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SirT1 modulates the estrogen–insulin-like growth factor-1 signaling for postnatal development of mammary gland in mice
Hongzhe Li1, 2,
Grace K Rajendran1, 2,
Ninning Liu1, 2,
Carol Ware3,
Brian P Rubin4, 5 and
Yansong Gu1, 2Email author
© Gu et al.; licensee BioMed Central Ltd. 2007
Published: 3 January 2007
Estrogen and insulin-like growth factor-1 (IGF-1) play important roles in mammary gland development and breast cancer. SirT1 is a highly conserved protein deacetylase that can regulate the insulin/IGF-1 signaling in lower organisms, as well as a growing number of transcription factors, including NF-κB, in mammalian cells. Whether SirT1 regulates the IGF-1 signaling for mammary gland development and function, however, is not clear. In the present study, this role of SirT1 was examined by studying SirT1-deficient mice.
SirT1-deficient (SirT1ko/ko) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1co/co) with CMV-Cre transgenic mice. Whole mount and histology analyses, immunofluorescence staining, immunohistochemistry, and western blotting were used to characterize mammary gland development in virgin and pregnant mice. The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region.
Both male and female SirT1ko/ko mice can be fertile despite the growth retardation phenotype. Virgin SirT1ko/ko mice displayed impeded ductal morphogenesis, whereas pregnant SirT1ko/ko mice manifested lactation failure due to an underdeveloped lobuloalveolar network. Estrogen implantation was sufficient to rescue ductal morphogenesis. Exogenous estrogen reversed the increased basal level of IGF-1 binding protein-1 expression in SirT1ko/ko mammary tissues, but not that of IκBα expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNFα treatment enhanced the level of the newly synthesized IκBα in SirT1ko/ko cells. SirT1 deficiency therefore affects the cellular response to multiple extrinsic signals.
SirT1 modulates the IGF-1 signaling critical for both growth regulation and mammary gland development in mice. SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis. These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells.
Mammary Gland Development
Ductal Epithelial Cell
SirT1 Gene
Ductal Morphogenesis
Epithelial Progenitor Cell
Mammalian SirT1 belongs to a family of nicotinamide adenine dinucleotide-dependent histone deacetylases [1, 2]. SirT1 is most closely related to yeast Sir2, the founding member of the evolutionarily conserved Sir2 family. Yeast Sir2 is required for silencing transcription at the telomeric region and mating type loci, and for suppression of ribosomal DNA recombination [3, 4]. The expression of an extra copy of Sir2 in either yeast mother cells or multicell organisms such as nematodes can significantly extend the lifespan [5, 6]. Inactivation of Sir2 enhances stress resistance and extends chronological lifespan of nondividing yeast cells, which is opposite to the requirement for Sir2 function in the reproductive lifespan [7]. Whether SirT1 regulates the reproductive lifespan and/or the chronological lifespan in mammals remains unknown.
Sir2 is an integral part of an evolutionarily conserved insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) system in worms (Caenorhabditis elegans), fruit flies (Drosophila), mice, and humans [8, 9]. The IIS system includes membrane-bound receptors, cytoplasmic kinases, and nuclear transcription factors. To maintain the proper expression of the effector genes for the IIS system, these conserved components form a sophisticated regulatory system, which centers on a family of forkhead transcription factors (forkhead box 'other' proteins (FoxOs)), and operates on two levels. On one level, SirT1-mediated protein deacetylation attenuates the transcriptional activity of nuclear FoxO transcription factors [10–12]. On the second level, the FoxO transcription factors can be sequestered within the cytoplasm when phosphorylated by activated Akt kinases in response to insulin and IGF-1 signals [13]. Conceivably, the IIS system senses the levels of insulin and IGF-1 and negatively regulates the expression of the effector genes. The IIS system is responsible for food storage, stress tolerance, and longevity in lower organisms, such as C. elegans [8, 9, 14]. In more advanced species, steroid hormones evolved to regulate the IIS system [15]. In mice and humans, the IGF-1 signaling of the IIS system mediates local effects for growth and hormonal regulation for multiple tissues, including mammary glands [16, 17].
Mammalian SirT1 has evolved to modify the activity of a growing number of transcription factors, including p53, NF-κB, and PGC-1α, suggesting that SirT1 functions in a wide range of cellular responses to stress, inflammation, and nutrients [18–21]. SirT1-deficient mice display characteristic phenotypes of perinatal death and growth retardation as well as other diverse phenotypes, such as eye defects, with varying severity [22, 23]. The underlying causal mechanism for these phenotypes, however, remains unknown. We recently generated SirT1-deficient (SirT1ko/ko) mice and found that both male and female SirT1ko/ko mice can be fertile, which is in contrast to the sterile phenotypes observed in one strain of SirT1-deficient mice [22]. This led to our study of the link between SirT1 and IGF-1 signaling using the mammary gland as a model organ.
The mammary gland is a unique organ because it develops after birth and undergoes dynamic changes throughout the reproductive lifespan of a female. At the onset of puberty, ovarian estrogen stimulates ductal morphogenesis during which mammary epithelial progenitor cells differentiate and proliferate while interacting with adipocytes and stromal cells within mammary fat pad [17, 24, 25]. Ovarian estrogen, in synergy with pituitary growth hormone (GH), stimulates stromal cells to produce local IGF-1. The local IGF-1, but not liver-produced systemic IGF-1, provides a paracrine signal for commencing ductal morphogenesis [26]. Mice lacking GH, estrogen, IGF-1, GH receptor, or estrogen receptor alpha (ERα) fail to undergo postnatal ductal morphogenesis [17, 24–31], indicating that both steroid hormones and IGF-1 are on the common pathway for a critical developmental checkpoint. Once the arborated ductal network is established, cycles of differentiation, proliferation, and death of secretory alveolar epithelium repeat with each pregnancy [17, 24, 25].
In the present article we report the finding and characterization of impeded ductal morphogenesis in virgin SirT1ko/ko mice and lactation failure in SirT1ko/ko mothers. The characterization of these phenotypes has identified a SirT1-dependent regulatory mechanism by which SirT1 modulates the effectiveness of the estrogen–IGF-1 signaling for mammary gland development. The estrogen–IGF-1 signaling is defined as the ovarian estrogen-regulated, stromal cell-produced local IGF-1 signal for stimulating mammary epithelial cells.
A previously described SirT1 targeting construct, KOII [23], was used to generate mice harboring a conditional targeted mutation in the SirT1 gene (SirT1co/co mice) (see Additional file 1). The breeding of SirT1co/co mice and CMV-Cre transgenic mice results in mice harboring a germline-transmitted deletion of exon 4 of the SirT1 gene (SirT1+/ko mice). Both SirT1co/co mice and SirT1+/co mice were used to establish breeding colonies for generating SirT1co/co and SirT1ko/ko mice, respectively. Both SirT1co/co mice and SirT1ko/ko mice were in a mixed 129SvJ/C57B6 background. Mice were housed in a special-pathogen-free facility and all procedures were approved by the University of Washington Animal Care and Use Committee. A PCR-based genotyping method was established to identify the wild-type, co, and ko loci of the SirT1 gene using three primers: 5' co primer, 5'-GGTTGACTTAGGTCTTGTCTG; 5' ko primer, 5'-AGGCGGATTTCTGAGTTCGA; 3' primer, 5'-CGTCCCTTGTAATGTTTCCC. Murine embryonic fibroblasts (MEFs) were isolated from the embryos between embryonic day 12.5 and embryonic day 14.5. The body weight was measured once a week.
Serum insulin-like growth factor-1
A commercial radioimmunoassay (ALPCO Inc., Windham, NH, USA) was used. Samples (300 μl) were prepared via an alcohol extraction followed by a 2-day disequilibrium incubation at 4°C. Samples were measured in duplicate and data were calculated as the samples were counted. Six standards ranging from 10 to 500 ng/ml were used to generate the standard curve. The lower limit of detection was 10 ng.
Survival assay
To determine the sensitivity of embryonic stem cells after ionizing radiation or hydrogen peroxide treatment, 300 embryonic stem cells were seeded onto a 10-cm plate for 24 hours. The plates were either exposed to a 137Cs source at indicated doses or were treated with embryonic stem media containing hydrogen peroxide at indicated concentrations for 30 minutes. Seven days after irradiation or hydrogen peroxide treatment, the embryonic stem colonies stained with crystal violet were counted.
Estrogen implantation
Estrogen pellets in the form of 17β-estradiol, at 0.1 mg, and the 21-day releasing time were obtained from Innovative Research (Sarasota, FL, USA). One pelletwas subcutaneously implanted in the subscapular region ofindividual female mice using a sterilizedmetal trochar (Innovative Research).
