Tollet P, Lahuna O, Ahlgren R, Mode A, Gustafsson J A
Department of Medical Nutrition Karolinska Institute Huddinge University Hospital, Sweden.
Mol Endocrinol. 1995 Dec;9(12):1771-81. doi: 10.1210/mend.9.12.8614413.
Expression of the rat CYP2C12 gene is liver specific and is induced by GH at the transcriptional level. In primary cultures of rat hepatocytes, GH inducibility of CYP2C12 and the presence of C/EBP alpha protein were demonstrated to be equally dependent on attachment of the cells to an extracellular matrix gel (Matrigel). Transient transfection of a C/EBP alpha expression vector into hepatocytes, cultured without Matrigel, increased the cellular P4502C12 messenger RNA levels 10-fold. Cotransfection studies using deletion constructs of the CYP2C12 promoter fused to the luciferase reporter gene localized the C/EBP alpha response to the region -250 to -180. Sequence comparisons and deoxyribonuclease I footprinting using rat liver nuclear extracts indicated two potential C/EBP binding sites in this region. Mutagenesis of the most upstream element (-229 to -207) abolished transactivation by C/EBP alpha. Using gel mobility supershift assays, this element was demonstrated to bind C/EBP alpha and C/EBP beta in liver nuclear extracts and in lysates from hepatocytes cultured on Matrigel. GH treatment of the cells did not alter the C/EBP protein levels or the C/EBP-binding activity to this element. Neither did GH increase the expression of CYP2C12 reporter gene constructs regardless of the presence of different amounts of cotransfected C/EBP alpha. We conclude that C/EBP alpha is a potent transactivator of the CYP2C12 gene and most likely contributes to its liver-specific expression. Although the results presented here do not exclude the possibility of a GH-enhanced transactivating ability of C/EBP alpha, the mechanism of GH-induced levels of P4502C12 is not through increased levels of C/EBP alpha or via enhanced DNA-binding activity of this transcription factor.
大鼠CYP2C12基因的表达具有肝脏特异性，且在转录水平上受生长激素（GH）诱导。在大鼠肝细胞原代培养中，已证明CYP2C12的GH诱导性和C/EBPα蛋白的存在同样依赖于细胞与细胞外基质凝胶（基质胶）的附着。将C/EBPα表达载体瞬时转染到未用基质胶培养的肝细胞中，可使细胞中P4502C12信使RNA水平增加10倍。使用与荧光素酶报告基因融合的CYP2C12启动子缺失构建体进行的共转染研究将C/EBPα反应定位到 -250至 -180区域。使用大鼠肝脏核提取物进行的序列比较和脱氧核糖核酸酶I足迹分析表明该区域有两个潜在的C/EBP结合位点。最上游元件（-229至 -207）的诱变消除了C/EBPα的反式激活作用。使用凝胶迁移超迁移分析，证明该元件在肝脏核提取物和在基质胶上培养的肝细胞裂解物中与C/EBPα和C/EBPβ结合。对细胞进行GH处理不会改变C/EBP蛋白水平或该元件的C/EBP结合活性。无论共转染不同量的C/EBPα，GH也不会增加CYP2C12报告基因构建体的表达。我们得出结论，C/EBPα是CYP2C12基因的有效反式激活因子，很可能有助于其肝脏特异性表达。尽管此处给出的结果不排除C/EBPα具有GH增强的反式激活能力的可能性，但GH诱导P4502C12水平的机制不是通过增加C/EBPα水平或通过增强该转录因子的DNA结合活性。
