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1.  GASP/WFIKKN Proteins: Evolutionary Aspects of Their Functions 
PLoS ONE  2012;7(8):e43710.
Growth and differentiation factor Associated Serum Protein (GASP) 1 and 2 are proteins known to be involved in the control of myostatin activity at least in vitro. Most deuterostome GASPs share a modular organization including WAP, follistatin/kazal, IGc2, two kunitz, and NTR domains. Based on an exon shuffling model, we performed independent phylogenetic analyses on these modules and assessed that papilin is probably a sister sequence to GASP with a divergence date estimated from the last common ancestor to bilateria. The final organization was acquired by the addition of the FS domain in early deuterostomes. Our study revealed that Gasp genes diverged during the first round of genome duplication in early vertebrates. By evaluating the substitution rate at different sites on the proteins, we showed a better conservation of the follistatin/kazal domain of GASP1 than GASP2 in mammals, suggesting a stronger interaction with myostatin. We also observed a progressive increase in the conservation of follistatin and kunitz domains from the ancestor of Ciona to early vertebrates. In situ hybridization performed on mouse embryos showed a weak Gasp1 expression in the formed somites at 10.5 dpc and in limb buds from embryonic E10.0 to E12.5. Similar results were obtained for zebrafish embryos. We propose a synthetic view showing possible interactions between GASP1 and myostatin and highlighting the role of the second kunitz domain in preventing myostatin proteolysis.
doi:10.1371/journal.pone.0043710
PMCID: PMC3427181  PMID: 22937083
2.  BRCA1 Regulates Follistatin Function in Ovarian Cancer and Human Ovarian Surface Epithelial Cells 
PLoS ONE  2012;7(6):e37697.
Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells.
doi:10.1371/journal.pone.0037697
PMCID: PMC3365892  PMID: 22685544
3.  Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin 
The Journal of Cell Biology  2012;197(7):997-1008.
Smad3/Akt/mTOR/S6K/S6RP signaling plays a critical role in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.
Follistatin is essential for skeletal muscle development and growth, but the intracellular signaling networks that regulate follistatin-mediated effects are not well defined. We show here that the administration of an adeno-associated viral vector expressing follistatin-288aa (rAAV6:Fst-288) markedly increased muscle mass and force-producing capacity concomitant with increased protein synthesis and mammalian target of rapamycin (mTOR) activation. These effects were attenuated by inhibition of mTOR or deletion of S6K1/2. Furthermore, we identify Smad3 as the critical intracellular link that mediates the effects of follistatin on mTOR signaling. Expression of constitutively active Smad3 not only markedly prevented skeletal muscle growth induced by follistatin but also potently suppressed follistatin-induced Akt/mTOR/S6K signaling. Importantly, the regulation of Smad3- and mTOR-dependent events by follistatin occurred independently of overexpression or knockout of myostatin, a key repressor of muscle development that can regulate Smad3 and mTOR signaling and that is itself inhibited by follistatin. These findings identify a critical role of Smad3/Akt/mTOR/S6K/S6RP signaling in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.
doi:10.1083/jcb.201109091
PMCID: PMC3384410  PMID: 22711699
4.  Differential expression of follistatin and FLRG in human breast proliferative disorders 
BMC Cancer  2009;9:320.
Background
Activins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases.
Methods
Paraffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma in situ (DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively.
Results
Follistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed.
Conclusion
The present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.
doi:10.1186/1471-2407-9-320
PMCID: PMC2749060  PMID: 19740438
5.  Follistatin promotes adipocyte differentiation, browning, and energy metabolism[S] 
Journal of Lipid Research  2014;55(3):375-384.
Follistatin (Fst) functions to bind and neutralize the activity of members of the transforming growth factor-β superfamily. Fst has a well-established role in skeletal muscle, but we detected significant Fst expression levels in interscapular brown and subcutaneous white adipose tissue, and further investigated its role in adipocyte biology. Fst expression was induced during adipogenic differentiation of mouse brown preadipocytes and mouse embryonic fibroblasts (MEFs) as well as in cold-induced brown adipose tissue from mice. In differentiated MEFs from Fst KO mice, the induction of brown adipocyte proteins including uncoupling protein 1, PR domain containing 16, and PPAR gamma coactivator-1α was attenuated, but could be rescued by treatment with recombinant FST. Furthermore, Fst enhanced thermogenic gene expression in differentiated mouse brown adipocytes and MEF cultures from both WT and Fst KO groups, suggesting that Fst produced by adipocytes may act in a paracrine manner. Our microarray gene expression profiling of WT and Fst KO MEFs during adipogenic differentiation identified several genes implicated in lipid and energy metabolism that were significantly downregulated in Fst KO MEFs. Furthermore, Fst treatment significantly increases cellular respiration in Fst-deficient cells. Our results implicate a novel role of Fst in the induction of brown adipocyte character and regulation of energy metabolism.
doi:10.1194/jlr.M039719
PMCID: PMC3934723  PMID: 24443561
mouse embryonic fibroblast; myostatin; brown fat; energy expenditure; uncoupling protein 1; mitochondria
6.  TMEFF2 Is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma 
PLoS ONE  2011;6(4):e18608.