Whole mount analysis
The inguinoabdominal mammary fat pads were spread on microscope slides, fixed in Carnoy's fixative overnight, hydrated and stained with carmine alum stain (Sigma, St. Louis, MO, USA) overnight, and were then dehydrated, treated with xylene to remove fat, and mounted with Secure Mount (Fisher Scientific, Pittsburgh, PA, USA) and cover slips.
Histological, immunohistochemical, and immunofluorescence analyses
The mice were given a single dose of bromodeoxyurdine (BrdU) at 100 g/kg body weight via intraperitoneal injection 2 hours before euthanasia. The mammary fat pads, as well as other tissues, were collected and fixed in 10% formalin solution (Fisher Scientific). Paraffin-embedded sections were prepared at 4 μm thickness followed by standard H & E staining for histological analysis and by immunohistochemical staining for BrdU-labeled cells (Sigma) and apoptotic cells (Promega, Madison, WI, USA). To detect the presence of mouse milk proteins, a rabbit anti-mouse milk specific protein antibody (Nordic Immunology, Tillburg, The Netherlands) was used for immunofluorescent staining, followed by Texas Red anti-rabbit secondary antibody (Molecular Probes) and DAPI staining for nuclei (Molecular Probe, Eugene, OR, USA). Microscopic analyses of all histological findings were carried out on an AxioVert 200M microscope with AxioVision 4.5 software (Carl Zeiss, München-Hallbergmoos, Germany).
Protein extracts were prepared from mammary tissues as well as from MEFs. The following primary antibodies were used: anti-SirT1 (Upstate Biotechology, Lake Placid, NY, USA), anti-IGF-1 binding protein-1 (IGFBP-1) (R&D Systems, Minneapolis, MN, USA), and anti-actin and anti-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The corresponding secondary antibodies were horseradish peroxidase-conjugated anti-rabbit (Pierce, Rockford, IL, USA), anti-rat, and anti-goat (Santa Cruz Biotechnology).
Growth retardation in SirT1ko/komice
The conditional targeted SirT1 mutant mice (SirT1co/co mice) carry an insertion mutation of the neomycin-resistant gene and lox sequences in the SirT1 gene flanking exon 4 that encodes a conserved Sir2 motif (Figure 1a). The mutation does not affect the expression of SirT1 in SirT1co/co mice (Figure 1b). As expected, SirT1co/co mice are phenotypically indistinguishable from wild-type mice. To convert the SirT1 co allele into the SirT1 ko allele, SirT1co/co mice were crossed with CMV-Cre transgenic mice to generate SirT1 heterozygotes carrying the SirT1+/ko, CMV-Cre+ genotype. The expression of the CMV-Cre transgene catalyzes the deletion of exon 4 in most lineages of cells, including germ cells (Figure 1a). This SirT1 ko allele should be identical to the previously described Δex4 mutation [23]. SirT1+/ko, Cre+ mice were backcrossed with wild-type mice to generate the mice harboring a germline-transmitted deletion mutation (that is, SirT1+/ko mice). The breeding of SirT1+/ko male mice and SirT1+/ko female mice resulted in SirT1ko/ko mice. The cells derived from either SirT1+/ko mice or SirT1ko/ko mice expressed a SirT1 mutant protein due to the inframe deletion of exon 4 (Figure 1b). This SirT1 mutant protein may not be functional, however, since SirT1+/ko mice are phenotypically indistinguishable from wild-type mice and SirT1Δex4/Δex4 mice were phenotypically identical to SirT1 null mice [23].
Generation and characterization of SirT1 co/co and SirT1 ko/ko mice. (a) Mouse SirT1 wild-type allele (SirT1+), conditional targeted allele (SirT1co), and knockout allele (SirT1ko). B, BamHI. (b) Western blot analysis of SirT1 expression in wild-type (+/+), conditional targeted (co/co), heterozygote (+/ko), and knockout (ko/ko) murine embryonic fibroblasts (MEFs) and mammary tissues (MG). (c) Growth retardation in surviving SirT1ko/ko mice (open bar) using sibling mice or age-matched mice of control genotypes (filled bar). (d) The serum levels of insulin-like growth factor-1 (IGF-1) in wild-type (+/+), SirT1+/ko (+/ko), and SirT1ko/ko (ko/ko) mice. (e) The survival curves of wild-type (WT), SirT1co/co, SirT1ko/ko, and Ku70-/- embryonic stem cells after ionizing radiation. (f) The survival curve of wild-type (WT), SirT1ko/ko, Ku70-/-, and Atm-/- embryonic stem cells after treatment with hydrogen peroxide.
Similar to other strains of SirT1-deficient mice, nearly two-thirds of SirT1ko/ko newborns die shortly after birth and the majority of surviving SirT1ko/ko mice manifest growth retardation (Table 1 and Figure 1c) [22, 23]. Growth retardation may result from a systemic defect in hormonal regulation, DNA double-strand break repair, or other causal mechanisms. We found that SirT1ko/ko mice have reduced levels of serum IGF-1 (Figure 1d). IGF-1 often acts as a local effector for pituitary GH. The serum level of GH in SirT1ko/ko mice, however, appeared to be within the normal range (data not shown). On the other hand, yeast Sir2 and its associated Sir complex, as well as mouse SirT6, have also been implicated in DNA double-strand break repair and the maintenance of chromosome stability [32–34]. We found that SirT1ko/ko embryonic stem cells do not have increased sensitivity to either ionizing radiation or hydrogen peroxide treatment when compared with wild-type cells, or with positive control cells such as Ku70-deficient or Atm-deficient cells (Figure 1e,f). Both Ku70-deficient mice and Atm-deficient mice display growth retardation phenotypes [35, 36]. The growth retardation phenotype in SirT1ko/ko mice is therefore probably not due to a defect in DNA damage repair, but rather results from a deficit in IGF-1 signaling.
Perinatal lethality, fertility, and lactation defect in SirT1ko/ko mice
Survivorsa
+/+
+/ko
ko/ko
ko/ko (n = 3)
More than 10 pairs of SirT1+/ko male mice and SirT1+/ko female mice were used for breeding. Data in parentheses indicate the number of pups of this genotype found dead within 3 days after their birth. aTotal number of surviving pups at weaning (postnatal day 21).
Lactation failure
Both male and female SirT1ko/ko mice can be fertile, which is in contrast with the reported sterile phenotype in one strain of SirT1-deficient mice [22]. When male SirT1ko/ko mice impregnated female SirT1+/ko mice, the number of surviving SirT1ko/ko offspring was reduced due to the partial perinatal lethal phenotype (Table 1). In a reciprocal approach, male SirT1+/ko mice can also impregnate female SirT1ko/ko mice (Table 1). After parturition, SirT1ko/ko mothers exhibited normal nursing behavior. The pups did not survive for more than 3 days after birth, however, unless they were immediately removed and put under the care of a foster mother. All pups died of dehydration or starvation as a result of lack of milk in their stomachs.
To determine whether the SirT1ko/ko mothers encountered a lactation defect, whole mount and histological analyses were used to characterize the morphological changes in the mammary glands. We found that virgin SirT1ko/ko mice displayed impeded ductal morphogenesis up to 9 months of age, while age-matched virgin wild-type mice displayed extensive ductal elongation and branching (Figure 2a). The absence of ductal morphogenesis persisted in pregnant SirT1ko/ko mice up to day 13 of pregnancy (Figure 2b). Despite the fact that pregnancy can induce ductal morphogenesis at the late stage of pregnancy, underdeveloped mammary glands in SirT1ko/ko female mice cause a severe deficit in milk production, as shown by histological analysis as well as the immunofluorescence analysis using an anti-milk antibody (Figure 2b,c).
Lactation failure in SirT1 ko/ko mice. (a) Whole mount analysis of the mammary tissues from virgin wild-type (+/+) and SirT1ko/ko mice (ko/ko). Scale bar = 2 mm. (b) Histological analysis of the mammary tissues harvested from wild-type (+/+) and SirT1ko/ko female mice on either day 13 of pregnancy (P13) or lactation day 1 (L1). Scale bars = 100 μm. (c) Immunofluorescence staining of milk production in the mammary tissues from wild-type (+/+) and SirT1ko/ko mice on L1. Red and smear stains, mouse milk proteins; blue dots, nuclear staining of adipocytes, epithelial cells, and other cells in mammary tissues. Scale bars = 100 μm.
