Follistatin mRNA Levels Research Articles

Effects of Growth Hormone on Muscle and Bone in Female Mice: Role of Follistatin.

The interactions between muscle and bone are noted in the clinical relationships between sarcopenia and osteoporosis. Myokines secreted from the skeletal muscles play roles in muscle-bone interactions related to various physiological and pathophysiological states. Although numerous evidence suggests that growth hormone (GH) influences both muscle and bone, the effects of GH on the muscle-bone interactions have remained unknown. We, therefore, investigated the influences of GH administration for 8 weeks on muscle and bone, including myokine expression, in mice with or without ovariectomy (OVX). GH administration significantly increased muscle mass in the whole body and lower limbs, as well as tissue weights of the extensor digitorum longus (EDL) and soleus muscles in mice with or without OVX. Moreover, it markedly increased grip strength in both mice. As for femurs, GH administration significantly increased cortical thickness and area in mice with or without OVX. Moreover, GH significantly blunted the decrease in the ratio of bone volume to tissue volume at the trabecular bone in mice with OVX. GH administration significantly decreased follistatin mRNA levels in the EDL, but not the soleus, muscles in mice with or without OVX, although it did not affect the other myokines examined. However, GH administration significantly elevated serum follistatin levels in mice. In conclusion, this study indicates that GH administration increases skeletal muscle mass and grip strength and cortical and trabecular bone-related parameters obtained by micro-computed tomography analyses in mice. However, myokine regulation might not be critical for the effects of GH on muscle and bone.

Lentivirus-Mediated Short Hairpin RNA for Follistatin Downregulation Suppresses Tumor Progression in Hypopharyngeal Carcinoma.

Follistatin (FST) inhibits the action of activin by interfering with the binding of activin to its receptor. Although the prognostic value of FST in various cancers has been investigated previously, studies rarely focused on hypopharyngeal carcinoma (HPC). In our study, the effect of FST expression on HPC tissues and cell lines was investigated. A total of 60 patients with HPC were recruited for this study. Levels of FST mRNA and protein were measured by quantitative polymerase chain reaction (PCR) and immunohistochemistry in HPC tissue samples and by qPCR in the HPC FaDu cells, as well as immortal nasopharyngeal epithelial cell line NP-69 cells. After silencing the FST expression in FaDu cells using lentivirus-mediated siRNA that was specific for FST mRNA, cell proliferation was determined by a Celigo assay. Tumor growth was monitored in nude mice and viability was determined by a methylthiazoletetrazolium assay. The ratio of cell cycle arrest and apoptosis was evaluated by flow cytometry. The colony formation ability was performed using Giemsa staining. In addition, wound healing and Transwell migration and invasion assays were performed for the analysis of cell motility. FST expression was significantly higher in human HPC tissue and FaDu cells than in normal tissue and NP-69 cells. A higher expression of FST in HPC samples was positively correlated with advanced tumors. Moreover, FST knockdown by shRNA significantly decreased cell growth, colony formation, migration and invasion. Furthermore, FST silencing increased the cell apoptosis percentage, and arrested cell cycle in the S phase in FaDu cells. In addition, FST silencing suppressed tumor growth in vivo. Our results indicated that the FST gene was associated with HPC progression and may serve as a potential therapeutic target for the treatment of HPC.

Grass Carp Follisatin: Molecular Cloning, Functional Characterization, Dopamine D1 Regulation at Pituitary Level, and Implication in Growth Hormone Regulation.

