Bone morphogenetic protein 2 regulates cell-cell communication by down-regulating connexin43 expression in luteinized human granulosa cells.

Does bone morphogenetic protein 2 (BMP2) regulate connexin43 (Cx43) and modulate cell-cell communication in luteinized human granulosa cells? BMP2 decreases gap junction intercellular communication (GJIC) of luteinized human granulosa cells by down-regulating Cx43 expression through an activin receptor-like kinase (ALK)2/ALK3-mediated Sma- and Mad-related protein (SMAD)-dependent signaling pathway. BMP2 and its putative receptors are highly expressed in the human corpus luteum and are involved in the process of luteolysis. Cx43-coupled gap junctions play a critical role in the development and maintenance of corpus luteum. This is a laboratory study conducted over a 1-year period. At least three independent experiments with three replicates were conducted and the experimental samples were compared with the appropriate vehicle controls for all of the inhibition-approach, concentration-dependent or time-course studies. SVOG cell line (immortalized human granulosa-lutein cells derived from in vitro fertilization patients in an academic research center) was used as the study model. The changes of Cx43 expression and levels of phosphorylated SMAD1/5/8 protein were evaluated after exposure to recombinant human BMP2. Real-time quantitative PCR and Western blot analysis were used to examine the specific mRNA and protein levels, respectively. The BMP/TGF-β type I receptor inhibitors (Dorsomorphin, DMH-1 and SB431542) and target depletion small interfering RNAs (ALK2, ALK3, ALK6 and SMAD4) were used to investigate the underlying molecular mechanisms. A scrape loading and dye transfer assay was used to evaluate the GJIC between the SVOG cells. Treatment with BMP2 down-regulated the expression of Cx43 and decreased the GJIC activity, whereas it increased the phosphorylated SMAD1/5/8 protein in SVOG cells (P < 0.05). These biological effects were abolished by pre-treatment with the BMP type I receptor inhibitors, Dorsomorphin and DMH-1 (P < 0.05), but not SB431542. Additionally, the individual or concomitant small interfering RNA-mediated knockdown of ALK2 and ALK3, but not ALK6 attenuated the BMP2-induced increases in phosphorylated SMAD1/5/8 and down-regulation of Cx43 expression (P < 0.05). The knockdown of SMAD4 completely abolished the BMP2-induced down-regulation of Cx43 expression (P < 0.05). This experimental study was conducted in an in vitro cell culture system, and may not reflect a realistic intra-ovarian environment. Our results suggested that BMP2 may be involved in the local modulation of cell-cell communication in the luteal phase. This study also represents the first comprehensive research of molecular mechanisms of BMP2 in the down-regulation Cx43 in luteinized human granulosa cells. Such data may provide valuable insights into ovarian physiology and benefit the development of potential therapeutic methods for patients suffering from luteal insufficiency. N/A. This research was supported by an operating grant from the China-Canadian Joint Health Research Initiative Grants Program to P.C.K. Leung and J.Z. Sheng. The authors declare no competing interest with the contents of this article.

Wu YT ，Chang HM ，Huang HF ，Sheng JZ ，Leung PC ... -  《-》

Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells.

Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell-cell communication in human granulosa cells? TGF-β1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway. TGF-β1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-β1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes. This is an experimental study which was performed over a 1-year period. Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-β1. An activin/TGF-β type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. TGF-β1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-β type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-β1 on Cx43 expression (mRNA and protein) was confirmed in primary cultures of human granulosa-lutein cells (P < 0.05). The small interfering RNA-mediated knockdown of ALK5, but not ALK4, abolished the TGF-β1-induced phosphorylation of SMAD2/3 and the up-regulation of Cx43. Furthermore, knockdown of SMAD2/3 or the common SMAD, SMAD4, abolished the stimulatory effects of TGF-β1 on Cx43 expression in SVOG cells. The TGF-β1-induced up-regulation of Cx43 contributed to the increase of GJIC between SVOG cells (P < 0.001). The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo. Our studies represent the first comprehensive research of molecular mechanisms of TGF-β1 in the regulation of Cx43 expression and GJIC in human granulosa cells and demonstrate that TGF-β1 may play a crucial role in the local modulation of cell-cell communication. Deepening our understanding of the molecular determinants will offer important insights into ovarian physiology and lead to the development of potential therapeutic methods for fertility regulation. This research was supported by an operating grant from the Canadian Institutes of Health Research to P.C.K.L. There are no conflicts of interest to declare. NA.