Pregnancy-induced mammary gland development
To characterize the developmental defect leading to lactation failure, we analyzed the mammary glands of SirT1ko/ko female mice on lactation day 1. Terminal end buds (TEBs) are club-shaped transitional structures (see Figure 3a, upper panel). In wild-type female mice, TEBs form and precede ductal elongation and branching at the onset of puberty [17, 24, 25]. When TEBs/ducts reach the edge of mammary fat pads, TEBs regress as shown in the virgin mice in Figure 3a (upper panel). Interestingly, the transitional TEBs can be readily identified in SirT1ko/ko female mice on lactation day 1, manifesting as either newly formed TEBs or as TEBs attached to developing ductal and alveolar structures (Figure 3a, lower panel). Moreover, SirT1ko/ko mice displayed varying degrees of ductal development, ranging from a lack of any ductal structure to a fully developed ductal network, the latter of which is comparable with the morphology and cellularity of wild-type mammary tissues on day 13 of pregnancy (Figure 3a). These observations indicated that pregnancy could rescue impeded ductal morphogenesis in virgin SirT1ko/ko mice.
Pregnancy-induced ductal morphogenesis. (a) Whole mount analysis of mammary gland development. Upper panel: wild-type (+/+) mice show terminal end buds (TEBs) at the onset of puberty, elongated ducts (virgin mice), site branching during pregnancy (P13), and lobuloalveolar structures for milk production on lactation day 1 (L1). Lower panel: virgin SirT1ko/ko (ko/ko) mice show impeded ductal morphogenesis. Pregnancy-induced ductal morphogenesis manifests transitional TEBs, ductal elongation, and side branching with variety in three SirT1ko/ko mice on L1. All scale bars = 200 μm. (b) Western blot analysis of both insulin-like growth factor-1 binding protein-1 (IGFBP-1) and IκBα in mammary tissues of male mice, virgin mice, and L1 female mice of wild type (+/+) and SirT1ko/ko (ko/ko). Actin is used as a loading control. Lower panel scores the estimated density of indicated lineages of cells in the protein extracts, which are based on the morphological analyses in (a).
Normal alveolar morphogenesis takes place under the control of additional hormones and growth factors during pregnancy and lactation [17, 24, 25]. These coordinated efforts are necessary to support a densely saturated lobuloalveolar system for secreting milk after parturition (see wild-type mice on lactation day 1 in Figures 2b and 3a). In some SirT1ko/ko mice, the alveolar morphogenesis was initiated, as indicated by the presence of a few secretory alveolar epithelial cells within scattered alveolar structures sprouted from a well-established ductal branching network (Figures 2c and 3a, lower panel). The extent of alveolar morphogenesis in SirT1ko/ko mice was inadequate, however, as shown by the severe deficit in milk production when compared with that in wild-type mice (Figure 2c).
Exogenous estrogen-induced ductal morphogenesis
To test whether the increased levels of estrogen in pregnant mice were sufficient to induce ductal morphogenesis in SirT1ko/ko mice, a single estrogen pellet was implanted in the subscapular region of each virgin SirT1ko/ko mouse and the mammary tissues were analyzed 14 and 21 days after the implantation. By day 14, wild-type mice displayed ductal side branching reminiscent of the morphological changes during early pregnancy (Figure 4a, left panel). In contrast, SirT1ko/ko mice showed TEBs and ductal elongation, which are the characteristic features of ductal morphogenesis in pubertal wild-type mice. By day 21, ductal elongation and side branching were restored in SirT1ko/ko mice and the extent of ductal morphogenesis was indistinguishable between wild-type and SirT1ko/ko female mice (Figure 4a, right panel). These observations clearly indicated that exogenous estrogen alone is sufficient to rescue ductal morphogenesis in virgin SirT1ko/ko mice. Furthermore, the presence of transitional TEBs on day 14 and of ductal side branching on day 21 suggested that the characteristic features of mammary gland development at puberty and during early pregnancy could be coupled in response to increasing levels of estrogen. Increased levels of estrogen, either during pregnancy or through implantation, were therefore sufficient to stimulate the differentiation of epithelial progenitor cells and ductal morphogenesis in virgin SirT1ko/ko mice.
Estrogen implantation stimulates ductal elongation and site branching. (a) Whole mount analysis of the mammary tissues from the virgin wild-type (+/+) and SirT1ko/ko mice implanted with estrogen pellets (+E2) on day 14 and day 21. Scale bars = 200 μm. (b) Western blot analysis of the expression of insulin-like growth factor-1 binding protein-1 (IGFBP-1) and IκBα, with actin as a loading control, in mammary tissues from the virgin wild-type (+/+) and SirT1ko/ko mice implanted with estrogen pellets on day 21.
Concurrent mammary epithelial cell proliferation and differentiation
The finding of pregnancy-induced mammary gland development prompted us to investigate how mammary epithelial progenitor cells in pregnant SirT1ko/ko mice can differentiate so rapidly. Both BrdU labeling and the TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay were used to measure the cell proliferation and apoptosis in the mammary tissues, respectively. We found in SirT1ko/ko mammary tissues that about one-third of the cells in TEBs were BrdU-positive as compared with the number of BrdU-positive cells in the newly differentiated ductal or alveolar epithelial cells (Figure 5a(upper panel), 5b). This BrdU-positive finding differs from that in wild-type mammary tissues, in which less than 3% of ductal epithelial cells and about 25% of alveolar epithelial cells were BrdU-positive. The newly generated ductal epithelial cells in SirT1ko/ko mice formed elongated ducts and side branches, while pushing TEBs towards the edge of mammary fat pad (Figure 3a, lower panel). The newly generated ductal epithelial cells can consequently further differentiate into a few observed alveolar cells in which milk proteins were detected (Figure 2c). These observations suggested that the proliferation and differentiation of epithelial progenitor cells, which is programmed to take place at different times of a female's reproductive life, could be concurrent at a late stage of pregnancy in SirT1ko/ko mice.
Mammary epithelial cell proliferation and apoptosis in SirT1 ko/ko mice on lactation day 1. (a) Upper panel: bromodeoxyurdine (BrdU) staining of the mammary sections from SirT1ko/ko (ko/ko) and wild-type (+/+) mice on day 13 of pregnancy (P13) and lactation day 1 (L1). From left to right, three terminal end buds (TEBs) at a scale of 100 μm, a single TEB, ducts, and alveolus of SirT1ko/ko mice at a scale of 20 μm, and ducts and alveolus of wild-type mice on P13 and L1 at a scale of 20 μm. Lower panel: TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay for apoptotic cells using sections neighbored to the sections for BrdU staining. (b) The quantitative analysis of BrdU-positive epithelial cells in TEBs, ducts, and alveoli of SirT1ko/ko mice on L1 (open bar) and wild-type mice on P13 (grey bar) and L1 (solid bar). (c) The quantitative analysis of TUNEL-positive cells in neighboring sections.
Apoptosis is a part of the self-renewal and remodeling processes in normal mammary tissues [17, 24, 25]. Apoptotic epithelial cells can be readily identified in both ductal and alveolar epithelium of SirT1ko/ko mice on lactation day 1 (Figure 5a, lower panel). The levels of apoptotic cells were significantly higher than those in wild-type mice on lactation day 1, but seemed to be closer to that in wild-type mice in mid-pregnancy (e.g. day 13 of pregnancy) (Figure 5c). The rush of proliferation and differentiation during pregnancy-rescued ductal development in SirT1ko/ko mice manifested multiple features on lactation day 1 that could reassemble some characteristic features of wild-type mice in mid-pregnancy. Successive pregnancies, however, failed to improve the survival rate of either SirT1+/ko pups or SirT1ko/ko pups (three or four pregnancies in 3 months for each of three SirT1ko/ko females tested), indicating that the lactation defect persisted. A SirT1 deficiency therefore affected ductal morphogenesis in virgin mice, and lobuloalveolar proliferation in parous mice.
Deregulated negative feedback signals
The characterization of lactation failure has revealed two developmental defects in SirT1ko/ko female mice. The impeded ductal morphogenesis in virgin SirT1ko/ko mice bore some resemblance to the defective ductal morphogenesis seen in mice lacking GH, estrogen, or IGF-1, as well as in mice harboring mutations in the corresponding receptors, such as the GH receptor and ERα [17, 24–31]. These previous studies have established the notion that GH and estrogen synergistically stimulate ductal morphogenesis, which is mediated by stromal cell-produced local IGF-1 in mammary tissues. Impeded ductal morphogenesis in virgin SirT1ko/ko mice is therefore probably due to a deficit in IGF-1 signaling.