Background
TMEFF2 is a protein containing a single EGF-like domain and two follistatin-like modules. The biological function of TMEFF2 remains unclear with conflicting reports suggesting both a positive and a negative association between TMEFF2 expression and human cancers.
Methodology/Principal Findings
Here we report that the extracellular domain of TMEFF2 interacts with PDGF-AA. This interaction requires the amino terminal region of the extracellular domain containing the follistatin modules and cannot be mediated by the EGF-like domain alone. Furthermore, the extracellular domain of TMEFF2 interferes with PDGF-AA–stimulated fibroblast proliferation in a dose–dependent manner. TMEFF2 expression is downregulated in human brain cancers and is negatively correlated with PDGF-AA expression. Suppressed expression of TMEFF2 is associated with its hypermethylation in several human tumor types, including glioblastoma and cancers of ovarian, rectal, colon and lung origins. Analysis of glioma subtypes indicates that TMEFF2 hypermethylation and decreased expression are associated with a subset of non-Proneural gliomas that do not display CpG island methylator phentoype.
Conclusions/Significance
These data provide the first evidence that TMEFF2 can function to regulate PDGF signaling and that it is hypermethylated and downregulated in glioma and several other cancers, thereby suggesting an important role for this protein in the etiology of human cancers.
doi:10.1371/journal.pone.0018608
PMCID: PMC3084709  PMID: 21559523
7.  Testosterone inhibits transforming growth factor-β signaling during myogenic differentiation and proliferation of mouse satellite cells: Potential role of follistatin in mediating testosterone action 
Testosterone (T) administration is associated with increased satellite cell number and skeletal muscle hypertrophy, although there is considerable heterogeneity in the response of different skeletal muscle groups to T in vivo. We investigated the effects of T on the growth and differentiation of satellite cells isolated from levator ani (LA) and gastrocnemius (gastroc) muscles. T up regulated follistatin (Fst) expression, but down regulated the mRNA and protein expression of a number of genes in the transforming growth factor-beta (TGF-β)-signaling pathway. Inhibition of Fst expression by small interfering RNA (siRNA) inhibited myogenic differentiation and blocked the pro-myogenic effects of T. Treatment of satellite cells with T or Fst up regulated the expression of Pax7 and PCNA, and increased their proliferation. T and Fst blocked TGF-β induced inhibition of growth and myogenic differentiation and down regulated TGF-β-dependent transcriptome in both LA and gastroc cells. We conclude that T stimulation of satellite cell proliferation and myogenic differentiation are associated with up regulation of Fst and inhibition of TGF-β-signaling.
doi:10.1016/j.mce.2011.11.019
PMCID: PMC3264813  PMID: 22138414
Transforming growth factor-β; follistatin; myostatin; myosin heavy chain II
8.  Differential Effects of Follistatin on Nonhuman Primate Oocyte Maturation and Pre-Implantation Embryo Development In Vitro1 
Biology of Reproduction  2009;81(6):1139-1146.
There is a vital need to identify factors that enhance human and nonhuman primate in vitro embryo culture and outcome, and to identify the factors that facilitate that objective. Granulosa and cumulus cells were obtained from rhesus monkeys that had either been FSH-primed (in vitro maturation [IVM]) or FSH and hCG-primed (in vivo maturation [VVM]) and compared for the expression of mRNAs encoding follistatin (FST), inhibin, and activin receptors. The FST mRNA displayed marginally decreased expression (P = 0.05) in association with IVM in the granulosa cells. The ACVR1B mRNA was more highly expressed in cumulus cells with IVM compared with VVM. Cumulus-oocyte complexes from FSH-primed monkeys exposed to exogenous FST during the 24-h IVM period exhibited no differences in the percentage of oocytes maturing to the metaphase II stage of meiosis compared to controls. However, embryos from these oocytes had significantly decreased development to the blastocyst stage. The effect of FST on early embryo culture was determined by exposing fertilized VVM oocytes to exogenous FST from 12 to 60 h postinsemination. FST significantly improved time to first cleavage and embryo development to the blastocyst stage compared with controls. The differential effects of exogenous FST on embryo development, when administered before and after oocyte maturation, may depend on the endogenous concentration in cumulus cells and oocytes. These results reveal evolutionary conservation of a positive effect of FST on embryogenesis that may be broadly applicable to enhance in vitro embryogenesis, with potential application to human clinical outcome and livestock and conservation biology.
