Yu-Ching Chen1, Hsun-Ming Chang2, Jung-Chien Cheng2, Horng-Der Tsai3, Cheng-Hsuan Wu3, Peter C K Leung4. 1. Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada Department of Obstetrics and Gynaecology, Changhua Christian Hospital, Changhua, Taiwan. 2. Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada. 3. Department of Obstetrics and Gynaecology, Changhua Christian Hospital, Changhua, Taiwan. 4. Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada peter.leung@ubc.ca.
Abstract
STUDY QUESTION: Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell-cell communication in human granulosa cells? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: This is an experimental study which was performed over a 1-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS FOR CAUTION: The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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. TRIAL REGISTRATION NUMBER: NA.
STUDY QUESTION: Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell-cell communication in human granulosa cells? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: This is an experimental study which was performed over a 1-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS FOR CAUTION: The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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. TRIAL REGISTRATION NUMBER: NA.
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