REGULATION OF HEPATIC P450S BY ANTICHOLESTEROL DRUGS

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Emerging evidence suggests that the complex processes of sterol biosynthesis and xenobiotic metabolism by enzymes of the cytochrome P450 superfamily are inextricably intertwined. Such an interrelationship has important implications for the safety and efficacy of the growing arsenal of "anti-cholesterol" drugs that is being developed to lower patients' plasma cholesterol levels and thereby treat or prevent coronary artery disease. The hypothesis of this proposal is three-fold: (1) Anti-cholesterol drugs that inhibit sterol biosynthesis, such as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase or squalene synthase, induce rat hepatic CYP2B1 gene expression by causing depletion of the critical cellular sterols that suppress CYP2B1 expression in the basal steady state, which results in activation of sterol regulatory element binding proteins (SREBP), followed by transcriptional activation of the CYP2B1 gene through specific 5'- flanking sequences contained within the phenobarbital responsive unit. (2) Anti-cholesterol drugs, such as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT) or squalene cyclase, that promote accumulation of specific cellular sterols, such as 24(S),25-epoxycholesterol, induce CYP3A23 gene expression through a mechanism whereby the accumulated sterols activate the LXRalpha nuclear receptor, resulting in transcriptional activation of the CYP3A23 gene through a specific sterol-responsive 5'-flanking sequence. (3) HMG-CoA reductase inhibitors induce CYP4A gene expression through a mechanism that requires increased fatty acid biosynthesis, activation of the peroxisome proliferation associated receptor alpha (PPARalpha) and transcriptional activation of the CYP4A1 gene through a peroxisome proliferator responsive element. The specific aims of this proposal are to (1) define the effects of chemical inhibitors of key steps of the cholesterol biosynthesis and esterification pathways on P450 expression in primary cultured rat hepatocytes, and to relate patterns of gene expression to changes in levels of specific cellular sterols, (2) identify the 5'-flanking sequence(s) that confer HMG-CoA reductase inhibitor-and squalene synthase inhibitor-inducible transcriptional activation to the CYP2B1 gene, and to determine whether this regulation is mediated through an SREBP transcription factor, (3) identify the 5'- flanking sequence(s) that confer sterol-, ACAT inhibitor-and squalene cyclase inhibitor-inducible transcriptional activation to the CYP3A23 gene, and to determine whether this regulation is mediated through the LXRalpha nuclear receptor and (4) determine whether HMG-CoA reductase inhibitor-inducible CYP4A1 induction occurs through a mechanism that requires elevated fatty acid biosynthesis and activation of PPARalpha. These studies will provide information with practical implications for the development of improved anti-cholesterol drugs, and will illuminate mechanisms whereby a cell recognizes, responds to, and metabolizes foreign chemicals.