Hsun-Ming Chang1, Jung-Chien Cheng, Peter C K Leung. 1. Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Room 317, 950 West 28th Avenue, Vancouver, British Columbia, Canada.
Abstract
CONTEXT: 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. OBJECTIVE: 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. DESIGN: 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. SETTING: The study was conducted in an academic center. MAIN OUTCOME MEASURES: 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. RESULTS: 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. CONCLUSION: 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.
CONTEXT: 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. OBJECTIVE: 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. DESIGN: 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. SETTING: The study was conducted in an academic center. MAIN OUTCOME MEASURES: 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. RESULTS: 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. CONCLUSION: 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.
Authors: Yilin Mao; Tara Nguyen; Ryan S Tonkin; Justin G Lees; Caitlyn Warren; Simon J O'Carroll; Louise F B Nicholson; Colin R Green; Gila Moalem-Taylor; Catherine A Gorrie Journal: Exp Brain Res Date: 2017-07-19 Impact factor: 1.972