Follistatin Transcription Research Articles

Downregulation of activin-signaling gene expression in passaged normal human dermal fibroblasts.

Activins are members of the transforming growth factor-β (TGF-β) superfamily and play important roles in proliferation, differentiation, and apoptosis of various target cells. We investigated changes of activin, activin receptor (ActR), and Smad-signaling gene expression with increasing passage number in normal human dermal fibroblasts. The expression of mRNA and protein was measured by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis from passage numbers 5 to 15. Activin A and follistatin transcript levels increased with increasing passage number. ActR types IA, IB, IIA and IIB mRNA levels decreased at high passage number. The levels of Smad2, 3 and 4 protein decreased with increasing passage number, which also attenuated phosphorylation of Smad2 and 3 protein expression. Smad7 was enhanced with increasing passage number. These results suggest that expression of activin-signaling in aging normal human dermal fibroblasts increases activin A and follistatin, whereas ActR-Smad signaling is decreased.

Silica nanoparticle releases SIRT6-induced epigenetic silencing of follistatin.

Follistatin (FST) plays a protective role during silica nanoparticle (SiO2 NP) exposure. SiO2 NP treatment induces FST transcription with an unknown mechanism. We herein reported that SIRT6, one of the sirtuin family members, induced epigenetic silencing of FST. The expression of FST was elevated after SIRT6 knockdown while reduced after SIRT6 overexpression. Chromatin immunoprecipitation revealed a direct interaction between SIRT6 with FST promoter. Knockdown of SIRT6 increased both Ac-H3K9 level and Ac-H3K56 level at FST promoter region. SiO2 NP treatment de-stabilized SIRT6 mRNA and reduced SIRT6 expression, leading to the activation of FST transcription. Finally, over-expression of SIRT6 increased SiO2 NP-induced apoptosis. Collectively, this study provided evidence that SIRT6 is a negative regulator of FST transcription and participates in the regulation of cell survival during silica nanoparticle exposure.

Epigenetic Regulators Modulate Muscle Damage in Duchenne Muscular Dystrophy Model.

Histone acetyl transferases (HATs) and histone deacetylases (HDAC) control transcription during myogenesis. HDACs promote chromatin condensation, inhibiting gene transcription in muscle progenitor cells until myoblast differentiation is triggered and HDACs are released. HATs, namely CBP/p300, activate myogenic regulatory and elongation factors promoting myogenesis. HDAC inhibitors are known to improve regeneration in dystrophic muscles through follistatin upregulation. However, the potential of directly modulating HATs remains unexplored. We tested this possibility in a well-known zebrafish model of Duchenne muscular dystrophy. Interestingly, CBP/p300 transcripts were found downregulated in the absence of Dystrophin. While investigating CBP rescuing potential we observed that dystrophin-null embryos overexpressing CBP actually never show significant muscle damage, even before a first regeneration cycle could occur. We found that the pan-HDAC inhibitor trichostatin A (TSA) also prevents early muscle damage, however the single HAT CBP is as efficient even in low doses. The HAT domain of CBP is required for its full rescuing ability. Importantly, both CBP and TSA prevent early muscle damage without restoring endogenous CBP/p300 neither increasing follistatin transcripts. This suggests a new mechanism of action of epigenetic regulators protecting dystrophin-null muscle fibres from detaching, independent from the known improvement of regeneration upon damage of HDACs inhibitors. This study builds supporting evidence that epigenetic modulators may play a role in determining the severity of muscle dystrophy, controlling the ability to resist muscle damage. Determining the mode of action leading to muscle protection can potentially lead to new treatment options for muscular dystrophies in the future.

Transcriptional activation of follistatin by Nrf2 protects pulmonary epithelial cells against silica nanoparticle-induced oxidative stress.

