Wei P, Ahn Y I, Housley P R, Alam J, Vedeckis W V
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112, USA.
J Steroid Biochem Mol Biol. 1998 Jan;64(1-2):1-12. doi: 10.1016/s0022-1759(97)89907-3.
The glucocorticoid receptor (GR) is a ligand-dependent transcription factor capable of stimulating and inhibiting the expression of target genes. To better understand the biological action of glucocorticoids and the function of GR, we have utilized the tetracycline (Tc)-regulated mammalian expression system to develop a novel cell line, E8.2/GR3, derived from GR null mouse L929 fibroblasts, that exhibits conditional expression of rat GR. The intracellular concentration of rGR in E8.2/GR3 cells--from undetectable levels to levels more than 10-fold greater than that observed in wild-type L929 cells--could be manipulated by varying the Tc concentration in the culture media. Similarly, dexamethasone (DEX)-dependent transactivation of the mouse mammary tumor virus long terminal repeat and transrepression of the cadmium-induced activity of the mouse heme oxygenase-1 gene enhancer, SX2, were strictly dependent on the presence of rGR, and the levels of these activities could be modulated by Tc. Similar levels of Tc, and thus rGR, were required for half-maximal transactivation and transrepression whereas a 6-fold lower concentration of DEX was required for half-maximal transrepression than for transactivation. RU486 inhibited both DEX-dependent transactivation and transrepression. DEX decreased the steady-state level of rGR mRNA and protein in a Tc dependent manner. DEX also induced morphological changes in E8.2/GR3 cells that were dependent on rGR as no alterations were observed in the presence of Tc. These cells provide a powerful system for examining the various activities of GR, particularly as a function of different intracellular receptor concentrations.
糖皮质激素受体（GR）是一种依赖配体的转录因子，能够刺激和抑制靶基因的表达。为了更好地理解糖皮质激素的生物学作用以及GR的功能，我们利用四环素（Tc）调控的哺乳动物表达系统，从GR基因敲除的小鼠L929成纤维细胞中开发出一种新型细胞系E8.2/GR3，该细胞系可实现大鼠GR的条件性表达。通过改变培养基中Tc的浓度，可以调控E8.2/GR3细胞中rGR的细胞内浓度，使其从不可检测的水平提高到比野生型L929细胞中观察到的水平高10倍以上。同样，小鼠乳腺肿瘤病毒长末端重复序列的地塞米松（DEX）依赖性反式激活以及镉诱导的小鼠血红素加氧酶-1基因增强子SX2活性的反式抑制，都严格依赖于rGR的存在，并且这些活性水平可以通过Tc进行调节。半最大反式激活和反式抑制所需的Tc水平以及因此的rGR水平相似，而半最大反式抑制所需的DEX浓度比反式激活低6倍。RU486抑制DEX依赖性的反式激活和反式抑制。DEX以Tc依赖的方式降低rGR mRNA和蛋白质的稳态水平。DEX还诱导E8.2/GR3细胞发生形态变化，这种变化依赖于rGR，因为在存在Tc的情况下未观察到改变。这些细胞为研究GR的各种活性提供了一个强大的系统，特别是作为不同细胞内受体浓度的函数。
