Huang Y S, Chuang D T
Department of Biochemistry and the Biochemistry and Molecular Biology Graduate Program, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.
Biochem J. 1999 May 1;339 ( Pt 3)(Pt 3):503-10.
The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3. 0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s).
哺乳动物线粒体支链2-氧代酸脱氢酶（BCOD）复合物受可逆磷酸化（失活）/去磷酸化（激活）循环的调节。在本研究中，研究了糖皮质激素对大鼠肝癌细胞系（H4IIE和FTO-2B）以及大鼠体内BCOD激酶mRNA水平的影响。在H4IIE细胞中，发现地塞米松在培养48小时后可显著降低BCOD激酶mRNA的稳态浓度，这与BCOD复合物去磷酸化形式增加2倍相关。地塞米松处理未影响H4IIE细胞中激酶mRNA的半衰期。因此，地塞米松处理导致的稳态激酶mRNA水平降低并非由mRNA稳定性变化引起，这增加了在基因转录水平进行调节的可能性。为了鉴定激酶启动子中的负糖皮质激素反应元件，在存在或不存在地塞米松的情况下培养的H4IIE细胞中检测了3.0 kb启动子区域的嵌套缺失构建体。在瞬时或稳定转染时均未观察到启动子活性的显著差异。数据表明糖皮质激素反应元件位于3.0 kb启动子区域之外。在生理水平上，腹腔注射地塞米松的大鼠肝脏BCOD激酶mRNA水平降低。这种作用具有肝脏特异性，在其他组织中未检测到。这些结果表明，糖皮质激素对激酶基因表达的下调是通过肝脏特异性或富集的转录因子介导的。