Silica nanoparticles (SiO2 NPs) cause oxidative stress in respiratory system. Meanwhile, human cells launch adaptive responses to overcome SiO2 NP toxicity. However, besides a few examples, the regulation of SiO2 NP-responsive proteins and their functions in SiO2 NP response remain largely unknown. In this study, we demonstrated that SiO2 NP induced the expression of follistatin (FST), a stress responsive gene, in mouse lung tissue as well as in human lung epithelial cells (A549). The levels of Ac-H3(K9/18) and H3K4me2, two active gene markers, at FST promoter region were significantly increased during SiO2 NP treatment. The induction of FST transcription was mediated by the nuclear factor erythroid 2-related factor 2 (Nrf2), as evidenced by the decreased FST expression in Nrf2-deficient cells and the direct binding of Nrf2 to FST promoter region. Down-regulation of FST promoted SiO2 NP-induced apoptosis both in cultured cells and in mouse lung tissue. Furthermore, knockdown of FST increased while overexpression of FST decreased the expression level of NADPH oxidase 1 (NOX1) and NOX5 as well as the production of cellular reactive oxygen species (ROS). Taken together, these findings demonstrated a protective role of FST in SiO2 NP-induced oxidative stress and shed light on the interaction between SiO2 NPs and biological systems.

ERRβ signalling through FST and BCAS2 inhibits cellular proliferation in breast cancer cells

Background:The overexpression of oestrogen-related receptor-β (ERRβ) in breast cancer patients is correlated with improved prognosis and longer relapse-free survival, and the level of ERRβ mRNA is inversely correlated with the S-phase fraction of cells from breast cancer patients.Methods:Chromatin immunoprecipitation (ChIP) cloning of ERRβ transcriptional targets and gel supershift assays identified breast cancer amplified sequence 2 (BCAS2) and Follistatin (FST) as two important downstream genes that help to regulate tumourigenesis. Confocal microscopy, co-immunoprecipitation (CoIP), western blotting and quantitative real-time PCR confirmed the involvement of ERRβ in oestrogen signalling.Results:Overexpressed ERRβ induced FST-mediated apoptosis in breast cancer cells, and E-cadherin expression was also enhanced through upregulation of FST. However, this anti-proliferative signalling function was challenged by ERRβ-mediated BCAS2 upregulation, which inhibited FST transcription through the downregulation of β-catenin/TCF4 recruitment to the FST promoter. Interestingly, ERRβ-mediated upregulation of BCAS2 downregulated the major G1-S transition marker cyclin D1, despite the predictable oncogenic properties of BCAS2.Interpretation:Our study provides the first evidence that ERRβ, which is a coregulator of ERα also acts as a potential tumour-suppressor molecule in breast cancer. Our current report also provides novel insights into the entire cascade of ERRβ signalling events, which may lead to BCAS2-mediated blockage of the G1/S transition and inhibition of the epithelial to mesenchymal transition through FST-mediated regulation of E-cadherin. Importantly, matrix metalloprotease 7, which is a classical mediator of metastasis and E-cadherin cleavage, was also restricted as a result of ERRβ-mediated FST overexpression.

Spatio-temporal expression pattern and fasting response of follistatin gene in teleost Larimichthys crocea

Follistatin is a multifunctional regulatory protein involved in numerous physiological activities. To better understand its functions in Larimichthys crocea (L. crocea), the entire cDNA sequence of follistatin from L. crocea was cloned, the transcript abundance of follistatin was measured at different development stages and in different gender. A fasting-refeeding protocol was also performed to correlate follistatin tissue expression with nutritional status in the juvenile stage of L. crocea. The results showed that follistatin transcripts were weakly detected before gastrula stage (9 h post-fertilization; hpf), and at the highest level at myogenesis stage (15 hpf) and 30 days after hatching (30 dah). In adult fish, follistatin transcripts were observed in all the ten tissues examined and most abundant in eye, gill, muscle, brain and intestine. Moreover, the abundance of follistatin transcripts in most tissues was influenced by the age and gender. During fasting and refeeding period, the regulatory response of follistatin in muscle, liver, gonad, spleen and brain of L. crocea varied greatly. Altogether, these findings support the evidence that follistatin plays a profound role in the teleost development and that it is an indispensable factor for the survival of teleosts when challenged with environmental stresses like fasting.

