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.