The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells

AIM
To explore the signalling pathways involved in aldosterone-induced inflammation and fibrosis in rat vascular smooth muscle cells (VSMCs).


METHODS
Using Western blotting and real-time RT-PCR, we investigated the effects of aldosterone on the expression of cyclooxygenase-2 (Cox-2) and IL-6, two important proinflammatory factors, and TGFβ1, a critical profibrotic factor, in VSMCs.


RESULTS
Aldosterone treatment significantly increased the expression of Cox-2 and IL-6 and activation of p38MAPK and NF-κB. The expression of both Cox-2 and IL-6 could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone and the p38MAPK inhibitor SB203580. Also, the rapid phosphorylation of p38MAPK could be suppressed by SB203580 but not by spironolactone, implicating in nongenomic effects of aldosterone. Similar to SB203580 and spironolactone, the NF-κB inhibitor α-p-tosyl-L-lysine chloromethyl ketone (TLCK) markedly attenuated expression of Cox-2, indicating that MR, p38MAPK and NF-κB are associated with aldosterone-induced inflammatory responses. Furthermore, aldosterone enhanced expression of TGFβ1 in rat VSMCs. This result may be related to activation of the MR/ERK-Sp1 signalling pathway because PD98059, an ERK1/2 inhibitor, significantly blocked the rapid phosphorylation of ERK1/2 and function of Sp1 and led to reduced expression of TGFβ1. Spironolactone was also shown to significantly inhibit TGFβ1 and Sp1 expression but not ERK1/2 phosphorylation.


CONCLUSION
These results suggest that aldosterone-induced inflammatory responses and fibrotic responses may be mediated by the MR/p38MAPK-NF-κB pathways and the MR/ERK-Sp1 pathways in VSMCs, respectively.


Introduction
It has recently been recognized that aldosterone can exert direct effects on the vasculature, a non-epithelial tissue, and lead to vascular inflammation, fibrosis, remodelling, and finally cardiovascular diseases [1] . In hypertensive rats, there is evidence that aldosterone-salt treatment caused obvious coronary inflammatory lesions that were characterized by monocyte and macrophage infiltration and increased expression of cyclooxygenase-2 (Cox-2) and osteopontin. The administration of eplerenone, a selective aldosterone blocker, attenuated vascular and myocardial injury and Cox-2 and osteopontin expression [2,3] . Meanwhile, eplerenone significantly reduced the aortic expression of IL-β1, IL-6, and tumour necrosis factor alpha (TNF-α), and the mineralocorticoid receptor (MR) antagonist spironolactone prevented aortic collagen accumulation induced by aldosterone in spontaneously hypertensive rats [4,5] . A more recent study using aortic tissues from aldosteronetreated hypertensive rats further revealed that aldosterone increased vascular inflammatory changes, angiotensin II content, and the expression of angiotensin-converting enzyme, vascular proinflammatory genes (osteopontin, MMP-2, ICAM-1, VCAM-1, PDGF-A, VEGF, and PAI-1) and oxidative stress genes (4-hydroxy-2-neonenal, p47phox, p22phox, and gp91phox), the expression of which were all abolished by eplerenone [6] . These findings suggest that aldosterone plays Zhu CJ et al Acta Pharmacologica Sinica npg an important role in regulating inflammation and fibrosis in the vascular system. But so far, the intracellular signalling mechanisms underlying the aldosterone-induced vascular injury have not been completely elucidated.
Several studies have examined the signalling pathways in vascular cells that regulate the inflammatory/profibrotic effects induced by aldosterone. Jaffe et al reported that aldosterone activates the expression of endogenous genes (collagen types I and III, IL-16 and cytotoxic T-lymphocyte-associated protein 4) via functional MRs in human coronary VSMCs [7] . Callera et al observed that aldosterone rapidly increases the phosphorylation of p38 MAP kinase (p38MAPK) and extracellular signal-regulated kinase (ERK) and significantly increases NADPH oxidase activity and collagen synthesis in VSMCs from both Wistar-Kyoto rats and spontaneously hypertensive rats, all of which are mediated through c-Src-dependent pathways [8,9] . Also, Ishizawa et al reported that aldosterone stimulates rat VSMC proliferation via the activation of the big MAP kinase 1 and is not inhibited by cycloheximide [10] . Recently, two studies demonstrated that in mouse VSMCs, aldosterone-induced activation of ERK1/2, c-Jun NH 2 -terminal protein kinase (JNK) and nuclear factor (NF)-κB depends on the activity of angiotensin type 1a receptors [11] , and that the proinflammatory phenotype promoted by aldosterone in aged rat VSMCs is mediated by ERK1/2 and epidermal growth factor receptor-dependent pathways [12] . These data suggest that intracellular signalling pathway(s) that regulate aldosteronestimulated vascular effects could involve receptors, protein kinases, nuclear factors, oxidant factors, and other unknown factors.
In the present study, we provide new evidence that aldosterone induces the expression of inflammatory factors, Cox-2 and IL-6 through the MR-p38 and NF-κB pathways and induces expression of transforming growth factor-β1 (TGFβ1), a profibrotic factor, through the MR-ERK1/2 and Sp1 pathways in rat VSMCs.