In addition to this early defect, pregnancy-induced mammary gland development in SirT1ko/ko mice appeared to be arrested at the beginning of lobuloalveolar development, which is reminiscent of the phenotype in mice lacking IκB kinase alpha (IKKα) activity for NF-κB activation [37]. IKKα-deficient mice exhibit normal ductal morphogenesis at the onset of puberty and exhibit normal ductal side branching during early pregnancy. Owing to the absence of NF-κB-dependent cyclin D1 expression, however, IKKα-deficient mice display defective alveolar epithelial cell proliferation and fail to lactate [37]. While SirT1 negatively regulates the transcription activity of FoxOs and NF-κB in mammalian cells [10, 11, 20], SirT1ko/ko mice displayed the loss-of-function phenotypes. We hypothesized that SirT1 deficiency deregulates the expression of negative feedback signals and thereby desensitizes the cells to various types of stimulation. Indeed, among the many FoxOs and NF-κB-regulated genes, IGFBP-1 and IκBα encode negative feedback signals for IGF-1 signaling and NF-κB activation, respectively. We found that the basal levels of both IGFBP-1 and IκBα were increased in adipose tissues from virgin SirT1ko/ko mice and in ductal epithelial cells from SirT1ko/ko mice on lactation day 1 (Figure 3b). Meanwhile, the expression pattern of IGFBP-1 and IκBα in wild-type mammary tissues, shown in Figure 3b, was in agreement with the findings of others [16, 38, 39]. Namely, the expression of IGFBP-1 was downregulated during pregnancy, whereas NF-κB activity was increased at a late stage of pregnancy. The loss of SirT1 therefore increased the basal level of both IGFBP-1 and IκBα in multilineages of cells, and potentially increased thresholds for activating SirT1-modulated signaling pathways.
SirT1 deficiency did not affect the ability of mammary epithelial progenitor cells to differentiate into functional alveolar epithelial cells despite the lactation failure (Figure 2c). SirT1 deficiency may therefore alter the homeostasis of the signals for ductal morphogenesis and/or the cellular response to the signals. Increased expression of both IGFBP-1 and IκBα in adipose tissues and mammary epithelial cells could simply reflect a global deregulation of gene expression in SirT1ko/ko cells in which the activity of both FoxOs and NF-κB is increased. IGFBP-1 is a potent inhibitor of IGF-1 in vivo [40], suggesting that the elevated basal level of IGFBP-1 in adipose tissues in SirT1ko/ko mice could reduce the effect of IGF-1. Moreover, deregulated IGFBP-1 expression, but not deregulated IκBα expression, was reversed in response to increased levels of estrogen in the mammary fat pads after implantation (Figure 4b). The finding is relevant to the fact that the expression of IGFBP-1 is downregulated as the level of estrogen increases in pregnant wild-type mice (Figure 3b). Increased levels of estrogen therefore stimulate the production of stromal cell-derived local IGF-1, which overcomes the IGFBP-1 barrier in SirT1ko/ko mice and, ultimately, reverses the increased levels of IGFBP-1 in mammary tissues.
It was noted that increased levels of estrogen and local IGF-1 did not interfere with the expression of IκBα (Figure 4b). To determine whether deregulated IκBα expression in SirT1ko/ko cells can be reversed, we treated MEFs with TNFα and measured the kinetics of IκBα expression. Three independent lines of SirT1ko/ko MEFs were used, which displayed varying basal levels of increased IκBα expression. Following IκBα degradation induced by TNFα, the levels of newly synthesized IκBα in all three SirT1ko/ko MEFs were always higher than that of wild-type MEFs (Figure 6). This in vitro finding implies that SirT1 deficiency affects NF-κB signaling when the IκB kinase activation is normal.
SirT1 deficiency derails NF-κB signaling in response to TNFα stimulation. (a) Western blot analysis of IκBα expression in SirT1ko/ko (ko/ko) and wild-type (+/+) murine embryonic fibroblasts (MEFs) within the first hour after TNFα stimulation. Actin is used as a loading control. (b) The relative levels of newly synthesized IκBα in three independent SirT1ko/ko MEF lines (red circles, blue and green triangles) and wild-type MEFs after TNFα stimulation. For each line of MEFs, the unit of IκBα protein at each time point is relative to the unit 0 minutes after normalization with actin.
Our current system might not be suitable for studying the effects of SirT1 deficiency on NF-κB activation in vivo. NF-κB signaling exhibits distinct biological responses because the expression of IκBα depends on NF-κB activation while the other isoforms of IκB (β and ε) are independent of NF-κB feedback [41]. For example, TNFα stimulation induces dampened oscillatory behavior after the first hour, whereas lipopolysacharide treatment leads to stable NF-κB activation [42, 43]. The NF-κB signaling in the developing mammary gland is under the temporal control of RANK signaling and IKKα activation [37]. The finding of increased levels of newly synthesized IκBα within the first hour indicated that SirT1 deficiency could disrupt the temporal control of NF-κB signaling to all types of stimulation (Figure 6). It remains unknown, however, whether the reduced IGF-1 signaling would affect the efficacy of RANK signaling and IKKα activation in developing mammary glands. The fact that individual SirT1ko/ko mice manifested varying degrees of ductal morphogenesis (Figure 3a) makes it difficult to dissect the potential compound effect of SirT1 deficiency on both upstream IKKα activation and downstream NF-κB signaling. Other experimental systems, such as mammary epithelial cell-specific SirT1ko/ko female mice, may be used to address this issue.
Our study of SirT1ko/ko mice has unveiled a regulatory mechanism by which SirT1 modulates the efficacy of estrogen-stimulated local IGF-1 signaling for ductal morphogenesis. SirT1 deficiency alters the homeostasis of gene expression and attenuates the efficacy of IGF-1 signaling. As a result, SirT1ko/ko mice manifest partial perinatal lethality and lactation failure phenotypes, implicating the role of evolutionarily conserved SirT1 in both postnatal survival and offspring survival of mammals.
SirT1 balances the homeostasis of IGF-1 and IGFBP-1 in vivo
SirT1 is an integral part of the IIS system that checks and balances the efficacy of insulin and IGF-1 signals. The IGF-1 signaling of the IIS system is required for survival after birth. As illustrated in Figure 7a, the function of SirT1 parallels the linear IGF-1/IGF-1 receptor/phosphoinositide-3'-OH kinase/Akt signaling pathway, both of which can negatively regulate the transcription activity of FoxOs. Mice harboring targeted mutations in either IGF-1 or the IGF-1 receptor gene exhibit perinatal lethality and growth retardation [44, 45]. In the IGF-1 receptor-expressing cells, the binding of insulin and/or IGF-1 activates the phosphoinositide-3'-OH kinase and Akt kinase cascade for survival [8, 9, 13]. The phosphoinositide-3'-OH kinase/Akt signaling pathway is also highly conserved from worms to mammals, and functions to modulate energy metabolism and lifespan in lower organisms. The mammalian Akt kinase family has three members, Akt1–Akt3, which provide specificity and versatility. Mice lacking both the Akt1 and Akt2 genes display perinatal lethal and growth retardation phenotypes that are strikingly similar to that of either IGF-1 or IGF-1 receptor-deficient mice [46]. In this regard, SirT1ko/ko mice, as well as other strains of SirT1-deficienct mice, also exhibit perinatal lethal and growth retardation phenotypes (Figure 1c and Table 1) [22, 23]. The consistent observation of perinatal lethal and growth retardation phenotypes from our study and studies with mice lacking IGF-1, the IGF-1 receptor, or Akt1/Akt2 suggests that upregulated FoxO activity unleashes the expression of downstream effector genes that lead to the birth-related stress. This birth-related stress may be related to the switch from a maternal level of IGF-1 to a neonatal level of IGF-1 because the surviving SirT1ko/ko mice can live into adulthood and can reproduce despite the growth retardation phenotype (Figure 1c). In this context, at least one of the FoxO-deficient mice strains, FoxO3 (Foxo3a/FKHRL1)-deficient mice, grow normally and develop functional mammary glands [47].
SirT1 modulates estrogen–insulin-like growth factor-1 signaling for ductal morphogenesis: a model. (a) The insulin/insulin-like growth factor-1 (IGF-1) signaling system: both SirT1 and Akt kinases can negatively regulate the transcription activity of forkhead box 'other' protein (FoxO) proteins. SirT1 deficiency deregulates the expression of insulin-like growth factor-1 binding protein-1 (IGFBP-1), which may exert autocrine and/or paracrine effects to inhibit IGF-1. (b) Mammary epithelial precursor cells (EPC) express IGF-1 receptor (IGF-1R) and can differentiate into estrogen receptor (ER)-negative ductal epithelial cells (DEC-I) in response to maternal, circulating, or estrogen-stimulated, stromal cell (S)-derived local IGF-1. At the onset of puberty, ovarian estrogen, in synergy with growth hormone (GH), enhances the production of local IGF-1 and stimulates the differentiation of DEC-I to ER-positive ductal epithelial cells (DEC-II). SirT1 deficiency deregulates the expression of IGFBP-1 in adipose tissues (a), however, which attenuates the efficacy of the IGF-1 signaling and causes impeded ductal morphogenesis in virgin SirT1ko/ko mice. Either pregnancy or exogenous estrogen can overcome the impeded ductal morphogenesis in virgin SirT1ko/ko mice and can stimulate the differentiation of EPC.
