Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of speci®c G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in signi®cant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C 2 -ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGFstimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G i -coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.

Anti-Jo-1 antibodies (AJoA), which bind to and inhibit the activity of histidyl-transfer RNA synthetase (HRS), are found in a genetically and clinically distinct subset of myositis patients. This specificity suggests that understanding the antigenic epitopes and immunoregulation governing the production of AJoA may result in clues to disease pathogenesis. Limited digestion of human HRS by V8 protease resulted in four major antigenic polypeptides of 35, 34, 21, and 20 kD; digestion with subtilisin gave four fragments of the same sizes and two additional major antigenic polypeptides of 28 and 17 kD. Sera from 12 AJoA positive patients reacted indistinguishably with these proteolytic fragments by Western blotting, and AJoA elution studies suggested a common epitope(s) on all six. Isoelectric focusing showed a different polyclonal pattern of AJoA in each patient, although serial analyses in individual patients revealed stable AJoA spectrotypes over years of observation. Enzymelinked immunosorbant assays showed that the AJoA response was mainly restricted to the IgG1 heavy chain isotype. The levels of IgG1 AJoA varied in proportion to disease activity over time but were independent of total IgG1 levels, and three patients became AJoA negative as their myositis remitted after treatment. These findings suggest that AJoA are induced by an antigen-driven mechanism, bind to a common epitope or epitopes on HRS, and are modulated by an immune response closely linked to that which is responsible for myositis in these patients. (J. Clin. Invest. 1990. 85:468-475.) anti-histidyltRNA synthetase * autoantibody subclass * dermatomyositisi immunomodulation * polymyositis

In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor ␣ (TNF-␣) without affecting GTP cyclohydrolase I (GT-PCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH 4 ), a cofactor required for iNOS activity. TNF-␣ 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-␣ 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-␣. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-␣, 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. 5,6,7, 1 is the obligate cofactor for the aromatic L-amino hydroxylases and is also required for activity of all nitric-oxide synthase isoforms (reviewed in Ref. 1). It has been proposed that BH 4 may also have cofactorindependent roles (2), including inhibition of cytokine-induced apoptosis (3) and stimulation of dopamine release (4). Cellular levels of BH 4 are regulated by the activity of GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the BH 4 biosynthetic pathway (5). Its expression is increased by proinflammatory cytokines, such as IFN-␥ and TNF-␣, and is coordinately regulated with cytokine-inducible nitric-oxide synthase (iNOS) (6). BH 4 binds to NOS monomers, promoting their dimerization and subsequent activation (7), and recent crystallographic analysis suggests that it may play a direct role in the NOS reaction in a radical form (8). 6(R)-GTPCH, a homodecamer of 30-kDa subunits arranged as two pentamers facing one another (9), catalyzes the rearrangement of GTP to dihydroneopterin triphosphate. This intermediate is then converted to BH 4 in two subsequent reactions catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, respectively, neither of which are rate-limiting. The signal transduction pathways that regulate induction of GTPCH are not wel...

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-␣ (TNF-␣) concomitantly potentiated the stimulation of nitric oxide (NO) and BH 4 production induced by IFN-␥ and interleukin-1␤. 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-␣. 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-␣ on NO production and iNOS expression and activity in C6 cells. Surprisingly, although TNF-␣ increased BH 4 synthesis and GTPCH activity, neither BH 4 nor GTPCH expression was affected by C 2 -ceramide or sphingomyelinase in IFN-␥-and interleukin-1␤-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-␣ or ceramide. Treatment with the antioxidant pyrrolidine dithiocarbamate, which is known to inhibit NF-B and sphingomyelinase in C6 cells, or with the peptide SN-50, which blocks translocation of NF-B to the nucleus, inhibited TNF-␣-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.

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