Oxidized low density lipoprotein inhibits inducible nitric oxide synthase, GTP cyclohydrolase I and transforming growth factor beta gene expression in rat macrophages.
Several studies have already demonstrated that oxidized- LDL decreases nitric oxide (NO) generation by cytokine-stimulated macrophages. However, the mechanisms of such an inhibition have not been yet elucidated. NO generation by inducible nitric oxide synthase (iNOS) is dependent on the presence of cofactors for NO generation, tetrathydrobiopterin (BH4) among them. The NO generation by these cells is also regulated by some endogenous inhibitors, like TGF-beta. Therefore, the aim of our recent study was to investigate the influence of ox-LDL on the expression of iNOS and GTP cyclohydrolase I (GTP-CH I), the key enzyme involved in the BH4 synthesis as well as the ox-LDL effect on TGF-beta expression in rat macrophages stimulated with IFNgamma (250 U/ml) and LPS (500 ng/ml). Macrophages, activated in this way, express iNOS, GTP-CH I, and TGF-beta mRNA. This expression was inhibited when the macrophages were preincubated for 24 hours with ox-LDL (100 microg/ml). Quantitative PCR revealed about 10-fold inhibition of iNOS gene expression by ox-LDL. As a consequence of down-regulation of iNOS and GTP-CH I genes, almost 3-fold diminished generation of NO2- by rat macrophages was observed. An inhibition of the TGFbeta mRNA expression was also found. Our studies indicate that decreased NO generation by ox-LDL treated macrophages may be the result of the diminished expression of both iNOS and GTP-CH I genes. This effect may be mediated by the activity of certain endogenous inhibitors of gene expression, however, our studies exclude the TGF-beta as a candidate for this activity.
Dulak
J
J
Department of Clinical Biochemistry, Collegium Medicum, Jagiellonian University, Kraków, Poland. mbdulak@cyf-kr.edu.pl
Polus
M
M
Guevara
I
I
Hartwich
J
J
Wybrańska
I
I
Krzesz
R
R
Dembińska-Kieć
A
A
D000818:Animals; D002478:Cells, Cultured; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000302:isolation & purification; D051381:Rats; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics
D008070:Lipopolysaccharides; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C099252:oxidized low density lipoprotein; D009569:Nitric Oxide; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D006136:GTP Cyclohydrolase