Abstract
Livers of aryl hydrocarbon receptor (AHR)-null mice have high levels of retinoic acid (RA), retinol, and retinyl palmitate. Hepatic accumulation of RA in these mice may be responsible in part for the hepatic phenotype characterized by small liver size and fibrosis. The increased levels of hepatic RA may be due to decreased metabolism of RA to 4-hydroxyretinoic acid. To identify the P450 isoform(s) involved in RA metabolism, liver microsomes from AHR-null and wild-type mice were subjected to Western blotting and probed with antibodies to rat P450s that cross-react with murine forms. Signal intensity in Western blots probed with anti-rat CYP2C6 antibodies correlated with levels of RA 4-hydroxylation. Furthermore, this anti-rat CYP2C6 antibody inhibited RA 4-hydroxylase activity of wild-type mouse liver microsomes to the levels of AHR-null mouse liver. When used to screen a mouse liver cDNA expression library, this antibody exclusively recognized the murine P450 CYP2C39. Catalytic assays of five recombinant mouse CYP2Cs expressed in Escherichia coli revealed that only CYP2C39 was competent for RA 4-hydroxylation (K(m) = 812.3 nm and V(max) 47.85 (fmol/min/pmol P450)). Real time reverse transcriptase-PCR used to assess the Cyp2C39 mRNA expression showed decreased levels (30%) of this transcript in AHR-null compared with wild-type liver, consistent with decreased protein levels observed by Western blot analysis using an antibody to a CYP2C39-specific peptide. These data show that CYP2C39 catalyzes RA catabolism and thus possibly controls RA levels in mouse liver. Down-regulation of Cyp2C39 is hypothesized to be responsible for the liver phenotype in the AHR-null mouse.
Highlights
Livers of aryl hydrocarbon receptor (AHR)-null mice have high levels of retinoic acid (RA), retinol, and retinyl palmitate
Probing Western immunoblots of AHR-null and wild-type mouse liver microsomes with a panel of antibodies raised against rat P450s could be used to identify an antibody that would cross-react with the RA-hydroxylating P450 and to highlight differences in this P450 between AHR-null and wildtype mice
Proteins that were cross-reactive to the antibody to rat CYP2C6 were decreased in AHR-null livers relative to wild type (37% decrease was estimated by densitometric analysis by ImageQuant 5.1 software)
Summary
Vol 279, No 5, Issue of January 30, pp. 3434 –3438, 2004 Printed in U.S.A. ITS DOWN-REGULATION OFFERS A MOLECULAR BASIS FOR LIVER RETINOID ACCUMULATION AND FIBROSIS IN ARYL HYDROCARBON RECEPTOR-NULL MICE*. To identify the P450 isoform(s) involved in RA metabolism, liver microsomes from AHR-null and wild-type mice were subjected to Western blotting and probed with antibodies to rat P450s that cross-react with murine forms. Real time reverse transcriptase-PCR used to assess the Cyp2C39 mRNA expression showed decreased levels (30%) of this transcript in AHR-null compared with wild-type liver, consistent with decreased protein levels observed by Western blot analysis using an antibody to a CYP2C39-specific peptide These data show that CYP2C39 catalyzes RA catabolism and possibly controls RA levels in mouse liver. Given that AHR ligands can reduce tissue retinoid content, and that the AHR can influence gene expression even in the absence of exogenous ligand, it seemed likely that loss of the AHR might affect retinoid homeostasis This was confirmed by our observation that livers from AHR-null mice exhibit a 3-fold increase in retinol esters, retinol and retinoic acid, relative to wild-type livers [6].
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