title: Nitric oxide and interleukin-1β stimulate the proteasome-independent degradation of the retinoic acid hydroxylase CYP2C22 in primary rat hepatocytes.
abstract: CYP2C22 was recently described as a retinoic acid-metabolizing cytochrome P450 enzyme whose transcription is induced by all-trans-retinoic acid (atRA) in hepatoma cells (Qian L, Zolfaghari R, and Ross AC (2010) J Lipid Res 51:1781-1792). We identified CYP2C22 as a putative nitric oxide (NO)-regulated protein in a proteomic screen and raised specific polyclonal antibodies to CYP2C22 to study its protein expression. We found that CYP2C22 is a liver-specific protein that was not significantly induced by activators of the pregnane X receptor, constitutive androstane receptor, or peroxisome proliferator-activated receptor-α, but was downregulated to <25% of control by the aryl hydrocarbon receptor agonist β-naphthoflavone in cultured rat hepatocytes. CYP2C22 protein and its mRNA both were induced by atRA in hepatocytes, with EC50 of 100-300 nM, whereas the maximal extent of mRNA induction was twice that of the protein. CYP2C22 protein, but not its mRNA, was rapidly downregulated in hepatocytes by interleukin-1 (IL-1) or NO-donating compounds, and the downregulation by IL-1 was blocked by inhibition of NO synthases. The NO donor (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate reduced the half-life of CYP2C22 from 8.7 to 3.4 hours in the presence of cycloheximide, demonstrating that NO-dependent downregulation is due to stimulated proteolysis. No intermediate degradation products were detected. However, this degradation was insensitive to inhibitors of calpains or the canonical proteasomal or lysosomal pathways, indicating that NO-dependent degradation of CYP2C22 proceeds via a novel pathway.
authors.affiliation: Department of Pharmacology, Emory University, Atlanta, Georgia (C.-m.L., B.-s.L., E.T.M.); and Department of Pharmaceutics, University of Washington, Seattle, Washington (S.L.A., N.I.).
authors.forename: Choon-myung
authors.initials: CM
authors.lastname: Lee
authors.forename: Bang-sub
authors.initials: BS
authors.lastname: Lee
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Arnold
authors.forename: Nina
authors.initials: N
authors.lastname: Isoherranen
authors.forename: Edward T
authors.initials: ET
authors.lastname: Morgan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D015536:Down-Regulation / Q000502:physiology*; D057809:HEK293 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D053583:Interleukin-1beta / Q000502:physiology*; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D061251:Primary Cell Culture; D046988:Proteasome Endopeptidase Complex / Q000502:physiology; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D053583:Interleukin-1beta; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C552837:cytochrome P-450 2C22, rat; D046988:Proteasome Endopeptidase Complex