Follistatin Is Induced by Ionizing Radiation and Potentially Predictive of Radiosensitivity in Radiation-Induced Fibrosis Patient Derived Fibroblasts

Follistatin is a potent regulator of the inflammatory response and binds to and inhibits activin A action. Activin A is a member of the TGFβ protein superfamily which has regulatory roles in the inflammatory response and in the fibrotic process. Fibrosis can occur following cell injury and cell death induced by agents such as ionizing radiation (IR). IR is used to treat cancer and marked fibrotic response is a normal tissue (non-tumour) consequence in a fraction of patients under the current dose regimes. The discovery and development of a therapeutic to abate fibrosis in these radiosensitive patients would be a major advance for cancer radiotherapy. Likewise, prediction of which patients are susceptible to fibrosis would enable individualization of treatment and provide an opportunity for pre-emptive fibrosis control and better tumour treatment outcomes. The levels of activin A and follistatin were measured in fibroblasts derived from patients who developed severe radiation-induced fibrosis following radiotherapy and compared to fibroblasts from patients who did not. Both follistatin and activin A gene expression levels were increased following IR and the follistatin gene expression level was lower in the fibroblasts from fibrosis patients compared to controls at both basal levels and after IR. The major follistatin transcript variants were found to have a similar response to IR and both were reduced in fibrosis patients. Levels of follistatin and activin A secreted in the fibroblast culture medium also increased in response to IR and the relative follistatin protein levels were significantly lower in the samples derived from fibrosis patients. The decrease in the follistatin levels can lead to an increased bioactivity of activin A and hence may provide a useful measurement to identify patients at risk of a severe fibrotic response to IR. Additionally, follistatin, by its ability to neutralise the actions of activin A may be of value as an anti-fibrotic for radiation induced fibrosis.

Granulosa cell tumor mutant FOXL2C134W suppresses GDF-9 and activin A-induced follistatin transcription in primary granulosa cells

A single somatic FOXL2 mutation (FOXL2C134W) was identified in almost all granulosa cell tumor (GCT) patients. In the pituitary, FOXL2 and Smad3 coordinately regulate activin stimulation of follistatin transcription. We explored whether a similar regulation occurs in the ovary, and whether FOXL2C134W has altered activity. We show that in primary granulosa cells, GDF-9 and activin increase Smad3-mediated follistatin transcription. In contrast to findings in the pituitary, FOXL2 negatively regulates GDF-9 and activin-stimulated follistatin transcription in the ovary. Knockdown of endogenous FOXL2 confirmed this inhibitory role. FOXL2C134W displayed enhanced inhibitory activity, completely ablating GDF-9 and activin-induced follistatin transcription. GDF-9 and activin activity was lost when either the smad binding element or the forkhead binding element were mutated, indicating that both sites are required for Smad3 actions. This study highlights that FOXL2 negatively regulates follistatin expression within the ovary, and that the pathogenesis of FOXL2C134W may involve an altered interaction with Smad3.

Essential but differential role of FOXL2wt and FOXL2C134W in GDF-9 stimulation of follistatin transcription in co-operation with Smad3 in the human granulosa cell line COV434

The FOXL2C134W mutation has been identified in virtually all adult granulosa cell tumors (GCTs). Here we show that the exogenous FOXL2 expression is necessary for GDF-9 stimulation of follistatin transcription in the human GCT cell line, COV434 that lacks endogenous FOXL2 expression. Interestingly, in the presence of Smad3 co-expression, FOXL2C134W negated GDF-9 stimulation of follistatin transcription. However, mutation of the Smad binding element (SBE) located in the intronic enhancer elements in the follistatin gene restored normal FOXL2 activity to FOXL2C134W, thus the altered activity of FOXL2C134W is dependent on the ability of Smad3 to directly bind the SBE. Mutation of the FOXL2 binding element (FBE) or the FBE and SBE completely prevented GDF-9 activity, suggesting that the FBE is essential for GDF-9 stimulation in COV434. Overall, our study supports the view that altered interaction of FOXL2C134W with co-factors may underlie the pathogenesis of this mutation in GCTs.

P5-07-07: Follistatin Suppresses In Vitro Growth of Breast Cancer Cells and Its Reduced Expression in Breast Cancer Associated with Poor Differentiation and Prognosis.

Abstract Background: Bone morphogenetic protein (BMP) belongs to TGF-β superfamily, being widely involved in prenatal and postnatal development and homeostasis of various tissues and organs. BMP signaling has been implicated in tumorigenesis and disease progression of certain malignancies. Recent studies demonstrated a profound role played Noggin, a BMP antagonist in bone metastasis from breast cancer. Current study examined expression of Follistatin, another BMP antagonist in a breast cancer cohort, and its relevance to pathological and clinical parameters. Methods: Expression of Follistatin was examined in breast cancer tissues (n=93) and normal background tissues (n=30) using quantitative real time PCR and immunhistochemistry (IHC). The breast tissue samples were collected immediately after surgery. The clinical follow-up was routinely performed after surgery. The median follow-up period was 120 months (June 2004). Full-length human Follistatin coding sequence was cloned into a plasmid vector. Following transfection and verification, effect of Follistatin overexpression on cell growth was examined using an in vitro growth assay. Results: IHC revealed cytoplamic staining of Follistatin in mammary epithelial cells in breast tissues. The staining appeared to be weaker in breast cancer, but there was no significant difference in its transcripts levels compared with that of normal background tissues. Follistatin transcripts were decreased in both moderately differentiated and poorly differentiated tumors, p=0.0165 and p=0.0169 in comparison with well differentiated tumors respectively. Transcript levels of Follistatin in primary tumors tended to be lower in the patients with poor prognosis (33.8±14.7) including those patients had local recurrence, metastases and died of breast cancer, p=0.09 when compared with that of patients remained disease free (117.0±46.0). Its expression appeared particularly lower in the patients died of the disease (21.8±12.0), p=0.05 v.s. disease free. Over-expression of Follistatin exerted inhibitory effect on in vitro growth of MDA-MB-231 cells. Conclusions: Decreased expression of Follistatin in primary tumors of breast cancer correlates with poorer differentiation and mortality. Follistatin suppresses in vitro growth of breast cancer cells. It suggests that Follistatin is a negative regulator for tumor growth and disease progression of breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-07.

Developmental Changes in Pituitary Adenylate Cyclase Activating Polypeptide Expression during the Perinatal Period: Possible Role in Fetal Gonadotroph Regulation

Normal reproductive functioning may require secretion of LH independently of FSH. Variation in GnRH pulse frequency and inhibin negative feedback are mechanisms for differential gonadotropin regulation; however, the first instance of differential regulation in rats is during fetal development, prior to the establishment of GnRH connections, when LH accumulates appreciably 2-4 d prior to FSH. Pituitary adenylate cyclase activating polypeptide (PACAP) can differentially regulate the gonadotropins in vitro by stimulating alpha-subunit transcription, lengthening LHbeta transcripts and decreasing FSHbeta mRNA levels, probably through stimulation of follistatin transcription. These experiments are the first to examine whether PACAP influences gonadotroph function in perinatal pituitaries. In vivo, pituitary PACAP mRNA and peptide levels were high at embryonic d 19 and declined by 94 and 85%, respectively, after parturition. This was accompanied by a decrease of 65 and 96% in total follistatin and follistatin-288 mRNAs. These changes were temporally associated with a 20- and 6.5-fold rise in FSHbeta and GnRH receptor mRNAs, respectively, with no significant increase in LHbeta mRNA. In pituitary cell cultures from fetal and postnatal male rats, PACAP mRNA levels were likewise highest in fetal cultures in which the PACAP 6-38 antagonist decreased alpha-subunit and increased FSHbeta mRNA. PACAP 6-38 also reduced basal and GnRH-stimulated LH secretion with little effect on FSH. These data support the hypothesis that PACAP expressed at high levels in the fetal pituitary stimulates alpha-subunit expression and LH secretion and restrains FSH synthesis relative to LH and that a decline in PACAP allows for the neonatal rise in FSH and GnRH receptor because follistatin is decreased.

