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Smad7 is a transforming growth factor-beta–inducible mediator of apoptosis in granulosa cells
Smad7 is a transforming growth factor-beta–inducible mediator of apoptosis in granulosa cells Marisol Quezada, Ph.D., Jikui Wang, Ph.D., Valerie Hoang, M.S., and Elizabeth A. McGee, M.D. Department of Obstetrics and Gynecology and Institute for Women's Health, Virginia Commonwealth University, Richmond, Virginia
Objective: To determine the functional role of Smad7 in granulosa cells. Design: Granulosa cell culture and molecular biological techniques were used to investigate regulation and function of Smad7. Setting: Research laboratory. Animal(s): C57bl/j hybrid mouse. Intervention(s): Primary mouse granulosa cells were isolated and grown in culture for all messenger RNA expression experiments. Smad7 promoter constructs were evaluated with a luciferase reporter system in SIGC cells to determine sites activating Smad7 expression. Main Outcome Measure(s): Overexpression (Smad7 complementary DNA) and downregulation (Smad7 small interfering RNA) of Smad7 in primary mouse granulosa cells were used to evaluate the functional role of Smad7 in granulosa cells. Result(s): Smad7 expression was upregulated by treatment with transforming growth factor-b (TGF-b) but not activin or activation of the cyclic adenosine monophosphate pathway. The promoter of Smad7 was activated by TGF-b. Truncation of the promoter or mutation of the Smad response element at 141 eliminated TGF-b activation of the promoter. Smad3 was not specifically required for TGF-b–stimulated expression of Smad7, though activation of the TGFBR1 receptor was. When Smad7 was overexpressed in granulosa cells, apoptosis was markedly increased. When Smad7 expression was reduced with small interfering RNA, then the TGF-b–induced apoptosis was blocked. Conclusion(s): Smad7 mediates apoptosis induced by TGF-b in mouse granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis. (Fertil SterilÒ 2012;97:1452–9. Ó2012 by American Society for Reproductive Medicine.) Key Words: Smad7, TGF-b, apoptosis, folliculogenesis
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n mammals, fertility depends on progressive development of the egg-containing follicles through a process known as folliculogenesis. Follicles that do not progress to ovulation undergo atresia and are lost from the pool of growing follicles. The fate of each follicle and its enclosed oocyte is determined by both local and systemic factors regulating the cellular events within the follicle that determine whether it continues to grow and develop or undergoes atresia via apoptosis (1, 2). The transforming growth factor-b (TGF-b) family of growth factors playscritical roles in maintaining cell growth and differentiation in the ovary (3). Classically, TGF-b signals via
heteromeric complexes of type II and type I serine/threonine kinase receptors and their interacting Smad partners. The TGF-b ligand binding to the type II receptor facilitates the initiation of signaling with phosphorylation of the type I receptor and subsequent phosphorylation of Smad2 and Smad3 (4). Activated Smad2/3 bind with the common mediator, Smad4, and translocate to the nucleus to regulate ligandspecific gene expression. Smad complexes interact with other nuclear transcription factors, although Smad3 can also bind DNA directly (4, 5). Within the ovary, Smad2 and Smad3 expression are stage specific (6). Female mice deficient in Smad3 have reduced responsiveness to FSH
Received December 8, 2011; revised March 13, 2012; accepted March 14, 2012. M.Q. has nothing to disclose. J.W. has nothing to disclose. V.H. has nothing to disclose. E.A.M. has nothing to disclose. Supported by NICHD HD 045700 and D43 TW0076962. Reprint requests: Elizabeth A. McGee, M.D., Virginia Commonwealth University, MCV Campus, Sanger Hall, P.O. Box 980034, Richmond, Virginia 23298-0034 (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 6, June 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.03.024 1452
stimulation and reduced ability of FSH to up-regulate its own receptor, resulting in impaired folliculogenesis (7). Smad6 and Smad7 are ligand-induced inhibitory Smads, which function as negative regulators of Smad activity (8). Smad7 is a general antagonist for both TGF-b and bone morphogenetic protein (BMP) family signaling, in contrast to Smad6, which plays a more specific role in the BMP pathway (9). Smad7 antagonizes TGF-b signaling through multiple mechanisms, playing pivotal roles in embryonic development and adult homoeostasis (10, 11). Altered expression of Smad7 is associated with several human disease processes, including cancer, tissue fibrosis, and inflammatory diseases (12). Though Smad7 mRNA has been localized in mouse oocytes (13), this is the first report of localization, regulation, and function of Smad7 in granulosa cells. Transforming growth factor-b has been described as an inhibitory factor for ovarian folliculogenesis (3). VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® Previously we reported that TGF-b is a potent proapoptotic factor for preantral follicles in culture (14). Additionally, Smad7 causes apoptosis in podocytes, HaCaT keratinocytes, and cortical neurons (11). Because Smad7 is regulated by TGF-b in other systems, we have considered a role for Smad7 in TGF-b–mediated granulosa cell apoptosis.
MATERIALS AND METHODS Animals and Granulosa Cell Culture All animal experiments were performed in accord with National Institutes of Health guidelines and with institutional approval. For primary granulosa cell culture, ovaries from 25- to 28-day-old female mice were removed, dissected free of connective tissue, and incubated as described by Campbell (15). Granulosa cells were harvested, pelleted, resuspended, and cultured as previously described (7). Cells were plated at approximately 40% confluence in McCoy's medium containing 10% fetal bovine serum (FBS) and Pen/Strep at 37 C. The medium was changed to serum-free McCoy's with 0.1% bovine serum albumin (BSA) and 10 mL/mL Insulin, transferrin, selenium (ITSþ) for 24 h before the experiments. All reagents and inhibitors were purchased from Sigma, except for TGF-b1 and activin A, which were from R&D Systems.
Histology and Immunostaining For histologic examination, 5-mm paraffin sections were rehydrated, quenched, and incubated with 10% goat serum before incubation with polyclonal rabbit antibodies against Smad6 and Smad7 (1:100 and 1:500, respectively; Zymed). A biotinylated anti-rabbit IgG antibody was the secondary (Vector Laboratories), and slides were incubated with Vector Laboratories elite ABC reagent for 30 minutes before detection with diaminobenzidine using the SuperPicTure Kit (Zymed).
Quantification of Cell Viability and Apoptosis Cell proliferation was verified using the Cell Growth Determination Kit from Sigma (according to the manufacturer's instructions). Absorbance levels were compared with a standard curve from dilutions of a known number of granulosa cells. All the experiments were performed in quadruplicate. For apoptosis studies, cells were fixed in 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. Cells were then analyzed using a DeadEnd colorimetric TUNEL system (Promega) according to the manufacturer's protocol. The percentage of apoptotic nuclei was determined by counting 100 cells on each of four different coverslips per group and scoring each cell as stained or not stained. Groups were unknown to the person scoring the cells. Experiments were performed in duplicate. Apoptotic index was calculated as number of positive stained cells/number of total cells.
tary DNA (cDNA) synthesis was performed with SuperScript III First-Strand System for RT–polymerase chain reaction (PCR) (Invitrogen). The TaqMan assay system was used for messenger RNA (mRNA) evaluation using an ABI 7500 Thermocycler (Applied Biosystems). Specific 6-carboxyfluorescein (FAM)-labeled probes for mouse Smad7, Smad6, TATA-box binding protein (TBP), and plasminogen activator inhibitor-1 (PAI-1) were purchased from Applied Biosystems. The details of this assay are provided in Supplemental Materials and Methods, available online.
Smad7 Promoter-Luciferase Reporters Mouse Smad7 promoter-luciferase reporter constructs were created using the pGL-3 vector system (Promega). For promoter activity experiments constructs were transfected into a well-characterized spontaneously immortalized rat granulosa cell line, SIGC (16–18). The genomic DNA fragment containing the 4.3-kb Smad7 flanking region was amplified from genomic DNA (19). The detailed strategies to create the truncated and mutated constructs are described in Supplemental Materials and Methods.
Overexpression of Smad7 in Primary Granulosa Cells Mouse Smad7 full-length (insert size 1.3 kb, called S7FL) and truncated (insert size 0.67 kb, called S7T) constructs were kindly provided by Dr. Peter ten Dijke (8). Primary granulosa cells were transfected with these constructs and controls using FuGENE 6 (Roche Diagnostics) as described in Supplemental Materials and Methods.
siRNA in Primary Granulosa Cells Small interfering RNA (siRNA)-Smad2, siRNA-Smad7, or AllStars Negative Control siRNA (Qiagen) were transfected into primary granulosa cells using FuGENE 6. One day after transfection, cells were treated with TGF-b (1 ng/mL) for 24 hours. After TGF-b treatment, triplicate wells were prepared for analysis by TUNEL, PCR, or immunofluorescence. Transfection efficiency in each experiment was monitored using Cell Death Control siRNA (Qiagen). Knockdown was verified by immunostaining and measuring the mean intensity of fluorescence per cell on the coverslips. Smad7 down-regulation exceeded 60% in each replicate, whereas Smad2 downregulation exceeded 90%.
Data Analysis Experiments were repeated at least three times, and results were reported as the mean SEM. Data were subjected to analysis of variance and post hoc testing with Tukey's honestly significant difference test when more than two groups were compared. Student's t test was used for single comparisons. Statistical significance was accepted at P< .05.
RNA Isolation and Real-Time PCR Analysis Ribonucleic acid was isolated from granulosa cells using the PureLink MiniKit (Invitrogen) according to the manufacturer's protocol. The reverse transcription (RT) complemenVOL. 97 NO. 6 / JUNE 2012
RESULTS In contrast to Smad6, Smad7 is expressed in granulosa cells and is regulated by TGF-b. 1453
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY To determine the cellular localization of Smad7 protein in the ovary, we performed immunohistochemistry on ovarian sections from 23-day-old and cycling mice. Representative images in Figure 1A and B show the contrasting patterns of expression for Smad6 and Smad7. Smad6 expression is limited primarily to the cytoplasm of the oocytes (Fig. 1A). Smad7 protein expression was detected in oocytes and granulosa cells from preantral and antral follicles. Strong Smad7 staining was also observed in the ovarian surface epithelium and in endothelial cells lining vessels. Smad7 was seen in both cytoplasm and the nucleus of scattered cells within the follicles. However, in the corpora lutea, Smad7 was more focused as nuclear staining, though some cells also had cytoplasmic staining (Fig. 1B). We next determined the effect of TGFb treatment on the expression of the inhibitory Smads in granulosa cells in culture. As shown in Figure 1D, TGF-b treatment of granulosa cells increased Smad7 mRNA expression more than 2.5-fold (P< .05). The expression of Smad7 was both dose and time dependent. In dose–response studies, Smad7 expression was maximal at a dose of 10 ng/mL (Supplemental Fig. 1A and B). Smad7 expression increased up to 24 h, where it remained stable to 72 h. Although Smad6 mRNA was detectable in cultured granulosa cells, TGF-b treatment did not increase Smad6 expression.
