Abstract
The induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16 alpha-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation. In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5'-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN. Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used. Analysis of both of the "footprints" (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element. A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter 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 receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays. Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used. We conclude 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.
Highlights
CYP3A1, a member of the cytochrome P450 supergene family, is prominently induced in liver microsomes of rats or in primary cultures of adult rat hepatocytes treated with the synthetic glucocorticoid, dexamethasone and, paradoxically, by such antiglucocorticoids as pregnenolone 16␣-carbonitrile (PCN)1 [1,2,3,4,5]
Tests of agonist-antagonist relationships demonstrated that rates of de novo synthesis of immunoreactive CYP3A1 protein and accumulation of CYP3A1 mRNA were stimulated synergistically when hepatocyte cultures were incubated in the presence of dexamethasone plus PCN, even though the expression of tyrosine aminotransferase (TAT) was inhibited by this same protocol [3,4,5]
To investigate the role of CYP3A1 transcription we established a system for transient expression of 1.5 kilobases of DNA 5Ј-flanking the CYP3A1 gene2 fused to a chloramphenicol acetyltransferase (CAT) reporter plasmid in primary cultures of adult rat hepatocytes that maintain synergistic inducibility of hepatocellular CYP3A1 mRNA by dexamethasone plus PCN [5]
Summary
CYP3A1, a member of the cytochrome P450 supergene family, is prominently induced in liver microsomes of rats or in primary cultures of adult rat hepatocytes treated with the synthetic glucocorticoid, dexamethasone and, paradoxically, by such antiglucocorticoids as pregnenolone 16␣-carbonitrile (PCN)1 [1,2,3,4,5]. Regulation of the Rat CYP3A1 Gene duced by treatment of the cultures with dexamethasone or with PCN and was induced synergistically by treatment with dexamethasone plus PCN while expression of a transfected control plasmid containing the classical glucocorticoid inducible MMTV gene was induced by dexamethasone, was not induced by PCN, and was only slightly induced by dexamethasone plus PCN [5]. These results establish that the unique features of CYP3A1 regulation by glucocorticoids largely involve effects on CYP3A1 gene transcription. Finding that the sequence lacks a classical GRE, fails to bind GR, but binds to a pattern of liver nuclear proteins from control or dexamethasone-treated rats that appears the same, we conclude that activation of CYP3A1 gene transcription by glucocorticoids may involve a novel indirect interaction of GR with CYP3A1
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