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.     

The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide-mediated expression of inducible nitric oxide synthase

Lipopolysaccharide (LPS) and interferon-gamma (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C8-ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-kappaB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C8-ceramide. As the dominant negative ras also reduced NF-kappaB transactivity, NF-kappaB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-kappaB activation under inflammatory conditions.     

Immunomodulatory cytokines suppress epithelial nitric oxide production in inflammatory bowel disease by acting on mononuclear cells

Inducible nitric oxide synthase (iNOS) activity in colonic epithelial HT-29 cells is modulated by the T-cell-derived cytokines IL-4 and IL-13, but is not affected by IL-10 despite its effect in models of colitis. We studied the effects of these cytokines on nitric oxide (NO) production by colonic tissue. IL-10 and IL-4 but not IL-13 suppressed the NO production and iNOS expression by inflamed tissue and cytokine-stimulated noninflamed tissue from patients with ulcerative colitis, whereas the three cytokines suppressed NO production in cytokine-stimulated biopsies from controls. To examine why colonic biopsies and HT-29 cells respond differently to immunomodulatory cytokines, a coculture of mixed mononuclear monocytes (MMC) and HT-29 cells was studied. Treatment of HT-29 cells with conditioned medium from IFN-γ/LPS-stimulated MMC produced significant amounts of NO, which suggested the presence of an MMC-derived soluble factor modifying epithelial NO production. Pretreatment of IFN-γ/LPS-stimulated MMC with IL-10 and IL-4 but not IL-13 suppressed NO production by HT-29 cells. Interestingly, pretreatment of HT-29 cells with IL-1 receptor antagonist suppressed the IFN-γ/LPS-stimulated MMC-induced NO production. These results suggest that immunomodulatory cytokines might exert an inhibitory effect on NO up-regulation by colonic epithelium via the inhibition of MMC-derived soluble mediators, such as IL-1.     

Induction of MRP1 and gamma-glutamylcysteine synthetase gene expression by interleukin 1beta is mediated by nitric oxide-related signalings in human colorectal cancer cells

Treatment of human colorectal cancer cells HT29 with interleukin 1beta (IL-1beta) induces expression of the multidrug resistance protein (MRP1) gene encoding the ATP-dependent glutathione S-conjugate export (GS-X) pump and the gamma-glutamylcysteine synthetase (gamma-GCSh) gene encoding heavy (catalytic) subunit of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the biosynthesis of glutathione (GSH). The induction can be suppressed by N(G)-methyl-L-arginine, a specific inhibitor of nitric oxide synthase (NOS). These results suggest that IL-1beta-mediated MRP1 and gamma-GCSh induction involve nitric oxide (NO) -related signaling. Further supports to the involvement of NO in the induction of MRP1 and gamma-GCSh expression are made by the following observations. (i) Expression of MRP1 and gamma-GCSh genes were induced by treating the cells with NO donors, i.e., S-nitro-N-acetyl-D,L-penicillamide (SNAP) and S-nitroso-L-glutathione, in a concentration-dependent manner. (ii) Ectopic expression of inducible NOS (iNOS) activity by transfecting expressible recombinant iNOS cDNA encoding functional iNOS but not the nonfunctional version resulted in elevated expression of MRP1 and gamma-GCSh. We also demonstrated that HT-29 cells treated with either 1L-1beta or SNAP induced ceramide production, and addition of C2 or C6 ceramides into cultured HT-29 cells resulted in induction of gamma-GCSh but not MRP1 expression. Collectively, our results demonstrate that induction of MRP1 and gamma-GCSh by IL-1beta is regulated, at least in part, by an NO-related signaling, and induction of gamma-GCSh is by NO-related ceramide signaling.     

Ceramide induces the dephosphorylation and inhibition of constitutively activated Akt in PTEN negative U87mg cells

In the present study, treatment of the PTEN negative U87MG human glioblastoma cell line with C2-ceramide resulted in a dose- and time-dependent decrease in the constitutive phosphorylation of Akt at threonine 308 and serine 473. The C2-ceramide induced dephosphorylation of Akt correlated with a 90-95% reduction in the Akt kinase activity. Exposure to C2-ceramide did not affect the basal or PDGF activated levels PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3), indicating PI3-K activity was not inhibited. Additionally, treatment of cells with the PI3-K inhibitor wortmannin and C2-ceramide resulted in an enhanced rate of Akt dephosphorylation versus either agent alone. Finally, treatment of cells with the phosphatase inhibitors okadaic acid or calyculin A prevented the C2-ceramide induced dephosphorylation and inhibition of Akt activity. These data demonstrate the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implicate the activation of ceramide activated protein phosphatase, rather than decreased PI3-K activity, as the mechanism of inhibition.     

