Chiang C H, Cheng K W, Igarashi S, Nathwani P S, Leung P C
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada.
J Clin Endocrinol Metab. 2000 Oct;85(10):3828-39. doi: 10.1210/jcem.85.10.6886.
Estrogen is one of the major sex steroid hormones that is produced from the human ovary, and its actions are established to be a receptor-mediated process. Despite the demonstration of estrogen receptor (ER) expression, little is known regarding the regulation of ER in the human ovary. In the present study we investigated the expression and hormonal regulation of ERalpha and ERbeta in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ERalpha and ERbeta messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ERalpha is expressed at a relatively lower level than ERbeta. Basal expression studies indicated that ERalpha mRNA levels remain unchanged, whereas ERbeta mRNA levels increased with time in culture in vitro, suggesting that ERbeta is likely to play a dynamic role in mediating estrogen action in hGLCs. The regulation of ERalpha and ERbeta expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ERalpha (45%; P < 0.01) and ERbeta (40%; P < 0.01) mRNA levels. The hCG-induced decrease in ERalpha and ERbeta expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10 micromol/L) treatment. Additional studies using a specific protein kinase A (PKA) inhibitor (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt) and an adenylate cyclase inhibitor (SQ 22536) further implicated the involvement of the cAMP/PKA signaling pathway in hCG action in these cells. The hCG-induced decrease in ERalpha and ERbeta mRNA levels was prevented in the presence of these inhibitors. Next, the effect of GnRH on ER expression was studied. Sixty-eight percent (P < 0.001) and 60% (P < 0.001) decreases in ERalpha and ERbeta mRNA levels, respectively, were observed after treatment with 0.1 micromol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHa-induced down-regulation of ERalpha and ERbeta expression, suggesting the involvement of PKC in GnRH signal transduction in hGLCs. In agreement with the semiquantitative RT-PCR results, Western blot analysis detected a decrease in ERalpha and ERbeta proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 micromol/L), 8-bromo-cAMP (1 mmol/L), forskolin (10 micromol/L), or phorbol 12-myristate 13 acetate (10 micromol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17beta-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 micromol/L GnRHa for 24 h before 17beta-estradiol administration. In summary, we observed a differential expression of ERalpha and ERbeta mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ERalpha and ERbeta gene expression suggests that hCG and GnRH may contribute to the control of granulosa-luteal cell function. Furthermore, our data suggest that the effects of hCG and GnRH on ERalpha and ERbeta expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively.
雌激素是人体卵巢产生的主要性甾体激素之一，其作用是一个受体介导的过程。尽管已证实雌激素受体（ER）的表达，但关于人卵巢中ER的调节知之甚少。在本研究中，我们调查了人颗粒黄体细胞（hGLCs）中ERα和ERβ的表达及激素调节。使用逆转录聚合酶链反应（RT-PCR）扩增，在hGLCs中检测到了ERα和ERβ信使核糖核酸（mRNA）。Northern印迹分析显示，ERα的表达水平相对低于ERβ。基础表达研究表明，ERα mRNA水平保持不变，而ERβ mRNA水平在体外培养过程中随时间增加，这表明ERβ可能在介导hGLCs中的雌激素作用方面发挥动态作用。研究了人绒毛膜促性腺激素（hCG）对ERα和ERβ表达的调节。hCG处理（10 IU/mL）显著降低了ERα（45%；P < 0.01）和ERβ（40%；P < 0.01）的mRNA水平。8-溴环磷腺苷（8-bromo-cAMP，1 mmol/L）和福斯可林（forskolin，10 μmol/L）处理模拟了hCG诱导的ERα和ERβ表达降低。使用特异性蛋白激酶A（PKA）抑制剂（3',5'-环磷酸腺苷硫代磷酸酯，Rp-异构体三乙铵盐）和腺苷酸环化酶抑制剂（SQ 22536）的进一步研究进一步表明，cAMP/PKA信号通路参与了hCG在这些细胞中的作用。在这些抑制剂存在的情况下，hCG诱导的ERα和ERβ mRNA水平降低被阻止。接下来，研究了促性腺激素释放激素（GnRH）对ER表达的影响。用0.1 μmol/L GnRH激动剂（GnRHa）处理后，分别观察到ERα和ERβ mRNA水平降低了68%（P < 0.001）和60%（P < 0.001）。用蛋白激酶C（PKC）抑制剂（GF109203X）对细胞进行预处理完全逆转了GnRHa诱导的ERα和ERβ表达下调，表明PKC参与了hGLCs中GnRH信号转导。与半定量RT-PCR结果一致，蛋白质印迹分析检测到用hCG（10 IU/mL）, GnRH（0.1 μmol/L）, 8-溴环磷腺苷（1 mmol/L）, 福斯可林（10 μmol/L）或佛波酯（10 μmol/L）处理后hGLCs中ERα和ERβ蛋白水平降低。在功能上，我们证明了17β-雌二醇在体外抑制hGLCs中孕酮的产生，并且在给予17β-雌二醇之前用10 IU/mL hCG或0.1 μmol/L GnRHa预处理24小时可消除这种抑制作用。总之，我们观察到体外hGLCs中ERα和ERβ mRNA的差异表达。hCG和GnRHa诱导的ERα和ERβ基因表达下调表明，hCG和GnRH可能参与了颗粒黄体细胞功能的控制。此外，我们的数据表明，hCG和GnRH对hGLCs中ERα和ERβ表达的影响分别部分由PKA和PKC信号通路的激活介导。