TGF-b Stimulates Smad7 Promoter Activity To further understand the mechanism by which TGF-b increases Smad7 mRNA, we evaluated whether TGF-b can increase the transcriptional activity of the Smad7 promoter.
As shown in Figure 2A, TGF-b treatment (1 ng/mL) significantly activated a Smad7 promoter/luciferase reporter system transfected into a rat granulosa cell line (SIGC). In contrast, activin treatment did not increase Smad7 mRNA expression levels (Supplemental Fig. 1C) or promoter activation up to a dose of 200 ng/mL (Fig. 2A). Furthermore, neither forskolin nor 8-Bromoadenosine-30 , 50 -cyclic monophosphate (8-BrcAMP) (Fig. 2A) nor FSH (Supplemental Fig. 1D) modulated the TGF-b–stimulated increase in Smad7.
Identification of the TGF-b–Responsive Region of the Mouse Smad7 Promoter Examination of the 5' flanking region of the mouse Smad7 gene sequence revealed six palindromic Smad-binding elements (SBE) sites. Additional information regarding the analysis of the Smad7 promoter is provided in Supplemental Materials and Methods and in Supplemental Figure 3. Smad7 promoter deletion constructs were generated to identify the TGF-b–responsive region. The SIGC cells were transfected with these constructs generated from the full-length mouse Smad7 promoter, and their basal and stimulated activity was analyzed. Although a 3.8-fold induction was observed with fulllength construct, the induction was decreased by deleting the region from 4039 to 150 and was abolished by deletion from 4039 to 118 (Fig. 2B). Mutation of the most proximal putative TGF-b site in a longer promoter construct significantly reduced the TGF-b inducibility of the Smad7 promoter, providing further evidence that this cis element is
FIGURE 1
Localization and regulation of Smad6 and Smad7 in the ovary. (A) Smad6 is highly expressed in the cytoplasm of the oocytes of growing follicles. (B) Smad7 is expressed in the granulosa cells and oocytes of growing follicles and in corpora lutea. (C) Negative control is absence of primary antibody. PAF ¼ preantral follicle; AF ¼ antral follicle; CL ¼ corpus luteum. (D) Effect of TGF-b (1 ng/mL) on Smad6 and Smad7 mRNA from primary granulosa cells cultured. Mean SEM; aP<.05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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FIGURE 2
Transforming growth factor-b responsiveness of the Smad7 promoter. (A) SIGC cells were transfected with a 4.3-kb mouse Smad7 promoter reporter construct and treated as indicated. Transforming growth factor-b treatment increased luciferase activity, but activin and stimulators of PKA did not. (B) Putative SBE sites of Smad7 promoter (black circles). The progressive 50 deletion fragments are represented by grey bars. Mutated SBE is depicted by the black rectangle. Mean SEM; a s b,b s cP<.05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
the primary determinant of TGF-b responsiveness in the context of granulosa cells.
TGF-b Can Up-regulate Smad7 in the Absence of Either Smad2 or Smad3 It has been shown that SB431542 is a specific inhibitor of the type I receptors for TGF-b and inhibits the phosphorylation of Smad2/3 induced by TGF-b (20–23). The addition of the TGF-b receptor antagonist SB431542 blocked TGF-b– stimulated Smad7 promoter activation (Fig. 3A) and mRNA expression (Fig. 3B). We also used a specific inhibitor of Smad3 phosphorylation (SIS3) in these studies (24, 25). The inhibition of Smad3 did not prevent TGF-b stimulation of Smad7 promoter activation or mRNA (Fig. 3C and D), although it did reduce the expression of PAI-1 mRNA, used as a control of SIS3 efficacy (Supplemental Fig. 2A). We confirmed these results using granulosa cells that do not express VOL. 97 NO. 6 / JUNE 2012
Smad3 (hatched bars), from Smad3-deficient mice (7). Additionally, the reduction of Smad2 with siRNA in wild-type granulosa cells did not prevent TGF-b stimulation of the Smad7 promoter (grey bars, Fig. 3D). As shown in Figure 3D, all the cell types maintained the ability to up-regulate Smad7 promoter activity in response to TGF-b. Together, these results provide evidence that neither the presence of Smad2 nor of Smad3 is specifically required for Smad7 up-regulation by TGF-b, but activation of the type I receptor is required.
Overexpression of Smad7 Promotes Apoptosis, Whereas Reduction of Smad7 Prevents TGF-b– Induced Apoptosis Because TGF-b is a potent proapoptotic factor for preantral follicles (14) and Smad7 expression is increased by TGF-b in granulosa cells, we evaluated the biologic effect of increased Smad7 expression in granulosa cells. The verification 1455
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FIGURE 3
Neither Smad2 nor Smad3 are specifically required for up-regulation of Smad7. (A) SIGC cells transfected with Smad7 promoter were incubated for 24 hours with TGF-b (1 ng/mL) in and SB431542 10 mM or vehicle control. (B) Smad7 mRNA expression in primary granulosa cells treated as above. (C) SIGC cells transfected with the Smad7 promoter and incubated for 24 hours with or without TGF-b (1 ng/mL) in the presence of the specific inhibitor of Smad3 phosphorylation (SIS3, 10 mM) or vehicle control. Luciferase activity was normalized against activity of a cotransfected renilla vector. Bars represent mean SEM from three different experiments. (D) Smad7 mRNA expression in primary granulosa cells from wild-type or from Smad3 knockout mice (hatched bars) or from wild-type primary cells transfected with siRNA for Smad2 (grey bars) incubated 24 hours with the treatments before mRNA analysis as above. Mean SEM; a s bP<.05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
of expression and function of Smad7 protein is detailed in Supplemental Results (Supplemental Fig. 4). First, we determined the effect of overexpression of Smad7 on viable cell number in primary granulosa cell cultures using a tetrazolium salt assay (7). The overexpression of Smad7 (S7FL) decreased cell viability compared with cells transfected with control vector (empty vector, EV) at 6 and 24 hours (Fig. 4A). To determine whether this decrease in cell viability corresponded to an increase in apoptosis, we transfected the cells as before and performed a TUNEL assay to label free DNA ends. The overexpression of S7FL resulted in increased apoptosis of granulosa cells relative to cells transfected with EV (Fig. 4B). Together, these results suggest that the up-regulation of Smad7 can induce apoptosis in granulosa cells. To evaluate whether Smad7 is required for TGF-b induction of apoptosis in granulosa cells, we also performed TUNEL assay on cells that were transfected with siRNA for Smad7 and compared the apoptotic index with cells transfected with control siRNA. Transforming growth factor-b–stimulated 1456
apoptosis was significantly decreased by reduction of Smad7 expression (Fig. 4C). Coverslips were immunostained for Smad7 with each experiment, and the mean intensity of fluorescence per cell was reduced to 40% of basal expression. Thus overexpression of Smad7 induces apoptosis, and knockdown of Smad7 expression prevents TGF-b–stimulated apoptosis in granulosa cells.