Follistatin supplementation of culture medium after fertilization improves primate embryo development, while addition during in vitro maturation decreases oocyte developmental potential.
doi:10.1095/biolreprod.109.077198
PMCID: PMC2802231  PMID: 19641179
embryo; gene expression; gene regulation; granulosa cells; oocyte development; ovary; rhesus macaque
9.  Quadrupling Muscle Mass in Mice by Targeting TGF-ß Signaling Pathways 
PLoS ONE  2007;2(8):e789.
Myostatin is a transforming growth factor-ß family member that normally acts to limit skeletal muscle growth. Mice genetically engineered to lack myostatin activity have about twice the amount of muscle mass throughout the body, and similar effects are seen in cattle, sheep, dogs, and a human with naturally occurring loss-of-function mutations in the myostatin gene. Hence, there is considerable interest in developing agents capable of inhibiting myostatin activity for both agricultural and human therapeutic applications. We previously showed that the myostatin binding protein, follistatin, can induce dramatic increases in muscle mass when overexpressed as a transgene in mice. In order to determine whether this effect of follistatin results solely from inhibition of myostatin activity, I analyzed the effect of this transgene in myostatin-null mice. Mstn−/− mice carrying a follistatin transgene had about four times the muscle mass of wild type mice, demonstrating the existence of other regulators of muscle mass with similar activity to myostatin. The greatest effect on muscle mass was observed in offspring of mothers homozygous for the Mstn mutation, raising the possibility that either myostatin itself or a downstream regulator may normally be transferred from the maternal to fetal circulations. These findings demonstrate that the capacity for increasing muscle growth by manipulating TGF-ß signaling pathways is much more extensive than previously appreciated and suggest that muscle mass may be controlled at least in part by a systemic mode of action of myostatin.
doi:10.1371/journal.pone.0000789
PMCID: PMC1949143  PMID: 17726519
10.  Myostatin inhibition by a follistatin-derived peptide ameliorates the pathophysiology of muscular dystrophy model mice 
Acta Myologica  2008;27(1):14-18.
Summary
Gene-targeted therapies, such as adeno-associated viral vector (AAV)-mediated gene therapy and cell-mediated therapy using myogenic stem cells, are hopeful molecular strategies for muscular dystrophy. In addition, drug therapies based on the pathophysiology of muscular dystrophy patients are desirable. Multidisciplinary approaches to drug design would offer promising therapeutic strategies. Myostatin, a member of the transforming growth factor-β superfamily, is predominantly produced by skeletal muscle and negatively regulates the growth and differentiation of cells of the skeletal muscle lineage. Myostatin inhibition would increase the skeletal muscle mass and prevent muscle degeneration, regardless of the type of muscular dystrophy. Myostatin inhibitors include myostatin antibodies, myostatin propeptide, follistatin and follistatin-related protein. Although follistatin possesses potent myostatin-inhibiting activity, it works as an efficient inhibitor of activins. Unlike myostatin, activins regulate the growth and differentiation of nearly all cell types, including cells of the gonads, pituitary gland and skeletal muscle. We have developed a myostatin-specific inhibitor derived from follistatin, designated FS I-I. Transgenic mice expressing this myostatin-inhibiting peptide under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. mdx mice were crossed with FS I-I transgenic mice and any improvement of the pathological signs was investigated. The resulting mdx/FS I-I mice exhibited increased skeletal muscle mass and reduced cell infiltration in muscles. Muscle strength was also recovered in mdx/FS I-I mice. Our data indicate that myostatin inhibition by this follistatin-derived peptide has therapeutic potential for muscular dystrophy.
PMCID: PMC2859604  PMID: 19108572
Myostatin; follistatin; muscular dystrophy
11.  Expression of SET Protein in the Ovaries of Patients with Polycystic Ovary Syndrome 
Background. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. It suggested that SET may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome (PCOS). In this study, expression and cellular localization of SET protein were investigated in human polycystic and normal ovaries. Method. Ovarian tissues, six normal ovaries and six polycystic ovaries, were collected during transsexual operation and surgical treatment with the signed consent form. The cellular localization of SET protein was observed by immunohistochemistry. The expression levels of SET protein were analyzed by Western Blot. Result. SET protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both PCOS ovarian tissues and normal ovarian tissues. The level of SET protein expression in polycystic ovaries was triple higher than that in normal ovaries (P < 0.05). Conclusion. SET was overexpressed in polycystic ovaries more than that in normal ovaries. Combined with its localization in theca cells, SET may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in PCOS.
doi:10.1155/2013/367956
PMCID: PMC3686144  PMID: 23861679
12.  The Effects of Transforming Growth Factor-β2 on the Expression of Follistatin and Activin A in Normal and Glaucomatous Human Trabecular Meshwork Cells and Tissues 
Purpose.
