Phosphorylation of a constrained azacyclic FTY720 analog enhances anti-leukemic activity without inducing S1P receptor activation.

The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720's anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.

Dual role of sphingosine kinase-1 in promoting the differentiation of dermal fibroblasts and the dissemination of melanoma cells.

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

Thiazolidinedione-dependent activation of sphingosine kinase 1 causes an anti-fibrotic effect in renal mesangial cells.

BACKGROUND AND PURPOSE: PPARγ agonists [thiazolidinediones (TZDs)] are known to exert anti-fibrotic effects in the kidney. In addition, we previously demonstrated that sphingosine kinase 1 (SK-1) and intracellular sphingosine-1-phosphate (S1P), by reducing the expression of connective tissue growth factor (CTGF), have a protective role in the fibrotic process. EXPERIMENTAL APPROACH: Here, we investigated the effect of TZDs on intracellular sphingolipid levels and the transcriptional regulation of SK-1 in mesangial cells to evaluate potential novel aspects of the anti-fibrotic capacity of TZDs. KEY RESULTS: Stimulation with the TZDs, troglitazone and rosiglitazone, led to increased S1P levels in rat mesangial cells. This was paralleled by increased SK-1 activity as a consequence of direct effects of the TZDs on SK-1 expression. GW-9662, a PPARγ antagonist, inhibited the stimulating effect of TZDs on SK-1 mRNA and activity levels and intracellular S1P concentrations. Furthermore, SK-1 up-regulation by TZDs was functionally coupled with lower amounts of pro-fibrotic CTGF. SK-1 inhibition with SKI II almost completely abolished this effect in a dose-dependent manner. Moreover, the CTGF lowering effect of TZDs was fully blocked in MC isolated from SK-1 deficient mice (SK-1(-/-) ) as well as in glomeruli of SK-1(-/-) mice compared with wild-type mice treated with TRO and RSG. CONCLUSION AND IMPLICATIONS: These data show that TZD-induced SK-1 up-regulation results in lower amounts of CTGF, demonstrating novel facets for the anti-fibrotic effects of this class of drugs.

On-chip non-invasive and label-free cell discrimination by impedance spectroscopy.

OBJECTIVES: Many flow-cytometric cell characterization methods require costly markers and colour reagents. We present here a novel device for cell discrimination based on impedance measurement of electrical cell properties in a microfluidic chip, without the need of extensive sample preparation steps and the requirement of labelling dyes. MATERIALS AND METHODS, RESULTS: We demonstrate that in-flow single cell measurements in our microchip allow for discrimination of various cell line types, such as undifferentiated mouse fibroblasts 3T3-L1 and adipocytes on the one hand, or human monocytes and in vitro differentiated dendritic cells and macrophages on the other hand. In addition, viability and apoptosis analyses were carried out successfully for Jurkat cell models. Studies on several species, including bacteria or fungi, demonstrate not only the capability to enumerate these cells, but also show that even other microbiological life cycle phases can be visualized. CONCLUSIONS: These results underline the potential of impedance spectroscopy flow cytometry as a valuable complement to other known cytometers and cell detection systems.

FTY720 suppresses interleukin-1beta-induced secretory phospholipase A2 expression in renal mesangial cells by a transcriptional mechanism.

BACKGROUND AND PURPOSE: FTY720 is a potent immunomodulatory prodrug that is converted to its active phosphorylated form by a sphingosine kinase. Here we have studied whether FTY720 mimicked the action of sphingosine-1-phosphate (S1P) and exerted an anti-inflammatory potential in renal mesangial cells. EXPERIMENTAL APPROACH: Prostaglandin E(2) (PGE(2)) was quantified by an enzyme-linked immunosorbent-assay. Secretory phospholipase A(2) (sPLA(2)) protein was detected by Western blot analyses. mRNA expression was determined by Northern blot analysis and sPLA(2)-promoter activity was measured by a luciferase-reporter-gene assay. KEY RESULTS: Stimulation of cells for 24 h with interleukin-1beta (IL-1beta) is known to trigger increased PGE(2) formation which coincides with an induction of the mRNA for group-IIA-sPLA(2) and protein expression. FTY720 dose-dependently suppressed IL-1beta-induced IIA-sPLA(2) protein secretion and activity in the supernatant. This effect is due to a suppression of cytokine-induced sPLA(2) mRNA expression which results from a reduced promoter activity. As a consequence of suppressed sPLA(2) activity, PGE(2) formation is also reduced by FTY720. Mechanistically, the FTY720-suppressed sPLA(2) expression results from an activation of the TGFbeta/Smad signalling cascade since inhibition of the TGFbeta receptor type I by a specific kinase inhibitor reverses the FTY720-mediated decrease of sPLA(2) protein expression and sPLA(2) promoter activity. CONCLUSIONS AND IMPLICATIONS: In summary, our data show that FTY720 was able to mimic the anti-inflammatory activity of TGFbeta and blocked cytokine-triggered sPLA(2) expression and subsequent PGE(2) formation. Thus, FTY720 may exert additional in vivo effects besides the well reported immunomodulation and its anti-inflammatory potential should be considered.

Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase-1 expression and activity.

BACKGROUND AND PURPOSE: Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved. EXPERIMENTAL APPROACH: MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate. KEY RESULTS: Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration. CONCLUSIONS AND IMPLICATIONS: These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.

Nitric oxide and mechanisms of redox signalling: matrix and matrix-metabolizing enzymes as prime nitric oxide targets.

One of the greatest biomedical breakthroughs of the twentieth century was the discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO). NO has received special attention ever since: besides its potent vasodilatory and vasoprotective effects, NO was identified as a key player in innate immunity and was found to act as an unconventional type of neurotransmitter. This article focuses on mechanisms of NO signalling that form the basis of functional cell responses to accommodate changes in the cellular microenvironment. Redox-based regulation of signal transduction and, on a more long-term scale, changes in gene expression will be exemplified by NO-modulation of matrix components and matrix-metabolizing enzymes. It seems to be a safe bet that ongoing analyses of NO signalling and gene expression will provide a wealth of promising therapeutic targets in human diseases.

Interleukin-1beta induces chronic activation and de novo synthesis of neutral ceramidase in renal mesangial cells.

The lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163-166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1beta treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1beta treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1beta and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1beta-stimulated neutral ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novo synthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions.

Superoxide potently induces ceramide formation in glomerular endothelial cells.

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2-30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.

Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release.

Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short-chain ceramide analogs for 30 and 60 min leads to a concentration-dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen-activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide-induced arachidonic acid release. By use of photoactivatable ceramide analogs, D- and L-[125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramides (TID-ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D- and L-TID-ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting 'hinge' domain of cPLA2, efficiently bound D- and L-TID-ceramides, whereas the catalytic domain did not interact with TID-ceramides. In vitro binding assays reveal that stearoyl-arachidonyl-phosphatidylcholine (SAPC)-liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC-liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates cPLA2 action in inflammatory diseases.

Amplification of IL-1 beta-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-kappa B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways.

The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1beta-induced matrix metalloproteinase (MMP-9) expression in glomerular mesangial cells (MC). Here we report that exogenously administrated superoxide, generated by the hypoxanthine/xanthine oxidase system (HXXO) or by the redox cycler 2, 3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of IL-1beta-primed, steady state, MMP-9 mRNA level and an increase in gelatinolytic activity in the conditioned medium. Superoxide generators alone were ineffective. Cytokine-induced steady state mRNA levels of TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the 5'-flanking region of the rat MMP-9 gene proved a transcriptional regulation of MMP-9 expression by superoxide. HXXO augmented the IL-1beta-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-kappaB and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1beta. Moreover, specific inhibitors of the classical extracellular signal-regulated kinase (ERK) pathway and p38 mitogen-activated protein kinase (MAPK) cascade, partially reversed the HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore, IL-1beta-triggered phosphorylation of all three MAPKs, including p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced by superoxide. Our data identify superoxide as a costimulatory factor amplifying cytokine-induced MMP-9 expression by interfering with the signaling cascades leading to the activation of AP-1 and NF-kappaB.

Nitric oxide augments release of chemokines from monocytic U937 cells: modulation by anti-inflammatory pathways.

