Anti-Mullerian Hormone Regulates Stem Cell Factor via cAMP/PKA Signaling Pathway in Human Granulosa Cells by Inhibiting the Phosphorylation of CREB
Anti-Mullerian Hormone Regulates Stem Cell Factor via cAMP/PKA Signaling Pathway in Human Granulosa Cells by Inhibiting the Phosphorylation of CREB
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DOI：
10.1007/s43032-019-00033-4
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发表时间：
2020-01-01
影响因子：
2.9
通讯作者：
Hu, Rong
中科院分区：
文献类型：
--
作者：
Fu, Yun-Xing;Wang, Fei-Miao;Hu, Rong
抗苗勒氏管激素 (AMH) 通过人颗粒细胞 (GC) 中的 cAMP/PKA 信号通路下调干细胞因子 (SCF) 的水平。关于相互作用的分子机制的信息很少。本研究旨在确定 AMH 是否通过 cAMP-PKA-CREB 信号通路在人 GC 中调节 SCF 的表达。在本研究中，我们验证了 cAMP 反应元件结合蛋白 (CREB) 与人 GC 中 SCF 启动子的结合。此外，测试了CREB对SCF启动子的影响，并使用截短以及SCF启动子突变和CREB突变的测定来鉴定CREB与SCF启动子结合的位点。为了研究AMH、SCF启动子和CREB之间的相关性，将pGL-Basic-SCF+CREB分别转染到过表达的AMH GCs（AMH-high GCs）、低表达的AMH GCs（AMH-low GCs）和正常GCs（GCs）中。最后，在高 AMH 和低 AMH 的 GC 中进行免疫荧光、双重免疫染色和蛋白质印迹，以证实 AMH 通过抑制 GC 中 CREB (pCREB) 的磷酸化来介导 SCF 表达的调节。结果表明CREB与SCF启动子相互作用并显着增强SCF的转录水平。 CREB结合位点位于SCF基因启动子的318-321 bp处。 AMH 通过 GC 中的 PKA 信号通路磷酸化 CREB 来抑制 SCF 的表达。这些发现提供了对 AMH 抑制卵泡生长的分子机制的深入了解，这将有助于开发一种新疗法。
Anti-Mullerian hormone (AMH) downregulates the level of stem cell factor (SCF) via the cAMP/PKA signaling pathway in human granulosa cells (GCs). Little information is available on the molecular mechanism underlying the interaction. This study is aimed at determining whether AMH regulates expression of SCF via the cAMP-PKA-CREB signaling pathway in human GCs. In the present study, we verified the binding of cAMP-response element-binding protein (CREB) to promoter of SCF in human GCs. Furthermore, the effect of CREB was tested on the SCF promoter, and the site of CREB binding to SCF promoter was identified using truncations as well as assays of SCF-promoted mutation and CREB mutation. To investigate the correlation among AMH, SCF promoter, and CREB, pGL-Basic-SCF+CREB was transfected into overexpressed AMH GCs (AMH-high GCs), low expressed AMH GCs (AMH-low GCs), and normal GCs (GCs), respectively. Finally, immunofluorescence, double immunostaining, and Western blot were carried out in AMH-high and AMH-low GCs to confirm the AMH-mediated regulation of SCF expression by inhibiting the phosphorylation of CREB (pCREB) in GCs. Results indicated CREB interacted with SCF promoter and significantly enhanced the transcription level of SCF. The CREB binding site was localized at 318-321 bp of SCF gene promote. AMH inhibits the expression of SCF by phosphorylation of CREB via the PKA signaling pathway in GCs. These findings provide an in-depth understanding of the molecular mechanism underlying AMH suppressing the follicle growth, which would aid in the development of a novel therapy.