Gaiddon C, Tian J, Loeffler J P, Bancroft C
URA 1446 du CNRS, Laboratoire de Physiologie Generale, Universite Louis Pasteur, Strasbourg, France.
Endocrinology. 1996 Apr;137(4):1286-91. doi: 10.1210/endo.137.4.8625901.
Constitutively active mutations of the G protein alpha(S) subunit are detected at a high frequency in human pituitary adenomas that secrete GH or PRL. It seems possible that over-expression of the pituitary cell-specific transcription factor Pit-1/GHF-1 (Pit-1) gene in response to active alpha(S) subunits contributes to the formation of these adenomas. We have examined whether expression in pituitary cells of one of these constitutively active alpha(S) subunits, Q227L-alpha(S), stimulates expression directed by the Pit-1 promoter. Transient expression of Q227L-alpha(S) yielded a strong stimulation of a target Pit-1 promoter-chloramphenicol acetyl transferase (CAT) construct, (-200)Pit-1-CAT. Expression of wild-type alpha(S) or an inactive alpha(S) mutant yielded, respectively, reduced or no stimulation of CAT activity. A dominant inhibitor of protein kinase A (PKA), RAB, blocked almost completely either forskolin (FSK) or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that PKA is required for the action of Q227L-alpha(S) on the Pit-1 promoter. The Pit-1 promoter contains a binding site for Pit-1 and two CREB binding sites. Mutation of the Pit-1 binding site reduced but did not eliminate either FSK or Q227L-alpha(S) stimulation of Pit-1 promoter activity, implying a partial but incomplete requirement for this element for a PKA-mediated response to Q227L-alpha(S). The CREB dominant inhibitor S133A-CREB yielded a partial reduction in either FSK or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that one or both of the Pit-1 promoter adenosine 3'5'-monophosphate response element binding protein (CREB) binding sites is/are also required for a complete PKA-mediated response to Q227L-alpha(S). The observation that S133A-CREB completely blocked the response to FSK or Q227L-alpha(S) of a Pit-1 promoter containing a mutated site PitB1 implies that the binding sites for Pit-1 and CREB account for all of the response elements for FSK or alpha(S) in the Pit-1 promoter.
在分泌生长激素（GH）或催乳素（PRL）的人垂体腺瘤中，高频检测到G蛋白α（S）亚基的组成型活性突变。垂体细胞特异性转录因子Pit-1/GHF-1（Pit-1）基因可能因活性α（S）亚基而过度表达，这有助于这些腺瘤的形成。我们研究了这些组成型活性α（S）亚基之一Q227L-α（S）在垂体细胞中的表达是否会刺激Pit-1启动子指导的表达。Q227L-α（S）的瞬时表达对靶标Pit-1启动子-氯霉素乙酰转移酶（CAT）构建体（-200）Pit-1-CAT产生了强烈刺激。野生型α（S）或无活性α（S）突变体的表达分别导致CAT活性的降低或无刺激。蛋白激酶A（PKA）的显性抑制剂RAB几乎完全阻断了福斯可林（FSK）或Q227L-α（S）对（-200）Pit-1-CAT表达的刺激，这意味着PKA是Q227L-α（S）作用于Pit-1启动子所必需的。Pit-1启动子包含一个Pit-1结合位点和两个CREB结合位点。Pit-1结合位点的突变降低但未消除FSK或Q227L-α（S）对Pit-1启动子活性的刺激，这意味着该元件对PKA介导的对Q227L-α（S）的反应有部分但不完全的需求。CREB显性抑制剂S133A-CREB使FSK或Q227L-α（S）对（-200）Pit-1-CAT表达的刺激部分降低，这意味着Pit-1启动子腺苷3'5'-单磷酸反应元件结合蛋白（CREB）结合位点中的一个或两个对于PKA介导的对Q227L-α（S）的完整反应也是必需的。观察到S133A-CREB完全阻断了含有突变位点PitB1的Pit-1启动子对FSK或Q227L-α（S）的反应，这意味着Pit-1和CREB的结合位点构成了Pit-1启动子中FSK或α（S）的所有反应元件。