Activin is involved in pituitary hormone regulation and its pituitary actions can be nullified by local production of its binding protein follistatin. In our recent study with grass carp, local release of growth hormone (GH) was shown to induce activin expression at pituitary level, which in turn could exert an intrapituitary feedback to inhibit GH synthesis and secretion. To further examine the activin/follistatin system in the carp pituitary, grass carp follistatin was cloned and confirmed to be single-copy gene widely expressed at tissue level. At the pituitary level, follistatin signals could be located in carp somatotrophs, gonadotrophs, and lactotrophs. Functional expression also revealed that carp follistatin was effective in neutralizing activin’s action in stimulating target promoter with activin-responsive elements. In grass carp pituitary cells, follistatin co-treatment was found to revert activin inhibition on GH mRNA expression. Meanwhile, follistatin mRNA levels could be up-regulated by local production of activin but the opposite was true for dopaminergic activation with dopamine (DA) or its agonist apomorphine. Since GH stimulation by DA via pituitary D1 receptor is well-documented in fish models, the receptor specificity for follistatin regulation by DA was also investigated. Using a pharmacological approach, the inhibitory effect of DA on follistatin gene expression was confirmed to be mediated by pituitary D1 but not D2 receptor. Furthermore, activation of D1 receptor by the D1-specific agonist SKF77434 was also effective in blocking follistatin mRNA expression induced by activin and GH treatment both in carp pituitary cells as well as in carp somatotrophs enriched by density gradient centrifugation. These results, as a whole, suggest that activin can interact with dopaminergic input from the hypothalamus to regulate follistatin expression in carp pituitary, which may contribute to GH regulation by activin/follistatin system via autocrine/paracrine mechanisms.

Activin/follistatin system in grass carp pituitary cells: - Regulation by local release of growth hormone and luteinizing hormone and its functional role in growth hormone synthesis and secretion.

Gonadotrophin regulation by activin/follistatin system is well-documented, but the corresponding effect on growth hormone (GH) has not been fully characterized and with little information available in lower vertebrates, especially in fish models. In grass carp, local interactions of GH and luteinizing hormone (LH) can induce GH release and gene expression at pituitary level via autocrine/paracrine mechanisms. To shed light on the role of activin/follistatin system in GH regulation by local actions of GH and LH, grass carp activin βA and βB were cloned, shown to be single-copy genes expressed in the pituitary, and confirmed to encode activin proteins capable of transactivating promoter with activin-responsive elements. In grass carp pituitary cells, activin A and B were effective in reducing GH secretion and GH cell content with concurrent drop in GH mRNA level whereas the opposite was true for follistatin, the activin-binding protein known to neutralize the effects of endogenous activin. Treatment with activin A and B not only could suppress basal but also inhibit GH mRNA expression induced by GH and human chorionic gonadotropin (hCG), a functional analogue of LH in fish model. Apparently, down-regulation of GH mRNA by activin was mediated by reducing GH transcript stability with concurrent inhibition on GH promoter activity via the SMAD pathway. In reciprocal experiments, GH treatment was found to up-regulate activin βA, activin βB and follistatin mRNA levels in carp pituitary cells but the opposite was noted by removing endogenous GH with GH antiserum. Interestingly, parallel treatment with hCG could also inhibit basal as well as GH-induced activin βA, activin βB and follistatin gene expression. These results, as a whole, indicate that the pituitary activin/follistatin system can serve as a regulatory target for local interactions of GH and LH and contribute to GH regulation by autocrine/paracrine mechanisms in the carp pituitary.

Role of follistatin in muscle and bone alterations induced by gravity change in mice.

Interactions between muscle and bone have been recently noted. We reported that the vestibular system plays crucial roles in the changes in muscle and bone induced by hypergravity in mice. However, the details of the mechanisms by which gravity change affects muscle and bone through the vestibular system still remain unknown. Here, we investigated the roles of humoral factors linking muscle to bone and myostatin-related factors in the hypergravity-induced changes in muscle and bone in mice with vestibular lesions (VL). Hypergravity elevated serum and mRNA levels of follistatin, an endogenous inhibitor of myostatin, in the soleus muscle of mice. VL blunted the hypergravity-enhanced levels of follistatin in the soleus muscle of mice. Simulated microgravity decreased follistatin mRNA level in mouse myoblastic C2C12 cells. Follistatin elevated the mRNA levels of myogenic genes as well as the phosphorylation of Akt and p70S6 kinase in C2C12 cells. As for bone metabolism, follistatin antagonized the mRNA levels of osteogenic genes suppressed by activin A during the differentiation of mesenchymal cells into osteoblastic cells. Moreover, follistatin attenuated osteoclast formation enhanced by myostatin in the presence of receptor activator of nuclear factor-κB ligand in RAW 264.7 cells. Serum follistatin levels were positively related to bone mass in mouse tibia. In conclusion, the present study provides novel evidence that hypergravity affects follistatin levels in muscle through the vestibular system in mice. Follistatin may play some roles in the interactions between muscle and bone metabolism in response to gravity change.

