OBJECTIVE: To determine the functional role of Smad7 in granulosa cells. DESIGN: Granulosa cell culture and molecular biological techniques were used to investigate regulation and function of Smad7. SETTING: Research laboratory. ANIMAL(S): C57bl/j hybrid mouse. INTERVENTION(S): Primary mouse granulosa cells were isolated and grown in culture for all messenger RNA expression experiments. Smad7 promoter constructs were evaluated with a luciferase reporter system in SIGC cells to determine sites activating Smad7 expression. MAIN OUTCOME MEASURE(S): Overexpression (Smad7 complementary DNA) and downregulation (Smad7 small interfering RNA) of Smad7 in primary mouse granulosa cells were used to evaluate the functional role of Smad7 in granulosa cells. RESULT(S): Smad7 expression was upregulated by treatment with transforming growth factor-β (TGF-β) but not activin or activation of the cyclic adenosine monophosphate pathway. The promoter of Smad7 was activated by TGF-β. Truncation of the promoter or mutation of the Smad response element at -141 eliminated TGF-β activation of the promoter. Smad3 was not specifically required for TGF-β-stimulated expression of Smad7, though activation of the TGFBR1 receptor was. When Smad7 was overexpressed in granulosa cells, apoptosis was markedly increased. When Smad7 expression was reduced with small interfering RNA, then the TGF-β-induced apoptosis was blocked. CONCLUSION(S): Smad7 mediates apoptosis induced by TGF-β in mouse granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis.
OBJECTIVE: To determine the functional role of Smad7 in granulosa cells. DESIGN: Granulosa cell culture and molecular biological techniques were used to investigate regulation and function of Smad7. SETTING: Research laboratory. ANIMAL(S): C57bl/j hybrid mouse. INTERVENTION(S): Primary mouse granulosa cells were isolated and grown in culture for all messenger RNA expression experiments. Smad7 promoter constructs were evaluated with a luciferase reporter system in SIGC cells to determine sites activating Smad7 expression. MAIN OUTCOME MEASURE(S): Overexpression (Smad7 complementary DNA) and downregulation (Smad7 small interfering RNA) of Smad7 in primary mouse granulosa cells were used to evaluate the functional role of Smad7 in granulosa cells. RESULT(S): Smad7 expression was upregulated by treatment with transforming growth factor-β (TGF-β) but not activin or activation of the cyclic adenosine monophosphate pathway. The promoter of Smad7 was activated by TGF-β. Truncation of the promoter or mutation of the Smad response element at -141 eliminated TGF-β activation of the promoter. Smad3 was not specifically required for TGF-β-stimulated expression of Smad7, though activation of the TGFBR1 receptor was. When Smad7 was overexpressed in granulosa cells, apoptosis was markedly increased. When Smad7 expression was reduced with small interfering RNA, then the TGF-β-induced apoptosis was blocked. CONCLUSION(S): Smad7 mediates apoptosis induced by TGF-β in mouse granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis.
Authors: Yang Gao; Samantha Duran; John P Lydon; Francesco J DeMayo; Robert C Burghardt; Kayla J Bayless; Laurent Bartholin; Qinglei Li Journal: Biol Reprod Date: 2014-12-10 Impact factor: 4.285