FoxL2 and Smad3 Coordinately Regulate Follistatin Gene Transcription

Follistatin is a transcriptional target and a modulator of activin action. Through an autocrine/paracrine loop, activin controls follistatin levels and thus regulates its own bioavailability. In gonadotropic alphaT3-1 cells, activin induces follistatin transcription primarily through the action of Smad3 at an intronic Smad-binding element (SBE1). Using a proteomics approach, we searched for endogenous alphaT3-1 proteins that participate in SBE1-mediated transcription. We identified FoxL2, a member of the forkhead family, as a candidate modulator of SBE1 function. Mutations of FoxL2 are associated with the blepharophimosis/ptosis/epicanthus inversus syndrome characterized with craniofacial defects and premature ovarian failure. FoxL2 localizes to alpha-glycoprotein subunit- and follicle-stimulating hormone beta-positive cells of the adult mouse pituitary and is present in alphaT3-1 and LbetaT2 cells, but its pituitary actions remain largely unknown. We have determined that FoxL2 binds to a forkhead-binding element (FKHB) located just downstream of the SBE1 site of the follistatin gene and functions as a Smad3 partner to drive SBE1-mediated transcription in alphaT3-1 cells treated with activin. Chromatin immunoprecipitation assays confirm that endogenous FoxL2 and Smad3 are recruited to the intronic enhancer of the follistatin gene where the SBE1 and FKHB sites are located. Exogenous FoxL2 enhances SBE1-mediated transcription, and short hairpin RNA-mediated knockdown of endogenous FoxL2 protein compromises this effect in alphaT3-1 cells. FoxL2 directly associates with Smad3 but not Smad2 or Smad4. This association between Smad3 and FoxL2 is mediated by the MH2 domain of Smad3 and is dependent on an intact forkhead domain in FoxL2. Altogether, these observations highlight a novel role for FoxL2 and suggest that it may function as a transcriptional regulator and a coordinator of Smad3 targets.

GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells

Hedgehog (HH) signaling in the epidermis is primarily mediated by the zinc finger transcription factors GLI1 and GLI2. Exquisite regulation of HH/GLI signaling is crucial for proper specification of the epidermal lineage and development of its derivatives, whereas dysregulation of HH/GLI signaling disrupts tissue homeostasis and causes basal cell carcinoma (BCC). Similarly, bone morphogenetic proteins (BMPs) and activins have been described as key signaling factors in the complex regulation of epidermal fate decisions, although their precise interplay with HH/GLI is largely elusive. Here we show that, in human epidermal cells, expression of the activin/BMP antagonist follistatin (FST) is predominantly up-regulated by the HH effector GLI2. Consistently, we found strong FST expression in the outer root sheath of human hair follicles and BCC. Detailed promoter analysis showed that two sequences with homology to the GLI consensus binding site are required for GLI2-mediated activation. Interestingly, activation of the FST promoter is highly GLI2-specific, because neither GLI1 nor GLI3 can significantly increase FST transcription. GLI2 specificity requires the presence of a 518-bp fragment in the proximal FST promoter region. On the protein level, sequences C-terminal to the zinc finger are responsible for GLI2-specific activation of FST transcription, pointing to the existence of GLI-interacting cofactors that modulate GLI target specificity. Our results reveal a key role of GLI2 in activation of the activin/BMP antagonist FST in response to HH signaling and provide new evidence for a regulatory interaction between HH and activin/BMP signaling in hair follicle development and BCC.