To compare follistatin (FST) and activin (Act) expression in normal and glaucomatous trabecular meshwork (TM) cells and tissues and determine if exogenous TGF-β2 regulates the expression of FST and Act in TM cells.
Methods.
Total RNA was isolated from TM cell strains, and mRNA expression for FST 317/344 isoforms and Act was determined via RT-PCR and quantitative PCR (qPCR). Western immunoblotting and immunocytochemistry determined FST and Act A protein levels in normal TM (NTM) and glaucomatous TM (GTM) cells. Cells were treated with recombinant human TGF-β2 protein at 0 to 10 ng/mL for 0 to 72 hours. qPCR, Western immunoblotting, immunocytochemistry, and ELISA immunoassay were utilized to determine changes in FST and Act A mRNA and protein levels. In addition, NTM and GTM tissue samples were examined by immunohistochemistry for expression of FST, FST 315, FST 288, and Act A.
Results.
Both FST mRNA and protein levels were significantly elevated in GTM cells. FST mRNA transcripts FST 317/344 were also significantly elevated in GTM cells. Immunohistochemistry showed FST levels were significantly elevated in GTM tissues. Exogenous TGF-β2 significantly induced FST mRNA and protein expression. Immunohistochemistry demonstrated that Act A protein levels were significantly higher in NTM tissues compared to GTM tissues.
Conclusions.
FST is elevated in GTM cells and tissues. FST is known to be an inhibitor of bone morphogenetic proteins (BMPs), which, coupled with the ability of TGF-β2 to upregulate FST levels, may indicate a possible role of FST in the pathogenesis of glaucoma. These results suggest that additional endogenous molecules in human TM may regulate TGF-β2 signaling via inhibition of BMP family members.
This study identifies the expression of TGF-β/BMP signaling molecules in the TM.
doi:10.1167/iovs.12-10292
PMCID: PMC3487490  PMID: 23010638
13.  Therapeutic Impact of Follistatin-Like 1 on Myocardial Ischemic Injury in Preclinical Models 
Circulation  2012;126(14):1728-1738.
Background
Acute coronary syndrome is a leading cause of death in developed countries. Follistatin-like 1 (FSTL1) is a myocyte-derived secreted protein that is upregulated in the heart in response to ischemic insult. Here, we investigated the therapeutic impact of FSTL1 on acute cardiac injury in small and large preclinical animal models of ischemia/reperfusion and dissected its molecular mechanism.
Methods and Results
Administration of human FSTL1 protein significantly attenuated myocardial infarct size in a mouse or pig model of ischemia/reperfusion, which was associated with a reduction of apoptosis and inflammatory responses in the ischemic heart. Administration of FSTL1 enhanced the phosphorylation of AMP-activated protein kinase in the ischemia/reperfusion–injured heart. In cultured cardiac myocytes, FSTL1 suppressed apoptosis in response to hypoxia/reoxygenation and lipopolysaccharide-stimulated expression of proinflammatory genes through its ability to activate AMP-activated protein kinase. Ischemia/reperfusion led to enhancement of bone morphogenetic protein-4 expression and Smad1/5/8 phosphorylation in the heart, and FSTL1 suppressed the increased phosphorylation of Smad1/5/8 in ischemic myocardium. Treating cardiac myocytes with FSTL1 abolished the bone morphogenetic protein-4 –stimulated increase in apoptosis, Smad1/5/8 phosphorylation, and proinflammatory gene expression. In cultured macrophages, FSTL1 diminished lipopolysaccharide-stimulated expression of proinflammatory genes via activation of AMP-activated protein kinase and abolished bone morphogenetic protein-4 – dependent induction of proinflammatory mediators.
Conclusions
Our data indicate that FSTL1 can prevent myocardial ischemia/reperfusion injury by inhibiting apoptosis and inflammatory response through modulation of AMP-activated protein kinase– and bone morphogenetic protein-4 – dependent mechanisms, suggesting that FSTL1 could represent a novel therapeutic target for post-myocardial infarction, acute coronary syndrome.
doi:10.1161/CIRCULATIONAHA.112.115089
PMCID: PMC3548325  PMID: 22929303
apoptosis; inflammation; ischemia; myocytes; cardiac; reperfusion
14.  Systemic administration of Follistatin288 increases muscle mass and reduces fat accumulation in mice 
Scientific Reports  2013;3:2441.