IGFBP-1 is one of six IGF-1 binding proteins, but is the only one that can be negatively regulated by insulin [48]. IGF-1 binding proteins inhibit the action of local IGF-1 in a spatial and temporal manner [40, 49]. In mammalian cells, FoxO3 can directly bind to the promoter of the IGFBP-1 gene and activate the transcription [50–52]. SirT1 acts to negatively regulate the activity of nuclear FoxO3 and to decrease the expression of IGFBP-1 when the IGF-1 signal is low (Figure 7a). Increased levels of IGFBP-1 can diminish the action of IGF-1 signals in vivo. In that way, SirT1 balances the homeostasis of IGF-1 and IGFBP-1 for varying levels of IGF-1 signals.
The homeostasis of IGF-1 and IGFBP-1 clearly affects fertility, as shown by the infertility phenotype in both IGF-1-deficient mice and IGFBP-1 transgenic mice [28, 40, 49]. The serum level of estrogen in IGF-1-deficient female mice appears significantly reduced, whereas in ERα-deficient mice, which are also infertile, there is a threefold increase [53]. In contrast, SirT1ko/ko mice can be fertile despite the fact that the fertility was reduced as compared with that of wild-type females (Table 1). Although our preliminary assessment indicated that the serum level of estrogen in SirT1ko/ko female mice was not overtly altered, it remains possible that a subtle irregularity or an individual variation of the estrogen level or ERα expression might be attributed to the reduced fertility. An alternative explanation for reduced fertility is that SirT1 deficiency deregulates the activity of FoxO3 and manifests an opposite effect of FoxO3 deficiency. FoxO3-deficient mice exhibit early depletion of ovarian follicles, which is reminiscent of premature ovarian failure in women [47, 54]. Premature ovarian failure manifests as early cessation of ovarian function and as premature menopause caused by genetic mutations, chemo/radiation therapy, or other unknown factors. It is possible that SirT1 deficiency counterbalances normal release of ovarian follicles and reduces the fertility in mice.
SirT1 modulates the efficacy of the IGF-1 signal for ductal morphogenesis
We hypothesize that, in virgin SirT1ko/ko mice, increased levels of IGFBP-1 in adipose tissues effectively diminish IGF-1 signals, including the estrogen–IGF-1 signaling, and halt the differentiation of mammary epithelial progenitor cells (Figure 7b). Several results of this study provided support for this hypothesis. First, the expression of IGFBP-1 is increased in mammary fat pads from virgin SirT1ko/ko mice, in which there is no evidence of any duct (Figures 2a and 3a). Second, both pregnancy and estrogen implantation can rescue impeded ductal morphogenesis in SirT1ko/ko mice (Figures 3a and 4a), demonstrating that increased levels of estrogen can enhance the production of local IGF-1. Moreover, the varying degrees of rescue among individual mice, as well as individual mammary fat pads of the same mouse, also indicated that local IGF-1 must be halted in SirT1ko/ko mice (Figure 3a, and data not shown). Finally, increased levels of estrogen specifically activate the IGF-1 signaling in mammary tissues, as shown by the reversal of IGFBP-1 expression in SirT1ko/ko mice after estrogen implantation (Figure 4b). These results indicate that a gradient of IGF-1 signals, including both circulating and local IGF-1, may regulate mammary gland development at embryonic, postnatal, and reproductive stages, and that increased expression of IGFBP-1 decreases the efficacy of IGF-1 signals.
Mammary development may undergo estrogen/IGF-1-independent phases and estrogen/IGF-1-dependent phases, which encompasses a late stage of embryonic development to the onset of puberty and involves maternal IGF-1, circulating IGF-1, and estrogen-stimulated local IGF-1. By embryonic day 16.5, mammary mesenchymes are connected to primitive fat pads and begin to proliferate, which results in rudimentary ducts [17, 25, 55, 56]. We propose that this process marks the transition from estrogen/IGF-1-independent mammary stem cells to IGF-1-dependent epithelial progenitor cells, and that maternal IGF-1 is sufficient to stimulate the differentiation of epithelial progenitor cells to estrogen-independent ductal epithelial cells but not estrogen-dependent epithelial cells (Figure 7b). This explains why the development of rudimentary ducts is normal in either IGF-1 or ERα knockout mice and that TEBs form and regress immediately before and after birth, respectively [17, 26, 27]. During the prepubertal period, the rudimentary ducts grow isometrically under the influence of circulating IGF-1. Like maternal IGF-1, circulating IGF-1 is not sufficient to induce the differentiation of estrogen-independent ductal epithelial cells into estrogen-dependent epithelial cells, which will express ERα and become estrogen dependent (Figure 7b). At the onset of puberty, ovarian estrogen, in synergy with pituitary GH, causes a surge in the production of stromal cell-derived local IGF-1 and stimulates the differentiation of estrogen-independent ductal epithelial cells to estrogen-dependent epithelial cells, which begins postnatal development of mammary gland (Figure 7b). Impeded ductal morphogenesis results in virgin SirT1ko/ko mice because local IGF-1 is apparently not sufficient to overcome increased IGFBP-1 in mammary adipose tissues. SirT1 therefore positively regulates the efficacy of the estrogen–IGF-1 signaling for ductal epithelial cell proliferation and differentiation.
The potential effect of modulating SirT1 activity
Breast cancer results from the accumulation of inherited and/or somatic mutations. Lifetime exposure to estrogen is a major risk factor for breast cancer, and a number of lifestyle factors, such as diet, body fat, alcohol consumption, exercise, parity, and hormone replacement therapy, may influence a woman's exposure to estrogen. The compound personal risk to a woman is difficult to assess because the molecular link between these factors and the effect of estrogen is poorly understood. The result of this study demonstrated that SirT1 positively modulates the efficacy of the estrogen–IGF-1 signal. This suggests that if SirT1 serves as a lifetime sensor to dietary restriction and acute withdrawal of nutrients [14, 57], its activity could ultimately influence the risk of breast cancer. Interestingly, an epidemiological study of Dutch famine in 1945 indicated that an exposure to famine at prepubertal age increased the risk of breast cancer, most clearly seen in women who never gave birth [58]. Moreover, the serum levels of estrogen and IGF-1 were increased among those exposed to the famine, which suggests that an epigenetic adaptation phenomenon may take place. Assessing the potential correlation between the enzymatic activity of SirT1 and the risk of breast cancer is therefore of great interest in order to identify feasible targets for chemoprevention against breast cancer.
Several dietary polyphenols as well as small molecules have been identified as either agonists or antagonists of SirT1 [59, 60]. The pharmacological effects of these compounds in humans remain elusive given that SirT1 targets multiple transcription factors and exerts pleiotropic effects. Nonetheless, our study of SirT1ko/ko mice demonstrated the potential utility of SirT1 antagonists. Specifically, reducing SirT1 activity alters the homeostasis of cellular responses, including deregulation of the expression of IGFBP-1 and IκBα, which can attenuate the stimulation from the estrogen–IGF-1 signaling and potentially desensitize mammary epithelial cells to NF-κB activation. SirT1 is therefore a candidate target for chemoprevention against breast cancer.
The study of mammary gland development is an excellent model system for unraveling how SirT1 modulates the efficacy of the estrogen–IGF-1 signaling and regulates the timing of ductal morphogenesis. These new mechanistic insights may also aid in understanding the role of SirT1 in breast cancer as well as in other organs in which local IGF-1 plays an important role.
Atm:
BrdU:
bromodeoxyurdine
CMV:
cytomegalovirus
ERα:
estrogen receptor alpha
FoxO:
forkhead box 'other' protein
GH:
IGF-1:
insulin-like growth factor-1
insulin-like growth factor-1 binding protein-1
H & E:
hemotoxylin and eosin
IIS:
insulin/insulin-like growth factor-1 signaling
IκBα:
inhibitors of NF-κB alpha subunit
IKKα:
IκB kinase alpha
MEF:
murine embryonic fibroblast
NF:
nuclear factor
receptor activator of NF-κB
Sir:
silencing information regulator
TEB:
terminal end bud
TNF:
tumor necrosis factor.
