Role of tetrahydrobiopterin availability in the regulation of nitric-oxide synthase expression in human mesangial cells.
Human mesangial cells express an inducible form of nitric-oxide synthase (iNOS) after treatment with cytokines. Tetrahydrobiopterin (BH4), an essential cofactor for NOS, is required for cytokine-induced NO generation. We report here that BH4 is necessary not only for the activity but also for the expression of iNOS in human mesangial cells. Inhibition of de novo BH4 synthesis with 2,4-diamino-6-hydroxypyrimidine (DAHP) significantly attenuated iNOS activity as well as mRNA and protein expression in response to interleukin 1beta plus tumor necrosis factor alpha (IL-1beta/TNF-alpha). In contrast, sepiapterin, which provides BH4 through the pterin salvage pathway, strongly potentiated IL-1beta/TNF-alpha-induced iNOS expression and abrogated the inhibitory effect of DAHP. Inhibition of the pterin salvage pathway with methotrexate abolished sepiapterin potentiation of iNOS induction but did not alter the effect of IL-1beta/TNF-alpha. Determination of intracellular pteridines confirmed that sepiapterin markedly raised BH4 content, an effect that was blocked by methotrexate. These results suggest that BH4 availability plays an important role in the regulation of iNOS expression. The effect of BH4 appears to be mediated, at least in part, by an increase in mRNA stability, as indicated by the observation that DAHP shortened, whereas sepiapterin prolonged the half-life of IL-1beta/TNF-alpha-induced iNOS mRNA. Taken together, our results suggest that the biosynthesis of BH4 contributes to cytokine induction of iNOS expression in human mesangial cells through the stabilization of iNOS mRNA.
Saura
M
M
Centro de Investigaciones Biológicas and the Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain.
Pérez-Sala
D
D
Cañada
F J
FJ
Lamas
S
S
D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005920:Glomerular Mesangium / Q000201:enzymology*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007700:Kinetics; D019331:NG-Nitroarginine Methyl Ester; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D009573:Nitrites / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D011621:Pteridines / Q000494:pharmacology; D011622:Pterins; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D013400:Sugar Acids / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C017182:5-dehydro-3-deoxy-D-arabino-heptulosonic acid-7-phosphate; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D009573:Nitrites; D011621:Pteridines; D011622:Pterins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013400:Sugar Acids; D014409:Tumor Necrosis Factor-alpha; D009599:Nitroprusside; D001708:Biopterins; D001120:Arginine; C016727:sepiapterin; D019001:Nitric Oxide Synthase; C003402:sapropterin; D019331:NG-Nitroarginine Methyl Ester