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.

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.

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.

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 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.

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.

Identification of ceramide targets in interleukin-1 and tumor necrosis factor-alpha signaling in mesangial cells.

An increasing number of cell-surface receptors have been shown to trigger sphingomyelin turnover and generation of the lipid signaling molecule ceramide. Ceramide plays a role in mediating cellular responses as diverse as inflammation, differentiation, gene expression, growth suppression, and apoptosis. A radioiodinated, photoaffinity-labeling analog of ceramide ([125I]TID-ceramide) was used to identify downstream signaling targets of ceramide. Ceramide was found to bind specifically to and activate protein kinase c-Raf, leading to subsequent activation of the extracellular signal-regulated kinase (ERK) module in mesangial cells. We found also that ceramide binds to and differentially modulates the activity of distinct protein kinase C isoenzymes. These data are discussed in the context of interleukin 1beta-induced inflammatory gene expression in mesangial cells.

Feedback regulation of extracellular ATP-stimulated phosphoinositide hydrolysis by protein kinase C-alpha in bovine glomerular endothelial cells.

In glomerular endothelial cells, extracellular ATP stimulates a phospholipase C with subsequent hydrolysis of polyphosphoinositides and an increase in cytosolic free Ca2+ concentration ([Ca2+]i). Short-term (30 min) pretreatment of endothelial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C (PKC), decreases the ATP-stimulated phosphoinositide degradation and Ca2+ mobilization. However, this inhibition was lost after incubating the cells for four hours with TPA. Longer-term pretreatment (10 to 48 hr) even potentiated ATP-induced phosphoinositide breakdown and Ca2+ mobilization. In addition, pretreating the cells for 30 minutes with the specific PKC inhibitor Ro 31-8220 dose-dependently increased ATP-stimulated phosphoinositide hydrolysis, thus clearly indicating a regulatory role for PKC in the inositol lipid signaling pathway in glomerular endothelial cells. By using specific antibodies recognizing the different PKC isoenzymes, it is observed that glomerular endothelial cells express five isoenzymes: PKC-alpha, -delta, -epsilon, -zeta and -theta. No PKC-beta, -gamma, -eta and -mu isoenzymes were detected. On exposure to TPA, a complete depletion of PKC-alpha is observed within four hours. In contrast, PKC-epsilon was more resistant to phorbol ester, and even after 48 hours of TPA treatment, only 60% of PKC-epsilon was down-regulated. PKC-theta decreased very slowly from the cytosol (47% left after 24 hr of phorbol ester treatment) and translocated to the Triton X100-insoluble fraction. Moreover, PKC-delta and PKC-zeta were not significantly affected by 48 hours of phorbol ester incubation. Thus, only PKC-alpha is depleted with a kinetic that corresponds to the loss of feedback inhibition of ATP-stimulated phosphoinositide turnover. In the next step, [Ca2+]i changes were measured in single cells loaded with Fura-2 after microinjection of neutralizing PKC isoenzyme-specific antibodies. Injection of antibodies specific for PKC-alpha potently increased Ca2+ mobilization in response to ATP stimulation when compared to cells injected with buffer only or antibodies specific for PKC-epsilon. These results provide evidence that PKC-alpha mediates feedback inhibition of ATP-stimulated phosphoinositide hydrolysis in glomerular endothelial cells.

Activation of protein kinase C subtypes alpha, gamma, delta, epsilon, zeta, and eta by tumor-promoting and nontumor-promoting agents.

