Detection of protein and mRNA of various components of the NADPH oxidase complex in an immortalized human chondrocyte line.
The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 x 10(-2) nmol/min/10(6) cells (S.E.M. = +/-0.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox and p47-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.
Moulton
P J
PJ
Faculty of Applied Sciences, University of the West of England, Bristol.
Hiran
T S
TS
Goldring
M B
MB
Hancock
J T
JT
D001483:Base Sequence; D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology; D002356:Cartilage / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000032:analysis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002273:Carcinogens; D018076:DNA, Complementary; D007375:Interleukin-1; D007476:Ionophores; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013481:Superoxides; D015847:Interleukin-4; D015759:Ionomycin; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate