Divergence in regulation of nitric-oxide synthase and its cofactor tetrahydrobiopterin by tumor necrosis factor-alpha. Ceramide potentiates nitric oxide synthesis without affecting GTP cyclohydrolase I activity.
Synthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a required cofactor for inducible nitric-oxide synthase (iNOS) activity, is usually coordinately regulated with iNOS expression. In C6 glioma cells, tumor necrosis factor-alpha (TNF-alpha) concomitantly potentiated the stimulation of nitric oxide (NO) and BH(4) production induced by IFN-gamma and interleukin-1beta. Expression of both iNOS and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the BH(4) biosynthetic pathway, was also markedly increased, as were their activities and protein levels. Ceramide, a sphingolipid metabolite, may mediate some of the actions of TNF-alpha. Indeed, we found that bacterial sphingomyelinase, which hydrolyzes sphingomyelin and increases endogenous ceramide, or the cell permeable ceramide analogue, C(2)-ceramide, but not C(2)-dihydroceramide (N-acetylsphinganine), significantly mimicked the effects of TNF-alpha on NO production and iNOS expression and activity in C6 cells. Surprisingly, although TNF-alpha increased BH(4) synthesis and GTPCH activity, neither BH(4) nor GTPCH expression was affected by C(2)-ceramide or sphingomyelinase in IFN-gamma- and interleukin-1beta-stimulated cells. It is likely that increased BH(4) levels results from increased GTPCH protein and activity in vivo rather than from reduced turnover of BH(4), because the GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, blocked cytokine-stimulated BH(4) accumulation. Moreover, expression of the GTPCH feedback regulatory protein, which if decreased might increase GTPCH activity, was not affected by TNF-alpha or ceramide. Treatment with the antioxidant pyrrolidine dithiocarbamate, which is known to inhibit NF-kappaB and sphingomyelinase in C6 cells, or with the peptide SN-50, which blocks translocation of NF-kappaB to the nucleus, inhibited TNF-alpha-dependent iNOS mRNA expression without affecting GTPCH mRNA levels. This is the first demonstration that cytokine-stimulated iNOS and GTPCH expression, and therefore NO and BH(4) biosynthesis, may be regulated by discrete pathways. As BH(4) is also a cofactor for the aromatic amino acid hydroxylases, discovery of distinct mechanisms for regulation of BH(4) and NO has important implications for its specific functions.
Vann
L R
LR
Laboratory of Cellular and Molecular Regulation, NIMH, National Institutes of Health, Bethesda, Maryland 20892, USA.
Twitty
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Spiegel
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Milstien
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D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D006136:GTP Cyclohydrolase / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D014409:Tumor Necrosis Factor-alpha; D001708:Biopterins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D006136:GTP Cyclohydrolase; C003402:sapropterin