The study is supported by grants from the American Cancer Society (RSG-04-019-01-CNE) and the Nathan Shock Center of Excellence in the Biology of Aging at the University of Washington (to YG). The authors thank Jennifer Blasi, Thomas Gernon, Janet Leath, Jennifer Lee, Bryce Sopher, Loraine Warner, and Heather-Marie Wilson for their contributions in the early phases of the study, and Dr Fred Alt, Dr Mary Helen Barcellos-Hoff, Dr Mark Groudine, Dr Warren Ladiges, Dr George Martin, Dr Peter Rabinovitch, and Dr Jeffrey Schwartz for their support and advice.
13058_2006_1597_MOESM1_ESM.pdf Additional file 1: A pdf file containing the materials and methods for the generation of SirT1ko/ko mice. This supporting file documented the details of materials and methods used to generate both SirT1co/co mice and SirT1ko/ko mice. (PDF 205 KB)
13058_2006_1597_MOESM2_ESM.tiff Authors' original file for figure 1
HL characterized the phenotypes of the SirT1 mutant mice, participated in the design of the study, and helped to draft the manuscript. GKR and NL helped to characterize the phenotypes. CW generated the SirT1 mutant embryonic stem cells and mice. BPR helped analyze the histology data. YG conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.
Department of Radiation Oncology, University of Washington School of Medicine, Seattle, WA 98195, USA
Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA
Department of Comparative Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA
Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195, USA
Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH 44195, USA
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The marshmallows are a fresh product so at their best for up to three weeks.


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I am using Processing with MadMapper (a projection mapping program) via syphon. I already found the code (below) to get the processing talk to MadMapper via Syphon which works. BUT the thing is I have no idea how that works and therefore I can't really customize my Processing sketch code.
Do you always have to write "PGraphics" before canvas? What is it?
If you can please let me know I would really appreciate it. Thanks!
// Create syhpon server to send frames out.
So, a lot of these are basic Processing questions likely better served on the processing forums, and not specific to Syphon.
PGraphics is a variable type, processing graphics, its the 'canvas' type that Processing uses in this case.
Id suggest you hit up the processing forum and docs to understand how to configure /customize your drawing! Good luck!


Have you ever wondered when the first Novena was prayed? Look no farther than chapters 1 and 2 of the Acts of the Apostles to see that the disciples were gathered together in the Upper Room on the Day of Pentecost with Mary waiting and praying for the coming of the Holy Spirit, the promised Gift of the Lord. They had been there since Ascension Thursday praying the Church's very first novena! Despite the modern anomaly of moving Ascension to a Sunday, the Church repeats that original novena every year in preparation for the coming of the Third Person of the Blessed Trinity, the Sanctifier.
First, make time to pray in silence every day. This seems so simple, but busy people will know that it easier to say than to do. Jesus advised, "Go to your room, close the door and pray to your Father in secret." Indeed, that is what we need to do. Find the Upper Room of your life, whether it be your own private space, a Blessed Sacrament chapel at the local parish or just the focused "sanctuary of the heart" in a quiet moment and place; and spend some significant down time in silence so that you develop spiritual ears to hear the Voice that only speaks in silence. A minimum of 15 minutes each day in silence will train busy people to have more discerning hearts. That practice of a quarter of an hour in silence will undoubtedly lead the heart to want to spend more time with the Beloved every day.
Second, pray specifically for what you need. This means that you must identify the concrete needs of your life and present them to God as St. Paul says in Ephesians, "in every form of prayer and petition." God loves to answer prayers, but His answers can only be recognized by those who know exactly what they want! No need is too big. God will temper our prayer if it does not fully correspond to His Will. No need is too small either. God will also lift our spirits by answering our needs either by giving us just enough for the task at hand or showing us that what we ask for is less than what He really wants to give us. Either way, ask specifically and petition as many prayers as you and your loved ones need to a God who has an infinite storehouse of goodness ready to bestow on those who ask with childlike trust.
Finally, pray for the seven-fold Gift of the Holy Spirit. Even if you don't know what to pray for, the Eternal Spirit does. All good things in heaven and earth are contained in that One Gift. Pray to receive the One who satisfies all our needs in a new and special way this Pentecost. He gives gifts that cannot be exhausted: Wisdom, Understanding, Knowledge, Counsel, Piety, Fear of the Lord and Fortitude. Jesus also said, "Will not the Father give the Holy Spirit to anyone who asks him?" (Lk 11:11-13) Indeed He will!


Early the next morning, the Izambard household was teeming with activities as both adults and children scampered about packing their overnight bags. Sebastien set about making arrangements for the Il Divo private jet to take him and Ginny to Paris immediately after dinner and back two days later. He couldn't book any tickets for the commercial flight as it was a weekend and all flights were fully booked. The kids didn't forget to bring their favourite toys with them as they happily got into the car and waited for their parents to load their bags into the trunk.
Thirty minutes later, Sam stood at the door to welcome the guests. The kids said a quick hello to her and immediately ran into the house, only to be met by Urs coming out of the playroom.
"Hello, Lily and Nick. Are you happy to be staying here for two nights?" he asked warmly as they stopped to acknowledge him. Both of them nodded their heads impatiently and he laughed, "Ok, run along, they are waiting for you inside." Soon, kiddy laughter and chatter could be heard coming out of the playroom.
"Thanks for doing this for us, Urs," Ginny gave him a hug.
"No problem, Ginny. Just enjoy yourself," and he gave her a cheeky wink. Ginny blushed and Sam jab him in the side before pulling Ginny into the kitchen to help with the breakfast.
"I hope you haven't eaten breakfast cause I've prepared something for you guys as well," she said when they got into the kitchen.
"I wonder what my husband told your husband," Ginny mumbled absentmindedly as she looked at what Sam had prepared for breakfast: toasts, scrambled eggs, sunny side ups, ham, bacon and pancakes that were in different shapes like Mickey Mouse, heart, and normal round shapes. She idly wondered how Sam had managed to make so much so early in the morning. Then she noticed the apple pie baking in the oven and her eyes widened.
"Does it matter, Gin? He's only trying to secure some precious time for the two of you and that's very important in view of the tour they're going to do soon, don't you think?" Sam replied as she set about taking out the pie and cutting it into slices. She instructed Ginny to help bring the food into the dining room where Urs had already set the table.
When everything was laid out on the table, the kids were summoned to have their hands washed before joining the adults at the table.
"Oh look, Mommy, a Mickey Mouse pancake," Lily's eyes were round with wonder as she looked at the special pancake.
"My Mommy made those. Here, use this chocolate fudge to make the eyes and mouth," Angel instructed her best friend and both set to work decorating their pancakes. Grace had problem doing hers and big sister Angel had to help her out. Nick tried his luck at decorating his piece but made a mess. Tony observed them all before deciding it wasn't worth the trouble and proceed to take a bite at his Mickey Mouse, starting with one ear first.
Sebastien laughed softly as he observed the kids having fun with their food. "Do you always make something special for them?" he asked Sam.
"Yes. I hope you don't mind the extra work with my kids around here," Sebastien stated apologetically.
"So she and Carlos coming to stay over?" Urs asked.
"What's he doing having breakfast with a friend and leaving Joanne alone in the apartment?" Ginny was disgusted at the thought, especially since she felt that being apart constantly was the root of their earlier problems.
"It's a friend from a Spanish tv station who flew in for some business," Sam explained.
"Could be Carmen Calderón, the VP of the tv station that wanted us to star in a soap opera in exchange for helping Joanne sort out her contractual problem," Sebastien explained.
"Well, I hope he remembers to keep it professional by not going into her hotel room," Ginny huffed.
Carlos sat in the hotel café opposite Carmen, smiling enchantingly at something that Carmen said. In his mind he was thinking about Joanne being left in the apartment alone. He had insisted that she come with him to meet Carmen but she declined, saying that Carmen probably wanted to discuss their appearance on the TV program.
Joanne was right though and they had just gone through the schedules and were now making small talk.
"So what are you doing this evening, Carlito?" Carmen asked in their native tongue.
"I see. So there's no way I could get you to show me around town, is there?" she asked with the raise of one finely trimmed eyebrow.
"Well, I'm free till maybe three, then I have to go back and pick up my wife. We need to go grab some wine for the dinner. Can't go empty handed even though we're really like a family now," he explained.
She did not seem to be flirting with him and he relaxed a little before replying, "I'd be honored to be your host, Carmen. Just let me make a little phone call to my wife and tell her to expect me at three." Carmen smiled her agreement and he proceeded to call Joanne.
"Oh, but I was thinking of going over there earlier since Seb and his family are already there. They are leaving the kids there while they fly to Paris for two nights," Joanne sounded a little disappointed.