Expression, Regulation, and Functional Characterization of FST Gene in Porcine Granulosa Cells

ABSTRACTProliferation, differentiation, and estrogen secretion of granulosa cells are the key factors affecting the estrous after weaning in sows. The objective of this study was to evaluate the expression of Follistatin (FST) in the ovary of Xiushui Hang and Duroc sows at weaning and estrus, the effect of FSH on transcript abundance of FST gene in granulosa cells and the role of FST gene in the weaning to estrus using siRNAs targeted to FST gene. In the present study, expression of the FST mRNA was evaluated by real time PCR. The FST mRNA levels showed a reduction from weaning to the estrus in both Xiushui Hang and Duroc sows, and the mRNA levels in Duroc ovary was higher than in Xiushui Hang sows at the beginning of estrus. Granulosa cells were obtained from the two largest follicles around follicular deviation, FST expression was decreased sharply after treatment with FSH (250 ng/ml). Knockdown of FST by siRNA in porcine granulosa cells significantly increased cell proliferation and estrogen secretion. These results indicate that FST gene is a negative regulator of follicle growth and function during the weaning-estrus interval.

The effect of eight weeks resistance and aerobic training on myostatin and follistatin expression in cardiac muscle of rats.

Introduction: The clinical studies have shown that the myostatin gene expression and its serum density occur more frequently in heart patients than in healthy individuals. The purpose of this study is to investigate the influence of 8-week resistance and aerobic exercise on the myostatin and follistatin gene expression of myocardium muscle of healthy male Wistar rats. Methods: In this experimental study, 20 five-week-old adult Wistar rats (250 ± 26.5 g) were divided into three groups: healthy control group (n = 6), resistance exercise group (n = 7), and aerobic exercise group (n = 7). The resistance and aerobic exercise plan consisted of 8 weeks and 3 sessions per week. The resistance exercise group performed climbing a one-meter 26-stair ladder with a slope of 85 degrees for 3 sets of 5 repetitions per session. The aerobic exercise group performed running at a speed of 12 meters per minute for 30 minutes during the first sessions gradually increasing up to a speed of 30 meters per minute for 60 minutes during the final sessions (equivalent to 70% to 80% of maximum oxygen consumption). The differences between the groups were evaluated using a one-way analysis of variance (ANOVA) test. When appropriate, LSD post-hoc test was used. The significance level for the study was less than 0.05. Results: The results of this study shows that after 8 weeks of exercise, there is no significant difference between myostatin mRNA gene expression levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise (P = 0.172, F = 1.953). However, the mean differences between follistatin mRNA levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise are statistically significant (F = 38.022, P = 0.001). Furthermore, the ratio of follistatin to myostatin mRNA gene expression of the heart muscle (P = 0.001, F = 10.288) shows significant difference among the three groups. Conclusion: Our results indicate that the resistance and aerobic exercise could cause a decrease in myostatin and an increase in follistatin levels, thus preventing many muscular physiological disorders such as arthritis and muscle weakness.

Upregulation of activin-B and follistatin in pulmonary fibrosis - a translational study using human biopsies and a specific inhibitor in mouse fibrosis models.

BackgroundActivins are members of the TGF-ß superfamily of growth factors. First, we identified by expression array screening that activin-B and follistatin are upregulated in human idiopathic pulmonary fibrosis (IPF). Next, we wanted to clarify their specific role in lung fibrosis formation.MethodsWe used specific antibodies for activin-A and -B subunits and follistatin to measure and localize their levels in idiopathic pulmonary fibrosis and control lung biopsies. To inhibit activin signaling, we used soluble activin type IIB receptor fused to the Fc portion of human IgG1 (sActRIIB-Fc) in two different mouse models of pulmonary fibrosis.ResultsActivin-B and follistatin mRNA levels were elevated in the human IPF lung. Immunoreactivity to activin-A, -B and follistatin localized predominantly to the hyperplastic, activated alveolar epithelium, but was also seen in inflammatory cells. Mice treated with sActRIIB-Fc showed increased skeletal muscle mass and a clear reduction in alveolar cell counts in bronchoalveolar lavage fluid, but no significant antifibrotic effect in the lung was observed.ConclusionsThe upregulation of activin-B and follistatin in IPF is a novel finding. Our results indicate that activin inhibition is not an efficient tool for antifibrotic therapy, but could be useful in reducing alveolar cellular response to injury. Activin-B and follistatin levels may be useful as biomarkers of IPF.

Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

Evidence supports local roles for transforming growth factor β superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signalling receptors is likely modulated by extracellular binding proteins (BP). In this study, we compared ex vivo expression of four BPs (chordin, gremlin, noggin and follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1-18 mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P < 0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P < 0.01) gremlin and follistatin mRNA abundance, with a significant cell-type × follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P < 0.05) in GC of large 'E-active' than 'E-inactive' follicles while follistatin mRNA level was higher (P < 0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP-BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin or FSHR mRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin and gremlin mRNA levels up to 20-fold (P < 0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signalling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signalling.

Altered sex hormone concentrations and gonadal mRNA expression levels of activin signaling factors in hatchling alligators from a contaminated Florida lake

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly.

Role of Follistatin in Promoting Adipogenesis in Women

Follistatin is a glycoprotein that binds and neutralizes biological activities of TGFbeta superfamily members including activin and myostatin. We previously identified by expression profiling that follistatin levels in white adipose tissue (WAT) were regulated by obesity. The objective of the study was to elucidate the role of follistatin in human WAT and obesity. We measured secreted follistatin protein from WAT biopsies and fat cells in vitro. We also quantified follistatin mRNA expression in sc and visceral WAT and in WAT-fractionated cells and related it to obesity status, body region, and cellular origin. We investigated the effects of follistatin on adipocyte differentiation of progenitor cells in vitro. Women (n = 66) with a wide variation in body mass index were recruited by advertisement and from a clinic for weight-reduction therapy. WAT secreted follistatin in vitro. Follistatin mRNA levels in sc but not visceral WAT were decreased in obesity and restored to nonobese levels after weight reduction. Follistatin mRNA levels were high in the stroma-vascular fraction of WAT and low in adipocytes. Recombinant follistatin treatment promoted adipogenic differentiation of progenitor cells and neutralized the inhibitory action of myostatin on differentiation in vitro. Moreover, activin and myostatin signaling receptors were detected in WAT and adipocytes. Follistatin is a new adipokine important for adipogenesis. Down-regulated WAT expression of follistatin in obesity may counteract adiposity but could, by inhibiting adipogenesis, contribute to hypertrophic obesity (large fat cells) and insulin resistance.

Peroxisome Proliferator-activated Receptor γ Down-regulates Follistatin in Intestinal Epithelial Cells through SP1

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) down-regulates the expression of follistatin mRNA in intestinal epithelial cells in vivo. The mechanism of PPARgamma-mediated down-regulation of follistatin was investigated using non-transformed, rat intestinal epithelial cells (RIE-1). RIE cells expressed activin A, the activin receptors ActRI and ActRII, and the follistatin-315 mRNA. RIE-1 cells responded to endogenous activin A, and this response was antagonized by follistatin, as evidenced by changes in cell growth and regulation of an activin-responsive reporter. Using RIE-1 cells, we show that activation of PPARgamma by rosiglitazone reduced follistatin mRNA levels in a dose- and concentration-dependent manner. Down-regulation of follistatin by rosiglitazone required the DNA binding domain of PPARgamma and was dependent upon dimerization with the retinoid X receptor. Inhibition of follistatin expression by rosiglitazone was not associated with decreased follistatin mRNA stability, suggesting that regulation may be at the promoter level. Analysis of the follistatin promoter revealed consensus binding sites for AP-1, AP-2, and Sp1. Targeting the AP-1 pathway with SP600125, an inhibitor of JNK, and TAM67, a dominant negative c-Jun, had no effect on PPARgamma-mediated down-regulation of follistatin. However, the follistatin promoter was dramatically regulated by Sp1, and this regulation was inhibited by PPARgamma expression. Knockdown of Sp1 expression relieved repression of follistatin levels by rosiglitazone. Moreover, PPARgamma was found to interact with Sp1 and repress its transcriptional activation function. Collectively, our data indicate that repression of Sp1 transcriptional activity by PPARgamma is the underlying mechanism responsible for PPARgamma-mediated regulation of follistatin expression.