DISCUSSION Smad7 is an important inhibitor of TGF-b superfamily signaling. Because Smad7 is up-regulated by TGF-b itself, it can function in a negative feedback loop. However, recent studies in other cell types suggest that the role of Smad7 may be much more complex than just negative feedback. Although Smad7 has been reported to be present in immature oocytes (13), we have shown that Smad7 is expressed in granulosa cells of growing follicles and mediates apoptosis induced by TGF-b. Moreover, we have reported that Smad7 is up-regulated by TGF-b at least partially through VOL. 97 NO. 6 / JUNE 2012
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Smad7 overexpression induces apoptosis, whereas TGF-b–induced apoptosis requires Smad7 expression. Primary granulosa cells transfected with full-length Smad7 cDNA (S7FL) or EV and treated with TGF-b (1 ng/mL). (A) Cell viability determined by tetrazolium salt assay. (B) Programmed cell death was assessed with TUNEL. At both 6 and 24 hours S7FL cells were overwhelmingly more apoptotic than EV cells. (C) Assay (TUNEL) of primary granulosa cells treated with TGF-b (black bars) or vehicle (white bar). Cells were transfected with siRNA Smad7 or control siRNA before TGF-b treatment. Mean SEM; a s b,c s dP< .05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
a canonical Smad regulatory element in the proximal promoter of Smad7. Transforming growth factor-b regulates Smad7 expression but not Smad6 in many cell types (11). The localization of Smad6 and Smad7 in follicles is consistent with different roles for the two inhibitors. Smad6 function is largely thought to be limited to the BMP pathway, whereas Smad7 has much wider function in both BMP and TGF-b pathways. Smad7 is thought to inhibit TGF-b signaling by competing with Smad2/3 for binding to the type I receptor, enhancing ubiquitination of Smad complexes, and inhibiting Smad/DNA complexes in the nucleus (26). Specific roles of Smad7 may be cell and stage specific (11, 12). VOL. 97 NO. 6 / JUNE 2012
It is interesting that activin did not regulate Smad7 expression in granulosa cells. The differential response to activin and TGF-b does, however, explain the different effects seen with TGF-b and activin treatment of preantral follicles in serum-free culture; activin treatment results in growth and differentiation, whereas TGF-b treatment results in overwhelming apoptosis (14). This differential activin/TGF-b response is also consistent with a report of differential activation of a Smad7 promoter construct in fibroblast cells (27). Failure of FSH or cAMP activation of the promoter was also interesting because TGF-b and FSH often act additively or synergistically in the ovary (28, 29). Although many of the functions of TGF-b and activin and even the 1457
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY gonadotropins overlap during follicle development (1), this unique role for TGF-b in up-regulating Smad7 provides the possibility of independent functions for TGF-b, distinct from activin or gonadotropin. The Smad7 promoter contains several putative Smad regulatory sites. However, deletions of most of these sites did not result in decreased activation of the construct by TGF-b. Deletion of the canonical SBE site at 141 bp reduced this stimulation to near-basal levels. The importance of this site in granulosa cells was confirmed with mutation of the site in a longer construct. The use of chemical and genetic inhibitors of either Smad2 or Smad3 demonstrates that neither are specifically required for Smad7 up-regulation by TGF-b, but activation of the type I receptor is required. This is much different than the more specific requirement for Smad3 in the regulation of the FSH receptor (7). However, more studies are needed to definitively show whether there is a role for the noncanonical signaling pathways activated by the type I receptor in Smad7 regulation. Requirements for Smad2 and/or Smad3 have differed in other reports of Smad7 regulation. Our results are consistent with the activation of the mouse promoter in murine fibroblasts and human hepatoma cell lines (30, 31), in which both Smad2 and Smad3 were functional in activating the Smad7 promoter. Smad3 has been reported to be the preferential activator of Smad7 in a human promoter expressed in mouse embryonic fibroblast cells (32). It is not clear whether the variable results in promoter activation were species specific or related to methodologic differences. We used a mouse promoter in a rat cell line. However, our studies confirmed the promoter-based experiments with evaluation of mRNA expression in primary granulosa cell culture. Other signaling pathways, including noncanonical TGF-b pathways, may be involved in the transcriptional regulation of Smad7 expression in granulosa cells. In silico analysis identified many other transcription factor–binding sequences that are present in the promoter region of Smad7 (Supplemental Fig. 3). Additionally, transcription factors such as AP-1, specificity protein 1 (SP1), and activating transcription factor 2 (ATF-2) have been shown to bind to Smad complexes in vitro and enhance or otherwise modify the extent of TGFb–induced transcription in other tissue types (27, 31–33). The combinations of transcription factors also seem to be cell and even developmental stage specific, promoting stimulation or suppression of Smad7 expression. Further studies are needed to elucidate these possible complex interactions in Smad7 regulation during folliculogenesis and their roles in regulating apoptosis. Overexpression of Smad7 induces increased apoptosis, whereas decreased Smad7 expression reduces apoptosis induced by TGF-b in granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis. Overexpression of Smad7 protein increased apoptosis at both 6 hours and 24 hours after transfection relative to controls. Although not a true representation of TGF-b–stimulated effects, because the Smad7 was driven by a constitutive promoter and highly expressed within the cytoplasm of the cells, the study does clearly demonstrate that increase of Smad7 protein is capable of inducing apoptosis of granulosa cells. A reduction of 1458
Smad7 expression by 60% was sufficient to block the TGFb–induced apoptosis in primary granulosa cells. Together, these data provide strong evidence that Smad7 is the mediator of TGF-b–induced apoptosis of granulosa cells. Regulation of folliculogenesis is very complex. Many follicles begin growth, yet very few continue along the developmental pathway to ovulation. The role of gonadotropins and the cAMP/protein kinase A (PKA) pathway in follicle growth and the intrafollicular regulation of the balance of PKA activation in follicle selection and dominance have long been known (1). Likewise, additional factors functioning as autocrine and paracrine hormones modulating follicle development have long been recognized. Though folliculogenesis research has traditionally focused on complete blocks in follicle development, human infertility should more appropriately be described as subfertility, whereby follicles can grow and may ovulate, but less efficiently or with poorer egg quality compared with normal. For example, in mice, Smad3 is necessary for normal rates of follicle development with appropriately mature oocytes. Yet follicles can still grow and even ovulate in the absence of Smad3, although the females are infertile (7). Smad7 is regulated by TGF-b. It is also expressed in granulosa cells and is able to inhibit the functions of Smad2 and Smad3. Moreover, Smad7 could also inhibit granulosa cell proliferation through inhibition of the GDF9/BMP15 pathway (34). If Smad7 promotes apoptosis over survival, the expression level of Smad7 present in a follicle could be a determinant of follicle fate. If the balance between activators and inhibitors of Smad7 results in increased Smad7, then an increase in granulosa apoptosis would be expected with concomitant decreased granulosa cells mass, decreased aromatase expression, and reduced oocyte quality. In contrast, if Smad7 is reduced, then within a follicle there would be less granulosa cell loss to apoptosis—resulting in increased granulosa cell mass and increased aromatase and other markers of follicle health. Smad3 would not be as inhibited and follicle-stimulating hormone receptor (FSHR) might be more effective in upregulating its own receptor. Thus there are several ways that dysregulated Smad7 could be a factor in the more subtle presentations of subfertility in humans. Dysfunction of individual proteins responsible for subfertility has been difficult to identify because redundant layers of regulation, stage-specific and temporal differences in molecular function, and even cross-talk between pathways confuse the findings in investigations of systems. Yet knowledge of the range of functions of a key pleiotropic molecule like Smad7 will allow interpretation of more advanced studies such as pathway activation analyses, or predicting which combinations of abnormalities found in genome-wide association studies might result in identifiable phenotypes (35).
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Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, et al. SB431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 2002;62:65–74. Hjelmeland MD, Hjelmeland AB, Sathornsumetee S, Reese ED, Herbstreith MH, Laping NJ, et al. SB-431542, a small molecule transforming growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility. Mol Cancer Ther 2004;3:737–45. Mimura K, Kono K, Hanawa M, Kanzaki M, Nakao A, Ooi A, et al. Trastuzumab-mediated antibody-dependent cellular cytotoxicity against esophageal squamous cell carcinoma. Clin Cancer Res 2005;11:4898–904. Jinnin M, Ihn H, Tamaki K. Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta1-induced extracellular matrix expression. Mol Pharmacol 2006;69:597–607. Gilchrist RB, Ritter LJ. Differences in the participation of TGH (beta) superfamily signalling pathways mediating porcine and murine cumulus cell expansion. Reproduction 2011;142:647–57. Yan X, Liu Z, Chen Y. Regulation of TGF-beta signaling by Smad7. Acta Biochim Biophys Sin (Shanghai) 2009;41:263–72. €ttinger EP. von Gersdorff G, Susztak K, Rezvani F, Bitzer M, Liang D, Bo Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta. J Biol Chem 2000; 275:11320–6. Dodson WC, Schomberg DW. The effect of transforming growth factor-beta on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology 1987;120:512–6. Ke FC, Chuang LC, Lee MT, Chen YJ, Lin SW, Wang PS, et al. The modulatory role of transforming growth factor beta1 and androstenedione on folliclestimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells. Biol Reprod 2004;70:1292–8. Denissova NG, Pouponnot C, Long J, He D, Liu F. Transforming growth factor beta-inducible independent binding of SMAD to the Smad7 promoter. Proc Natl Acad Sci U S A 2000;97:6397–402. Brodin G, Ahgren A, ten Dijke P, Heldin CH, Heuchel R. Efficient TGF-beta induction of the Smad7 gene requires cooperation between AP-1, Sp1, and Smad proteins on the mouse Smad7 promoter. J Biol Chem 2000; 275:29023–30. €ttinger EP. Smad proSchiffer M, von Gersdorff G, Bitzer M, Susztak K, Bo teins and transforming growth factor-beta signaling. Kidney Int Suppl 2000;77:S45–52. Jungert K, Buck A, Buchholz M, Wagner M, Adler G, Gress TM, et al. SmadSp1 complexes mediate TGFbeta-induced early transcription of oncogenic Smad7 in pancreatic cancer cells. Carcinogenesis 2006;27:2392–401. Reader KL, Heath DA, Lun S, McIntosh CJ, Western AH, Littlejohn RP, et al. Signalling pathways involved in the cooperative effects of ovine and murine GDF9þBMP15-stimulated thymidine uptake by rat granulosa cells. Reproduction 2011;142:123–31. McGee EA, Strauss JF. Predicting ovarian futures: the contribution of genetics. In: Kim S, editor. The textbook of fertility preservation. Cambridge, United Kingdom: Cambridge University Press; 2011.
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SUPPLEMENTAL MATERIALS AND METHODS RNA Isolation and Real-Time PCR Analysis Ribonucleic acid was isolated from granulosa cells using the Pure Link Mini Kit (Invitrogen) according to the manufacturer's protocol. The RT cDNA synthesis was performed with a Super Script III First-Strand System (Invitrogen) for RTPCR under the conditions described by the manufacturer. For real-time PCR, the TaqMan assay system was used (Applied Biosystems) with specific FAM-labeled probes for mouse Smad7 (catalog no. Mm00484741-m1), mouse Smad6 (Mn00484738-ml), TBP (Mm01277045-m1), and PAI-1 (Mm00435858-m1). The real-time PCR reaction was completed on an ABI 7500 Thermocycler (Applied Biosystems) with 2 minutes at 50 C (AmpErase UNG activation), 10 minutes at 95 C (AmpliTaq Gold DNA polymerase activation), and 40 cycles each with 15 seconds at 95 C (melting) and 1 minute at 60 C (annealing/extension). The cycle threshold (CT) value was determined for each reaction (run in duplicate) using Sequence Detection Software version 1.7a (Applied Biosystems). The CT values were normalized with the housekeeping gene TBP and expressed as fold change of the control using the DDCT method (1).
