CCAAT ENHANCER-BINDING PROTEIN-ALPHA-DEPENDENT TRANSACTIVATION OF CYP2C12 IN RAT HEPATOCYTES/

Expression of the rat CYP2C12 gene is liver specific and is induced by GH at the transcriptional level. In primary cultures of rat hepatocyt es, 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 Matr igel, increased the cellular P4502C12 messenger RNA levels 10-fold. Co transfection studies using deletion constructs of the CYP2C12 promoter fused to the luciferase reporter gene localized the C/EBP alpha respo nse to the region -250 to -180. Sequence comparisons and deoxyribonucl ease I footprinting using rat liver nuclear extracts indicated two pot ential C/EBP binding sites in this region. Mutagenesis of the most ups tream element (-229 to -207) abolished transactivation by C/EBP alpha. Using gel mobility supershift assays, this element was demonstrated t o bind C/EBP alpha and C/EBP beta in liver nuclear extracts and in lys ates from hepatocytes cultured on Matrigel. GH treatment of the cells did not alter the C/EBP protein levels or the C/EBP-binding activity t o this element. Neither did GH increase the expression of CYP2C12 repo rter gene constructs regardless of the presence of different amounts o f 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 throu gh increased levels of C/EBP alpha or via enhanced DNA-binding activit y of this transcription factor.