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.

Anterior Pituitary Gene Expression with Reproductive Aging in the Female Rat1

Although reproductive aging in women is classically attributed to loss of ovarian follicles, recent data have suggested that the entire hypothalamic-pituitary-ovarian axis undergoes functional changes with time. The aim of this study was to characterize age-related changes in pituitary gene expression for factors with known importance for gonadotroph function, including 1) steroid hormone receptors (Esr and Pgr), 2) orphan nuclear receptors [Nr5a1 (steroidogenic factor-1) and Nr5a2 (liver receptor homologue-1)], and 3) pituitary-derived polypeptides (activin, inhibin, and follistatin), as well as 4) gonadotropin subunits and 5) GnRH receptors. We chose to utilize a middle-aged rat model for these studies. Young (Y; 3-mo-old) and middle-aged (MA; 9- to 12-mo-old) rats were ovariectomized, primed with estradiol, and injected with progesterone to induce an LH surge. The mRNA levels for the gonadotropin subunits and GnRH receptors were decreased in middle-aged females relative to young animals. Nr5a1 and follistatin mRNA levels were significantly greater in Y versus MA animals following ovariectomy. Furthermore, steroid-induced regulation of these genes was lost in the MA animals. Regulation of the Nr5a2, Inhba, and Inhbb transcripts was also limited to the young animals. In contrast, there were no significant differences in the mRNA levels of Esr or Pgr family members between age groups at any time point. Although this in vivo model normalizes ovarian steroid levels, it does not control for potential differences in GnRH stimulation with aging. Therefore, in a second set of experiments, we used an in vitro perifusion system to compare the effects of pulsatile GnRH in the two age groups. Nr5a1 mRNA expression was greater in Y than MA animals and was significantly decreased by GnRH pulses in both age groups. Follistatin mRNA levels increased significantly with GnRH treatment in Y animals but were not significantly changed in the MA females. Taken together, these data demonstrate gene-specific blunting of pituitary gene expression postovariectomy and during the steroid-induced surge in middle-aged rats. We propose that age-related changes in pituitary physiology may contribute to reproductive senescence.

Regulation of Follicle-Stimulating Hormone Secretion by the Interactions of Activin-A, Dexamethasone and Testosterone in Anterior Pituitary Cell Cultures of Male Rats

This study was designed to evaluate the effects of glucocorticoids and gonadal steroids on the expression of inhibin/activin subunits and follistatin of the anterior pituitary and test the hypothesis that resulting changes in the local activin/inhibin/follistatin tone contribute to steroid effects on follicle stimulating hormone (FSH) production from gonadotropes. In primary cell cultures of male rat anterior pituitaries, dexamethasone (DEX) or testosterone (T) stimulated FSH secretion and FSHβ mRNA and their effects were additive with activin-A. Follistatin (FS288) and inhibin-A antagonized the rise in FSH secretion both in the absence and presence of exogenous activin-A. Despite the similarity in their action on FSH production, DEX and T had opposite effects on follistatin mRNA levels. Follistatin mRNA levels of cultured rat anterior pituitary cells were elevated upon the addition of DEX but attenuated by T. On the other hand, both DEX and T suppressed inhibin/activin βB mRNA levels while only DEX affected βA mRNA. In these cells, activin-A stimulated follistatin and inhibin/activin βB mRNA levels but had no effect on βA. Together, DEX and activin-A caused a further increase in follistatin mRNA levels while T attenuated the effect of activin-A alone. Both steroids attenuated the effect of activin-A on βB mRNA accumulation. These results support the possibility that DEX and T, possibly acting on different subsets of anterior pituitary cells, use distinct mechanisms to modify the local activin/inhibin/follistatin circuitry and thereby upregulate FSH production from the anterior pituitary gonadotropes.