Smad7 Promoter-Luciferase Reporters Mouse Smad7 promoter-luciferase reporter constructs were created using the pGL-3 basic vector system (Promega). A genomic DNA fragment containing the 4.3-kb Smad7 flanking region was amplified by PCR using WT female mouse genomic DNA (2). The 4039/þ208 promoter fragment was then subcloned into the NheI/XhoI sites of the pGL-3 basic vector. The SBE sites were identified within the 4.3-kb Smad7 promoter, using ALGGEN-PROMO (a virtual laboratory for the study of transcription factor–binding sites in DNA sequences; http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit. cgi?dirDB¼TF_8.3) and DBD: transcription factor prediction database (www.transcriptionfactor.org). Deletion mutants of the Smad7 upstream sequence fragment were generated using enzyme restriction. Smad7 promoter constructs with an SBE deletion were generated using the QuickChange II XL site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's protocol. The sequence with the SBE site gtctagac was changed to GTCTTCTG. The Smad7 promoter construct mutations were verified by sequencing. To examine Smad7 promoter activity, the luciferase reporter constructs were transfected into a well-characterized spontaneously immortalized rat granulosa cell line, SIGC (3, 4), which was kindly provided by Dr. Robert Burghardt, Texas A&M University, College Station, Texas (5). Cells were cultured in Dulbecco's modified Eagle medium/F-12 with 5% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin (all from Invitrogen) and were seeded at a density of 3 104 cells per well in 24-well plates 1 day before transfection. Briefly, full-length Smad7 promoter or deletion constructs (500 ng per well) plus 10 ng of internal control renilla DNA were transfected using FuGENE 6 (Roche Diagnostics) according to the manufacturer's protocol. Cells 1459.e1
were serum starved for 8 hours before incubation with or without 1 ng/mL of TGF-b for 24 hours. The DualLuciferase Reporter Assay System (Promega) was used according to the manufacturer's protocol. Luciferase activity was normalized relative to the activity of pGL-3 basic vector. Results were expressed as the ratio of firefly luciferase/renilla at equal amounts of protein. Each experiment represents the mean of two replicates and was repeated at least three times using two independent plasmid DNA preparations.
Transfection in Primary Granulosa Cells Mouse Smad7 full-length (insert size 1.3 kb, called S7FL) and truncated (insert size 0.67 Kb, called S7T) constructs were kindly provided by Dr. Peter ten Dijke (6). Primary granulosa cells were transfected using FuGENE 6 according to the manufacturer's protocol. Cells were plated in McCoy's 5A medium containing 10% FBS and Pen/Strep at 37 C in a humid atmosphere containing 5% CO2. The medium was changed to serum-free McCoy's 5A medium containing 0.1% BSA and 10 mL/mL ITSþ culture supplement for 24 hours before performing the transfection. After transfection, medium and unattached cells were removed, and media was changed to McCoy's 5A medium containing 10% FBS and Pen/Strep overnight. Media was changed to serum-free McCoy 5A medium containing 0.1% BSA and 10 mL/mL ITSþ culture supplement for 24 hours before experiments. Expression of Smad7 protein after transfection of granulosa cells was confirmed by both Western analysis and immunocytochemistry. Transfection efficiency was verified histologically with each experiment (Supplemental Fig. 4B) and routinely exceeded 50%. The intensity of Smad7 protein expression per cell increased more than 1.5-fold in S7FL-transfected cells compared with mock-transfected (NT) or not-transfected cells (Supplemental Fig. 4C). Primary granulosa cells cultured on coverslips were transfected with siRNA-Smad2, siRNA-Smad7, or with a AllStars Negative siRNA Control (a nonsilencing control, consisting of siRNAs that have no known homology to mammalian genes, to control for nonspecific silencing effects) (Qiagen) using FuGENE 6 according to the manufacturer's instructions. One day after transfection, cells were treated with TGF-b (1 ng/mL) for 24 hours. After TGF-b treatment, triplicate wells were prepared for analysis by TUNEL staining, PCR analysis, or immunofluorescence. Transfection efficiency in each experiment was monitored using AllStars Cell Death Control siRNA. Knockdown of the Smads was verified by immunostaining measuring the mean intensity of fluorescence per cell on the coverslips. The intensity of Smad2 and Smad7 protein expression per cell decreased more than 95% and 60%, respectively, compared with the negative siRNA control (Supplemental Fig. 2B and C).
SUPPLEMENTAL RESULTS To confirm that Smad7 was indeed overexpressed and that the expressed protein was functional, the following studies VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® were performed. Primary granulosa cells were transfected with mouse Smad7 full-length (insert size 1.3 kb, called S7FL), truncated (insert size 0.67 kb, S7T), EV, or NT. Overexpression of Smad7 protein was verified by measuring the mean intensity of fluorescence per cell on the coverslips subjected to immunostaining that were run with each experiment. Smad7 protein expression exceeded 1.5fold in cells transfected with full-length Smad7 (S7FL) compared with the negative control (EV) (Supplemental Fig. 4C). The S7FL cells have increased staining in the cytoplasm relative to EV cells, where a lower level of staining is seen predominantly in the nucleus (Supplemental Fig. 4B). The functionality of the Smad7 produced in transfected cells was confirmed by the reduction of TGF-b– stimulated PAI-1 expression in S7FL. Neither NT, EV, nor S7T was able to alter PAI-1 expression (Supplemental Fig. 4A).
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SUPPLEMENTAL REFERENCES 1. 2.
3.
4.
5.
6.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and (-Delta Delta C(T)) method. Methods 2001;25:402–8. Liu X, Chen Q, Kuang C, Zhang M, Ruan Y, Xu ZC, et al. A 4.3 kb Smad7 promoter is able to specify gene expression during mouse development. Biochim Biophys Acta 2007;1769:149–52. Peluso JJ, Pappalardo A, Fernandez G, Wu CA. Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells. Endocrinology 2004;145:3014–22. Geles KG, Freiman RN, Liu WL, Zheng S, Voronina E, Tjian R. Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks. Proc Natl Acad Sci U S A 2006;103:2594–9. Stein LS, Stoica G, Tilley R, Burghardt RC. Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation. Cancer Res 1991;51:696–706. Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, et al. 1997 Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature 1997;389:631–5.
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Regulation of Smad7 in granulosa cells. (A) Transforming growth factor-b dose–response effect on Smad7 mRNA expression in mouse granulosa cell primary culture. (B) Transforming growth factor-b dose–response effect on Smad7 promoter activity in SIGC cells transfected with the 4.3-kb mouse Smad7 promoter reporter construct. Luciferase activity was normalized against activity of a cotransfected renilla vector. In both, Smad7 expression was maximal at a dose of 10 ng/mL. Bars represent mean SEM from three different experiments. (C) Smad7 mRNA expression in primary granulosa cells treated with activin A (200 ng/mL). Activin treatment did not increase Smad7 mRNA expression levels. Data were normalized with the housekeeping gene TBP and expressed as fold change of the control using the DDCT method. Results represent mean SEM from three different experiments. (D) Smad7 mRNA expression in primary granulosa cells treated with FSH (1 mIU/mL) and/or TGF-b. Treatment with FSH did not modify either basal or TGF-b–stimulated Smad7 mRNA expression. Data were normalized with the housekeeping gene TBP and expressed as fold change of the control using the DDCT method. Results represent mean SEM from three different experiments. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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Additional data confirming inhibitor and siRNA effects on gene expression. (A) Expression of PAI-1 mRNA in primary granulosa cells treated with SIS3 (10 mM). SIS3 preincubation blocked TGF-b–stimulated PAI-1 increase, confirming that SIS3 is able to function as an inhibitor in granulosa cells. (B) Primary granulosa cells cultured on coverslips were transfected with siRNA-Smad2. (C) Primary granulosa cells were also transfected with siRNASmad7. The intensity of Smad2 and Smad7 protein expression per cell decreased markedly compared with the negative control. Bars represent mean SEM from three different experiments. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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SUPPLEMENTAL FIGURE 3
Smad7 promoter. Schematic map of mouse Smad7 promoter region with putative cis-regulatory elements from (500 to þ280 bp), which contains three palindromic SBE sites. Androgen receptor (AR), nuclear factor of activated T cells (NFAT), specificity protein 1 (SP1), specificity protein 3 (SP3), peroxisome proliferator-activated receptor-a (PPARa), and activator protein 1 (AP1) were additional transcription factor–binding sites that were adjacent to the SBE sites. The inset sequence spans from 332 to 85 bp and shows the most proximal SBE and its 3-bp spacing from the E-box and overlapping AP-1 and c-Myc sites. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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SUPPLEMENTAL FIGURE 4
Smad7 overexpression reduces TGF-b–stimulated PAI-1 expression. (A) Primary granulosa cells were transfected with full-length Smad7 (S7FL), truncated Smad7 (S7T), Smad7, EV, or NT and subject to 24 hours of TGF-b treatment (1 ng/mL). Treatment with TGF-b increased the expression of PAI-1 mRNA for NT, EV, and S7T groups. However, the cells transfected with full-length Smad7 exhibited a diminished TGF-b– stimulated PAI-1 expression, demonstrating the functionality of this construct in our system. (B) Immunocytochemical analysis for Smad7 in mouse granulosa primary culture after Smad7 full-length transient transfection (S7FL) or transfection with empty vector (EV). The S7FL cells have increased cellular staining for Smad7 relative to EV cells, where staining is seen only at a low level, predominantly in the nucleus. Right panels are of blue DAPI staining for the nucleus (original magnification, 200). (C) Smad7 protein expression exceeded 1.5-fold in cells transfected with full-length Smad7 (S7FL) compared with the negative control, cells transfected with EV. Bars represent mean Smad7 intensity of fluorescence per cell. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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Objective: To determine the functional role of Smad7 in granulosa cells. Design: Granulosa cell culture and molecular biological techniques were used to investigate regulation and function of Smad7. Setting: Research laboratory. Animal(s): C57bl/j hybrid mouse. Intervention(s): Primary mouse granulosa cells were isolated and grown in culture for all messenger RNA expression experiments. Smad7 promoter constructs were evaluated with a luciferase reporter system in SIGC cells to determine sites activating Smad7 expression. Main Outcome Measure(s): Overexpression (Smad7 complementary DNA) and downregulation (Smad7 small interfering RNA) of Smad7 in primary mouse granulosa cells were used to evaluate the functional role of Smad7 in granulosa cells. Result(s): Smad7 expression was upregulated by treatment with transforming growth factor-b (TGF-b) but not activin or activation of the cyclic adenosine monophosphate pathway. The promoter of Smad7 was activated by TGF-b. Truncation of the promoter or mutation of the Smad response element at 141 eliminated TGF-b activation of the promoter. Smad3 was not specifically required for TGF-b–stimulated expression of Smad7, though activation of the TGFBR1 receptor was. When Smad7 was overexpressed in granulosa cells, apoptosis was markedly increased. When Smad7 expression was reduced with small interfering RNA, then the TGF-b–induced apoptosis was blocked. Conclusion(s): Smad7 mediates apoptosis induced by TGF-b in mouse granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis. (Fertil SterilÒ 2012;97:1452–9. Ó2012 by American Society for Reproductive Medicine.) Key Words: Smad7, TGF-b, apoptosis, folliculogenesis
I
n mammals, fertility depends on progressive development of the egg-containing follicles through a process known as folliculogenesis. Follicles that do not progress to ovulation undergo atresia and are lost from the pool of growing follicles. The fate of each follicle and its enclosed oocyte is determined by both local and systemic factors regulating the cellular events within the follicle that determine whether it continues to grow and develop or undergoes atresia via apoptosis (1, 2). The transforming growth factor-b (TGF-b) family of growth factors playscritical roles in maintaining cell growth and differentiation in the ovary (3). Classically, TGF-b signals via
heteromeric complexes of type II and type I serine/threonine kinase receptors and their interacting Smad partners. The TGF-b ligand binding to the type II receptor facilitates the initiation of signaling with phosphorylation of the type I receptor and subsequent phosphorylation of Smad2 and Smad3 (4). Activated Smad2/3 bind with the common mediator, Smad4, and translocate to the nucleus to regulate ligandspecific gene expression. Smad complexes interact with other nuclear transcription factors, although Smad3 can also bind DNA directly (4, 5). Within the ovary, Smad2 and Smad3 expression are stage specific (6). Female mice deficient in Smad3 have reduced responsiveness to FSH
Received December 8, 2011; revised March 13, 2012; accepted March 14, 2012. M.Q. has nothing to disclose. J.W. has nothing to disclose. V.H. has nothing to disclose. E.A.M. has nothing to disclose. Supported by NICHD HD 045700 and D43 TW0076962. Reprint requests: Elizabeth A. McGee, M.D., Virginia Commonwealth University, MCV Campus, Sanger Hall, P.O. Box 980034, Richmond, Virginia 23298-0034 (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 6, June 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.03.024 1452
stimulation and reduced ability of FSH to up-regulate its own receptor, resulting in impaired folliculogenesis (7). Smad6 and Smad7 are ligand-induced inhibitory Smads, which function as negative regulators of Smad activity (8). Smad7 is a general antagonist for both TGF-b and bone morphogenetic protein (BMP) family signaling, in contrast to Smad6, which plays a more specific role in the BMP pathway (9). Smad7 antagonizes TGF-b signaling through multiple mechanisms, playing pivotal roles in embryonic development and adult homoeostasis (10, 11). Altered expression of Smad7 is associated with several human disease processes, including cancer, tissue fibrosis, and inflammatory diseases (12). Though Smad7 mRNA has been localized in mouse oocytes (13), this is the first report of localization, regulation, and function of Smad7 in granulosa cells. Transforming growth factor-b has been described as an inhibitory factor for ovarian folliculogenesis (3). VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® Previously we reported that TGF-b is a potent proapoptotic factor for preantral follicles in culture (14). Additionally, Smad7 causes apoptosis in podocytes, HaCaT keratinocytes, and cortical neurons (11). Because Smad7 is regulated by TGF-b in other systems, we have considered a role for Smad7 in TGF-b–mediated granulosa cell apoptosis.
