Hamernik D L, Keri R A, Clay C M, Clay J N, Sherman G B, Sawyer H R, Nett T M, Nilson J H
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Mol Endocrinol. 1992 Oct;6(10):1745-55. doi: 10.1210/mend.6.10.1280329.
The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.
人和牛α亚基基因的近端5'-侧翼区域可使异源报告基因在转基因小鼠中实现垂体特异性表达。为了研究这些启动子区域是否也包含细胞特异性表达和激素调节所需的调控元件，我们使用了三系独立的转基因小鼠。两系转基因小鼠包含嵌合基因，该嵌合基因由1.6千碱基对（kbp）的人类α亚基近端5'-侧翼序列或315碱基对的牛α亚基近端5'-侧翼序列与编码氯霉素乙酰转移酶（CAT）的细菌基因相连组成。第三系转基因小鼠包含人类α亚基基因近端1.6 kbp的5'-侧翼序列与编码β-半乳糖苷酶（β-gal；Hαβ-gal转基因小鼠）的细菌lacZ基因相连。激素替代模式表明，人和牛的α-CAT转基因均受促性腺激素释放激素（GnRH）调节，提示它们在促性腺激素细胞中表达。因此，人和牛α亚基基因的近端5'-侧翼区域必定包含赋予促性腺激素细胞特异性表达和对GnRH反应性的调控元件。与人类α亚基启动子不同，牛α亚基启动子缺乏功能性的环磷酸腺苷（cAMP）反应元件，提示细胞特异性和GnRH转录信号的转导通过不依赖cAMP反应元件的途径发生。促甲状腺激素细胞也表达糖蛋白激素α亚基基因。然而，用丙硫氧嘧啶和三碘甲状腺原氨酸（T3）进行的激素替代模式未能改变人或牛α-CAT转基因小鼠垂体中的CAT活性。由于甲状腺激素反应元件已定位在人类α亚基基因的近端5'-侧翼区域，这些数据提示α-CAT转基因缺乏将表达导向促甲状腺激素细胞的足够信息。通过对Hαβ-gal转基因小鼠垂体进行免疫细胞化学研究获得了这种可能性的直接证据。β-半乳糖苷酶活性出现在促性腺激素细胞中，而不出现在促甲状腺激素细胞中。因此，我们得出结论，不同且可分离的调控元件介导α亚基基因在促性腺激素细胞和促甲状腺激素细胞中的表达。
