Squalestatin 1-inducible expression of rat CYP2B: Evidence that an endogenous isoprenoid is an activator of the constitutive androstane receptor

Because our previous studies indicated that squalestatin 1 treatment induces CYP2B expression in primary cultures of rat hepatocytes as a direct consequence of squalene synthase inhibition, we investigated possible underlying mechanisms. Cotransfection of cultured Sprague-Dawley male rat hepatocytes with each of the three sterol regulatory element binding protein (SREBP) transcription factors failed to induce luciferase expression from a squalestatin 1-responsive CYP2B1 reporter plasmid. Squalestatin 1 treatment of primary hepatocyte cultures from male Wistar-Kyoto rats produced a greater induction of CYP2B mRNA than occurred in cultures from female rats, consistent with the previously demonstrated response dimorphism that has been attributed to differences in constitutive androstane receptor (CAR) levels. Cotransfection of female Wistar-Kyoto rat hepatocyte cultures with plasmid expressing either mouse or rat CAR restored squalestatin 1-inducible CYP2B1-reporter expression. Cotransfection of Sprague-Dawley rat hepatocyte cultures with plasmid expressing rat CAR lacking the C-terminal AF-2 subdomain inhibited squalestatin 1-inducible CYP2B1-reporter expression. Squalestatin 1-mediated CYP2B mRNA induction in rat hepatocyte cultures was completely abolished by pretreatment with the 3-hydroxymethyl-3-glutaryl CoA reductase inhibitor pravastatin and was rescued by mevalonate supplementation, whereas phenobarbital-mediated induction was unaffected by these treatments. Finally, direct addition of trans, trans-farnesol to the culture medium caused the rapid induction of CYP2B mRNA. These results indicate that squalestatin 1 treatment induces CYP2B expression, not by inhibiting sterol synthesis and activating SREBPs, but by evoking the accumulation of an endogenous isoprenoid and activating CAR.