The present study describes the physiological response associated with daily subcutaneous injection of mice with recombinant follistatin288. This systemic administration of follistatin288 increases the follistatin levels in serum, indicating that the protein enters the circulation. The data suggest that a dose-dependent increase in body lean mass also occurs, together with an increase in muscle mass, possibly as a result of an increase in the size of the muscle fibers. After thirteen weeks of treatment, metabolic changes were observed; additionally, the switching of muscle fiber types was also apparent through myosin heavy chain remodeling, implying that changes are occurring at the molecular level. Furthermore, an increase in the muscle mass was associated with a significant decrease in the body fat mass. Overall, this study raises the possibility for the use of follistatin288 as an agent to treat muscle wasting diseases and/or to restrict fat accumulation by systemic administration of the protein.
doi:10.1038/srep02441
PMCID: PMC3743061  PMID: 23942549
15.  Effects of the activin A-myostatin-follistatin system on aging bone and muscle progenitor cells 
Experimental gerontology  2012;48(2):290-297.
The activin A-myostatin-follistatin system is thought to play an important role in the regulation of muscle and bone mass throughout growth, development, and aging; however, the effects of these ligands on progenitor cell proliferation and differentiation in muscle and bone are not well understood. In addition, age-associated changes in the relative expression of these factors in musculoskeletal tissues have not been described. We therefore examined changes in protein levels of activin A, follistatin, and myostatin (GDF-8) in both muscle and bone with age in C57BL6 mice using ELISA. We then investigated the effects of activin A, myostatin and follistatin on the proliferation and differentiation of primary myoblasts and mouse bone marrow stromal cells (BMSCs) in vitro. Myostatin levels and the myostatin:follistatin ratio increased with age in the primarily slow-twitch mouse soleus muscle, whereas the pattern was reversed with age in the fast-twitch extensor digitorum longus muscle. Myostatin levels and the myostatin: follistatin ratio increased significantly (+75%) in mouse bone marrow with age, as did activin A levels (+17%). Follistatin increased the proliferation of primary myoblasts from both young and aged mice, whereas myostatin increased proliferation of younger myoblasts but decreased proliferation of older myoblasts. Myostatin reduced proliferation of both young and aged BMSCs in a dose-dependent fashion, and activin A increased mineralization in both young and aged BMSCs. Together these data suggest that aging in mice is accompanied by changes in the expression of activin A and myostatin, as well as changes in the response of bone and muscle progenitor cells to these factors. Myostatin appears to play a particularly important role in the impaired proliferative capacity of muscle and bone progenitor cells from aged mice.
doi:10.1016/j.exger.2012.11.004
PMCID: PMC3678732  PMID: 23178301
sarcopenia; myoblasts; bone marrow stromal cells; proliferation; differentiation
16.  Growth Differentiation Factor 9 (GDF9) Suppresses Follistatin and Follistatin-Like 3 Production in Human Granulosa-Lutein Cells 
PLoS ONE  2011;6(8):e22866.
Background
We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin βB-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.
Methodology
hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin βB-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test.
Principal Findings
GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin βB-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin βB-subunit mRNA and inhibin B production were attenuated by adding FST.
Conclusion
GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin βB-subunit mRNA expression and inhibin B production in hGL cells.
doi:10.1371/journal.pone.0022866
PMCID: PMC3148233  PMID: 21829661
17.  Functional genetic polymorphisms and female reproductive disorders: Part I: polycystic ovary syndrome and ovarian response 
Human Reproduction Update  2008;14(5):459-484.
BACKGROUND
The identification of polymorphisms associated with a disease can help to elucidate its pathogenesis, and this knowledge can be used to improve prognosis for women with a particular disorder, such as polycystic ovary syndrome (PCOS). Since an altered response to ovarian stimulation is also a characteristic of the disease, further knowledge about its aetiology could help in defining the parameters that determine the response of an individual to ovarian stimulation.
METHODS
PubMed and EMBASE databases were systematically searched for gene association studies published until the end of August 2007, using search criteria relevant to PCOS and ovarian response to stimulation. Data from additional papers identified through hand searches were also included; 139 publications were reviewed.
RESULTS
Several genes involved in ovarian function and metabolism are associated with increased susceptibility to PCOS, but none is strong enough to correlate alone with susceptibility to the disease, or response to therapy. A single-nucleotide polymorphism in exon 10 of the FSH receptor (FSHR) gene, FSHR p.N680S, was consistently identified as having a significant association with ovarian response to FSH.
CONCLUSIONS
No consistent association between gene polymorphism and PCOS could be identified. The FSHR gene may play a significant role in the success of ovarian stimulation, and can be used as a marker to predict differences in FSHR function and ovarian response to FSH. Genotyping the FSHR p.N680S polymorphism may provide a means of identifying a population of poor responders before in vitro fertilization procedures are initiated.
doi:10.1093/humupd/dmn024
PMCID: PMC2515090  PMID: 18603647
female reproduction; genetic polymorphisms; polycystic ovary syndrome
18.  Hypoxia enhances the expression of follistatin-like 3 in term human trophoblasts 
Placenta  2007;29(1):51-57.
