synapse Formation

How membrane receptors initiate signal transduction upon ligand binding is a matter of intense scrutiny. The T cell receptor complex (TCR-CD3) is composed of TCR alpha/beta ligand binding subunits bound to the CD3 subunits responsible for signal transduction. Although it has long been speculated that TCR-CD3 may undergo a conformational change, confirmation is still lacking. We present strong evidence that ligand engagement of TCR-CD3 induces a conformational change that exposes a proline-rich sequence in CD3 epsilon and results in recruitment of the adaptor protein Nck. This occurs earlier than and independently of tyrosine kinase activation. Finally, by interfering with Nck-CD3 epsilon association in vivo, we demonstrate that TCR-CD3 recruitment of Nck is critical for maturation of the immune synapse and for T cell activation.

Coactivator SRC-2-dependent metabolic reprogramming mediates prostate cancer survival and metastasis.

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2-driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer.

Cancer-associated fibroblasts promote hepatocellular carcinoma metastasis through chemokine-activated hedgehog and TGF-β pathways.

Fibroblasts are rich in the surrounding microenvironment of hepatocellular carcinoma (HCC) because most HCCs occur in fibrotic or cirrhotic livers. However, the role of cancer-associated fibroblasts (CAFs) in HCC metastasis remains obscure. Here, we reported that CAFs promote the migration and invasion of HCC cells in vitro and facilitate the HCC metastasis to the bone, brain and lung in NOD/SCID mice. The RayBio human chemokine antibody array revealed that CAFs secret higher levels of CCL2, CCL5, CCL7 and CXCL16 than peri-tumor fibroblasts. CCL2 and CCL5 increase the migration but not the invasion of HCC cells, while CCL7 and CXCL16 promote both migration and invasion of HCC cells. Moreover, CCL2 and CCL5 stimulate the activation of the hedgehog (Hh) pathway, while CCL7 and CXCL16 enhance the activity of the transforming growth factor-β (TGF-β) pathway in HCC cells. The neutralizing antibodies of chemokines notably attenuate the effect of CAFs on HCC metastasis and compromised the activation of Hh and TGF-β pathways in HCC cells. In summary, CAF-secreted CCL2, CCL5, CCL7 and CXCL16 promote HCC metastasis through the coordinate activation of Hh and TGF-β pathways in HCC cells.

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion.

A crucial role for interleukin (IL)-1 in the induction of IL-17–producing T cells that mediate autoimmune encephalomyelitis

It was recently demonstrated that interleukin (IL)-23–driven IL-17–producing (ThIL-17) T cells mediate inflammatory pathology in certain autoimmune diseases. We show that the induction of antigen-specific ThIL-17 cells, but not T helper (Th)1 or Th2 cells, by immunization with antigens and adjuvants is abrogated in IL-1 receptor type I–deficient (IL-1RI−/−) mice. Furthermore, the incidence of experimental autoimmune encephalomyelitis (EAE) was significantly lower in IL-1RI−/− compared with wild-type mice, and this correlated with a failure to induce autoantigen-specific ThIL-17 cells, whereas induction of Th1 and Th2 responses was not substantially different. However, EAE was induced in IL-1RI−/− mice by adoptive transfer of autoantigen-specific cells from wild-type mice with EAE. IL-23 alone did not induce IL-17 production by T cells from IL-1RI−/− mice, and IL-23–induced IL-17 production was substantially enhanced by IL-1α or IL-1β, even in the absence of T cell receptor stimulation. We demonstrate essential roles for phosphatidylinositol 3-kinase, nuclear factor κB, and novel protein kinase C isoforms in IL-1– and IL-23–mediated IL-17 production. Tumor necrosis factor α also synergized with IL-23 to enhance IL-17 production, and this was IL-1 dependent. Our findings demonstrate that IL-1 functions upstream of IL-17 to promote pathogenic ThIL-17 cells in EAE.

Bioenergetic aspects of apoptosis, necrosis and mitoptosis

In this review I summarize interrelations between bioenergetic processes and such programmed death phenomena as cell suicide (apoptosis and necrosis) and mitochondrial suicide (mitoptosis). The following conclusions are made. (I) ATP and rather often mitochondrial hyperpolarization (i.e. an increase in membrane potential, ΔΨ) are required for certain steps of apoptosis and necrosis. (II) Apoptosis, even if it is accompanied by ΔΨ and [ATP] increases at its early stage, finally results in a ΔΨ collapse and ATP decrease. (III) Moderate (about three-fold) lowering of [ATP] for short and long periods of time induces apoptosis and necrosis, respectively. In some types of apoptosis and necrosis, the cell death is mediated by a ΔΨ-dependent overproduction of ROS by the initial (Complex I) and the middle (Complex III) spans of the respiratory chain. ROS initiate mitoptosis which is postulated to rid the intracellular population of mitochondria from those that are ROS overproducing. Massive mitoptosis can result in cell death due to release to cytosol of the cell death proteins normally hidden in the mitochondrial intermembrane space.