"Why don't we stay there for the night then? Or even two nights. It's time we make use of the EC. I heard from David it's got everything we might need," Carlos suggested, all the while keeping an eye on Carmen, hoping that she wouldn't think he was being rude making such a long phone call.
"Yes, my love. Three, I promise." With that they ended the conversation and he breathed a sigh of relief.
Carmen noticed that and gave a little laugh, "Stressed, Carlos? You should be, after what went on between the two of you." She proceeded to ask what he planned to do about the rumors that were spreading about Joanne and he told her about Joanne's decision not to confront Felix or the press about it.
"But don't let me see that guy again or I swear I'll break his neck," Carlos said vehemently.
"Tsk, tsk! You should rein in your fiery temper, Carlos Marin. You could do more harm than good for your reputation and hers," Carmen cautioned. Though Carlos still seethed with anger, he understood what Carmen was saying and Joanne's words came back to his mind.
"Ah yes, but she has more to offer you that I cannot. I'm glad we remained friends, Carlito. Otherwise you'd be looking for someone else and we'd have ended up in a bitter divorce." She touched his face tenderly, and he reached up to hold her hand and let it rest on his cheek.
"It would be so easy to fall in love with you again, Carmen. It's a good thing I fell in love with Joanne all over again after the fight. I'm not going to risk losing her again," his honey brown eyes sought her understanding.
"Why don't you join us for dinner tonight? The other guys would love to meet you too, I'm sure." He felt bad having to leave her alone after what they had just shared.
After dropping her off at the door of her hotel suite, they shared a brief kiss and said goodbye.
The moment he stepped out of the hotel entrance, he heaved another sigh of relief until he remembered he was in London, where Il Divo first started out. With the rumors of Joanne and Felix still rife, he and Joanne had both been hounded by some reporters wanting their story. Would there be any paparazzi trailing their every move? If so, then what he did with Carmen might be captured by them on camera. Deciding that honesty is the best policy, he decided to tell Joanne about his little escapade with Carmen, praying that she wouldn't blow her top.
"I'm back," Carlos announced as he stepped through the door of their luxurious apartment. He noticed two overnight bags by the door but no sign of Joanne. He walked into their bedroom and there she was, curled up in foetal position as she slept. He lowered himself onto the bed gently and kissed her cheek.
In half an hour, they were headed for the Buhlers' residence in their car. During the drive, Carlos talked about his meeting with Carmen and did a confession that they were more than just friends before he met Joanne. Joanne turned to look at him with astonishment in her eyes.
"We ended our relationship months before I met you. We agreed to remain friends when it was clear she didn't want to settle down, and that was what we've been all these years, just friends. We hardly ever met after that, except when I have to film something at her studio," he shrugged, trying to downplay his relationship with Carmen.
"That's why you're telling me all this right now, isn't it?" Joanne folded her arms and looked out the window. He sighed exasperatedly and rubbed a hand over his face as he tried to come up with something that would make her forgive him but he was loss for words.
Joanne could feel the sting of tears in her eyes as she looked out the car window. It wasn't the fact that Carmen was his old love that hurt her. It was him telling her about it right now that riled her; he must have felt guilty about what went on between them earlier that day that he felt a need to confess. And that kiss! Though it was nothing like what he did with Anna, still it stung her to know that he had kissed another woman. Whatever happened to his promise of no flirting?
They remained quiet all throughout the ride to their destination. The moment they stepped through the door, Sam immediately picked up on their moods. Greeting Carlos first, she hugged Joanne warmly and led her into the kitchen after telling Carlos that the guys were in the family room.
"Had an eventful ride, huh?" she said knowingly.
Joanne smiled wryly as she tried not to cry. Ginny noticed something wasn't right with her and immediately sat her down.
Ginny explained what Sebastien had told them earlier on. Hearing that, Joanne knew there was no way she could keep it from her best friends, so she told them the conversation she had in the car with Carlos.
"We know so," they both replied.
"What you going to do? Hit him on the head with a rolling pin?" Sam wisecracked.
"Ok, you have my email address," Ginny winked.
"Mine, too," Sam chipped in amid more giggles.
In the family room where the men gathered, there were some hors d'oeuvres of cheeses and cold cuts which Ginny and Sam had prepared for them, along with bottles of champagne that Sebastien brought to go around as they waited for dinner to be served.
The men had a somewhat similar conversation to the women's as Urs had noticed Carlos wasn't his usual self when he just sat on the chair facing the floor to ceiling window overlooking the garden. He suspected things weren't going right for him and Joanne so he probed.
Turning around to face all three Divos with a look that said "I did it again!', he gave them an account of what went on earlier in the day and his conversation with Joanne on the way here.
"Thanks, amigo. You're a big help," Carlos replied sarcastically.
"That's what he's asking you," Sebastien joined in the interrogation, raising an eyebrow as he waited for a reply, and then popped a piece of cheese in his mouth.
Carlos thought for a moment longer while his friends waited and observed his facial expressions, and David and Urs exchanged worried glances when they saw a frown appeared on his face.
"But that was years ago, she might have changed her mind now," Sebastien suggested.
"Right, so there's nothing to worry about," Urs concluded as he walked over and picked up a piece of ham and savored it. Then he went around refilling their champagne flutes with more of the drink.
"Wrong! There's Joanne to worry about. Now she's not speaking to me, which is why I'm like this, like a dead man all over again," Carlos looked so down and out that the guys couldn't help but sympathized with him.
"Yes, and if all else fails, you still have the five carat diamond ring as backup plan," Sebastien winked.
"I think he'll need to throw in a massive mansion with all the works this time," David teased, and both Sebastien and Urs chuckled.
"Friends who could laugh with you when everything is fine and rosy, and laugh at you when it's not," David joked. This time it got Carlos joining in a booming laugh with them all. With his spirit lifted by a little humour, he started enjoying the appetizers on the tray.
With the personal matters sort of settled, they got down to plan the release of their new album and worked out the venues of their next tour. Since they had all decided a tour was what they wanted, they all agreed to take the tour further than just the UK and Europe, which was the original plan.
David was satisfied that he now had a concrete plan and could tell Claire when he would be back again, and he made sure both LA and New York were included in the tour itinerary. Though it would be another couple of weeks before he could get away from work, he was happy to have that little escapade to look forward to. He missed Claire and Amelia so much that even the video chat he had with them didn't take away the longing to be with them in person.
Dinner got underway with the kids finally giving up their fun at the playground and joined the table after washing up. Angel requested to sit next to Uncle David and that started the whole kiddy gang resisting their usual sitting place; they all wanted to sit next to him. It took a while for the adults to sort them out.
"There's only one Uncle David, kids. You can't all sit next to him. How about sitting next to Uncle Carlos and Aunty Joanne?" Joanne suggested.
"Ok," Gracie accepted and went to stand with Joanne, allowing her hand to be held while her daddy moved her high chair to where she had chosen to sit. Carlos shifted his and Joanne's chair to allow a space in between for the little girl's chair.
"I want to sit with Daddy and Mommy," Anthony announced, and immediately went to drag his chair closer to where his daddy always sat, at the head of the table. Mommy looked at the others for a while and decided she would vacate her usual place at the other end of the table and sit at the side next to where her son would be sitting at the head of the table.
Next to Sam sat Angel, then David, then Lily. Sebastien sat at the other end of the table, and on the other side of the table facing Lily was Nick, then their mommy, then Joanne, Grace and Carlos.
Sitting arrangement settled, Urs and Sam went into the kitchen to bring out the food that Sam had spent the whole afternoon preparing with Ginny's help. There were food to cater to adults and children: ox tail and creamy tomato soup, roast chicken, stewed and roast beef, roast and mashed potatoes, mini sausages, boiled vegetables served with gravy, and mini Yorkshire puddings to soak up the gravy for the different types of roast meat.
Sebastien looked at the spread in front of him and complimented Sam, "You really are a great hostess, Sam. The dishes look delicious.
"Maybe next time we could have Spanish cuisine instead? Carlos?" David looked over at the Spaniard with a conspiratorial look.
"Si, si, next time my Joanne will make some delicious Spanish food. Her paella is simply delicioso," he leaned across Grace and looked at his wife, who sat quietly with a smile.
The guys knew he was getting slightly nervous by his use of Spanish words and Sam could see Urs' lips curled into a little smirk before he hid it behind his napkin. She gave him a glare and dared him to embarrass their guests further and he sobered up a little, cleared his throat and said what came to mind next, "Right, we'll have dinner once a month together, the wives will take turns to cook for us, except when we're touring." Then he raised his glass of wine in a toast to make it an official decree.