Hypoxic injury hinders placental differentiation and alters trophoblast gene expression. We tested the hypothesis that the expression of follistatin-like 3 (FSTL3), a member of the follistatin family of proteins, is modulated by hypoxia in primary human trophoblast (PHT). Using immunofluorescence of human term placental villi we detected the expression of FSTL3 protein in placental villi, primarily in trophoblasts. We verified this finding in cultured term PHT cells. Basal expression of FSTL3 transcript in cultured PHT cells, determined using quantitative PCR, was stable over the culture period. Importantly, when compared to culture in FiO2=20% or FiO2=8%, PHT cells cultured in FiO2 <1% exhibited a 4–6 fold increase in FSTL3 mRNA expression as early as 4 h in hypoxia. Whereas cellular FSTL3 protein was unchanged in hypoxia, we found that hypoxia increased the level of FSTL3 in the medium. Lastly, the exposure of PHT cells to either the hypoxia-mimetic cobalt chloride or the proline hydroxylase inhibitor dimethyloxaloylglycine upregulated the expression of FSTL3 transcript. Our data indicate that hypoxia enhances the expression of FSTL3 and its release from PHT cells. Our finding that hypoxia-mimetic agents enhance FSTL3 expression implicates HIF1α in this process.
doi:10.1016/j.placenta.2007.09.001
PMCID: PMC2237886  PMID: 17959243
19.  Activin-type II receptor B (ACVR2B) and follistatin haplotype associations with muscle mass and strength in humans 
Genetic variation in myostatin, a negative regulator of skeletal muscle, in cattle has shown remarkable influence on skeletal muscle, resulting in a double-muscled phenotype in certain breeds; however, DNA sequence variation within this gene in humans has not been consistently associated with skeletal muscle mass or strength. Follistatin and activin-type II receptor B (ACVR2B) are two myostatin-related genes involved in the regulation and signaling of myostatin. We sought to identify associations between genetic variation and haplotype structure in both follistatin and ACVR2B with skeletal muscle-related phenotypes. Three hundred fifteen men and 278 women aged 19–90 yr from the Baltimore Longitudinal Study of Aging were genotyped to determine respective haplotype groupings (Hap Groups) based on HapMap data. Whole body soft tissue composition was measured by dual-energy X-ray absorptiometry. Quadriceps peak torque (strength) was measured using an isokinetic dynamometer. Women carriers of ACVR2B Hap Group 1 exhibited significantly less quadriceps muscle strength (shortening phase) than women homozygous for Hap Group 2 (109.2 ± 1.9 vs. 118.6 ± 4.1 N·m, 30°/s, respectively, P = 0.036). No significant association was observed in men. Male carriers of follistatin Hap Group 3 exhibited significantly less total leg fat-free mass than noncarriers (16.6 ± 0.3 vs. 17.5 ± 0.2 kg, respectively, P = 0.012). No significant associations between these haplotype groups were observed in women. These results indicate that haplotype structure at the ACVR2B and follistatin loci may contribute to interindividual variation in skeletal muscle mass and strength, although these data indicate sex-specific relationships.
doi:10.1152/japplphysiol.01322.2006
PMCID: PMC2646094  PMID: 17347381
genetics; skeletal muscle; sex
20.  Modeling and structural analysis of cellulases using Clostridium thermocellum as template 
Bioinformation  2012;8(22):1105-1110.
Cellulase is one of the most widely distributed enzymes with wide application. They are involved in conversion of biomass into simpler sugars. Cellulase of Trichoderma longibrachiatum, a known cellulolytic fungus was compared with Clostridium thermocellum [AAA23226.1] cellulase. Blastp was performed with AAA23226.1 as query sequence to obtain nine similar sequences from NCBI protein data bank. The physicochemical properties of cellulase were analyzed using ExPASy’s ProtParam tool namely ProtParam, SOPMA and GOR IV. Homology modeling was done using SWISS MODEL and checked quality by RMSD values using VMD1.9.1. Active sites of each model were predicted using automated active site prediction server of SCFBio. Study revealed instability of cellulase of two eukaryotic strains namely Trichoderma longibrachiatum [CAA43059.1] and Melanocarpus albomyces [CAD56665.1]. The negative GRAVY score value of cellulases ensured better interaction and activity in aqueous phase. It was found that molecular weight (M. Wt) ranges between 25-127.56 kDa. Iso-electric point (pI) of cellulases was found to be acidic in nature. GOR IV and SOPMA were used to predict secondary structure of cellulase, which showed that random coil, was dominated. Neighbor joining tree with C. thermocellum [AAA23226.1] cellulase as root showed that cellulases of Thermoaerobacter subterraneus [ZP_07835928] and C. thermocellum [CAA4305.1] were more similar to eukaryotic cellulases supported by least boot strap values. Pseudoalteromonas haloplanktis cellulase was found to be the ideal model supported by least RMSD score among the predicted structures. Trichoderma longibrachiatum cellulase was found to be the best compared to other cellulases, which possess high number of active sites with ASN and THR rich active sites. CYS residues were also present ensuring stable interaction and better bonding. Hydrophilic residues were found high in active sites of all analyzed models and template.