Protein kinase C (PKC) subtypes alpha, gamma, delta, epsilon, zeta, and eta have been expressed using the baculovirus expression system. The partially purified PKC subtypes have been studied for their substrate specificities and phospholipid-independent activation by various chemically different nontumor- and tumor-promoting agents, as well as their inhibition of kinase activity by staurosporine and two related compounds. An endogenous PKC-like kinase activity of Sf9 cells was detected and analyzed for cofactor requirements and inhibition. Protamine sulfate was most efficiently phosphorylated by all of the PKC subtypes tested, although this phosphorylation was independent of phosphatidylserine (PS) and diacylglycerol (DAG) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Except for PKC-zeta, all subtypes tested phosphorylated myelin basic protein (MBP), histone, or a peptide derived from the pseudosubstrate region of PKC-alpha in a PS/DAG-dependent manner but to varying extents. Among the various agents tested, TPA most efficiently stimulated the kinase activities of the PKC subtypes in a phospholipid-dependent manner. Phorbol 12,13-dibutyrate (PDBu) was less effective than TPA but displayed no major difference among the subtypes. Activation of PKC-alpha by bryostatin-1 reached only half of the TPA response whereas the other subtypes were activated more effectively. The weak tumor promoter resiniferonol 9,13,14-orthophenyl acetate (ROPA) mainly stimulated PKC-alpha and PKC-gamma at 1 microM concentration, whereas PKC-epsilon and PKC-eta were much less activated. Sapintoxin D, mezerein, indolactam V, and resiniferatoxin at concentrations of 1-100 nM preferentially activated PKC-alpha in a DAG-like manner, whereas at 1 microM other subtypes were activated as well. Preferential activation of PKC-alpha was also noted for tinyatoxin and thapsigargin, but their mode of activation is unclear because these two compounds did not compete for the phorbol ester binding of the PKC subtypes as the other agents did. Of the three PKC inhibitors tested, staurosporine most efficiently inhibited kinase activity of the PKC subtypes, whereas K252a and CGP 41251 were at least 10 times less effective. However, K252a showed certain specificity for inhibition of PKC-alpha, and CGP 41251 failed to inhibit PKC-epsilon and PKC-zeta. Given the different substrate specificities and modes of activation by various tumor-promoting and nontumor-promoting agents, as well as the different sensitivities towards different inhibitors, our results indicate a divergence of individual PKC subtypes in signal transduction.

Stimulation by extracellular ATP and UTP of the stress-activated protein kinase cascade in rat renal mesangial cells.

1. Extracellular adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) have been shown to activate a nucleotide receptor (P2U receptor) in rat mesangial cells that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. This is followed by an increased activity of the mitogen-activated protein kinase cascade and cell proliferation. Here we show that ATP and UTP potently stimulate the stress-activated protein kinase pathway and phosphorylation of the transcription factor c-Jun. 2. Both nucleotides stimulated a rapid (within 5 min) and concentration-dependent activation of stress-activated protein kinases as measured by the phosphorylation of c-Jun in a solid phase kinase assay. 3. When added at 100 microM the rank order of potency of a series of nucleotide analogues for stimulation of c-Jun phosphorylation was UTP > ATP = UDP = ATP gamma S > 2-methylthio-ATP > beta gamma-imido-ATP = ADP > AMP = UMP = adenosine = uridine. Activation of stress-activated protein kinase activity by ATP and UTP was dose-dependently attenuated by suramin. 4. Down-regulation of protein kinase C-alpha, -delta and -epsilon isoenzymes by 24 h treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate did not inhibit ATP- and UTP-induced activation of c-Jun phosphorylation. Furthermore, the specific protein kinase C inhibitors, CGP 41251 and Ro 31-8220, did not inhibit nucleotide-stimulated c-Jun phosphorylation, suggesting that protein kinase C is not involved in ATP- and UTP-triggered stress-activated protein kinase activation. 5. Pretreatment of the cells with pertussis toxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP- and UTP-induced c-Jun phosphorylation. Furthermore, N-acetyl-cysteine completely blocked the activation of stress-activated protein kinase in response to extracellular nucleotide stimulation. 6. In summary, these results suggest that ATP and UTP trigger the activation of the stress-activated protein kinase module in mesangial cells by a pathway independent of protein kinase C but requiring a pertussis toxin-sensitive G-protein and tyrosine kinase activation.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1beta- and cAMP-induced nitric oxide synthase expression in rat renal mesangial cells.

Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) regulate mesangial cell proliferation and matrix production in vitro and in vivo and crucially participate in the pathogenesis of glomerulonephritis. We investigated whether PDGF-BB and bFGF influence nitric oxide (NO) production, another important effector molecule in inflammatory glomerular injury. Inducible NO synthase (iNOS) induction in rat glomerular mesangial cells has been described in response to two principal classes of activating signals comprising inflammatory cytokines such as interleukin 1beta (IL-1beta) or elevation of cyclic AMP (cAMP). Treatment of mesangial cells with IL-1beta induces iNOS activity measured as nitrite levels in cell culture supernatants. Coincubation of mesangial cells with PDGF-BB inhibits production of nitrite by approximately 95%. This effect can be reversed by the simultaneous incubation of PDGF-BB in the presence of calphostin C, a potent and selective inhibitor of protein kinase C. In contrast, incubation of cells in the presence of bFGF potentiates IL-1beta-induced production of NO and is functionally associated with an increased rate of apoptosis of mesangial cells. Western blot analyses reveal that PDGF-BB causes a decrease in the formation of iNOS protein which is preceded by decreases in iNOS mRNA steady state levels. bFGF drastically increases iNOS protein levels as well as the corresponding iNOS mRNA steady state levels. Nuclear run-on experiments reveal that PDGF-BB decreases the IL-1beta-induced transcription rate of the iNOS gene, whereas bFGF potentiates the transcriptional activity of the iNOS gene. Northern blot analyses demonstrate that bFGF strongly potentiates the formation of IL-1beta-induced IL-1 type I receptor mRNA levels, whereas PDGF-BB has no effect. Treatment of mesangial cells with the membrane-permeable cAMP analogue N6, O-2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP) markedly increases the production of nitrite. Whereas PDGF-BB does not affect cAMP-induced nitrite levels, bFGF strongly potentiates them. PDGF-BB alters neither cAMP-induced iNOS protein levels nor the corresponding iNOS mRNA steady state levels. By contrast, bFGF superinduces cAMP-stimulated iNOS protein and iNOS mRNA levels. These changes by bFGF are due to an increase in cAMP-induced transcriptional activity of the iNOS gene which is not affected by PDGF-BB. In summary, the results show that PDGF and bFGF differentially regulate iNOS expression in mesangial cells in a stimulus-specific way. The timely sequence of expression of PDGF and bFGF and of cytokines like IL-1 will crucially determine the amounts of NO produced and the functional consequences thereof in the course of progressive glomerular diseases.

Regulatory functions of protein kinase C isoenzymes in purinoceptor signalling in mesangial cells.

1. The expression and functional roles of protein kinase C (PKC) isoenzymes in purinoceptor signalling have been examined in rat renal mesangial cells. 2. It is observed that rat mesangial cells express four PKC isoenzymes, PKC-alpha, -delta, -epsilon and zeta, as determined by Western blot analysis. No PKC-beta, -gamma, -eta, -theta, or -mu isoforms were detected. 3. By using specific PKC inhibitors and down-regulation experiments we provide evidence that PKC alpha acts as a negative feedback regulator of ATP- and UTP-stimulated phosphoinositide turnover, whereas PKC epsilon triggers arachidonic acid release and subsequent prostaglandin synthesis and stimulates a phosphatidylcholine-hydrolysing phospholipase D. Moreover, PKC delta may activate the mitogen-activated protein kinase cascade and thus promote mesangial cell proliferation in response to extracellular ATP and UTP. 4. In summary our data identify mesangial cells in culture as an excellently characterized cell culture system with well-defined functions of PKC isoenzymes. Functional identification of PKC isoenzymes involved in specific cell responses is one of the most promising steps towards understanding of molecular mechanisms of cell regulation and identifies new targets for drug development.

Nitric oxide stimulates stress-activated protein kinases in glomerular endothelial and mesangial cells.

