Successful ovulation heavily relies on the proper expansion of COC under the control of gonadotropin and locally produced growth factors [1, 36]. Increasing evidence suggests that aberrant expression of BMPs and their downstream signaling may cause female infertility . Previous in vitro studies have shown that BMPs promote COC expansion by regulating the expression of several proteins related to COC expansion [37–41]. In the present study, we demonstrated that BMP2 increased hyaluronan production in hGL cells. These results are consistent with a previous study showing that BMP4, BMP6, BMP7 and BMP15 increase the production of hyaluronan in hGL cells . Additionally, our results showed that BMP2 treatment significantly upregulated the expression of HAS2, but not HAS1 and HAS3, in a dose and time-dependent manner. Similarly, animal studies showed that only HAS2 is upregulated in response to stimulation with gonadotropins, whereas HAS1 is slightly upregulated, and HAS3 is not affected by gonadotropins, indicating that HAS2 is the principal enzyme involved in the production of hyaluronan in hGL cells [42, 43].
Previous studies have shown that several members of the TGF-β superfamily can promote the production of hyaluronan by regulating the expression of HAS2 [41, 44, 45]. However, the precise mechanisms underlying this regulatory activity have not been determined. In the present study, we showed that CTGF mediates the BMP2-induced upregulation of HAS2 expression and hyaluronan synthesis in hGL cells. This conclusion is based on our results showing that BMP2 upregulates the expression of CTGF at the mRNA and protein levels as early as 1 h and 3 h, respectively. However, the mRNA level of HAS2 was not increased until up to 3 h after BMP2 treatment. Most importantly, overexpression of CTGF significantly increased the mRNA level of HAS2 and the production of hyaluronan, and knockdown of CTGF completely abolished the BMP2-induced upregulation of HAS2 expression and hyaluronan production in human granulosa cells. These findings indicate that CTGF is the downstream mediator of BMP2 in promoting hyaluronan production in hGL cells. Given the essential role of CTGF and hyaluronan in COC expansion, our studies further confirm that the modulation effect of BMP2 on CTGF expression and hyaluronan synthesis is an essential cellular mechanism regulating ovulation by modulating proper COC expansion during the late stage of follicle development. Nevertheless, we acknowledge that all of our results were based on in vitro cell experiments, with no evidence showing that BMP2 directly affects follicular COC expansion in vivo. Future animal studies aimed at addressing this issue will be of great interest to support our in vitro findings in the current study.
The activation of the TGF-β superfamily signaling pathway depends on the combination of the ligands and their related receptors. Upon ligand–receptors binding, type II receptors phosphorylate type I receptors to form an activated ligand/receptor complex, which in turn activates the downstream effector proteins, the R-SMADs . Compared with more than forty TGF-β superfamily members, only seven TGF-β type I and five TGF-β type II receptors have been identified. A large number of studies have demonstrated that the interaction modes between the ligands and their corresponding receptors are diverse in different tissues or cell types . Importantly, the mutation or aberrant expression of TGF-β superfamily members or receptors is associated with reproductive disorders . Thus, a detailed exploration of the interaction between ligands and their relevant receptors will help with understanding the underlying molecular mechanisms by which TGF-β superfamily members exert their cellular activities and the related pathological conditions. In general, three TGF-β type I receptors, including ALK2, ALK3 and ALK6, have been reported to bind to specific BMP ligands . In the current study, using pharmacological and siRNA-based knockdown approaches, we demonstrated that ALK2 and ALK3 receptors are involved in the BMP2-induced upregulation of CTGF expression in hGL cells. This result is consistent with our most recent study showing that BMP6 increases the expression of CTGF via both ALK2 and ALK3 receptors . A previous in vivo study demonstrated that the specific TGF-β type I receptor inhibitor dorsomorphin (a selective inhibitor of ALK2/ALK3/ALK6) completely attenuates the inductive effect of BMP2 on primordial follicle formation in mouse ovaries, whereas DMH-1 (a selective inhibitor of ALK2/ALK3) only partially abolishes the inductive effect of BMP2 . In contrast, our study showed that DMH-1 and dorsomorphin both completely abolished the BMP2-induced upregulation of CTGF expression in hGL cells, indicating that ALK2/ALK3 but not ALK6 is involved in the regulatory effect of BMP2. The discrepancy between two studies may explain the complexity of the BMP-receptor interaction, and the combination model of BMPs and their receptors is cell type- or species-dependent. In this regard, only ALK3 but not ALK2 is involved in the regulatory effect of BMP2 on cell invasion in human trophoblast cells . Collectively, these findings suggest that the activation of the BMP2 signaling pathway is dependent on specific receptors, which provides a new insight into molecular targets for treating female reproductive disorders.
In the canonical BMP signaling pathway, phosphorylated type I receptors recruit and phosphorylate the downstream R-SMAD proteins, which in turn form trimers with SMAD4 and translocate into the nucleus to regulate target gene expression . Data obtained from in vivo studies showed that granulosa cell-specific Smad4 knockout mice are subfertile and exhibit multiple defects in follicle development and ovulation, including disrupted steroidogenesis, impaired cumulus function, and premature luteinization . These results highlight the essential role of SMAD4 in follicular function. In this study, using specific siRNA-mediated knockdown approach, we showed that knockdown of endogenous SMAD4 completely reversed the BMP2-induced upregulation of CTGF expression and hyaluronan production, indicating that the SMAD4-derived signaling pathway is necessary for the regulatory effect of BMP2 on hyaluronan synthesis. Although we did not provide data showing that SMAD1/5/8 is involved in the BMP2-induced hyaluronan synthesis, our previous study demonstrated that BMP2 treatment induced the phosphorylation of SMAD1/5/8 in hGL cells and that BMP2 regulated its target gene expression via the SMAD1/5/8-SMAD4 signaling pathway [27, 50]. Taken together, our results suggest that the BMP2-induced upregulation of CTGF expression and hyaluronan synthesis is most likely mediated by the SMAD1/5/8-SMAD4 signaling pathway in hGL cells.
In conclusion, we have demonstrated that BMP2 upregulates the expression of CTGF and HAS2, which in turn increases the production of hyaluronan in hGL cells. Overexpression and siRNA-mediated knockdown results showed that CTGF is the regulator that mediates the BMP2-induced upregulation of HAS2 expression and hyaluronan synthesis in hGL cells. Additionally, both DMH-1 and dorsomorphin abolish the BMP2-induced upregulation of CTGF expression. Furthermore, ALK2 and ALK3, but not ALK6, are the functional receptors required for the inductive effect of BMP2 on CTGF expression. Taken together, our findings suggest that intraovarian BMP2 plays a vital role in regulating ovulation by modulating the production of hyaluronan in human granulosa cells.