Mouse liver CYP2C39 is a novel retinoic acid 4-hydroxylase. Its down-regulation offers a molecular basis for liver retinoid accumulation and fibrosis in aryl hydrocarbon receptor-null mice.
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.
Andreola
Fausto
F
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hayhurst
Graham P
GP
Luo
Gang
G
Ferguson
Stephen S
SS
Gonzalez
Frank J
FJ
Goldstein
Joyce A
JA
De Luca
Luigi M
LM
D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D005355:Fibrosis / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D000072516:Retinoic Acid 4-Hydroxylase; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014212:Tretinoin / Q000737:chemistry
D017931:DNA Primers; D018076:DNA, Complementary; D010455:Peptides; D000072317:Polychlorinated Dibenzodioxins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; C400322:cytochrome P-450 CYP2C subfamily; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C000607083:Cyp2c39 protein, mouse; D000072467:Cytochrome P450 Family 2; D000072516:Retinoic Acid 4-Hydroxylase