Cell culture
Rat VSMCs were maintained in 75-cm 2 tissue culture flasks in DMEM containing 10% fetal bovine serum, 100 IU/mL peni-cillin, and 100 IU/mL streptomycin at 37 °C with 95% air and 5% carbon dioxide. The cells were grown to 75% confluence and treated with vehicle or aldosterone at serial concentrations from 10 -9 to 10 -6 mol/L for the times indicated. Twenty four hours before the treatment, the medium was replaced with medium containing 0.4% serum. For the inhibitor studies, SB at 2×10 -5 mol/L, PD at 5×10 -5 mol/L, TLCK at 10 -4 mol/L, or spironolactone at 5×10 -5 mol/L was added to the culture medium 1 h prior to the addition of aldosterone.
The protein concentrations of the cytoplasmic and nucleoplasmic extracts were quantitated. The proteins were electrophoresed on 10% separating gels and analysed by the same methods described above.

Densitometric analysis
The density of the bands on the Western blots was quantified by scanning densitometry (LAS-3000 Fujifilm and Kodak Digital Science 1D software) and expressed as the normalized values to actin.

Statistical analysis
All values are expressed as the mean±SD. The data were analysed using the Student's t test. A P<0.05 was accepted as statistically significant.

Results
Aldosterone induces the expression of Cox-2 protein and IL-6 mRNA in VSMCs In rat VSMCs, aldosterone induced the expression of Cox-2 protein in a concentration-and time-dependent manner, from 1 to 1000 nmol/L and 30 min to 2 or 4 h, respectively ( Figure  1A). Aldosterone treatment also significantly enhanced IL-6 mRNA levels by 10-fold over the control group (Figure 2). These activities were blocked by the MR antagonist spironolactone and the p38MAPK inhibitor SB203580, but not by the ERK1/2 inhibitor PD98059, although PD98059 did reduce the levels to a certain extent ( Figure 1B and 2). TLCK had no inhibitory effects on IL-6 mRNA expression. These data suggest that aldosterone potentially elicits vascular inflammatory responses through the MR and protein kinase system.
Aldosterone increases the phosphorylation of p38MAPK SB203580 could significantly down-regulate the expression of Cox-2 and IL-6, implying that p38MAPK is involved in the inflammatory responses induced by aldosterone. Incubation of cultured VSMCs with aldosterone for 15 or 30 min markedly enhanced the phosphorylation of p38MAPK, which was obviously suppressed by SB203580 but not spironolactone ( Figure 3A), suggesting that MAPKs are involved in the signal transduction of direct vascular effects (nongenomic effects) induced by aldosterone.