MATERIALS AND METHODS Animals and Granulosa Cell Culture All animal experiments were performed in accord with National Institutes of Health guidelines and with institutional approval. For primary granulosa cell culture, ovaries from 25- to 28-day-old female mice were removed, dissected free of connective tissue, and incubated as described by Campbell (15). Granulosa cells were harvested, pelleted, resuspended, and cultured as previously described (7). Cells were plated at approximately 40% confluence in McCoy's medium containing 10% fetal bovine serum (FBS) and Pen/Strep at 37 C. The medium was changed to serum-free McCoy's with 0.1% bovine serum albumin (BSA) and 10 mL/mL Insulin, transferrin, selenium (ITSþ) for 24 h before the experiments. All reagents and inhibitors were purchased from Sigma, except for TGF-b1 and activin A, which were from R&D Systems.
Histology and Immunostaining For histologic examination, 5-mm paraffin sections were rehydrated, quenched, and incubated with 10% goat serum before incubation with polyclonal rabbit antibodies against Smad6 and Smad7 (1:100 and 1:500, respectively; Zymed). A biotinylated anti-rabbit IgG antibody was the secondary (Vector Laboratories), and slides were incubated with Vector Laboratories elite ABC reagent for 30 minutes before detection with diaminobenzidine using the SuperPicTure Kit (Zymed).
Quantification of Cell Viability and Apoptosis Cell proliferation was verified using the Cell Growth Determination Kit from Sigma (according to the manufacturer's instructions). Absorbance levels were compared with a standard curve from dilutions of a known number of granulosa cells. All the experiments were performed in quadruplicate. For apoptosis studies, cells were fixed in 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. Cells were then analyzed using a DeadEnd colorimetric TUNEL system (Promega) according to the manufacturer's protocol. The percentage of apoptotic nuclei was determined by counting 100 cells on each of four different coverslips per group and scoring each cell as stained or not stained. Groups were unknown to the person scoring the cells. Experiments were performed in duplicate. Apoptotic index was calculated as number of positive stained cells/number of total cells.
tary DNA (cDNA) synthesis was performed with SuperScript III First-Strand System for RT–polymerase chain reaction (PCR) (Invitrogen). The TaqMan assay system was used for messenger RNA (mRNA) evaluation using an ABI 7500 Thermocycler (Applied Biosystems). Specific 6-carboxyfluorescein (FAM)-labeled probes for mouse Smad7, Smad6, TATA-box binding protein (TBP), and plasminogen activator inhibitor-1 (PAI-1) were purchased from Applied Biosystems. The details of this assay are provided in Supplemental Materials and Methods, available online.
Smad7 Promoter-Luciferase Reporters Mouse Smad7 promoter-luciferase reporter constructs were created using the pGL-3 vector system (Promega). For promoter activity experiments constructs were transfected into a well-characterized spontaneously immortalized rat granulosa cell line, SIGC (16–18). The genomic DNA fragment containing the 4.3-kb Smad7 flanking region was amplified from genomic DNA (19). The detailed strategies to create the truncated and mutated constructs are described in Supplemental Materials and Methods.
Overexpression of Smad7 in Primary Granulosa Cells Mouse Smad7 full-length (insert size 1.3 kb, called S7FL) and truncated (insert size 0.67 kb, called S7T) constructs were kindly provided by Dr. Peter ten Dijke (8). Primary granulosa cells were transfected with these constructs and controls using FuGENE 6 (Roche Diagnostics) as described in Supplemental Materials and Methods.
siRNA in Primary Granulosa Cells Small interfering RNA (siRNA)-Smad2, siRNA-Smad7, or AllStars Negative Control siRNA (Qiagen) were transfected into primary granulosa cells using FuGENE 6. One day after transfection, cells were treated with TGF-b (1 ng/mL) for 24 hours. After TGF-b treatment, triplicate wells were prepared for analysis by TUNEL, PCR, or immunofluorescence. Transfection efficiency in each experiment was monitored using Cell Death Control siRNA (Qiagen). Knockdown was verified by immunostaining and measuring the mean intensity of fluorescence per cell on the coverslips. Smad7 down-regulation exceeded 60% in each replicate, whereas Smad2 downregulation exceeded 90%.
Data Analysis Experiments were repeated at least three times, and results were reported as the mean SEM. Data were subjected to analysis of variance and post hoc testing with Tukey's honestly significant difference test when more than two groups were compared. Student's t test was used for single comparisons. Statistical significance was accepted at P< .05.
RNA Isolation and Real-Time PCR Analysis Ribonucleic acid was isolated from granulosa cells using the PureLink MiniKit (Invitrogen) according to the manufacturer's protocol. The reverse transcription (RT) complemenVOL. 97 NO. 6 / JUNE 2012
RESULTS In contrast to Smad6, Smad7 is expressed in granulosa cells and is regulated by TGF-b. 1453
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY To determine the cellular localization of Smad7 protein in the ovary, we performed immunohistochemistry on ovarian sections from 23-day-old and cycling mice. Representative images in Figure 1A and B show the contrasting patterns of expression for Smad6 and Smad7. Smad6 expression is limited primarily to the cytoplasm of the oocytes (Fig. 1A). Smad7 protein expression was detected in oocytes and granulosa cells from preantral and antral follicles. Strong Smad7 staining was also observed in the ovarian surface epithelium and in endothelial cells lining vessels. Smad7 was seen in both cytoplasm and the nucleus of scattered cells within the follicles. However, in the corpora lutea, Smad7 was more focused as nuclear staining, though some cells also had cytoplasmic staining (Fig. 1B). We next determined the effect of TGFb treatment on the expression of the inhibitory Smads in granulosa cells in culture. As shown in Figure 1D, TGF-b treatment of granulosa cells increased Smad7 mRNA expression more than 2.5-fold (P< .05). The expression of Smad7 was both dose and time dependent. In dose–response studies, Smad7 expression was maximal at a dose of 10 ng/mL (Supplemental Fig. 1A and B). Smad7 expression increased up to 24 h, where it remained stable to 72 h. Although Smad6 mRNA was detectable in cultured granulosa cells, TGF-b treatment did not increase Smad6 expression.
TGF-b Stimulates Smad7 Promoter Activity To further understand the mechanism by which TGF-b increases Smad7 mRNA, we evaluated whether TGF-b can increase the transcriptional activity of the Smad7 promoter.
As shown in Figure 2A, TGF-b treatment (1 ng/mL) significantly activated a Smad7 promoter/luciferase reporter system transfected into a rat granulosa cell line (SIGC). In contrast, activin treatment did not increase Smad7 mRNA expression levels (Supplemental Fig. 1C) or promoter activation up to a dose of 200 ng/mL (Fig. 2A). Furthermore, neither forskolin nor 8-Bromoadenosine-30 , 50 -cyclic monophosphate (8-BrcAMP) (Fig. 2A) nor FSH (Supplemental Fig. 1D) modulated the TGF-b–stimulated increase in Smad7.