The adults all exchanged a look of disbelieve at the little girl's astuteness and Urs was slightly red-faced at being caught out by his daughter. David just threw back his head and laughed.
"Well, maybe by the time it's his turn he will have a wife," Sebastien recovered slightly to suggest this, trying not to burst out laughing too.
"Do you have a girlfriend, Uncle David?" Lily asked curiously.
"Because if you don't have a girlfriend then you can't have a wife, right Daddy?" Lily turned to Sebastien with a look of certainty in her eyes.
Sam gave him a blank look and shrugged. She was enjoying this conversation and thought it was about time he confessed to all of them about his little secret back in LA. Though Urs had briefly explained to her what David had told the men about Claire, she wanted to hear it straight from the horse's mouth.
Seeing that he wasn't going to get any help from his usual ally, he buried his face in his plate of food and started attacking the mashed potato there.
"Uncle David, aren't you going to tell us about Aunty Claire?" Angel wouldn't let go. She had overheard her parents' discussion about David and Claire over dinner the day the guys met at the club house. David's head shot up and stared at Urs accusingly.
"Well, don't look at me. It's going to come out sooner or later. And that's your Angel, you can run but you can never hide from her," Urs retorted to everyone's laughter, except the young ones who were quietly sitting there and wondered what all the fuss was about. Anthony picked up a piece of roast beef and munched on it when he decided he had waited long enough for nothing to happen.
Throwing in the towel, David sat back in his chair and begun, "Yes, Lily, I do have a girlfriend. Her name is Claire." He smiled sheepishly as he looked around the table. The other adults waited expectantly for the next question to pop up.
"Where is she now, Uncle David? Why didn't you bring her here?" Angel asked as she continued eating the broccoli she was holding with a fork.
"Can't you buy her a plane ticket or something?" Lily asked after swallowing a piece of roast chicken.
"She can't come as she has to take care of her daughter. She has a little girl named Amelia who's a few months younger than Tony and Grace," David replied as naturally as he could.
By now the other adults were close to choking on their food, Sebastien especially was turning so red in the face with stifled laughter that he had to bury his mouth in his napkin the whole time the girls posed all the sensible questions. Carlos muttered an 'Oh my god' before he too took his napkin to his face. Joanne busied herself by paying extra attention to little Gracie, wiping her mouth and bringing the drinking cup to her whenever the little girl asked for it, all the while with a huge grin on her face. Ginny did the same for Nick, trying not to look at David as that would have been a fatal mistake and she was sure of it.
Sam looked over to David and smiled sweetly. "You're on your own, brother," she whispered. Urs paid extra attention chewing at the roast beef in his mouth, but it wasn't easy as his lips had other idea, constantly curving up into a smirk.
"So is Aunty Claire looking for another daddy for Amelia? Is that why you are her boyfriend?" Angel asked as the little ones continued to eat.
"I don't know for sure if she wants a daddy for Amelia, but we like each other, so we're seeing each other," David gave up trying to be coy about his love life.
"Then you should marry her. Aunty Sandra is not here anymore so you don't have to worry about her being angry," Angel advised.
David heaved a sigh of relief when finally his two tormentors decided to let him off the hook. He looked over Angel's head and caught Sam's attention before sticking out his tongue at her. Tony and Urs saw it and Urs' little clone looked up at his daddy and giggled, bringing an indulgent smile to his daddy's face.
After dinner came dessert. As Sam brought out strawberry cheesecakes for adults and chocolate brownies topped with ice-cream for the kids, there were claps and words of approval from everyone.
"When are you cooking for us?" Grace asked as she looked up at Joanne.
"Well…" Joanne looked to Carlos for a little help.
That started all the small ones talking about their favorite food at once and among the din things like pizzas, ice-creams, chocolate cakes, hamburgers and hotdogs could be heard.
"Ok," came the unified reply.
Soon, the table was cleared and as the womenfolk were about to pile into the kitchen to begin washing up the dishes, Carlos volunteered his service and those of his brothers'.
"Why don't you lovely ladies take a break and let us men do the washing?" he wriggled his eyebrow and gave a cheesy smile. Sam and Ginny broke into a fit of giggles while Joanne just walked over to him and handed him the dish cloth.
Carlos followed Joanne out the kitchen entrance with his eyes while the other guys followed his gaze.
"Why?" Urs grinned, sort of knowing what was coming.
"Either to muffle the sound of his screams when she squeeze the truth out of him or the sound of their…'ahem'," Sebastien replied laughingly.
Carlos and Urs doubled over laughing along with David as the French one turned lobster red and glared at the youngest Divo. He turned around and begun attacking the dishes in the sink. The other three guys continued laughing, unperturbed that their friend clearly wasn't happy.
The three laughing Divos stopped laughing for a moment and looked up at her before they burst into a giggling fit.
"Alright! Enough! I'm leaving. Come on Ginny, we have to make the flight." Sebastien threw the dish cloth into the sink and walked away from his friends who were still laughing uncontrollably. He grabbed Ginny's hand and led her out of the kitchen amid her protests.
By now the three laughing men had sobered enough to follow the couple out of the kitchen and they were all stood in foyer.
"I'm sorry, Seb. It was a crude joke, please accept my sincere apology," David said with as much sincerity as he could muster, placing a hand over his heart and bowed.
Urs and Carlos went over to Sebastien and patted his back.
"Seb, we're brothers, no? So what's a little joke among brothers?" Carlos asked.
"Carlos is right. Although David might have gone overboard, I'm sure our relationship now is such that it can withstand a few crude jokes," it was Urs' turn to try to calm the fuming Frenchie down.
David went over and offered his hand and Sebastien took it and gave him a shoulder hug. The other two Divos followed and finally Ginny hugged her husband and gave him a little kiss to soothe his wounded ego.
"Can we go over to the EC where all the kids are? They're helping Joanne to settle in," Ginny asked.
"Ok, let's go. The others can do the dishes," Sebastien said with a devilish smile before leaving the house. Urs, Carlos and David shared a look in silent amusement.
"Nick, please stop jumping on the bed. That's for Uncle Carlos and Aunty Joanne to sleep in," Sam had her hands on her hips as she talked to Nick.
"But it's so fun," Nick continued jumping on the bed.
"I want my mommy!" he shouted.
Sebastien raised an eyebrow at the scene but Ginny just pulled him into Joanne's room to talk to Lily. Sam made sure the boy stayed where he was before joining them all. Nick sobbed a little and gave a little hiccup when his breath got caught in his throat.
"I'm sorry, Seb. I know it's hard to see your child being disciplined by someone other than yourself or Ginny, but it has to be done if he's to learn to respect authority," she put a hand on Sebastien arm and smiled apologetically.
"Yes, Mommy," Lily said in a small voice.
"Awww, Lily, there's no need to be afraid. You're here to have some fun and as long as you don't misbehave, you won't be punished at all," Sam tried to put her at ease. Lily nodded. Turning to Angel, Sam asked her to bring Lily to the living room to join the twins who were watching cartoon.
When the two girls left hand in hand, Sam turned to Joanne and told her the fridge had been stocked up for her. Joanne was still dazed at the first parenting lesson she got from Sam and could only nod her head. She had never come across a stubborn child and wondered if she could handle one the way Sam did.
"I'll be cooking dinner every night so come and join us," she said. Turning to Sebastien and Ginny, she told them not to worry about the kids and just enjoy their little break.
"Oh, it's not good saying goodbye to Nick now. Let me release him first." Sam quickly went out and told Nick he was released from detention, but not before explaining to him why he was being punished.
"Listen, Nick. Aunty Sam only punished you because you didn't listen to her. If you had done what she told you to do you wouldn't be punished at all, instead, you'd be having lots of fun. Do you understand what I'm saying, Nick?" Ginny put a finger under his chin and lifted his head up to face her. Nick nodded.
"That's my boy. Now, go and apologize to Aunty Sam for misbehaving," Ginny turned him in Sam's direction.
Sam smiled at the boy and opened her arms to him. He walked over slowly and mumbled, "I'm sorry, Aunty Sam." And he was rewarded with a hug and a kiss on the cheek.
Hearing the plan for the next day, his face lit up immediately and he ran over to his parents to kiss and say goodbye to them. They all made way to the living room where Sebastien and Ginny said goodbye to Lily and the rest of the kids.
Before Sebastien and Ginny left in their car, the three Divos had finished up their chores and were already waiting outside the house to bid them goodbye.
"Nope! Men's joke," he grinned.
Sighing, he admitted defeat and gave her an account of what David said about them. Instead of feeling humiliated by the crude joke, she laughed and commented that David was a smart one.
Sam is a good mother…. and maybe someday, Seb will be a stronger father. He is slowly learning what his children need as far as guidance.