Sarapura V D, Wood W M, Gordon D F, Ocran K W, Kao M Y, Ridgway E C
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Endocrinology. 1990 Sep;127(3):1352-61. doi: 10.1210/endo-127-3-1352.
The alpha-subunit gene of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Thus, this gene must contain elements that mediate expression and hormonal responses in different cell types. The localization of DNA regions important for expression and regulation of the alpha-subunit gene in thyrotrope cells has not previously been reported. In these studies luciferase expression constructs containing 1700 basepairs of 5' flanking DNA derived from the mouse alpha-subunit gene were introduced by electroporation into freshly dispersed cells from TSH-producing mouse pituitary tumors (TtT 97). This promoter functioned with greater efficiency in thyrotropes than in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Primer extension confirmed that transcription from the alpha-subunit constructs initiated at the same site as the endogenous gene. Studies using 5' truncations showed a progressive loss of alpha-subunit promoter activity in thyrotropes between -480 and -120, with regions upstream of -254 contributing substantially to expression in thyrotrope cells. Thyroid hormone inhibited alpha-subunit promoter activity in a dose-dependent fashion, although in vivo treatment of tumors with thyroid hormone before transfection was necessary to achieve maximal inhibition. Thyroid hormone inhibition of alpha-subunit promoter activity also occurred in GH4 cells, but no effect was observed in L-cells. Studies using 5' truncations localized a region responsible for thyroid hormone inhibition between -62 and +43, encompassing the TATA sequence and the transcriptional initiation site. When this region was compared to the thyroid hormone inhibitory regions of the alpha-subunit genes from other species and the mouse TSH beta-subunit gene, a 6-basepair motif, 5' (G/A)GTG(G/A)G 3', emerged as a possible consensus sequence for a thyroid hormone inhibitory element.
糖蛋白激素的α亚基基因通常在垂体促甲状腺细胞、促性腺细胞以及胎盘细胞中表达。因此，该基因必定包含介导不同细胞类型中基因表达和激素应答的元件。此前尚未报道对促甲状腺细胞中α亚基基因表达和调控起重要作用的DNA区域的定位。在这些研究中，通过电穿孔将含有源自小鼠α亚基基因的1700个碱基对5'侧翼DNA的荧光素酶表达构建体导入来自产生促甲状腺激素的小鼠垂体肿瘤（TtT 97）的新鲜分散细胞中。该启动子在促甲状腺细胞中的功能效率高于非促甲状腺垂体GH4细胞和L细胞成纤维细胞。引物延伸证实，α亚基构建体的转录起始于与内源基因相同的位点。使用5'端缺失的研究表明，在-480至-120之间，促甲状腺细胞中α亚基启动子活性逐渐丧失，-254上游区域对促甲状腺细胞中的表达有很大贡献。甲状腺激素以剂量依赖方式抑制α亚基启动子活性，尽管在转染前用甲状腺激素对肿瘤进行体内处理对于实现最大抑制是必要的。甲状腺激素对α亚基启动子活性的抑制在GH4细胞中也会发生，但在L细胞中未观察到影响。使用5'端缺失的研究将负责甲状腺激素抑制的区域定位在-62至+43之间，包括TATA序列和转录起始位点。当将该区域与来自其他物种的α亚基基因和小鼠促甲状腺激素β亚基基因的甲状腺激素抑制区域进行比较时，一个6碱基对基序5'（G/A）GTG（G/A）G 3'作为甲状腺激素抑制元件的可能共有序列出现。