Identification of the TGF-b–Responsive Region of the Mouse Smad7 Promoter Examination of the 5' flanking region of the mouse Smad7 gene sequence revealed six palindromic Smad-binding elements (SBE) sites. Additional information regarding the analysis of the Smad7 promoter is provided in Supplemental Materials and Methods and in Supplemental Figure 3. Smad7 promoter deletion constructs were generated to identify the TGF-b–responsive region. The SIGC cells were transfected with these constructs generated from the full-length mouse Smad7 promoter, and their basal and stimulated activity was analyzed. Although a 3.8-fold induction was observed with fulllength construct, the induction was decreased by deleting the region from 4039 to 150 and was abolished by deletion from 4039 to 118 (Fig. 2B). Mutation of the most proximal putative TGF-b site in a longer promoter construct significantly reduced the TGF-b inducibility of the Smad7 promoter, providing further evidence that this cis element is
FIGURE 1
Localization and regulation of Smad6 and Smad7 in the ovary. (A) Smad6 is highly expressed in the cytoplasm of the oocytes of growing follicles. (B) Smad7 is expressed in the granulosa cells and oocytes of growing follicles and in corpora lutea. (C) Negative control is absence of primary antibody. PAF ¼ preantral follicle; AF ¼ antral follicle; CL ¼ corpus luteum. (D) Effect of TGF-b (1 ng/mL) on Smad6 and Smad7 mRNA from primary granulosa cells cultured. Mean SEM; aP<.05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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FIGURE 2
Transforming growth factor-b responsiveness of the Smad7 promoter. (A) SIGC cells were transfected with a 4.3-kb mouse Smad7 promoter reporter construct and treated as indicated. Transforming growth factor-b treatment increased luciferase activity, but activin and stimulators of PKA did not. (B) Putative SBE sites of Smad7 promoter (black circles). The progressive 50 deletion fragments are represented by grey bars. Mutated SBE is depicted by the black rectangle. Mean SEM; a s b,b s cP<.05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
the primary determinant of TGF-b responsiveness in the context of granulosa cells.
TGF-b Can Up-regulate Smad7 in the Absence of Either Smad2 or Smad3 It has been shown that SB431542 is a specific inhibitor of the type I receptors for TGF-b and inhibits the phosphorylation of Smad2/3 induced by TGF-b (20–23). The addition of the TGF-b receptor antagonist SB431542 blocked TGF-b– stimulated Smad7 promoter activation (Fig. 3A) and mRNA expression (Fig. 3B). We also used a specific inhibitor of Smad3 phosphorylation (SIS3) in these studies (24, 25). The inhibition of Smad3 did not prevent TGF-b stimulation of Smad7 promoter activation or mRNA (Fig. 3C and D), although it did reduce the expression of PAI-1 mRNA, used as a control of SIS3 efficacy (Supplemental Fig. 2A). We confirmed these results using granulosa cells that do not express VOL. 97 NO. 6 / JUNE 2012
Smad3 (hatched bars), from Smad3-deficient mice (7). Additionally, the reduction of Smad2 with siRNA in wild-type granulosa cells did not prevent TGF-b stimulation of the Smad7 promoter (grey bars, Fig. 3D). As shown in Figure 3D, all the cell types maintained the ability to up-regulate Smad7 promoter activity in response to TGF-b. Together, these results provide evidence that neither the presence of Smad2 nor of Smad3 is specifically required for Smad7 up-regulation by TGF-b, but activation of the type I receptor is required.
Overexpression of Smad7 Promotes Apoptosis, Whereas Reduction of Smad7 Prevents TGF-b– Induced Apoptosis Because TGF-b is a potent proapoptotic factor for preantral follicles (14) and Smad7 expression is increased by TGF-b in granulosa cells, we evaluated the biologic effect of increased Smad7 expression in granulosa cells. The verification 1455
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FIGURE 3
Neither Smad2 nor Smad3 are specifically required for up-regulation of Smad7. (A) SIGC cells transfected with Smad7 promoter were incubated for 24 hours with TGF-b (1 ng/mL) in and SB431542 10 mM or vehicle control. (B) Smad7 mRNA expression in primary granulosa cells treated as above. (C) SIGC cells transfected with the Smad7 promoter and incubated for 24 hours with or without TGF-b (1 ng/mL) in the presence of the specific inhibitor of Smad3 phosphorylation (SIS3, 10 mM) or vehicle control. Luciferase activity was normalized against activity of a cotransfected renilla vector. Bars represent mean SEM from three different experiments. (D) Smad7 mRNA expression in primary granulosa cells from wild-type or from Smad3 knockout mice (hatched bars) or from wild-type primary cells transfected with siRNA for Smad2 (grey bars) incubated 24 hours with the treatments before mRNA analysis as above. Mean SEM; a s bP<.05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
of expression and function of Smad7 protein is detailed in Supplemental Results (Supplemental Fig. 4). First, we determined the effect of overexpression of Smad7 on viable cell number in primary granulosa cell cultures using a tetrazolium salt assay (7). The overexpression of Smad7 (S7FL) decreased cell viability compared with cells transfected with control vector (empty vector, EV) at 6 and 24 hours (Fig. 4A). To determine whether this decrease in cell viability corresponded to an increase in apoptosis, we transfected the cells as before and performed a TUNEL assay to label free DNA ends. The overexpression of S7FL resulted in increased apoptosis of granulosa cells relative to cells transfected with EV (Fig. 4B). Together, these results suggest that the up-regulation of Smad7 can induce apoptosis in granulosa cells. To evaluate whether Smad7 is required for TGF-b induction of apoptosis in granulosa cells, we also performed TUNEL assay on cells that were transfected with siRNA for Smad7 and compared the apoptotic index with cells transfected with control siRNA. Transforming growth factor-b–stimulated 1456
apoptosis was significantly decreased by reduction of Smad7 expression (Fig. 4C). Coverslips were immunostained for Smad7 with each experiment, and the mean intensity of fluorescence per cell was reduced to 40% of basal expression. Thus overexpression of Smad7 induces apoptosis, and knockdown of Smad7 expression prevents TGF-b–stimulated apoptosis in granulosa cells.
DISCUSSION Smad7 is an important inhibitor of TGF-b superfamily signaling. Because Smad7 is up-regulated by TGF-b itself, it can function in a negative feedback loop. However, recent studies in other cell types suggest that the role of Smad7 may be much more complex than just negative feedback. Although Smad7 has been reported to be present in immature oocytes (13), we have shown that Smad7 is expressed in granulosa cells of growing follicles and mediates apoptosis induced by TGF-b. Moreover, we have reported that Smad7 is up-regulated by TGF-b at least partially through VOL. 97 NO. 6 / JUNE 2012
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Smad7 overexpression induces apoptosis, whereas TGF-b–induced apoptosis requires Smad7 expression. Primary granulosa cells transfected with full-length Smad7 cDNA (S7FL) or EV and treated with TGF-b (1 ng/mL). (A) Cell viability determined by tetrazolium salt assay. (B) Programmed cell death was assessed with TUNEL. At both 6 and 24 hours S7FL cells were overwhelmingly more apoptotic than EV cells. (C) Assay (TUNEL) of primary granulosa cells treated with TGF-b (black bars) or vehicle (white bar). Cells were transfected with siRNA Smad7 or control siRNA before TGF-b treatment. Mean SEM; a s b,c s dP< .05. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
a canonical Smad regulatory element in the proximal promoter of Smad7. Transforming growth factor-b regulates Smad7 expression but not Smad6 in many cell types (11). The localization of Smad6 and Smad7 in follicles is consistent with different roles for the two inhibitors. Smad6 function is largely thought to be limited to the BMP pathway, whereas Smad7 has much wider function in both BMP and TGF-b pathways. Smad7 is thought to inhibit TGF-b signaling by competing with Smad2/3 for binding to the type I receptor, enhancing ubiquitination of Smad complexes, and inhibiting Smad/DNA complexes in the nucleus (26). Specific roles of Smad7 may be cell and stage specific (11, 12). VOL. 97 NO. 6 / JUNE 2012
It is interesting that activin did not regulate Smad7 expression in granulosa cells. The differential response to activin and TGF-b does, however, explain the different effects seen with TGF-b and activin treatment of preantral follicles in serum-free culture; activin treatment results in growth and differentiation, whereas TGF-b treatment results in overwhelming apoptosis (14). This differential activin/TGF-b response is also consistent with a report of differential activation of a Smad7 promoter construct in fibroblast cells (27). Failure of FSH or cAMP activation of the promoter was also interesting because TGF-b and FSH often act additively or synergistically in the ovary (28, 29). Although many of the functions of TGF-b and activin and even the 1457
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY gonadotropins overlap during follicle development (1), this unique role for TGF-b in up-regulating Smad7 provides the possibility of independent functions for TGF-b, distinct from activin or gonadotropin. The Smad7 promoter contains several putative Smad regulatory sites. However, deletions of most of these sites did not result in decreased activation of the construct by TGF-b. Deletion of the canonical SBE site at 141 bp reduced this stimulation to near-basal levels. The importance of this site in granulosa cells was confirmed with mutation of the site in a longer construct. The use of chemical and genetic inhibitors of either Smad2 or Smad3 demonstrates that neither are specifically required for Smad7 up-regulation by TGF-b, but activation of the type I receptor is required. This is much different than the more specific requirement for Smad3 in the regulation of the FSH receptor (7). However, more studies are needed to definitively show whether there is a role for the noncanonical signaling pathways activated by the type I receptor in Smad7 regulation. Requirements for Smad2 and/or Smad3 have differed in other reports of Smad7 regulation. Our results are consistent with the activation of the mouse promoter in murine fibroblasts and human hepatoma cell lines (30, 31), in which both Smad2 and Smad3 were functional in activating the Smad7 promoter. Smad3 has been reported to be the preferential activator of Smad7 in a human promoter expressed in mouse embryonic fibroblast cells (32). It is not clear whether the variable results in promoter activation were species specific or related to methodologic differences. We used a mouse promoter in a rat cell line. However, our studies confirmed the promoter-based experiments with evaluation of mRNA expression in primary granulosa cell culture. Other signaling pathways, including noncanonical TGF-b pathways, may be involved in the transcriptional regulation of Smad7 expression in granulosa cells. In silico analysis identified many other transcription factor–binding sequences that are present in the promoter region of Smad7 (Supplemental Fig. 3). Additionally, transcription factors such as AP-1, specificity protein 1 (SP1), and activating transcription factor 2 (ATF-2) have been shown to bind to Smad complexes in vitro and enhance or otherwise modify the extent of TGFb–induced transcription in other tissue types (27, 31–33). The combinations of transcription factors also seem to be cell and even developmental stage specific, promoting stimulation or suppression of Smad7 expression. Further studies are needed to elucidate these possible complex interactions in Smad7 regulation during folliculogenesis and their roles in regulating apoptosis. Overexpression of Smad7 induces increased apoptosis, whereas decreased Smad7 expression reduces apoptosis induced by TGF-b in granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis. Overexpression of Smad7 protein increased apoptosis at both 6 hours and 24 hours after transfection relative to controls. Although not a true representation of TGF-b–stimulated effects, because the Smad7 was driven by a constitutive promoter and highly expressed within the cytoplasm of the cells, the study does clearly demonstrate that increase of Smad7 protein is capable of inducing apoptosis of granulosa cells. A reduction of 1458