A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase

Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFalpha, sphingomyelinase, and C2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFalpha that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.     

Tumor Necrosis Factor-α Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.     

细胞渗透性神经酰胺可抑制大鼠离体黄体细胞甾体激素生成并诱导细胞凋亡

本文旨在观察神经酰胺对离体孵育的大鼠黄体细胞孕酮分泌及细胞凋亡的影响,以PMSG－hCG处理的雌性Wistar大鼠为模型,分离制备黄体细胞,将外源性细胞渗透性神经酰胺与黄体细胞共同孵育,分别用放免法和流式细胞仪分析神经酰胺对黄体细胞孕酮生成和凋亡的影响,同时还检测了一氧化氮合酶（NOS）活性和一氧化氮（NO）水平的变化,结果显示,神经酰胺可以剂量相关方式抑制hCG－诱导的孕酮分泌,而对基础孕酮没有显著影响,离体孵育12h的大鼠黄体细胞存在自发性凋亡,5umol/L神经酰胺能显著增加亡率（P＜0．05）,流式细胞仪分析可见增强的凋亡蜂,实验还发现,50umol/L神经酰胺能明显促进NOS活性（P＜0．01）和NO生成（P＜0．01）,结果提示,神经酰胺可能通过调节甾体激素生成和细胞凋亡而作为一种重要的信息分子参与黄体退化等卵巢的生理过程。     

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO₂ incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.     

On the presence of C2-ceramide in mammalian tissues: possible relationship to etherphospholipids and phosphorylation by ceramide kinase

C(2)-ceramide (N-acetyl-sphingenine) is often used as an analog to study ceramide-mediated cellular processes. According to Lee et al. [J. Biol. Chem. 271 (1996), 209-217], C(2)-ceramide is formed by an acetyl transfer from platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to sphingenine. To substantiate these unconfirmed findings, we (i) developed a method to quantify C(2)-ceramide and (ii) analyzed C(2)-ceramide levels in Pex5(-/-) mice, a model for Zellweger syndrome, in which the synthesis of ether lipids such as PAF is impaired. The presence of C(2)-ceramide could be established in brain (+/-10 pmol/g) and liver (+/-25 pmol/g) from control mice, and was approximately 5000-fold less than the main long-chain ceramide species. In Pex5(-/-) mice, C(2)-ceramide levels did not differ significantly compared to control tissues. Given the presence of a ceramide kinase in mammals, phosphorylation of C(2)-ceramide by human ceramide kinase (HsCERK) was tested. C(2)-ceramide appears to be a good substrate when albumin is used as carrier. In CHO cells overexpressing HsCERK, phosphorylation of exogenously added C(2)-ceramide could also be demonstrated. Our data indicate that C(2)-ceramide is present in mammalian tissues and can be converted to C(2)-ceramide-1-phosphate, in addition to other documented metabolic alterations, but does not seem to be linked to ether lipid metabolism.     

Amyloid-beta peptide enhances tumor necrosis factor-alpha-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimer's disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid-beta peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid-beta peptide 25-35 (Abeta) on proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha)-induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Abeta 25-35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF-alpha-induced iNOS expression and nitrite generation in OLGs. Abeta, TNF-alpha, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2-ceramide enhanced TNF-alpha-induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3-O-methyl-sphingomyelin (3-OMS), inhibited iNOS expression and nitrite production induced by TNF-alpha or by the combination of TNF-alpha and Abeta. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3-OMS also inhibited nuclear factor kappaB (NF-kappaB) binding activity induced by TNF-alpha or by the combination of TNF-alpha and Abeta. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF-alpha and the combination of TNF-alpha and Abeta.     

Immunomodulatory cytokines suppress epithelial nitric oxide production in inflammatory bowel disease by acting on mononuclear cells