Aldosterone stimulates NF-κB p65 expression
The role of the proinflammatory transcription factor NF-κB in the inflammatory responses induced by aldosterone was investigated. After the administration of aldosterone to VSMCs for  4 h, both the cytoplasmic and nucleoplasmic extracts were prepared, and the amount of NF-κB p65 protein was determined by Western blot analysis. As shown in Figure 4, the expression of p65 was almost undetectable in the cytoplasm but was expressed 6.7-fold over the control in the nucleoplasm, even after aldosterone stimulation, indicating that NF-κB mediates the inflammatory responses of aldosterone.

Inhibition of NF-κB and p38MAPK activities stimulated by aldosterone
Further studies revealed that pretreating cells with TLCK, a serine proteinase inhibitor of NF-κΒ, could significantly block Cox-2 protein expression induced by aldosterone in cultured VEMCs. Similar results were obtained using the p38MAPK inhibitor SB203580 and the MR receptor blocker spironolactone ( Figure 5). The results suggest that aldosteroneinduced inflammatory effects could be mediated through a MR-p38MAPK-NF-κΒ pathway.
Aldosterone induces the expression of TGFβ1 mRNA Aldosterone-stimulated inflammation probably results in fibrosis in vascular tissues. Thus, the mRNA expression of the profibrotic molecule TGFβ1 in rat VSMCs was investigated using quantitative real-time RT-PCR. As shown in Figure 6, aldosterone treatment increased the levels of TGFβ1 mRNA. This effect was significantly reduced by the ERK1/2 inhibitor PD98059 and the MR receptor antagonist spironolactone,     Aldosterone increases the phosphorylation of ERK1/2 Incubation of cultured VSMCs with aldosterone for 15 or 30 min markedly increased not only the phosphorylation of p38MAPK but also ERK1/2. The phosphorylation of ERK1/2 was strongly blocked by PD98059, while spironolactone had no effect ( Figure 3B), implying it was rapid, nongenomic effects of aldosterone. These data further demonstrate that MAPKs have a role in the proinflammatory and profibrotic effects of aldosterone.
Aldosterone stimulates Sp1 expression in the nucleoplasm The role of Sp1, a nuclear transcription factor, in profibrotic responses induced by aldosterone was also studied. As shown in Figure 7, aldosterone significantly increased Sp1 expression in the nucleoplasm, and this effect was blocked by PD98059 and spironolactone, which is similar to their effect on inhibiting TGFβ1 expression ( Figure 6). These results indicate that the profibrotic effects of aldosterone could be associated with the MR-ERKs-Sp1 pathway.