Smad7 expression by 60% was sufficient to block the TGFb–induced apoptosis in primary granulosa cells. Together, these data provide strong evidence that Smad7 is the mediator of TGF-b–induced apoptosis of granulosa cells. Regulation of folliculogenesis is very complex. Many follicles begin growth, yet very few continue along the developmental pathway to ovulation. The role of gonadotropins and the cAMP/protein kinase A (PKA) pathway in follicle growth and the intrafollicular regulation of the balance of PKA activation in follicle selection and dominance have long been known (1). Likewise, additional factors functioning as autocrine and paracrine hormones modulating follicle development have long been recognized. Though folliculogenesis research has traditionally focused on complete blocks in follicle development, human infertility should more appropriately be described as subfertility, whereby follicles can grow and may ovulate, but less efficiently or with poorer egg quality compared with normal. For example, in mice, Smad3 is necessary for normal rates of follicle development with appropriately mature oocytes. Yet follicles can still grow and even ovulate in the absence of Smad3, although the females are infertile (7). Smad7 is regulated by TGF-b. It is also expressed in granulosa cells and is able to inhibit the functions of Smad2 and Smad3. Moreover, Smad7 could also inhibit granulosa cell proliferation through inhibition of the GDF9/BMP15 pathway (34). If Smad7 promotes apoptosis over survival, the expression level of Smad7 present in a follicle could be a determinant of follicle fate. If the balance between activators and inhibitors of Smad7 results in increased Smad7, then an increase in granulosa apoptosis would be expected with concomitant decreased granulosa cells mass, decreased aromatase expression, and reduced oocyte quality. In contrast, if Smad7 is reduced, then within a follicle there would be less granulosa cell loss to apoptosis—resulting in increased granulosa cell mass and increased aromatase and other markers of follicle health. Smad3 would not be as inhibited and follicle-stimulating hormone receptor (FSHR) might be more effective in upregulating its own receptor. Thus there are several ways that dysregulated Smad7 could be a factor in the more subtle presentations of subfertility in humans. Dysfunction of individual proteins responsible for subfertility has been difficult to identify because redundant layers of regulation, stage-specific and temporal differences in molecular function, and even cross-talk between pathways confuse the findings in investigations of systems. Yet knowledge of the range of functions of a key pleiotropic molecule like Smad7 will allow interpretation of more advanced studies such as pathway activation analyses, or predicting which combinations of abnormalities found in genome-wide association studies might result in identifiable phenotypes (35).
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McGee EA, Hsueh AJ. Follicle recruitment and selection. Endocr Rev 2000; 21:200–14. Jin X, Xiao LJ, Zhang XS, Liu YX. Apoptosis in ovary. Front Biosci (Schol Ed) 2011;3:680–97. VOL. 97 NO. 6 / JUNE 2012
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Rosairo D, Kuyznierewicz I, Findlay J, Drummond A. Transforming growth factor-beta: its role in ovarian follicle development. Reproduction 2008; 136:799–809. Schmierer B, Hill CS. TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 2007;8:970–82. Itoh S, Itoh F, Goumans MJ, Ten Dijke P. Signaling of transforming growth factor-beta family members through Smad proteins. Eur J Biochem 2000; 267:6954–67. Xu J, Oakley J, McGee EA. Stage-specific expression of Smad2 and Smad3 during folliculogenesis. Biol Reprod 2002;66:1571–8. Gong X, McGee EA. Smad3 is required for normal follicular follicle-stimulating hormone responsiveness in the mouse. Biol Reprod 2009;81:730–8. Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, et al. 1997 Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature 1997;389:631–5. Park SH. Fine tuning and cross-talking of TGF-beta signal by inhibitory Smads. J Biochem Mol Biol 2005;38:9–16. Schiffer M, Schiffer LE, Gupta A, Shaw AS, Roberts IS, Mundel P, et al. Inhibitory smads and tgf-Beta signaling in glomerular cells. J Am Soc Nephrol 2002;13:2657–66. Yan X, Chen YG. Smad7: not only a regulator, but also a cross-talk mediator of TGF-b signaling. Biochem J 2011;434:1–10. Briones-Orta MA, Tecalco-Cruz AC, Sosa-Garrocho M, Caligaris C, MacíasSilva M. Inhibitory smad7: emerging roles in health and disease. Curr Mol Pharmacol 2011;4:141–53. Tian X, Halfhill AN, Diaz FJ. Localization of phosphorylated SMAD proteins in granulosa cells, oocytes and oviduct of female mice. Gene Expr Patterns 2010;10:105–12. McGee EA, Smith R, Spears N, Nachtigal MW, Ingraham H, Hsueh AJ. M€ ullerian inhibitory substance induces growth of rat preantral ovarian follicles. Biol Reprod 2001;64:293–8. Campbell KL. Ovarian granulosa cells isolated with EGTA and hypertonic sucrose: cellular integrity and function. Biol Reprod 1979;21:773–86. Peluso JJ, Pappalardo A, Fernandez G, Wu CA. Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells. Endocrinology 2004;145:3014–22. Geles KG, Freiman RN, Liu WL, Zheng S, Voronina E, Tjian R. Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks. Proc Natl Acad Sci U S A 2006;103:2594–9. Stein LS, Stoica G, Tilley R, Burghardt RC. Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation. Cancer Res 1991;51:696–706. Liu X, Chen Q, Kuang C, Zhang M, Ruan Y, Xu ZC, et al. A 4.3 kb Smad7 promoter is able to specify gene expression during mouse development. Biochim Biophys Acta 2007;1769:149–52. Laping NJ, Grygielko E, Mathur A, Butter S, Bomberger J, Tweed C, et al. Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542. Mol Pharmacol 2002;62:58–64.
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Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, et al. SB431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 2002;62:65–74. Hjelmeland MD, Hjelmeland AB, Sathornsumetee S, Reese ED, Herbstreith MH, Laping NJ, et al. SB-431542, a small molecule transforming growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility. Mol Cancer Ther 2004;3:737–45. Mimura K, Kono K, Hanawa M, Kanzaki M, Nakao A, Ooi A, et al. Trastuzumab-mediated antibody-dependent cellular cytotoxicity against esophageal squamous cell carcinoma. Clin Cancer Res 2005;11:4898–904. Jinnin M, Ihn H, Tamaki K. Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta1-induced extracellular matrix expression. Mol Pharmacol 2006;69:597–607. Gilchrist RB, Ritter LJ. Differences in the participation of TGH (beta) superfamily signalling pathways mediating porcine and murine cumulus cell expansion. Reproduction 2011;142:647–57. Yan X, Liu Z, Chen Y. Regulation of TGF-beta signaling by Smad7. Acta Biochim Biophys Sin (Shanghai) 2009;41:263–72. €ttinger EP. von Gersdorff G, Susztak K, Rezvani F, Bitzer M, Liang D, Bo Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta. J Biol Chem 2000; 275:11320–6. Dodson WC, Schomberg DW. The effect of transforming growth factor-beta on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology 1987;120:512–6. Ke FC, Chuang LC, Lee MT, Chen YJ, Lin SW, Wang PS, et al. The modulatory role of transforming growth factor beta1 and androstenedione on folliclestimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells. Biol Reprod 2004;70:1292–8. Denissova NG, Pouponnot C, Long J, He D, Liu F. Transforming growth factor beta-inducible independent binding of SMAD to the Smad7 promoter. Proc Natl Acad Sci U S A 2000;97:6397–402. Brodin G, Ahgren A, ten Dijke P, Heldin CH, Heuchel R. Efficient TGF-beta induction of the Smad7 gene requires cooperation between AP-1, Sp1, and Smad proteins on the mouse Smad7 promoter. J Biol Chem 2000; 275:29023–30. €ttinger EP. Smad proSchiffer M, von Gersdorff G, Bitzer M, Susztak K, Bo teins and transforming growth factor-beta signaling. Kidney Int Suppl 2000;77:S45–52. Jungert K, Buck A, Buchholz M, Wagner M, Adler G, Gress TM, et al. SmadSp1 complexes mediate TGFbeta-induced early transcription of oncogenic Smad7 in pancreatic cancer cells. Carcinogenesis 2006;27:2392–401. Reader KL, Heath DA, Lun S, McIntosh CJ, Western AH, Littlejohn RP, et al. Signalling pathways involved in the cooperative effects of ovine and murine GDF9þBMP15-stimulated thymidine uptake by rat granulosa cells. Reproduction 2011;142:123–31. McGee EA, Strauss JF. Predicting ovarian futures: the contribution of genetics. In: Kim S, editor. The textbook of fertility preservation. Cambridge, United Kingdom: Cambridge University Press; 2011.
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SUPPLEMENTAL MATERIALS AND METHODS RNA Isolation and Real-Time PCR Analysis Ribonucleic acid was isolated from granulosa cells using the Pure Link Mini Kit (Invitrogen) according to the manufacturer's protocol. The RT cDNA synthesis was performed with a Super Script III First-Strand System (Invitrogen) for RTPCR under the conditions described by the manufacturer. For real-time PCR, the TaqMan assay system was used (Applied Biosystems) with specific FAM-labeled probes for mouse Smad7 (catalog no. Mm00484741-m1), mouse Smad6 (Mn00484738-ml), TBP (Mm01277045-m1), and PAI-1 (Mm00435858-m1). The real-time PCR reaction was completed on an ABI 7500 Thermocycler (Applied Biosystems) with 2 minutes at 50 C (AmpErase UNG activation), 10 minutes at 95 C (AmpliTaq Gold DNA polymerase activation), and 40 cycles each with 15 seconds at 95 C (melting) and 1 minute at 60 C (annealing/extension). The cycle threshold (CT) value was determined for each reaction (run in duplicate) using Sequence Detection Software version 1.7a (Applied Biosystems). The CT values were normalized with the housekeeping gene TBP and expressed as fold change of the control using the DDCT method (1).