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.

Rat anterior pituitary folliculostellate cells are targets of interleukin-1beta and a major source of intrapituitary follistatin.

Folliculostellate cells of the anterior pituitary are postulated to be an important source of factors, such as follistatin, that regulate pituitary function by intercellular communication. To gain further insight into the function of this cell type, folliculostellate cells were enriched from cultured rat anterior pituitary cells, and an immortalized cell line designated FS/D1h was established and characterized. These FS/D1h cells express S100 immunoreactivity and produce IL-6 but not pituitary hormones such as GH, ACTH, FSH, and LH. Importantly, FS/D1h cells express large amounts of follistatin mRNA and secrete the protein, as quantified indirectly by the amount of [(125)I]activin A immunoprecipitated with a follistatin antiserum. The FS/D1h cells also express alpha, betaA, and betaB inhibin/activin subunit mRNAs, but whether they produce the corresponding activins and inhibins has not been determined. The response of FS/D1h cells to agents thought to modulate folliculostellate cell function was evaluated. IL-1beta (0.005-5 nM) stimulated the secretion of follistatin and increased mRNA expression. In parallel, IL-6 secretion was stimulated. Dexamethasone, pituitary adenylate cyclase-activating polypeptide(1-27), and lipopolysaccharide but not testosterone, 12-O-tetradecanoylphorbol-13-acetate, or forskolin also increased follistatin secretion. Surprisingly, activin had no effect on follistatin mRNA levels, despite the fact that FS/D1h cells express ActRII, ActRIIB, and ALK-4 (ActRIB). Activin, on the other hand, induced Smad7 mRNA accumulation and exerted an antiproliferative effect on FS/D1h cells. Altogether, these observations support the possibility that follistatin originating from folliculostellate cells participates in mediating the effects of IL-1beta, glucocorticoids, and other agents on the response of pituitary cells to activins.

Characterization of rat follistatin-related gene: effects of estrous cycle stage and pregnancy on its messenger RNA expression in rat reproductive tissues.

Follistatin-related gene (FLRG) was first identified as a target of a chromosomal translocation in a human B-cell leukemia. Because FLRG protein binds to activins and bone morphogenetic proteins, FLRG is postulated to be a regulator of these growth factors. However, physiological aspects of FLRG are unclear. To elucidate the physiology of FLRG, we examined expression of FLRG in reproductive tissues of the rat. FLRG mRNA was abundantly expressed in the placenta. FLRG mRNA was also expressed in the ovary, uterus, testis, lung, adrenal gland, pituitary, kidney, small intestine, and heart. During the second half of pregnancy, expression of FLRG in the placenta continuously increased, whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter. FLRG was also expressed in decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20. In contrast, follistatin mRNA was highly expressed in the decidua on Day 12, continuously decreased until Day 16, and then remained at relatively low levels thereafter. During the rat estrous cycle, levels of ovarian FLRG mRNA fluctuated diurnally, with highest levels during daytime, and did not change relative to the day of the estrous cycle. The present results suggest that FLRG may play a role in the regulation of reproductive events.

Follistatin overexpression in rodent liver tumors: a possible mechanism to overcome activin growth control.

The activin-follistatin system is a potent growth regulatory system of liver tissue homeostasis. Activin A inhibits hepatocellular DNA synthesis and induces cell death. Follistatin binds activin and sequesters it from the signaling pathway. Consistently, follistatin has been reported to act as an inducer of DNA synthesis in the liver. Using RNase protection analysis, we studied the expression of follistatin in rat and mouse liver tumors as a possible mechanism to overcome activin growth control. Approximately 40% of the tumors (nine of 24 each), most of them hepatocellular carcinomas, displayed increased levels of follistatin mRNA when compared to tumor-surrounding liver tissue. The degree of overexpression was highly variable but independent of the carcinogen treatment that animals had received. It was also independent from the histological stage of malignancy and further found in rat liver adenomas. Follistatin expression was also observed in cell lines derived from human hepatocellular carcinomas. Overexpression of follistatin may represent a unique strategy of hepatic tumors to overcome the inhibitory action of a growth factor, activin, by decreasing its local bioavailability.