doi:10.6026/97320630081105
PMCID: PMC3523225  PMID: 23251045
21.  Serum 25-Hydroxyvitamin D Levels, phosphoprotein enriched in diabetes gene product (PED/PEA-15) and leptin-to-adiponectin ratio in women with PCOS 
Background
Polycystic ovary syndrome (PCOS) is frequently associated with hypovitaminosis D. Vitamin D is endowed with pleiotropic effects, including insulin resistance (IR) and apoptotic pathway. Disruption of the complex mechanism that regulated ovarian apoptosis has been reported in PCOS. Phosphoprotein enriched in diabetes gene product (PED/PEA-15), an anti-apoptotic protein involved in type 2 diabetes mellitus (T2DM), is overexpressed in PCOS women, independently of obesity. Leptin-to-adiponectin ratio (L/A) is a biomarker of IR and low-grade inflammation in PCOS. The aim of the study was to investigate the levels of 25-hydroxy vitamin D (25(OH)D), and L/A, in association with PED/PEA-15 protein abundance, in both lean and overweight/obese (o/o) women with PCOS.
Patients and Methods
PED/PEA-15 protein abundance and circulating levels of 25(OH)D, L/A, sex hormone-binding globulin, and testosterone were evaluated in 90 untreated PCOS patients (25 ± 4 yrs; range 18-34) and 40 healthy controls age and BMI comparable, from the same geographical area. FAI (free androgen index) and the homeostasis model assessment of insulin resistance (HoMA-IR) index were calculated.
Results
In o/o PCOS, 25(OH)D levels were significantly lower, and L/A values were significantly higher than in lean PCOS (p < 0.001), while there were no differences in PED/PEA-15 protein abundance. An inverse correlation was observed between 25(OH)D and BMI, PED/PEA-15 protein abundance, insulin, HoMA-IR, FAI (p < 0.001), and L/A (p < 0.05). At the multivariate analysis, in o/o PCOS L/A, insulin and 25(OH)D were the major determinant of PED/PEA-15 protein abundance (β = 0.45, β = 0.41, and β = -0.25, respectively).
Conclusions
Lower 25(OH)D and higher L/A were associated to PED/PEA-15 protein abundance in PCOS, suggesting their involvement in the ovarian imbalance between pro-and anti-apoptotic mechanisms, with high L/A and insulin and low 25(OH)D levels as the main determinants of PED/PEA-15 protein variability. Further studies, involving also different apoptotic pathways or inflammatory cytokines and granulosa cells are mandatory to better define the possible bidirectional relationships between 25(OH)D, PED/PEA-15 protein abundance, leptin and adiponectin in PCOS pathogenesis.
doi:10.1186/1743-7075-8-84
PMCID: PMC3293764  PMID: 22112520
25-hydroxyvitamin D; PED/PEA-15; Leptin-to-adiponectin ratio; PCOS; apoptosis
22.  A Peptide of SPARC Interferes with the Interaction between Caspase8 and Bcl2 to Resensitize Chemoresistant Tumors and Enhance Their Regression In Vivo 
PLoS ONE  2011;6(11):e26390.
SPARC, a matricellular protein with tumor suppressor properties in certain human cancers, was initially identified in a genome-wide analysis of differentially expressed genes in chemotherapy resistance. Its exciting new role as a potential chemosensitizer arises from its ability to augment the apoptotic cascade, although the exact mechanisms are unclear. This study further examines the mechanism by which SPARC may be promoting apoptosis and identifies a smaller peptide analogue with greater chemosensitizing and tumor-regressing properties than the native protein. We examined the possibility that the apoptosis-enhancing activity of SPARC could reside within one of its three biological domains (N-terminus (NT), the follistatin-like (FS), or extracellular (EC) domains), and identified the N-terminus as the region with its chemosensitizing properties. These results were not only confirmed by studies utilizing stable cell lines overexpressing the different domains of SPARC, but as well, with a synthetic 51-aa peptide spanning the NT-domain. It revealed that the NT-domain induced a significantly greater reduction in cell viability than SPARC, and that it enhanced the apoptotic cascade via its activation of caspase 8. Moreover, in chemotherapy resistant human colon, breast and pancreatic cancer cells, its chemosensitizing properties also depended on its ability to dissociate Bcl2 from caspase 8. These observations translated to clinically significant findings in that, in-vivo, mouse tumor xenografts overexpressing the NT-domain of SPARC had significantly greater sensitivity to chemotherapy and tumor regression, even when compared to the highly-sensitive SPARC-overexpressing tumors. Our results identified an interplay between the NT-domain, Bcl2 and caspase 8 that helps augment apoptosis and as a consequence, a tumor's response to therapy. This NT-domain of SPARC and its 51-aa peptide are highly efficacious in modulating and enhancing apoptosis, thereby conferring greater chemosensitivity to resistant tumors. Our findings provide additional insight into mechanisms involved in chemotherapy resistance and a potential novel therapeutic that specifically targets this devastating phenomenon.
doi:10.1371/journal.pone.0026390
PMCID: PMC3206029  PMID: 22069448
23.  Ubiquitous Gasp1 overexpression in mice leads mainly to a hypermuscular phenotype 
BMC Genomics  2012;13:541.