A Smad-binding Element in Intron 1 Participates in Activin-dependent Regulation of the Follistatin Gene

Follistatins exert critical autocrine or paracrine control in many tissues by binding and bio-neutralizing activin and several other transforming growth factor-beta ligands. In the pituitary, activin acts locally to induce follistatin expression and thus modulate its own actions. This local feedback loop safeguards against excessive activin signaling and maintains the necessary balance of activin and follistatin tone. To better understand the mechanisms underlying the activation of follistatin by activin A, follistatin transcription was evaluated in gonadotrope-derived alphaT3-1 cells. Transient transfection experiments established that follistatin-luciferase plasmids that incorporate up to 2.86 kb of the upstream region of the rat follistatin gene are not induced by activin A in alphaT3-1 cells. On the other hand, plasmids that incorporate intron 1 are responsive to activin A and induced by a constitutively active form of ALK4. These experiments ultimately identified a conserved Smad-binding element (SBE1) in intron 1, between +1791 and +1795. In alphaT3-1 cells treated with activin A, SBE1 preferentially recruits Smad3, but not Smad2, and mediates Smad3-dependent activation of follistatin transcription. shRNA knockdown of endogenous Smad3 in these cells compromises SBE1-mediated transcription in response to activin A and interferes with its ability to positively regulate follistatin mRNA levels. The findings of the current work illustrate the critical role of intron 1 of the follistatin gene in mediating Smad-dependent effects of activin and regulating the expression level of this gene in some cell types, such as pituitary cells of gonadotrope lineage.

Molecular cloning and characterization of follistatin in the gilthead sea bream, Sparus aurata

Follistatin (FST) is an activin-binding protein that neutralizes the activity of activin. FST also binds other members of the transforming growth factor-beta (TGF-beta) superfamily, including myostatin (MSTN). We report herein on the isolation and characterization of a full-length cDNA sequence predicted to encode FST in a marine fish, the gilthead sea bream Sparus aurata. The deduced amino acid sequence of sea bream FST (saFST) is highly conserved to the counterpart sequences in other vertebrates and contains the N-terminal domain and three FST domains. The deduced mature saFST shows 81-86% identity with FSTs from other vertebrates. It is 290 amino acids long, similar to other fish FSTs and the short isoform of Xenopus FST but longer by two residues than mammalian FST288. Ontogeny of MSTN (a TGF-beta superfamily member and a negative growth regulator of skeletal muscle in mammals), and FST (known to bind MSTN) gene expression revealed the presence of both transcripts throughout larval development. However, a different expression pattern was found in earlier developmental stages; while MSTN could not be detected prior to the day of hatching, FST transcript was detected in embryos 12 h post-fertilization, confirming its role during vertebrate embryonic development. Both FST and MSTN were expressed in many adult tissues, with variable levels of expression, including muscle. Recombinant saFST inhibited saMSTN activity in a reporter gene assay, indicating a similar effect to that reported in mammals.

Expression of activin pathway genes in granulosa cells of dominant and subordinate bovine follicles

We examined the association between the expression profiles of genes of the activin signalling pathway and ovarian follicular dominance in cattle. In monovular species such as cattle, one ovarian follicle of a cohort is selected to become dominant, whereas all others (i.e. the subordinate follicles) eventually succumb to apoptosis. We showed that Inhibin-βA, coding for the βA chain found in the A isoforms of activin, Inh-α coding for the inhibin-specific α chain, and the activin antagonist follistatin were expressed at higher levels in dominant follicle granulosa cells from Day 3.5 (ovulation = Day 0). Before selection, Inh-βA but not Inh-α was significantly correlated with potential dominant follicles, as defined by high aromatase expression and follicular fluid estrogen concentrations. Follistatin expression marked the largest follicles at Day 1.5, but displayed large variation in levels among cows. The third inhibin gene, Inh-βB, could only be detected at very low levels from Day 7 and thus was unlikely to play a prominent role in activin/inhibin signalling in cattle during these stages. There was a decrease in activin tone ( P = 0.07) specifically in the aromatase-high/dominant follicles, as measured by the ratio of Inh-βA to Inh-α plus follistatin transcripts between Days 1.5 and 7. Messenger RNA for both activin type II receptors and the nuclear effector Smad2 were detected in granulosa cells, consistent with an autocrine role for activin signalling. Additionally, expression of the putative activin target genes Smad2 and FSHreceptor were, respectively, either strongly ( P < 0.001) or weakly ( P = 0.09) associated with dominant follicles.