Chen YC ，Chang HM ，Cheng JC ，Tsai HD ，Wu CH ，Leung PC ... -  《-》

Theca-derived BMP4 and BMP7 down-regulate connexin43 expression and decrease gap junction intercellular communication activity in immortalized human granulosa cells.

Chang HM ，Cheng JC ，Leung PC 《-》

ALK2/ALK3-BMPR2/ACVR2A Mediate BMP2-Induced Downregulation of Pentraxin 3 Expression in Human Granulosa-Lutein Cells.

Bone morphogenetic protein 2 (BMP2) belongs to the transforming growth factor-β superfamily and plays a critical role in regulating ovarian follicle function. Currently, the role of BMP2 during cumulus expansion remains to be determined. The aim of this study was to investigate the effect of BMP2 on the regulation of pentraxin 3 (PTX3) expression (the major component of cumulus expansion) and the underlying mechanisms in human granulosa-lutein (hGL) cells. Both primary and immortalized hGL cells were used as research models. Our results showed that treatment with BMP2 significantly suppressed the basal and luteinizing hormone-induced upregulation of PTX3. In addition, BMP2 stimulated the phosphorylation of SMAD1/5/8, and this effect was abolished by the addition of BMP type I receptor inhibitors, dorsomorphin homolog 1, and dorsomorphin but not SB431542. Moreover, the knockdown of activin receptorlike kinase 2/3 or BMP receptor type II/activin receptor type IIB receptors completely reversed the BMP2-induced phosphorylation of SMAD1/5/8 and restored PTX3 expression. Similarly, the knockdown of SMAD4 completely reversed the suppressive effect of BMP2 on the expression of PTX3. These results improve our understanding of the molecular mechanisms of BMP2 signaling. Our findings suggest that BMP2 may be involved in the regulation of cumulus expansion during the periovulatory stage.

Bai L ，Chang HM ，Cheng JC ，Chu G ，Leung PCK ，Yang G ... -  《-》

Bone morphogenetic protein 6 affects cell-cell communication by altering the expression of Connexin43 in human granulosa-lutein cells.

Connexin 43 (Cx43)-coupled gap junctions in granulosa cells play an important role in follicular development, oocyte maturation, and corpus luteum maintenance. Bone morphogenetic protein 6 (BMP6) is highly expressed in human oocytes and granulosa cells and is involved in the regulation of female reproduction. Currently, whether oocyte- and granulosa cell-derived BMP6 affects the expression of Cx43 and its related gap junction intercellular communication (GJIC) activity in human granulosa cells remains unknown. In this study, we demonstrate that BMP6 treatment significantly suppressed the expression of Cx43 in both primary and immortalized (SVOG) human granulosa-lutein cells. Using both pharmacological inhibitors and small interfering RNA-mediated knockdown approaches, we demonstrate that ALK2 and ALK3 BMP type I receptors are involved in BMP6-induced suppressive effects on Cx43 expression and GJIC activity in SVOG cells. Furthermore, these cellular activities are most likely mediated by the SMAD1/SMAD5-SMAD4-dependent signaling pathway. Notably, the ChIP analyses demonstrated that phosphorylated SMADs could bind to human Cx43 promoter. Our findings provide new insight into the molecular mechanisms by which an intrafollicular growth factor regulates cell-cell communication in human granulosa cells.

Wu HC ，Chang HM ，Yi Y ，Sun ZG ，Lin YM ，Lian F ，Leung PCK ... -  《-》