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Abstract
In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor alpha (TNF-alpha) without affecting GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a cofactor required for iNOS activity. TNF-alpha also stimulates sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (SPP), a further metabolite of ceramide. Several clones of C6 cells, expressing widely varying levels of sphingosine kinase, were used to examine the role of SPP in regulation of GTPCH and BH(4) biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-alpha on GTPCH expression and activity and BH(4) biosynthesis. In contrast, enforced expression of sphingosine kinase had no effect on iNOS expression or NO formation. Furthermore, N,N-dimethylsphingosine, a potent sphingosine kinase inhibitor, completely eliminated the increased GTPCH activity and expression induced by TNF-alpha. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-alpha, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH(4) biosynthesis. However, both SPP and dihydro-SPP markedly stimulated ERK 1/2 in C6 cells, which express cell surface SPP receptors. Interestingly, although this ERK activation was blocked by PD98059, which also reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity. Collectively, these results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH(4) levels.
Highlights
§ Both authors contributed to this work. ʈ Present address: Dept. of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0614. ** To whom correspondence should be addressed: LCMR, NIMH, National Institutes of Health, Bldg. 36, Rm. 2A-11, Bethesda, MD 20892
It is well established that inducible nitric-oxide synthase (iNOS) induction is dependent on the redox-sensitive transcription factor NF-B, we previously showed that NF- was not required for induction of GTP cyclohydrolase I (GTPCH) in rat C6 glioma cells, a convenient astrocyte model, when stimulated with combinations of proinflammatory cytokines (12)
Overexpression of Sphingosine Kinase in C6 Cells Increases SPP Levels—Previously, we found that, TNF-␣-induced expression of iNOS and subsequent NO production were mediated in part by increases in ceramide levels, GTPCH expression and activity and levels of the iNOS cofactor BH4 were not increased in parallel (12)
Summary
Vol 277, No 15, Issue of April 12, pp. 12649 –12656, 2002 Printed in U.S.A. Involvement of Sphingosine Kinase in TNF-␣-stimulated Tetrahydrobiopterin Biosynthesis in C6 Glioma Cells*. This ERK activation was blocked by PD98059, which reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity These results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH4 levels. In certain cells, such as human umbilical vein endothelial cells reverse transcriptase; SPP, sphingosine-1-phosphate; TNF-␣, tumor necrosis factor ␣; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ceramide, N-acyl-sphingosine; EB, extraction buffer; DTT, dithiothreitol; SPHK, sphingosine kinase; DMS, N,N-dimethylsphingosine; LDH, lactate dehydrogenase; FGF-2, fibroblast growth factor 2; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; GPCR, G protein-coupled receptor. We found that, C6 cells can secrete SPP, only SPP generated intracellularly in response to TNF-␣ stimulated GTPCH activity and BH4 biosynthesis
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