Inducible nitric oxide synthase (iNOS) activity in colonic epithelial HT-29 cells is modulated by the T-cell-derived cytokines IL-4 and IL-13, but is not affected by IL-10 despite its effect in models of colitis. We studied the effects of these cytokines on nitric oxide (NO) production by colonic tissue. IL-10 and IL-4 but not IL-13 suppressed the NO production and iNOS expression by inflamed tissue and cytokine-stimulated noninflamed tissue from patients with ulcerative colitis, whereas the three cytokines suppressed NO production in cytokine-stimulated biopsies from controls. To examine why colonic biopsies and HT-29 cells respond differently to immunomodulatory cytokines, a coculture of mixed mononuclear monocytes (MMC) and HT-29 cells was studied. Treatment of HT-29 cells with conditioned medium from IFN-γ/LPS-stimulated MMC produced significant amounts of NO, which suggested the presence of an MMC-derived soluble factor modifying epithelial NO production. Pretreatment of IFN-γ/LPS-stimulated MMC with IL-10 and IL-4 but not IL-13 suppressed NO production by HT-29 cells. Interestingly, pretreatment of HT-29 cells with IL-1 receptor antagonist suppressed the IFN-γ/LPS-stimulated MMC-induced NO production. These results suggest that immunomodulatory cytokines might exert an inhibitory effect on NO up-regulation by colonic epithelium via the inhibition of MMC-derived soluble mediators, such as IL-1.     

Inhibitory effect of nitric oxide on the induction of cytochrome P450 3A4 mRNA by 1,25-dihydroxyvitamin D3 in Caco-2 cells

Cytochrome P450 (CYP)-dependent drug metabolism decreases in vivo and in cultured hepatocytes under various immunostimulatory conditions. Nitric oxide (NO) released during inflammation is presumed to be involved in this phenomenon. CYP3A4, which is abundant in the liver and small intestine and participates in the metabolism of various drugs, is known to be induced by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in the colon carcinoma cell line Caco-2. In this study we examined whether NO affected CYP3A4 gene expression induced by 1,25(OH)₂D₃ in Caco-2 cells. Induction of CYP3A4 mRNA by 1,25(OH)₂D₃ was suppressed in a dose-dependent manner by treatment with the NO donors NOR-4 (15–500 μM) or S-nitroso-N-acetyl-penicillamine (30 μM-1 mM), which spontaneously release NO. These results indicated that NO has an inhibitory effect on the induction of CYP3A4 mRNA by 1,25(OH)₂D₃ in Caco-2 cells. Treatment with the guanylate cyclase inhibitor ODQ failed to prevent the inhibition of induction of CYP3A4 mRNA by 1,25(OH)₂D₃. 8-Bromo cGMP had no effect on 1,25-(OH)₂D₃-induced CYP3A4 gene expression. Therefore, the suppression of CYP3A4 mRNA by NO might be mediated through a guanylate cyclase-independent pathway.     

Ceramide-induced inhibition of Akt is mediated through protein kinase Czeta: implications for growth arrest.

We recently demonstrated that ceramide-coated balloon catheters limit vascular smooth muscle cell (VSMC) growth after stretch injury in vivo. In that study, inhibition of VSMC growth was correlated with a decrease in phosphorylation of the cell survival kinase Akt (protein kinase B). Utilizing cultured A7r5 VSMCs, we have now examined the mechanism by which ceramide inhibits Akt phosphorylation/activation. Our initial studies showed that ceramide-induced inhibition of Akt phosphorylation was not mediated through diminution in phosphoinositide 3-kinase activity. As we have previously demonstrated that protein kinase Czeta (PKCzeta) is a target of ceramide, we proposed an alternative signaling mechanism by which ceramide induces inhibition of Akt through activation of PKCzeta. We demonstrate that C(6)-ceramide (but not the inactive analog dihydro-C(6)-ceramide) induced PKCzeta activity and also caused a selective increase in the association between Akt and PKCzeta, without affecting PKCepsilon, in A7r5 cells. In addition, the ability of ceramide to significantly decrease platelet-derived growth factor-induced Akt phosphorylation or cell proliferation was abrogated in A7r5 cells overexpressing a dominant-negative mutant of PKCzeta. Taken together, these data suggest that ceramide-mediated activation of PKCzeta leads to diminished Akt activation and consequent growth arrest in VSMCs. The therapeutic potential for ceramide to limit dysregulated VSMC growth has direct applicability to vascular diseases such as restenosis and atherosclerosis.     

Inhibition of proinflammatory cytokine-induced invasiveness of HT-29 cells by chitosan oligosaccharide

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Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium

Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny M, Liu J, Oh P, and Schnitzer JE, J Biol Chem 278: 4424-4430, 2003). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited N-SMase but not acid SMase activity, prevented mechanoactivation of N-SMase as well as downstream tyrosine and mitogen-activated protein kinases. Cell-permeable ceramide analogs (N-acetylsphingosine, C2-ceramide, and N-hexanoylsphingosine, C6-ceramide) but not the inactive dihydroderivatives D2-ceramide and D6-ceramide (N-acetylsphinganine and N-hexanoylsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins as well as mechanoactivation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3, which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induced serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger ceramide.