Smad7 Promoter-Luciferase Reporters Mouse Smad7 promoter-luciferase reporter constructs were created using the pGL-3 basic vector system (Promega). A genomic DNA fragment containing the 4.3-kb Smad7 flanking region was amplified by PCR using WT female mouse genomic DNA (2). The 4039/þ208 promoter fragment was then subcloned into the NheI/XhoI sites of the pGL-3 basic vector. The SBE sites were identified within the 4.3-kb Smad7 promoter, using ALGGEN-PROMO (a virtual laboratory for the study of transcription factor–binding sites in DNA sequences; http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit. cgi?dirDB¼TF_8.3) and DBD: transcription factor prediction database (www.transcriptionfactor.org). Deletion mutants of the Smad7 upstream sequence fragment were generated using enzyme restriction. Smad7 promoter constructs with an SBE deletion were generated using the QuickChange II XL site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's protocol. The sequence with the SBE site gtctagac was changed to GTCTTCTG. The Smad7 promoter construct mutations were verified by sequencing. To examine Smad7 promoter activity, the luciferase reporter constructs were transfected into a well-characterized spontaneously immortalized rat granulosa cell line, SIGC (3, 4), which was kindly provided by Dr. Robert Burghardt, Texas A&M University, College Station, Texas (5). Cells were cultured in Dulbecco's modified Eagle medium/F-12 with 5% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin (all from Invitrogen) and were seeded at a density of 3 104 cells per well in 24-well plates 1 day before transfection. Briefly, full-length Smad7 promoter or deletion constructs (500 ng per well) plus 10 ng of internal control renilla DNA were transfected using FuGENE 6 (Roche Diagnostics) according to the manufacturer's protocol. Cells 1459.e1
were serum starved for 8 hours before incubation with or without 1 ng/mL of TGF-b for 24 hours. The DualLuciferase Reporter Assay System (Promega) was used according to the manufacturer's protocol. Luciferase activity was normalized relative to the activity of pGL-3 basic vector. Results were expressed as the ratio of firefly luciferase/renilla at equal amounts of protein. Each experiment represents the mean of two replicates and was repeated at least three times using two independent plasmid DNA preparations.
Transfection in Primary Granulosa Cells Mouse Smad7 full-length (insert size 1.3 kb, called S7FL) and truncated (insert size 0.67 Kb, called S7T) constructs were kindly provided by Dr. Peter ten Dijke (6). Primary granulosa cells were transfected using FuGENE 6 according to the manufacturer's protocol. Cells were plated in McCoy's 5A medium containing 10% FBS and Pen/Strep at 37 C in a humid atmosphere containing 5% CO2. The medium was changed to serum-free McCoy's 5A medium containing 0.1% BSA and 10 mL/mL ITSþ culture supplement for 24 hours before performing the transfection. After transfection, medium and unattached cells were removed, and media was changed to McCoy's 5A medium containing 10% FBS and Pen/Strep overnight. Media was changed to serum-free McCoy 5A medium containing 0.1% BSA and 10 mL/mL ITSþ culture supplement for 24 hours before experiments. Expression of Smad7 protein after transfection of granulosa cells was confirmed by both Western analysis and immunocytochemistry. Transfection efficiency was verified histologically with each experiment (Supplemental Fig. 4B) and routinely exceeded 50%. The intensity of Smad7 protein expression per cell increased more than 1.5-fold in S7FL-transfected cells compared with mock-transfected (NT) or not-transfected cells (Supplemental Fig. 4C). Primary granulosa cells cultured on coverslips were transfected with siRNA-Smad2, siRNA-Smad7, or with a AllStars Negative siRNA Control (a nonsilencing control, consisting of siRNAs that have no known homology to mammalian genes, to control for nonspecific silencing effects) (Qiagen) using FuGENE 6 according to the manufacturer's instructions. One day after transfection, cells were treated with TGF-b (1 ng/mL) for 24 hours. After TGF-b treatment, triplicate wells were prepared for analysis by TUNEL staining, PCR analysis, or immunofluorescence. Transfection efficiency in each experiment was monitored using AllStars Cell Death Control siRNA. Knockdown of the Smads was verified by immunostaining measuring the mean intensity of fluorescence per cell on the coverslips. The intensity of Smad2 and Smad7 protein expression per cell decreased more than 95% and 60%, respectively, compared with the negative siRNA control (Supplemental Fig. 2B and C).
SUPPLEMENTAL RESULTS To confirm that Smad7 was indeed overexpressed and that the expressed protein was functional, the following studies VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® were performed. Primary granulosa cells were transfected with mouse Smad7 full-length (insert size 1.3 kb, called S7FL), truncated (insert size 0.67 kb, S7T), EV, or NT. Overexpression of Smad7 protein was verified by measuring the mean intensity of fluorescence per cell on the coverslips subjected to immunostaining that were run with each experiment. Smad7 protein expression exceeded 1.5fold in cells transfected with full-length Smad7 (S7FL) compared with the negative control (EV) (Supplemental Fig. 4C). The S7FL cells have increased staining in the cytoplasm relative to EV cells, where a lower level of staining is seen predominantly in the nucleus (Supplemental Fig. 4B). The functionality of the Smad7 produced in transfected cells was confirmed by the reduction of TGF-b– stimulated PAI-1 expression in S7FL. Neither NT, EV, nor S7T was able to alter PAI-1 expression (Supplemental Fig. 4A).
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SUPPLEMENTAL REFERENCES 1. 2.
3.
4.
5.
6.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and (-Delta Delta C(T)) method. Methods 2001;25:402–8. Liu X, Chen Q, Kuang C, Zhang M, Ruan Y, Xu ZC, et al. A 4.3 kb Smad7 promoter is able to specify gene expression during mouse development. Biochim Biophys Acta 2007;1769:149–52. Peluso JJ, Pappalardo A, Fernandez G, Wu CA. Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells. Endocrinology 2004;145:3014–22. Geles KG, Freiman RN, Liu WL, Zheng S, Voronina E, Tjian R. Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks. Proc Natl Acad Sci U S A 2006;103:2594–9. Stein LS, Stoica G, Tilley R, Burghardt RC. Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation. Cancer Res 1991;51:696–706. Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, et al. 1997 Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature 1997;389:631–5.
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Regulation of Smad7 in granulosa cells. (A) Transforming growth factor-b dose–response effect on Smad7 mRNA expression in mouse granulosa cell primary culture. (B) Transforming growth factor-b dose–response effect on Smad7 promoter activity in SIGC cells transfected with the 4.3-kb mouse Smad7 promoter reporter construct. Luciferase activity was normalized against activity of a cotransfected renilla vector. In both, Smad7 expression was maximal at a dose of 10 ng/mL. Bars represent mean SEM from three different experiments. (C) Smad7 mRNA expression in primary granulosa cells treated with activin A (200 ng/mL). Activin treatment did not increase Smad7 mRNA expression levels. Data were normalized with the housekeeping gene TBP and expressed as fold change of the control using the DDCT method. Results represent mean SEM from three different experiments. (D) Smad7 mRNA expression in primary granulosa cells treated with FSH (1 mIU/mL) and/or TGF-b. Treatment with FSH did not modify either basal or TGF-b–stimulated Smad7 mRNA expression. Data were normalized with the housekeeping gene TBP and expressed as fold change of the control using the DDCT method. Results represent mean SEM from three different experiments. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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Additional data confirming inhibitor and siRNA effects on gene expression. (A) Expression of PAI-1 mRNA in primary granulosa cells treated with SIS3 (10 mM). SIS3 preincubation blocked TGF-b–stimulated PAI-1 increase, confirming that SIS3 is able to function as an inhibitor in granulosa cells. (B) Primary granulosa cells cultured on coverslips were transfected with siRNA-Smad2. (C) Primary granulosa cells were also transfected with siRNASmad7. The intensity of Smad2 and Smad7 protein expression per cell decreased markedly compared with the negative control. Bars represent mean SEM from three different experiments. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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SUPPLEMENTAL FIGURE 3
Smad7 promoter. Schematic map of mouse Smad7 promoter region with putative cis-regulatory elements from (500 to þ280 bp), which contains three palindromic SBE sites. Androgen receptor (AR), nuclear factor of activated T cells (NFAT), specificity protein 1 (SP1), specificity protein 3 (SP3), peroxisome proliferator-activated receptor-a (PPARa), and activator protein 1 (AP1) were additional transcription factor–binding sites that were adjacent to the SBE sites. The inset sequence spans from 332 to 85 bp and shows the most proximal SBE and its 3-bp spacing from the E-box and overlapping AP-1 and c-Myc sites. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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SUPPLEMENTAL FIGURE 4
Smad7 overexpression reduces TGF-b–stimulated PAI-1 expression. (A) Primary granulosa cells were transfected with full-length Smad7 (S7FL), truncated Smad7 (S7T), Smad7, EV, or NT and subject to 24 hours of TGF-b treatment (1 ng/mL). Treatment with TGF-b increased the expression of PAI-1 mRNA for NT, EV, and S7T groups. However, the cells transfected with full-length Smad7 exhibited a diminished TGF-b– stimulated PAI-1 expression, demonstrating the functionality of this construct in our system. (B) Immunocytochemical analysis for Smad7 in mouse granulosa primary culture after Smad7 full-length transient transfection (S7FL) or transfection with empty vector (EV). The S7FL cells have increased cellular staining for Smad7 relative to EV cells, where staining is seen only at a low level, predominantly in the nucleus. Right panels are of blue DAPI staining for the nucleus (original magnification, 200). (C) Smad7 protein expression exceeded 1.5-fold in cells transfected with full-length Smad7 (S7FL) compared with the negative control, cells transfected with EV. Bars represent mean Smad7 intensity of fluorescence per cell. Quezada. Smad7 mediates TGF-b apoptosis in ovary. Fertil Steril 2012.
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