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Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

ABSTRACT: We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin ?(B)-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.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 ?(B)-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.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 ?(B)-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 ?(B)-subunit mRNA and inhibin B production were attenuated by adding FST.GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin ?(B)-subunit mRNA expression and inhibin B production in hGL cells.

SUBMITTER: Shi FT

PROVIDER: S-EPMC3148233 | biostudies-other | 2011

REPOSITORIES: biostudies-other

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Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

Shi Feng-Tao FT Cheung Anthony P AP Huang He-Feng HF Leung Peter C K PC

PloS one 20110801 8

<h4>Background</h4>We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β(B)-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.<h4>Methodology</h4>hGL cells from women undergoing in vitro fertilization (IV ...[more]

PMID: 21829661

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Project description:BackgroundOvarian granulosa cells are the predominant source of estradiol and progesterone biosynthesis in vivo. Rosiglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor gamma (PPAR gamma), is applied as the treatment of insulin resistance including women with PCOS. The aim of the study was to investigate the direct effects of rosiglitazone on steroidogenesis and proinflammatory factor production in human granulosa-lutein cells (GLCs).MethodsPrimary human GLCs were separated during in vitro fertilization and cultured in the presence of rosiglitazone, GW9662 (an antagonist of PPAR gamma) and hCG. The mRNA expression of key steroidogenic factors including 3beta- hydroxysteriod dehydrogenase (3beta-HSD), cytochrome P-450 scc (CYP11A1), cytochrome P-450 aromatase (CYP19A1), and steroidogenic acute regulatory protein (StAR) were detected by quantitative real-time PCR. Estradiol and progesterone levels in GLCs cultures were measured by chemiluminescence immunoassay, and the proinflammtory factors (TNFalpha and IL-6) in conditioned culture media were measured by ELISA.ResultsPPAR gamma mRNA levels increased up to 3.24 fold by rosiglitazone at the concentration of 30 microM compared to control (P<0.05). hCG alone or hCG with rosiglitazone had no significant effects on PPAR gamma mRNA levels. The CYP19A1 mRNA level at exposure to rosiglitazone alone showed a drop, but was not significantly reduced comparing to control. The expression levels of enzymes 3beta-HSD and CYP11A1 in all treatments did not alter significantly. The StAR mRNA expression at exposure to rosiglitazone was significantly increased comparing to control (P<0.05). The media concentrations of E2 and progesterone by rosiglitazone treatment showed a declining trend comparing to control or cotreatment with hCG, which did not reach significance. Most importantly, treatment with rosiglitazone decreased TNFalpha secretion in a statistically significant manner compared with control (P<0.05). The concentration of IL-6 following rosiglitazone exposure did not significantly decrease comparing to control.ConclusionIn cultured GLCs, rosiglitazone stimulated StAR expression, but did not significantly affect steroidogenic enzymes, as well as E2 and progesterone production. Moreover, rosiglitazone significantly decreased the production of TNFalpha in human GLCs, suggesting that PPAR gamma may play a role in the regulation of GLCs functions through inhibiting proinflammatory factors.

Project description:BackgroundTGF? signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGF? signaling.MethodsTo test the importance of TGF? signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGF? response (FET), or tumorigenic with TGF? response (FET?) or tumorigenic with abrogated TGF? response via introduction of dominant negative TGF?RII (FET?/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging.ResultsAbrogation of TGF? signaling through introduction of a dominant negative TGF? receptor II (TGF?RII) in non-metastatic FET? human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGF? signaling in FET?-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGF? signaling is a metastasis suppressor, we rescued TGF? signaling in CBS metastatic colon cancer cells that had lost TGF? receptor expression due to epigenetic repression. Restoration of TGF? signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGF? signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma.ConclusionsThe observations presented here indicate a metastasis suppressor role for TGF? signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGF? signaling may be imprudent in some patient populations with residual TGF? tumor suppressor activity.

Dataset Information

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

Publications

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

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