Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type.
Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type.
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DOI：
10.1210/mend.6.12.1337142
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发表时间：
1992-12
影响因子：
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通讯作者：
C. Keech;S. M. Jackson;S. Siddiqui;K. Ocran;A. Gutierrez-Hartmann
中科院分区：
文献类型：
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作者：
C. Keech;S. M. Jackson;S. Siddiqui;K. Ocran;A. Gutierrez-Hartmann
cAMP-蛋白激酶-A（PKA）途径高度调节泌乳素细胞功能和PRL基因表达。为了进一步了解cAMP/PKA调节大鼠（r）PRL启动子活性的分子机制，并确定cAMP调节是否是细胞类型特异性的，我们1）将与萤火虫荧光素酶报告基因连接的rPRL启动子的完整（-425）、内部和5 ′-缺失以及位点特异性突变体横切到垂体和非垂体细胞系中;（2）探讨cAMP-cAMP反应元件结合蛋白（CREB）通路在GH_4大鼠垂体细胞中的作用。数据显示，从-425到-116删除rPRL启动子并没有消除cAMP调节，这意味着近端元件，如基础转录元件（-112/-80）或垂体特异性足迹（FP）I（-67/-45），介导cAMP反应。然而，FP I或FP II内的核苷酸变化（-130/-120）并没有改变rPRL启动子对1 μ M毛喉素（FSK）的反应，尽管这些突变分别导致基础rPRL启动子活性降低77%和26%。此外，FP I元件的基础转录元件的内部缺失也未能影响rPRL启动子的cAMP调节，尽管这些缺失分别使基础启动子活性降低90%和93%。由于这些内部缺失构建体另外含有从-425至+73的rPRL启动子序列，包括上游垂体特异性FP III和IV，因此数据表明这些细胞特异性元件中的任何一个都能够将cAMP调节赋予近端rPRL启动子。为了直接测试rPRL启动子的cAMP反应仅限于垂体特异性细胞类型的含义，我们利用了在位置-116处截短的5 '-缺失突变体和FP II位点特异性突变体，因为含有这些rPRL启动子的构建体在非垂体细胞中具有活性。尽管6.6和18.5倍的刺激野生型rPRL启动子活性在非垂体细胞，分别，这些突变仍然完全不响应FSK治疗。为了证明cAMP-CREB通路在GC/GH4大鼠垂体细胞中是功能性的，从GC大鼠垂体细胞中亲和纯化CREB，并且DNase-I保护研究表明它不结合近端rPRL启动子。此外，人糖蛋白α亚基启动子诱导10倍FSK在GH4大鼠垂体细胞。（400字处截断摘要）
Pituitary lactotroph cell function and PRL gene expression are highly regulated by the cAMP-protein kinase-A (PKA) pathway. To further our understanding of the molecular mechanisms by which cAMP/PKA regulates rat (r) PRL promoter activity and to determine whether cAMP regulation is cell type specific, we 1) transected intact (-425), internal and 5'-deletion, and site-specific mutants of the rPRL promoter ligated to the firefly luciferase reporter gene into both pituitary and nonpituitary cell lines; and 2) assessed the role of the cAMP-cAMP response element-binding protein (CREB) pathway in GH4 rat pituitary cells. The data show that deleting the rPRL promoter from -425 to -116 did not abolish cAMP regulation, implying that proximal elements, such as the basal transcription element (-112/-80) or the pituitary-specific footprint (FP) I (-67/-45), mediate the cAMP response. However, nucleotide changes within FP I or FP II (-130/-120) did not alter the rPRL promoter response to 1 microM forskolin (FSK), despite the 77% and 26% reductions in basal rPRL promoter activity caused by these mutations, respectively. Furthermore, internal deletion of either the basal transcription element of FP I element also failed to affect cAMP regulation of the rPRL promoter, again despite the 90% and 93% reductions in basal promoter activity by these deletions, respectively. Since these internal deletion constructs otherwise contain rPRL promoter sequences from -425 to +73, including the up-stream pituitary-specific FPs III and IV, the data suggest that any one of these cell-specific elements is capable of imparting cAMP regulation to the proximal rPRL promoter. To directly test the implication that the cAMP response of the rPRL promoter is restricted to the pituitary-specific cell type, we took advantage of a 5'-deletion mutant truncated at position -116 and a FP II site-specific mutant, since constructs containing these rPRL promoters are active in nonpituitary cells. Despite the 6.6- and 18.5-fold stimulations over wild-type rPRL promoter activity in nonpituitary cells, respectively, these mutations remained completely unresponsive to FSK treatment. To document that the cAMP-CREB pathway was functional in GC/GH4 rat pituitary cells, CREB was affinity purified from GC rat pituitary cells, and DNase-I protection studies showed that it does not bind to the proximal rPRL promoter. Also, the human glycoprotein alpha-subunit promoter was induced 10-fold by FSK in GH4 rat pituitary cells.(ABSTRACT TRUNCATED AT 400 WORDS)