Exposure of rat glomerular mesangial cells and primary cultures of bovine glomerular endothelial cells to compounds releasing nitric oxide (NO), including MAHMA-NONOate, S-nitrosoglutathione, and spermine-NO, results in a time- and concentration-dependent activation of stress-activated protein kinases (SAPK) as measured by the phosphorylation of c-Jun in a solid phase kinase assay. Dibutyryl cGMP had no effect on SAPK activity. Pretreatment of the cells with the tyrosine kinase inhibitor genistein strongly attenuated NO-induced c-Jun phosphorylation. Furthermore, N-acetylcysteine markedly reduced the activation of SAPK in response to NO. These studies identify SAPK as a target for NO which may be critical for the NO-induced apoptosis of glomerular mesangial and endothelial cells.

Ceramide-binding and activation defines protein kinase c-Raf as a ceramide-activated protein kinase.

Interleukin 1 is the prototype of an inflammatory cytokine, and evidence suggests that it uses the sphingomyelin pathway and ceramide production to trigger mitogen-activated protein kinase (MAPK) activation and subsequent gene expression required for acute inflammatory processes. To identify downstream signaling targets of ceramide, a radioiodinated photoaffinity labeling analog of ceramide ([125I] 3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramide) was employed. It is observed that ceramide specifically binds to and activates protein kinase c-Raf, leading to a subsequent activation of the MAPK cascade. Ceramide does not bind to any other member of the MAPK module nor does it bind to protein kinase C-zeta. These data identify protein kinase c-Raf as a specific molecular target for interleukin 1 beta-stimulated ceramide formation and demonstrate that ceramide is a lipid cofactor participating in regulation of c-Raf activity.

Modulatory role of platelet-derived growth factor on cytokine-induced nerve growth factor synthesis in rat glomerular mesangial cells.

Recent evidence indicates that cytokines are potent inducers of nerve growth factor (NGF) expression in peripheral tissues and in brain. Cultured rat glomerular mesangial cells respond to interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) by increased NGF synthesis. We found that co-stimulation of rat glomerular mesangial cells with platelet-derived growth factor (PGDF-BB) and IL-1 beta/TNF-alpha significantly augments the IL-1 beta/TNF-alpha-induced NGF mRNA levels and NGF synthesis. In contrast, preincubation with PDGF-BB drastically reduces NGF gene expression and NGF protein synthesis in response to IL-1 beta/TNF-alpha stimulation. Thus our results indicate that PDGF-BB is a potent modulator of cytokine-induced NGF expression; its precise action is critically depending on the time at which the PDGF receptor is activated.

Translocation of protein kinase C isoenzymes by elevated extracellular Ca2+ concentration in cells from a human giant cell tumor of bone.

In this study we investigated the protein kinase C isoenzymes expressed by human osteoclast-like cells harvested from a giant cell tumor of bone (GCT23 cells), and by freshly isolated rat osteoclasts. Immunoblotting analysis revealed that the -alpha, -delta, and -epsilon, PKC isoforms, but not the -beta isoenzyme, are expressed by GCT23 cells. Immunofluorescence studies demonstrated that PKC-alpha, -delta, and -epsilon are homogeneously expressed by both mononuclear and multinucleated GCT23 cells, as well as by rat osteoclasts. Similar to authentic osteoclasts, GCT23 cells responded to an increase of extracellular Ca2+ concentration ([Ca2+]o) with a dose-dependent elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). An increase of [Ca2+]o stimulated the translocation of PKC-alpha from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca2+]o sensing. By contrast, PKC-delta was not altered by exposure to elevated [Ca2+]o, whereas PKC-epsilon underwent reciprocal translocation, disappearing from the insoluble fraction and increasing in the cytosol. The effects of PKC on GCT23 cell functions were investigated by treatment with phorbol 12-myristate, 13-acetate (PMA). We observed that activation of PKC by PMA failed to affect adhesion onto the substrate, but down-regulated the [Ca2+]o-induced [Ca2+]i increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.