Involvement of Sphingosine Kinase in TNF-α-stimulated Tetrahydrobiopterin Biosynthesis in C6 Glioma Cells

Abstract

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...

Pharmacology and Antitumor Activity of ABC294640, a Selective Inhibitor of Sphingosine Kinase-2

“…SK has also been linked to estrogen signaling (Sukocheva et al, 2003) and estrogen-dependent tumorigenesis in MCF-7 cells (Nava et al, 2002). Other pathways or targets to which SK activity has been linked in cancer include vascular endothelial growth factor signaling via the Ras and mitogen-activated protein kinase pathway (Shu et al, 2002), protein kinase C (Nakade et al, 2003), tumor necrosis factor ␣ (Vann et al, 2002), and caspase activation (Edsall et al, 2001). Angiogenic factors and processes, such as cell motility, mitogenesis in smooth muscle cells, endothelial cell differentiation, and growth factor signaling are also affected by SK and S1P (Lee et al, 1999).…”

Antitumor Activity of Sphingosine Kinase Inhibitors

“…SK has also been linked to estrogen signaling (Sukocheva et al, 2003) and estrogen-dependent tumorigenesis in MCF-7 cells (Nava et al, 2002). Other pathways or targets to which SK activity has been linked in cancer include vascular endothelial growth factor signaling, including the Ras and MAPK pathway (Shu et al, 2002), protein kinase C (Nakade et al, 2003), tumor necrosis factor ␣ (Vann et al, 2002), hepatocyte nuclear factor 1 and retinoic acid receptor ␣ (Osawa et al, 2001b), intracellular calcium (Wheldon et al, 2001), and caspase activation (Edsall et al, 2001). Angiogenic factors and processes, such as cell motility, mitogenesis in smooth muscle cells, endothelial cell differentiation, and growth factor signaling, are also affected by SK and S1P (Lee et al, 1999).…”

Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression

“…30,[32][33][34]36,43 In For personal use only. on April 29, 2019. by guest www.bloodjournal.org From the spi-1-transgenic proerythroblasts cultured under stress conditions, the activations of the PI3K/AKT and ERK pathways were abrogated by the SPHK1 inhibitor.…”