Discussion
In this study, we showed that aldosterone stimulation caused inflammatory responses in rat VSMCs and resulted in the enhanced expression of the inflammatory factors Cox-2 and IL-6. This effect was attenuated by the MR antagonist spironolactone (Figures 1 and 2). These results are consistent with previous reports demonstrating that aldosterone treatment in uninephrectomized rats elevated the levels of Cox-2 mRNA and coronary inflammatory lesions; these vascular inflammatory responses were reduced by the aldosterone blocker eplerenone [2,3] . The results indicated that the MR was part of the signalling pathway involved in the inflammatory responses induced by aldosterone. Further studies in our laboratory have demonstrated that aldosterone has two different effects: (1) phosphorylation of p38MAPK (rapid, nongenomic effect), which was suppressed by SB203580 but not by spironolactone (Figure 3), implying that the MR does not mediate this effect; (2) activation and nuclear translocation of NF-κB p65 (delayed genomic effect; Figure 4). In VSMCs, the activation of both p38MAPK and NF-κB might result in the production of chemokines and inflammatory cytokines [1,13] . Indeed, we observed a significant increase in the expression of Cox-2 and IL-6 ( Figure 5). Furthermore, in spontaneously hypertensive rats, aldosterone up-regulated NF-κB p50 expression and down-regulated IκB mRNA expression. This effect was accompanied by the enhanced expression of cytokines (such as IL-1β, IL-6, and TNF-α) [4] . Similarly, in VSMCs from both Wistar-Kyoto rats and spontaneously hypertensive rats, aldosterone-induced phosphorylation of the protein tyrosine kinase c-Src, results in the amplification of NAD(P)H-mediated generation of reactive oxygen species (ROS) and leads to the activation of ROS-sensitive transcription factors, such as NF-κB and activation protein-1 (AP-1), recruitment of inflammatory cells and elaboration of inducible NO synthase (iNOS), and, consequently, vascular damage [8][9][10] . Therefore, protein kinases (p38MAPK, Src) and transcription factors (NF-κB, AP-1) are two elements of the signalling pathway that mediate the inflammatory responses of aldosterone. Recent studies using siRNA in mouse VSMCs revealed that the activation of NF-κB requires not only the MR but also the AT1 receptor [11] .
The data from this study demonstrated that the aldosteroneinduced expression of inflammatory factors (Cox-2) could be significantly reduced by the MR blocker spironolactone, the p38 MAPK inhibitor SB203580, and the NF-κB inhibitor TLCK ( Figure 5), indicating that the MR-p38MAPK-NF-κB signalling pathway mediates the inflammatory responses induced by aldosterone. In this signalling pathway, the activation of p38MAPK may not require the MR because spironolactone did not inhibit phosphorylation of p38 MAPK (Figure 3). This result may be due to the direct, nongenomic effects of aldosterone, which may facilitate classical MR-mediated effects [1] . It is known that aldosterone induces fibrosis in rat heart and blood vessels [14] , and spironolactone prevents aortic collagen accumulation in spontaneously hypertensive rats [5,15] . These data imply that the MR is involved in vascular fibrosis, but the underlying, detailed molecular mechanism has not been completely elucidated. In the present study, we observed the effects of aldosterone on the profibrotic molecule TGFβ1 in rat VMSCs. Our data show that the incubation of VSMCs with aldosterone significantly increased the expression of TGFβ1 mRNA, which was then inhibited by spironolactone and PD98059 ( Figure 5), implying that ERK1/2 is also involved in vascular fibrosis. Similar results were reported by Kobayashi et al [14] in Dahl salt-sensitive hypertensive rats. In that study, treating the animals with eplerenone significantly decreased the phosphorylation of protein kinase C (PKC)βII, c-Src, npg ERK1/2, and p70S6 kinase (p70S6K), as well as the expression of TGFβ1 and type I collagen mRNA and protein, which suggests that aldosterone-induced profibrotic effects could be mediated through the PKCβII-c-Src (upstream regulator)-ERK1/2-p70S6K (downstream effector) pathway. Furthermore, studies by Callera et al [8,9] in VSMCs from spontaneously hypertensive rats demonstrated that aldosterone stimulation could enhance the phosphorylation of c-Src and MAPKs, and the incorporation of [ 3 H]-proline, a marker of collagen synthesis, and stimulatory effects of aldosterone on collagen synthesis was inhibited by PP2 (c-Src inhibitor) and SB212190 (p38MAPK inhibitor), confirming that the vascular profibrotic actions of aldosterone could be mediated through the c-Src/ MAPK signalling pathways. Additionally, it was reported that the epidermal growth factor receptor (EGFR) could be involved in mediating the profibrotic responses of aldosterone because the EGFR is involved in the development of fibrosis and vascular dysfunction [16,17] , and aldosterone-induced stimulation of ERK1/2 in VSMCs is dependent on the EGFR [18] . In this study, our data also show that aldosterone could induce rapid phosphorylation of ERK1/2 in rat VSMCs, which was suppressed by PD98059 but not spironolactone, implying that aldosterone has nongenomic effects ( Figure 3). Moreover, spironolactone and PD98059 inhibited the enhanced expression of TGFβ1 mRNA and Sp1 protein, suggesting that the MR-ERKs-Sp1 pathway could mediate aldosterone-activated profibrotic responses in rat VSMCs. Taken together, our studies provide new evidence on the proinflammatory and profibrotic effects of aldosterone (including genomic and nongenomic effects) that are mediated through the MR-MAPKs (ERK1/2, p38MAPK), transcription factors (Sp1, NF-κB) signalling pathways. Regulating the aldosterone-induced effects through these factors will potentially reduce vascular remodelling and damage. This effect could be beneficial and effective in the treatment of cardiovascular diseases.