Theca-Derived BMP4 and BMP7 Down-Regulate Connexin43 Expression and Decrease Gap Junction Intercellular Communication Activity in Immortalized Human Granulosa Cells

Abstract

Connexin43 (Cx43)-coupled gap junctions in granulosa cells play important roles in follicular and oocyte development and may be modulated by theca cell-derived bone morphogenic protein (BMP) 4 and BMP7. The aim of this study was to examine the effects of BMP4 and BMP7 on Cx43 expression in human granulosa cells and its potential mediation by the Smad-dependent pathway. An immortalized human granulosa (SVOG) cell was used to investigate Cx43 expression and gap junction intercellular communication (GJIC) activity after exposure to BMP4 and BMP7. A BMP type I inhibitor, dorsomorphin, and small interfering RNAs targeting Smad4 were used to verify the specificity of the effects. The study was conducted in an academic center. Extracts were prepared from cultured cells, the Cx43 mRNA levels were examined using RT-quantitative real-time PCR, and the levels of Cx43 protein and phosphorylated Smad1/5/8 were assayed using Western blot analyses. GJIC activities between SVOG cells were evaluated using a scrape loading and dye transfer assay. Treatment with BMP4 and BMP7 significantly decreased Cx43 mRNA and protein levels, as well as GJIC activities. These suppressive effects were attenuated by cotreatment with the BMP type I receptor inhibitor dorsomorphin. Furthermore, Smad4 knockdown reversed the effects of BMP4 and BMP7 on Cx43 expression. Theca cell-derived BMP4 and BMP7 down-regulate Cx43 expression and decrease GJIC activity in human granulosa cells. Our findings indicate that this biological effect is most likely mediated by a Smad-dependent pathway.