Background
Myostatin, a member of the TGFβ superfamily, is well known as a potent and specific negative regulator of muscle growth. Targeting the myostatin signalling pathway may offer promising therapeutic strategies for the treatment of muscle-wasting disorders. In the last decade, various myostatin-binding proteins have been identified to be able to inhibit myostatin activity. One of these is GASP1 (Growth and Differentiation Factor-Associated Serum Protein-1), a protein containing a follistatin domain as well as multiple domains associated with protease inhibitors. Despite in vitro data, remarkably little is known about in vivo functions of Gasp1. To further address the role of GASP1 during mouse development and in adulthood, we generated a gain-of-function transgenic mouse model that overexpresses Gasp1 under transcriptional control of the human cytomegalovirus immediate-early promoter/enhancer.
Results
Overexpression of Gasp1 led to an increase in muscle mass observed not before day 15 of postnatal life. The surGasp1 transgenic mice did not display any other gross abnormality. Histological and morphometric analysis of surGasp1 rectus femoris muscles revealed an increase in myofiber size without a corresponding increase in myofiber number. Fiber-type distribution was unaltered. Interestingly, we do not detect a change in total fat mass and lean mass. These results differ from those for myostatin knockout mice, transgenic mice overexpressing the myostatin propeptide or follistatin which exhibit both muscle hypertrophy and hyperplasia, and show minimal fat deposition.
Conclusions
Altogether, our data give new insight into the in vivo functions of Gasp1. As an extracellular regulatory factor in the myostatin signalling pathway, additional studies on GASP1 and its homolog GASP2 are required to elucidate the crosstalk between the different intrinsic inhibitors of the myostatin.
doi:10.1186/1471-2164-13-541
PMCID: PMC3575399  PMID: 23046573
24.  Impact of FTO genotypes on BMI and weight in polycystic ovary syndrome: a systematic review and meta-analysis 
Diabetologia  2012;55(10):2636-2645.
Aims/hypothesis
FTO gene single nucleotide polymorphisms (SNPs) have been shown to be associated with obesity-related traits and type 2 diabetes. Several small studies have suggested a greater than expected effect of the FTO rs9939609 SNP on weight in polycystic ovary syndrome (PCOS). We therefore aimed to examine the impact of FTO genotype on BMI and weight in PCOS.
Methods
A systematic search of medical databases (PubMed, EMBASE and Cochrane CENTRAL) was conducted up to the end of April 2011. Seven studies describing eight distinct PCOS cohorts were retrieved; seven were genotyped for SNP rs9939609 and one for SNP rs1421085. The per allele effect on BMI and body weight increase was calculated and subjected to meta-analysis.
Results
A total of 2,548 women with PCOS were included in the study; 762 were TT homozygotes, 1,253 had an AT/CT genotype, and 533 were AA/CC homozygotes. Each additional copy of the effect allele (A/C) increased the BMI by a mean of 0.19 z score units (95% CI 0.13, 0.24; p = 2.26 × 10−11) and body weight by a mean of 0.20 z score units (95% CI 0.14, 0.26; p = 1.02 × 10−10). This translated into an approximately 3.3 kg/m2 increase in BMI and an approximately 9.6 kg gain in body weight between TT and AA/CC homozygotes. The association between FTO genotypes and BMI was stronger in the cohorts with PCOS than in the general female populations from large genome-wide association studies. Deviation from an additive genetic model was observed in heavier populations.
Conclusions/interpretation
The effect of FTO SNPs on obesity-related traits in PCOS seems to be more than two times greater than the effect found in large population-based studies. This suggests an interaction between FTO and the metabolic context or polygenic background of PCOS.
doi:10.1007/s00125-012-2638-6
PMCID: PMC3433670  PMID: 22801903
BMI; FTO; Meta-analysis; Polycystic ovary syndrome; Systematic review; Weight
25.  Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries 
Molecular Human Reproduction  2009;15(12):829-841.
Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β-binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200–1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.
doi:10.1093/molehr/gap072
PMCID: PMC2776474  PMID: 19692420
fibrillin; latent-transforming growth factor β-binding protein; polycystic ovary syndrome; ovary

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