Fowkes R C, Sidhu K K, Sosabowski J K, King P, Burrin J M
Department of Endocrinology, Barts and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK.
J Mol Endocrinol. 2003 Oct;31(2):263-78. doi: 10.1677/jme.0.0310263.
Hormone regulation of anterior pituitary expression of the common glycoprotein hormone alpha-subunit (alphaGSU) is mediated by multiple response elements residing in the first -435 bp of the human promoter. In rat pituitary cells and mouse alphaT3-1 precursor gonadotrophs, the human alphaGSU promoter is strongly responsive to activators of the adenylyl cyclase/cAMP pathway, such as the hypothalamic releasing hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) and forskolin (an adenylyl cyclase activator). However, the role of PACAP and cAMP in regulating alphaGSU transcription in the more differentiated LbetaT2 gonadotroph is unclear. Here, we investigate the regulation of the human alphaGSU promoter by PACAP and forskolin in LbetaT2 and alphaT3-1 gonadotrophs. PACAP failed to stimulate alphaGSU promoter activity or cAMP production in LbetaT2 cells, in marked contrast to alphaT3-1 cells. LbetaT2 gonadotrophs expressed extremely low levels of any PACAP type 1 receptors (PAC(1)-R) isoform by RT-PCR and lacked PAC(1)-R by radioligand binding. Forskolin stimulated the alphaGSU promoter in LbetaT2 cells, but by less than 30% of the response seen in alphaT3-1 gonadotrophs. This blunted cAMP transcriptional effect was not due to different levels of cAMP generation, or altered expression of the cAMP target proteins CREB, Akt, CBP or ICER. However, only LbetaT2 cells showed detectable expression of the protein kinase A type IIalpha regulatory subunit. Binding of activating transcription factor-2 and phosphorylated CREB to the consensus CRE was observed in both LbetaT2 and alphaT3-1 gonadotrophs, yet forskolin failed to stimulate either CRE- or CREB-mediated transcription in LbetaT2 cells. Collectively, these data demonstrate the lack of functional PACAP receptors in LbetaT2 gonadotrophs, and a pronounced attenuation in the responsiveness of this differentiated gonadotroph cell line to cAMP stimulus.
垂体前叶中常见糖蛋白激素α亚基（αGSU）的激素调节是由人类启动子第一个-435 bp内的多个反应元件介导的。在大鼠垂体细胞和小鼠αT3-1前体促性腺激素细胞中，人类αGSU启动子对腺苷酸环化酶/cAMP途径的激活剂有强烈反应，如下丘脑释放激素、垂体腺苷酸环化酶激活多肽（PACAP）和福斯可林（一种腺苷酸环化酶激活剂）。然而，PACAP和cAMP在调节更分化的LβT2促性腺激素细胞中αGSU转录方面的作用尚不清楚。在这里，我们研究了PACAP和福斯可林对LβT2和αT3-1促性腺激素细胞中人类αGSU启动子的调节。与αT3-1细胞形成鲜明对比的是，PACAP未能刺激LβT2细胞中的αGSU启动子活性或cAMP产生。通过RT-PCR检测，LβT2促性腺激素细胞表达极低水平的任何PACAP 1型受体（PAC(1)-R）亚型，并且通过放射性配体结合缺乏PAC(1)-R。福斯可林刺激LβT2细胞中的αGSU启动子，但刺激程度不到αT3-1促性腺激素细胞中所见反应的30%。这种减弱的cAMP转录效应不是由于cAMP产生水平不同，也不是由于cAMP靶蛋白CREB、Akt、CBP或ICER的表达改变。然而，只有LβT2细胞显示出可检测到的蛋白激酶A IIα调节亚基的表达。在LβT2和αT3-1促性腺激素细胞中均观察到激活转录因子-2和磷酸化CREB与共有CRE的结合，但福斯可林未能刺激LβT2细胞中CRE或CREB介导的转录。总的来说，这些数据表明LβT2促性腺激素细胞中缺乏功能性PACAP受体，并且这种分化的促性腺激素细胞系对cAMP刺激的反应性明显减弱。
