P450 isoforms in a murine macrophage cell line, RAW264.7, and changes in the levels of P450 isoforms by treatment of cells with lipopolysaccharide and interferon-gamma.
The presence of P450 in a murine macrophage cell line, RAW264.7, was investigated to clarify the biological role and regulation of P450. Microsomes of RAW264.7 cells were isolated and subjected to immunoblotting with anti-rat CYP2A1, 2B1, and 4A2 antibodies. The microsomes gave staining bands with all these antibodies, suggesting the presence of mouse Cyp2a, 2b, and 4a isoforms in RAW264.7. RAW264. 7 cells were treated with typical inducers of P450 (phenobarbital, clofibrate, beta-naphthoflavone and 3-methylcholanthrene). None of these chemicals induced these P450s. Stimulation of RAW264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (INF-gamma) which increase inducible nitric oxide synthase (iNOS) and cytokines in cells decreased Cyp4a protein but not Cyp2a and 2b proteins. To identify P450 isoforms in RAW264.7, we used polymerase chain reaction (PCR) primers for mouse Cyp2a4, 2a12, 2b9/10, 4a10, and 4a12. Total RNA was isolated from these cells and converted to cDNA by reverse transcriptase. PCR was done with these primers and the amplified nucleotides were analyzed by a DNA sequencer. Only Cyp2b9/10 and 4a12 primers gave clear bands, although all primers gave clear bands from liver total RNA. Nucleotide sequences of these products amplified by PCR were identical with Cyp2b9 and 4a12. These findings indicate that Cyp2b9 and 4a12 were present in a macrophage cell line, RAW264.7, and the regulation of P450 by inducers and cytokine differed from that in liver.
Nakamura
M
M
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
Imaoka
S
S
Amano
F
F
Funae
Y
Y
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction; D015151:Immunoblotting; D007371:Interferon-gamma / Q000494:pharmacology*; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins
D007527:Isoenzymes; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007371:Interferon-gamma; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat