IDENTIFICATION OF A NOVEL DEXAMETHASONE RESPONSIVE ENHANCER IN THE HUMAN CYP3A5 GENE AND ITS ACTIVATION IN HUMAN AND RAT-LIVER CELLS

The human liver cytochromes P450 3A (CYP3As), orthologous to the rat g lucocorticoid inducible forms, are composed of at least four different ially expressed members. To begin the study of the molecular events in the glucocorticoid regulation of CYP3A5, we fused 5' sequences of CYP 3A5 to the chloramphenicol acetyltransferase gene in a vector that con tains the herpes simplex virus thymidine kinase promoter. In HepG2 cel ls, the largest 5'CYP3A5 gene fragment (1.4 kb) suppressed the TK prom oter. However, suppression was overcome by addition of 10 mu M dexamet hasone. A series of unidirectional deletions revealed a unique 219-bp fragment (-891 to -1109 bp upstream from the transcriptional start sit e) that conferred dexamethasone responsiveness on the TK promoter rega rdless of either the distance or orientation from the promoter and thu s appears to be an enhancer. Nucleotide sequence analysis of this CYP3 A5 enhancer revealed no consensus 15-bp glucocorticoid responsive elem ent (GRE) (GGTACANNNTGTTCT); however, two GRE ''half-sites'' (TGTTCT) were found separated by 160 bp. Although dexamethasone stimulated the CYP3A5 enhancer only 3-4-fold in HepG2 cells, the CYP3A5 enhancer was stimulated 7- and 12-fold in immortalized primary human hepatocytes an d primary rat hepatocyte cultures, respectively. The glucocorticoid re ceptor (GCR) seems to be indispensable to this process because 1) dexa methasone induction can be blocked by the antiglucocorticoid RU-486, 2 ) dexamethasone-dependent transcriptional activation of the CYP3A5 enh ancer in HepG2 cells required cotransfection of an expression vector c ontaining the intact GCR, yet 3) cotransfection with a plasmid that co ntains a mutation in the ligand binding domain of the GCR does not act ivate the CYP3A5 enhancer in the presence of dexamethasone. To further localize the dexamethasone responsive region of the 219-bp CYP3A5 enh ancer, it was subdivided and fused to the TKCAT expression vector. Tra nsfection analysis in HepG2 cells demonstrated that neither GRE half-s ite can independently confer dexamethasone responsiveness on the TK pr omoter. Block mutations of either of the two GRE half-sites or point m utations at specific GCR binding sites eliminates dexamethasone induci bility, demonstrating the half-sites need to interact. Electromobility shift assays indicate that the CYP3A5 5'-GRE half-site 1) specificall y binds purified GCR, 2) can displace binding of the GCR to a consensu s GRE, and 3) shifts a protein in HepG2 nuclear extracts that is super shifted by GCR antibody, demonstrating that this enhancer is an authen tic GRE. This is the first study to demonstrate that a member of the h uman CYP3A gene family contains an enhancer that binds the GCR and tha t this binding is critical to transcriptional activation by dexamethas one.