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SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells†.

ABSTRACT: Connective tissue growth factor (also known as CTGF or CCN2) is a secreted matricellular protein that belongs to the CCN family. With wide-ranging biological activities and tissue expression patterns, CTGF plays a critical role in regulating various cellular functions. In the female reproductive system, CTGF is highly expressed in granulosa cells in growing ovarian follicles and is involved in the regulation of follicular development, ovulation, and luteal function. In the mammalian ovary, bone morphogenetic protein 6 (BMP6) is an important intraovarian modulator of follicular development. In this study, we demonstrated that BMP6 treatment significantly increased the expression of CTGF in both primary and immortalized human granulosa cells. Using both pharmacological inhibitors and Small interfering RNA-mediated knockdown approaches, we showed that ALK2 and ALK3 type I receptors are required for BMP6-induced cellular activities. Furthermore, this effect is most likely mediated by a Sma- and Mad-related protein (SMAD)-dependent pathway. Our studies provide novel insight into the molecular mechanisms by which an intraovarian growth factor affects the production of another factor via a paracrine effect in human granulosa cells.

SUBMITTER: Liu S

PROVIDER: S-EPMC6736146 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells†.

Liu Shuang S Chang Hsun-Ming HM Yi Yuyin Y Yao Yuan-Qing YQ Leung Peter C K PCK

Biology of reproduction 20190801 2

Connective tissue growth factor (also known as CTGF or CCN2) is a secreted matricellular protein that belongs to the CCN family. With wide-ranging biological activities and tissue expression patterns, CTGF plays a critical role in regulating various cellular functions. In the female reproductive system, CTGF is highly expressed in granulosa cells in growing ovarian follicles and is involved in the regulation of follicular development, ovulation, and luteal function. In the mammalian ovary, bone ...[more]

PMID: 31210269

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Project description:Transforming growth factor-? (TGF-?) and connective tissue growth factor (CTGF) are often described as the initial pro-fibrotic mediators upregulated early in fibrosis models dependent on angiotensin II (Ang-II). In the present study, we explore the mechanistic link between TGF-? and CTGF expression by using a novel TGF-? trap.NIH/3T3 fibroblasts were subjected to TGF-? with or without TGF-? trap or 1D11 antibody, CTGF or CTGF plus TGF-? for six or 24 hours, and then used for quantitative real-time polymerase chain reaction (qRT-PCR) or immunocytochemistry. Male C57BL/6 mice were infused with Ang-II and randomly assigned TGF-? trap for six or 24 hours. Hearts were harvested for histological analyses, qRT-PCR and western blotting.Exogenous TGF-?-induced fibroblasts resulted in significant upregulation of CTGF, TGF-? and type I collagen transcript levels in vitro. Additionally, TGF-? promoted the differentiation of fibroblasts into ?-SMA+ myofibroblasts. CTGF expression was reduced by the addition of TGF-? trap or neutralizing antibody, confirming that its expression is dependent on TGF-? signaling. In contrast, exogenous CTGF did not appear to have an effect on fibroblast production of pro-fibrotic transcripts or fibroblast differentiation. Ang-II infusion in vivo led to a significant increase in TGF-? and CTGF mRNA expression at six and 24 hours with corresponding changes in Smad2 phosphorylation (pSmad2), indicative of increased TGF-? signaling. Ang-II animals that received the TGF-? trap demonstrated reduced CTGF mRNA levels and pSmad2 at six hours, suggesting that early CTGF expression is dependent on TGF-? signaling.We demonstrated that CTGF expression is dependent on TGF-? signaling both in vitro and in vivo in a model of myocardial fibrosis. This also suggests that early myocardial CTGF mRNA expression (six hours) after Ang-II exposure is likely dependent on latent TGF-? activation via the canonical Smad-dependent pathway in resident cardiac cells.

Project description:BACKGROUND:Adenomyosis is a medical condition defined by the abnormal presence of endometrial tissue within the myometrium, in which fibrosis occurs with new collagen deposition and myofibroblast differentiation. In this study, the effect of several mediators and growth factors on collagen expression was investigated on human endometrial stromal cells (fibroblasts) derived from adenomyotic endometrium. EXPERIMENTAL APPROACH:RT-PCR, Western blot analysis, pharmacological interventions and siRNA interference were applied to primary cultured human endometrial stromal cells (fibroblasts). Immunohistochemistry was used to analyze protein expression in adenomyotic endometrium tissue specimens. RESULTS:Of the tested mediators, transforming growth factor β1 (TGFβ1) and its isoforms were effective to induce collagen and connective tissue growth factor (CTGF) expression. Collagen and CTGF induction by TGFβ1 could be reduced by the inhibitors targeting DNA transcription, protein translation, and Smad2/3 signaling. Interestingly, TGFβ1 induced Smad2/3 phosphorylation and CTGF mRNA expression, but not collagen mRNA expression, suggesting that TGFβ1 mediates collagen expression through CTGF induction and Smad2/3 activation. In parallel, TGFβ1 and CTGF also induced expression of heat shock protein (HSP) 47, a protein required for the synthesis of several types of collagens. However, only CTGF siRNA knockdown, could compromise TGFβ1-induced collagen expression. Finally, the immunohistochemistry revealed vimentin- and α-SMA-positive staining for (myo)fibroblasts, TGFβ1, collagen, and CTGF in the subepithelial stroma region of human adenomyotic endometria. CONCLUSION AND IMPLICATIONS:We reveal here that TGFβ1, collagen, and CTGF are expressed in the stroma of adenomyotic endometria and demonstrate that TGFβ1 can induce collagen production in endometrium-derived fibroblasts through cellular Smad2/3-dependent signaling pathway and CTGF expression, suggesting that endometrial TGFβ may take part in the pathogenesis of adenomyosis and ectopic endometrium may participate in uterine adenomyosis.

Dataset Information

SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells†.

Publications

SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells†.

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