Transcriptional regulation of follistatin expression by GnRH in mouse gonadotroph cell lines: Evidence for a role for cAMP signaling

GnRH applied continuously or in pulses of high frequency increases follistatin, and thereby differentially regulates FSH and LH. This study was conducted in αT3-1 and LβT2 gonadotroph cells to begin to understand the signaling pathways through which GnRH stimulates follistatin synthesis. GnRH increased follistatin expression and stimulated a follistatin-LUC reporter in LβT2 cells, but was inactive in αT3-1 cells. GnRH also increased cAMP levels and stimulated a cAMP-responsive promoter only in LβT2 cells. Forskolin stimulated follistatin in both cell lines. GnRH activation of follistatin was blocked by the PKA inhibitor H89 and by over-expression of a dominant-negative inhibitor of CREB (A-CREB). Activation was also suppressed by PKC depletion, and was reduced by the PKC inhibitor bisindolylmaleimide. The MEK inhibitor PD98059 blocked activation by GnRH or forskolin implying that MAPK contributes to cAMP/PKA-mediated activation of follistatin. When LβT2 cells were transfected with follistatin-LUC together with A-CREB, and perifused with GnRH, activation was blocked during continuous GnRH, but stimulation by hourly GnRH pulses was unaffected. These experiments provide evidence that GnRH stimulates follistatin through multiple signaling pathways, and that cAMP-CREB activation is obligatory when GnRH is applied continuously. The finding that follistatin transcription was CREB-dependent with continuous but not pulsatile GnRH implies that the mode of ligand activation of GnRH receptors modifies the transcriptional response by changing the signaling network. These results provide a mechanism linking GnRH pulsatility to the differential control of FSH-β and LH-β gene expression through follistatin.

Stromal Cells of the Human Postmenopausal Ovary Display a Distinctive Biochemical and Molecular Phenotype

It has been postulated that the postmenopausal ovary, which consists chiefly of stromal cells, produces androgens, but studies of steroidogenic activity in these cells have given mixed results. To learn whether key steroidogenic enzymes involved in androgen synthesis are expressed in the postmenopausal ovarian stroma, stromal cells were isolated so that gene expression could be compared with that in cultured human theca cells and dermal fibroblasts. Genes involved in steroidogenesis include steroidogenic acute regulatory protein (StAR), P450scc (side-chain cleaving enzyme), 3β-hydroxysteroid dehydrogenase (3β-HSD), P450c17 (17-hydoxj1ase, 17,20 lyase), and P450c27. Several growth factor-binding proteins were also studied, including gremlin, IGF-binding protein-4, follistatin, and secreted frizzled-related proteins (sFRP)-1 and -4. In addition, the production of pregnenolone, progesterone, and hydroxysterol metabolites and the metabolism of 3 H-pregnenolone in ovarian stromal cells were examined. Isolated postmenopausal stromal cells had a stellate morphology distinct from the appearance of ovarian surface epithelial cells in culture. The stromal cells co-expressed both vimentin and cytokeratin. Transcript levels of StAR, P450scc, and 3β-HSD were 25- to 45-fold greater in theca cells than in stromal cells. Transcript levels of P450scc and 3β-HSD in stromal cells were similar to those in dermal fibroblasts, but StAR transcripts were much more abundant in stromal cells. Neither cell type expressed significant amounts of P450c17. The cholesterol-metabolizing enzyme P450c27 was expressed in both stromal and theca cells. There was no appreciable metabolism of pregnenolone in ovarian stromal cells, and no progesterone secretion was detected. Gremlin transcripts were 10-fold higher in ovarian stromal and fibroblast cells than in theca cells, but levels of IGF-binding protein-4 did not differ significantly. Follistatin transcripts were 24 times higher in stromal than in theca cells, and levels of sFRP-1 were 40-fold higher in stromal cells. Levels of sFRP-4 were 15 times higher than in theca cells and 80 times higher than in fibroblasts. Postmenopausal ovarian stromal cells have a biochemical/molecular phenotype distinct from that of fibroblasts. They do not appear to have substantial steroidogenic potential despite their ability to metabolize cholesterol to hydroxysterols. Expression of growth factor-binding proteins in stromal cells suggest that the stroma may help to limit the action of regulatory molecules in the premenopausal ovary.

