A NOVEL CIS-ACTING ELEMENT IN A LIVER CYTOCHROME-P450 3A GENE CONFERSSYNERGISTIC INDUCTION BY GLUCOCORTICOIDS PLUS ANTIGLUCOCORTICOIDS

The induction by dexamethasone of rat liver CYP3A1 differs from classi cal glucocorticoid gene regulation in part because both glucocorticoid s and antiglucocorticoids such as pregnenolone 16 alpha-carbonitrile ( PCN) induce CYP3A1 through transcriptional gene activation. In the pre sent study, we transiently expressed in primary cultures of rat hepato cytes plasmids consisting of CYP3A1 5'-flanking sequences fused to a c hloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp u pstream of the transcriptional start site that was inducible by treatm ent of the cultures with dexamethasone or PCN and was induced synergis tically by dexamethasone plus PCN. Nuclear extract from control rat li ver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extract s from dexamethasone-treated animals were used. Analysis of both of th e ''footprints'' (FP1 and FP2) failed to reveal a classical sequence f or the glucocorticoid-responsive element. A 33-bp element that include s FP1 sequences inserted into the chloramphenicol acetyltransferase re porter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78 -bp element. However, an Escherichia coli expressed glucocorticoid rec eptor protein failed to protect sequences within FP1 in DNase I footpr inting experiments and failed to change its mobility in gel shift assa ys. Moreover, as judged by the gel shift assay, the specific protein b inding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone treated rats were used. We conclu de that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA.