Laboratory of Reproductive Endocrinology, Dept. of Anatomy and Cell Biology, College of Medicine, Hanyang University, Seoul 133-791, Korea.
Int J Mol Sci. 2020 Oct 6;21(19):7385. doi: 10.3390/ijms21197385.
() plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates gene expression. mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated expression, indicating that is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on expression. Truncation of a expression construct to -715 bp (p-715/luc) had no effect on transcriptional activity, whereas deletion to -402 bp (p-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the -715/-500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at -698/-688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at -698/-688 bp is indispensable for activation and suggest that could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.
（）在广泛的细胞中从增殖到分化的转变中发挥重要作用。先前的研究表明，它在促黄体激素（LH）诱导的排卵前颗粒细胞（GC）黄体化过渡中起着关键作用。本研究使用培养的大鼠排卵前 GC 来研究 LH 调节基因表达的机制。处理后，基因的 mRNA 和蛋白迅速且短暂地被诱导，在 45 分钟后达到峰值，在 3 小时后降至基础水平。用蛋白质合成抑制剂环己酰亚胺预处理对 LH 刺激的表达没有影响，表明它是 LH 反应的即时早期基因。为了研究涉及 LH 诱导调节的信号通路，评估了蛋白激酶 A（PKA）和蛋白激酶 C（PKC）通路。PKA 激动剂，但不是 C 激酶激动剂，模拟 LH 诱导转录。此外，PKA 的特异性抑制剂消除了 LH 对表达的刺激作用。表达构建体的截断至-715 bp（p-715/luc）对转录活性没有影响，而删除至-402 bp（p-402/luc）则大大降低了它。ChIP 分析显示，内源性 Sp1 在体内与-715/-500 bp 区域结合，并且对 LH 的最大转录反应需要在-698/-688 bp 处的 Sp1 结合元件，该元件在小鼠、大鼠和人中高度保守。这些发现表明，通过 cAMP/PKA 通路激活，并且-698/-688 bp 处的假定 Sp1 结合元件对于激活是必不可少的，并且表明可能成为治疗由对 LH 激增的异常反应引起的黄体期不足的策略的靶标。