Expression and neural control of follistatin versus myostatin genes during regeneration of mouse soleus.

Follistatin and myostatin are two secreted proteins involved in the control of muscle mass during development. These two proteins have opposite effects on muscle growth, as documented by genetic models. The aims of this work were to analyze in mouse, by using in situ hybridization, the spatial and temporal expression patterns of follistatin and myostatin mRNAs during soleus regeneration after cardiotoxin injury, and to investigate the influence of innervation on the accumulation of these two transcripts. Follistatin transcripts could be detected in activated satellite cells as early as the first stages of regeneration and were transiently expressed in forming myotubes. In contrast, myostatin mRNAs accumulated persistently throughout the regeneration process as well as in adult control soleus. Denervation significantly affected both follistatin and myostatin transcript accumulation, but in opposite ways. Muscle denervation persistently reduced the levels of myostatin transcripts as early as the young myotube stage, whereas the levels of follistatin mRNA were strongly increased in the small myotubes in the late stages of regeneration. These results are discussed with regard to the potential functions of both follistatin, as a positive regulator of muscle differentiation, and myostatin, as a negative regulator of skeletal muscle growth. We suggest that the belated up-regulation of the follistatin mRNA level in the small myotubes of the regenerating soleus as well as the down-regulation of the myostatin transcript level after denervation contribute to the differentiation process in denervated regenerating muscle.

Evidence that PACAP and GnRH down-regulate follicle-stimulating hormone-β mRNA levels by stimulating follistatin gene expression: effects on folliculostellate cells, gonadotrophs and LβT2 gonadotroph cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates α-subunit transcription and lengthens LH-β mRNA transcripts, but reduces FSH-β mRNA levels in rat pituitary cell cultures. PACAP also stimulates follistatin transcription, an effect which may explain the decrease in FSH-β mRNA. To begin to investigate the cells in which PACAP activates the follistatin gene, quantitative in situ hybridization for follistatin mRNA combined with immunostaining for LHβ and S100 protein was performed. In control cultures, follistatin mRNA was expressed in 70% of gonadotrophs and in 47% of folliculostellate cells (S-100+). PACAP increased ( P<0.001) both the number of follistatin-expressing cells as well as the number of grains per cell in both gonadotrophs and folliculostellate cells, while GnRH only affected ( P=0.01) gonadotrophs. Follistatin and FSH-β gene expression in rat pituitary cultures were also measured by competitive quantitative RT-PCR and northern analysis, respectively. Both PACAP and GnRH increased ( P<0.05) follistatin gene expression and suppressed ( P<0.05) FSH-β mRNA, and the effect of PACAP together with GnRH on follistatin exceeded that of GnRH alone. PACAP regulation of follistatin and FSH-β gene expression was studied further in LβT2 cells that were found to express receptors for the specific PACAP receptor, PAC 1. Follistatin mRNA was undetectable in cultures exposed to control media, or stimulated with PACAP, GnRH or rh-activin-A. In contrast to the results in primary pituitary cultures, PACAP increased FSH-β mRNA in these follistatin-deficient cells. Moreover, using transient transfection, PACAP stimulated transcription of ovine-FSH-β-luciferase. GnRH likewise increased FSH-β mRNA and stimulated FSH-β gene transcription in LβT2 cells. Activin-A increased FSH-β gene expression dose-dependently, and activin induction of FSH-β mRNA was blocked completely by 3-fold excess follistatin. These results indicate that PACAP stimulates follistatin gene expression in both gonadotrophs and folliculostellate cells, and provide further evidence that follistatin is required for PACAP or continuous GnRH to down-regulate FSH-β mRNA. These experiments suggest a mechanism by which PACAP influences FSH production selectively by an autocrine effect on gonadotrophs and by a paracrine mechanism through folliculostellate cells that involves follistatin.