Nitric oxide (NO) appears to act as an inflammatory mediator on monocytic cells. Exogenous NO augmented release of chemokines from human promonocytic U937 cells and peripheral blood mononuclear cells. Pharmacological strategies aiming at modulation of NO-induced release of interleukin-8 (IL-8) were investigated in U937 cells in detail. Release of IL-8 was down-regulated by transforming growth factor beta2 (TGF-beta2), by the protein tyrosine-kinase inhibitor genistein, and via rises in intracellular cyclic AMP, generated by prostaglandin E(2), rolipram, pentoxifylline, forskolin, or dibutyryl-cyclic AMP. In addition, incubation with the synthetic glucocorticoid dexamethasone or suppression of activity of p38 mitogen-activated protein (MAP) kinases by SB-203580 modulated release of IL-8. Activation of p38 MAP kinases was confirmed by the demonstration of an augmented appearance of phosphorylated p38 in the presence of NO. The present data suggest that exposure to exogenous NO resembles activation of U937 cells by proinflammatory stimuli. The anti-inflammatory cytokine TGF-beta2, as well as anti-inflammatory or immunosuppressive agents such as genistein, pentoxifylline, rolipram, dexamethasone, and SB-203580 modulate inflammatory, chemokine-inducing actions of NO.

Extracellular nucleotides activate the p38-stress-activated protein kinase cascade in glomerular mesangial cells.

1. Extracellular ATP and UTP have been reported to activate a nucleotide receptor (P2Y2-receptor) that mediates arachidonic acid release with subsequent prostaglandin formation, a reaction critically depending on the activity of a cytosolic phospholipase A2. In addition, extracellular nucleotides trigger activation of the classical mitogen-activated protein kinase (MAPK) cascade and cell proliferation as well as of the stress-activated protein kinase (SAPK) cascade. 2. In this study, we report that ATP and UTP are also able to activate the p38-MAPK pathway as measured by phosphorylation of the p38-MAPK and its upstream activators MKK3/6, as well as phosphorylation of the transcription factor ATF2 in a immunocomplex-kinase assay. 3. Time courses reveal that ATP and UTP induce a rapid and transient activation of the p38-MAPK activity with a maximal activation after 5 min of stimulation which declined to control levels over the next 20 min. 4. A series of ATP and UPT analogues were tested for their ability to stimulate p38-MAPK activity. UTP and ATP were very effective analogues to activate p38-MAPK, whereas ADP and gamma-thio-ATP had only moderate activating effects. 2-Methyl-thio-ATP, beta gamma-imido-ATP, AMP, adenosine and UDP had no significant effects of p38-MAPK activity. In addition, the extracellular nucleotide-mediated effect on p38-MAPK was almost completely blocked by 1 mM of suramin, a putative P2-purinoceptor antagonist. 5. In summary, these results demonstrate for the first time that extracellular nucleotides are able to activate the MKK3/6- p38-MAPK cascade most likely via the P2Y2-receptor. Moreover, this finding implies that all three MAPK subtypes are signalling candidates for extracellular nucleotide-stimulated cell responses.

Nitric oxide stimulates chronic ceramide formation in glomerular endothelial cells.

Exposure of glomerular endothelial cells for 24 h to compounds releasing NO, including spermine-NO, MAHMA-NO, and S-nitroso-glutathione, results in a dose-dependent and delayed (after 24 h) increase in the lipid signaling molecule ceramide. This NO-induced stimulation occurs in a cGMP-independent fashion since the membrane-permeant cGMP analogue dibutyryl cGMP has no effect on chronic ceramide production. Short-term incubation of endothelial cells for 20 min reveals that NO and dibutyryl cGMP fail to stimulate an acute ceramide increase, whereas TNF-alpha, a well-known activator of sphingomyelinases, is able to acutely increase ceramide formation. Interestingly, N-oleoylethanolamine, an acidic ceramidase inhibitor, potentiates NO-induced chronic ceramide production, indicating that ceramide generation rather than ceramide metabolism is modulated by NO. Furthermore, NO-induced delayed ceramide formation is partially inhibited by the thiol-specific inhibitor iodoacetamide and the radical scavenger alpha-tocopherol, suggesting a regulatory role of thiol-containing enzymes and the involvement of a redox-sensitive mechanism. In addition, NO causes an increased DNA fragmentation in glomerular endothelial cells which is further enhanced by N-oleoylethanolamine and can be mimicked by exogenous ceramide. In summary, these results imply that ceramide represents an important mediator of NO-triggered chronic cell responses like apoptosis. Inhibition of ceramide synthesis may provide a new therapeutic approach to the treatment of pathological conditions involving increased NO formation.

Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells.

Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.

Inducible NO synthase: role in cellular signalling.

The discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO) was one of the most exciting discoveries of biomedical research in the 1980s. Besides its potent vasodilatory effects, NO was found under certain circumstances to be responsible for the killing of microorganisms and tumour cells by activated macrophages and to act as a novel, unconventional type of neurotransmitter. In 1992, Science picked NO as the 'Molecule of the Year', and over the past years NO has become established as a universal intercellular messenger that acutely affects important signalling pathways and, on a more long-term scale, modulates gene expression in target cells. These actions will form the focus of the present review.

Nitric oxide stimulates the stress-activated protein kinase p38 in rat renal mesangial cells.

Nitric oxide (NO) has gained increased attention as a diffusible universal messenger that plays a crucial role in the pathogenesis of inflammatory and autoimmune diseases. Recently, we reported that exogenous NO is able to activate the stress-activated protein kinase (SAPK) cascade in mesangial cells. Here, we demonstrate that exposure of glomerular mesangial cells to compounds releasing NO, including spermine-NO and (Z)-1- (N-methyl-N-[6-(N-methylammoniohexyl)amino]diazen)-1-ium-1,2-diolate (MAHMA-NO), results in an activation of the stress-activated p38-mitogen-activated protein kinase (p38-MAPK) cascade as measured by the phosphorylation of the activator of transcription factor-2 (ATF2) in an immunocomplex kinase assay. Activation of the p38-MAPK cascade by a short stimulation (10 min) with the NO donor MAHMA-NO causes a large increase in ATF2 phosphorylation that is several times greater than that observed after stimulation with interleukin-1beta, a well-known activator of the p38-MAPK pathway. Time course studies reveal that MAHMA-NO causes rapid and maximal activation of p38-MAPK after 10 min of stimulation and that activation declines to basal levels within 60 min. The longer-lived NO donor spermine-NO causes a comparable rapid activation of the p38-MAPK pathway; however, the increased activation state of p38-MAPK was maintained for several hours before control values were reattained after 24 h of stimulation. Furthermore, the NO donors also activated the classical extracellular signal-regulated kinase (ERK) p44-MAPK cascade as shown by phosphorylation of the specific substrate cytosolic phospholipase A2 in an immunocomplex kinase reaction. Both MAHMA-NO and spermine-NO cause a rapid activation of p44-MAPK after 10 min of stimulation. Interestingly, there is a second delayed peak of p44-MAPK activation after 4-24 h of stimulation with NO donors. These results suggest that there is a differential activation pattern for stress-activated and mitogen-activated protein kinases by NO and that the integration of these signals may lead to specific cell responses.

Negative regulation of interleukin-1beta-activated neutral sphingomyelinase by protein kinase C in rat mesangial cells.

Endogenous ceramide is produced by the action of acidic or neutral sphingomyelinases (SMase) in response to stimuli such as proinflammatory cytokines or other inducers of stress. Interleukin-1beta (IL-1beta) is known to stimulate ceramide formation in rat renal mesangial cells; however, the respective subtype of SMase and its regulation have not been investigated. We found that IL-1beta induced an increase in endogenous ceramide levels via the action of a neutral SMase but not an acidic SMase in rat mesangial cells. Cytokine-induced activation of neutral SMase was inhibited by stimulation of protein kinase C (PKC) by the phorbol ester TPA which caused a reduction of ceramide back to control levels. This inhibitory effect of TPA was reversed by the specific PKC-inhibitor Ro-318220. Long-term incubation (24 h) of mesangial cells with TPA, which downregulates PKC-alpha, -delta, and -epsilon isoenzymes, resulted in a recovery of IL-1beta-stimulated neutral SMase activity as well as ceramide formation. These data implicate an important modulatory function of PKC in ceramide production in IL-1beta-activated mesangial cells.

Protein kinase C phosphorylates and activates GTP cyclohydrolase I in rat renal mesangial cells.

GTP cyclohydrolase I is the rate-limiting enzyme in the de novo synthesis pathway of tetrahydrobiopterin which is an essential cofactor for all NO synthase isoforms. The expression of GTP cyclohydrolase I is regulated on a transcriptional level by a variety of cytokines like interleukin 1beta or tumor necrosis factor alpha. The present paper reports that stimulation of protein kinase C by angiotensin II, platelet-derived growth factor BB or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate triggers the phosphorylation and activation of GTP cyclohydrolase I. These data establish that in addition to transcriptional regulation, there is a prominent post-transcriptional modulation of enzyme activity.