Muñiz Luis C, Yehia Ghassan, Mémin Elisabeth, Ratnakar Pillarisetty V A L, Molina Carlos A
Department of Biochemistry and Molecular Biology and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, New Jersey 07103, USA.
Biol Reprod. 2006 Aug;75(2):279-88. doi: 10.1095/biolreprod.105.049486. Epub 2006 Apr 19.
Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.
细胞周期蛋白D2（Ccnd2）是卵泡发生的必需基因，因为小鼠中的无效突变会损害颗粒细胞对促卵泡激素（FSH）的增殖反应。Ccnd2信使核糖核酸（mRNA）在发情周期中由FSH诱导产生，并被促黄体生成素（LH）迅速抑制。然而，卵巢中细胞周期蛋白D2基因表达的反应元件和转录因子尚未完全明确。利用大鼠颗粒细胞原代培养物和永生化小鼠颗粒细胞，我们证明了通过蛋白激酶A（PKA）依赖性信号传导机制在转录水平上调节细胞周期蛋白D2的一种机制。细胞周期蛋白D2的启动子活性显示可被FSH和PKA的催化α亚基（PRKACA）诱导，并且这种活性可被诱导型环磷酸腺苷早期阻遏物（ICER）抑制，ICER是一种环磷酸腺苷反应元件（CRE）调节亚型。对小鼠、大鼠和人类细胞周期蛋白D2启动子的计算机分析确定了两个CRE结合蛋白位点，一个相对于翻译起始位点的保守近端元件和一个不太保守的远端元件。近端元件的突变极大地降低了PRKACA和ICER对启动子活性的影响，而远端元件的突变对启动子活性的降低没有作用。电泳迁移率变动分析和脱氧核糖核酸酶足迹分析证实ICER与近端元件结合，染色质免疫沉淀分析证明这种结合在体内发生。这些结果表明Ccnd2上游区域内的一个CRE（至少部分地）参与了细胞周期蛋白D2转录的刺激和抑制。最后，我们的数据表明ICER参与颗粒细胞增殖的调节，因为ICER的过表达导致PRKACA诱导的DNA合成受到抑制。
