Mast cells link immune sensing to antigen-avoidance behaviour.

The physiological functions of mast cells remain largely an enigma. In the context of barrier damage, mast cells are integrated in type 2 immunity and, together with immunoglobulin E (IgE), promote allergic diseases. Allergic symptoms may, however, facilitate expulsion of allergens, toxins and parasites and trigger future antigen avoidance1-3. Here, we show that antigen-specific avoidance behaviour in inbred mice4,5 is critically dependent on mast cells; hence, we identify the immunological sensor cell linking antigen recognition to avoidance behaviour. Avoidance prevented antigen-driven adaptive, innate and mucosal immune activation and inflammation in the stomach and small intestine. Avoidance was IgE dependent, promoted by Th2 cytokines in the immunization phase and by IgE in the execution phase. Mucosal mast cells lining the stomach and small intestine rapidly sensed antigen ingestion. We interrogated potential signalling routes between mast cells and the brain using mutant mice, pharmacological inhibition, neural activity recordings and vagotomy. Inhibition of leukotriene synthesis impaired avoidance, but overall no single pathway interruption completely abrogated avoidance, indicating complex regulation. Collectively, the stage for antigen avoidance is set when adaptive immunity equips mast cells with IgE as a telltale of past immune responses. On subsequent antigen ingestion, mast cells signal termination of antigen intake. Prevention of immunopathology-causing, continuous and futile responses against per se innocuous antigens or of repeated ingestion of toxins through mast-cell-mediated antigen-avoidance behaviour may be an important arm of immunity.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Therapeutic silencing of SMOC2 prevents kidney function loss in mouse model of chronic kidney disease.

Chronic kidney disease (CKD) is associated with substantial morbidity and mortality. We developed a mouse model that mimics human CKD with inflammation, extracellular matrix deposition, tubulointerstitial fibrosis, increased proteinuria, and associated reduction in glomerular filtration rate over time. Using this model, we show that genetic deficiency of SMOC2 or therapeutic silencing of SMOC2 with small interfering RNAs (siRNAs) after disease onset significantly ameliorates inflammation, fibrosis, and kidney function loss. Mechanistically, we found that SMOC2 promotes fibroblast to myofibroblast differentiation by activation of diverse cellular signaling pathways including MAPKs, Smad, and Akt. Thus, targeting SMOC2 therapeutically offers an approach to prevent fibrosis progression and CKD after injury.

Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis.

MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle's loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -ß1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.

Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways.

MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti-miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti-miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-ß-induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy.

Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury.

Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased approach to identify genes in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rarefaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.

MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.

Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. To understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models and identified 24 commonly up-regulated miRNAs. Among them, miR-21 was highly elevated in both animal models and in human transplanted kidneys with nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21(-/-) mice suffered far less interstitial fibrosis in response to kidney injury, a phenotype duplicated in wild-type mice treated with anti-miR-21 oligonucleotides. Global derepression of miR-21 target mRNAs was readily detectable in miR-21(-/-) kidneys after injury. Analysis of gene expression profiles up-regulated in the absence of miR-21 identified groups of genes involved in metabolic pathways, including the lipid metabolism pathway regulated by peroxisome proliferator-activated receptor-α (Pparα), a direct miR-21 target. Overexpression of Pparα prevented ureteral obstruction-induced injury and fibrosis. Pparα deficiency abrogated the antifibrotic effect of anti-miR-21 oligonucleotides. miR-21 also regulated the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation Mpv17l was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for antifibrotic therapies.

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage.

BACKGROUND AND PURPOSE: Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved. EXPERIMENTAL APPROACH: Three human carcinoma cell lines (colon HCT-116, breast MDA-MB-231, lung NCI-H358) were used, which were either transduced with shRNA constructs to deplete SK-1, or treated with a SK-1 inhibitor. Cell growth and viability were assayed by [(3) H]thymidine incorporation and colony formation. Reactive oxygen species (ROS) were measured by fluorescence and apoptosis by annexin V with flow cytometry. Proteins were analysed by Western blotting. DNA damage was induced by doxorubicin. KEY RESULTS: Knock-down of SK-1 by shRNA strongly inhibited DNA synthesis and colony formation of carcinoma cells. SK-1 knock-down (SK-1kd) cells revealed dysfunctional extracellular signal-regulated protein kinase and PKB/Akt cascades, and contained increased levels of ROS. After SK-1kd, treatment with doxorubicin increased DNA damage, measured by histone-2AX phosphorylation. Similar effects were found in cells with a SK-1 inhibitor and doxorubicin. The increased damage response in SK-1kd cells was accompanied by greater reduction of DNA synthesis and colony formation, and by more pronounced apoptosis. Addition of a NADPH oxidase inhibitor reduced the increased apoptosis in doxorubicin-treated SK-1kd cells. CONCLUSIONS AND IMPLICATIONS: SK-1kd in carcinoma cells triggered oxidative stress by increasing intracellular Ros production. Targeted inhibition of SK-1 represents a promising approach to sensitize cells to DNA damage and facilitate apoptosis upon doxorubicin treatment.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation.

BACKGROUND: Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation. RESULTS: We show here that the previously developed putative SK-1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II) displays an additional facet of action complementary to the known inhibition of enzymatic SK-1 activity. In various human cell lines including glomerular podocytes and mesangial cells, the human endothelial cell line EA.hy 926, and the lung cancer cell line NCI H358, SKI II reduced TGFbeta- and TPA-stimulated cellular SK-1 activity by downregulating SK-1 protein expression without affecting SK-1 mRNA expression. By using cycloheximide to block the de novo protein synthesis, the protein expression of SK-1 under untreated conditions was stable over 24h. Under SKI II treatment, the half-live drastically decreased to approximately 0.8h. Mechanistically, this degradation occurred through a lysosomal pathway and involved cathepsin B since the general lysosomal inhibitor chloroquine and the specific cathepsin B inhibitor CA-074ME were able to reverse the effect of SKI II. Surprisingly, in vitro SK-1 activity assays revealed only a very weak direct inhibitory effect of SKI II on SK-1 overexpressed HEK293 cell lysates. CONCLUSION: These data show for the first time that the previously developed SK inhibitor SKI II hardly inhibits SK-1 directly but rather acts by triggering the lysosomal degradation of SK-1 in various cell types. This finding discloses a new mode of action of SKI II and strongly suggests that additional direct targets of SKI II may exist other than SK-1.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression.

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

The immunomodulator FTY720 and its phosphorylated derivative activate the Smad signalling cascade and upregulate connective tissue growth factor and collagen type IV expression in renal mesangial cells.

1.--The immunomodulating agent FTY720 is a substrate for the sphingosine kinase and the phosphorylated form is able to bind to sphingosine 1-phosphate (S1P) receptors. In this study, we show that exposure of renal mesangial cells to phospho-FTY720 leads to a rapid and transient activation of several protein kinase cascades, including the mitogen- and stress-activated protein kinases. The nonphosphorylated FTY720 also increased MAPK phosphorylation, but with a reduced potency and a more delayed time course. In addition, phospho-FTY720 and FTY720 are able to increase phosphorylation of Smad proteins which are classical members of the transforming growth factor-beta (TGF-beta) signalling device, thus suggesting a crosstalk between FTY720 and TGF-beta signalling. 2.--Pretreatment with the S1P(3) receptor antagonist suramin inhibits FTY720 and phospho-FTY720-induced Smad phosphorylation, whereas pertussis toxin pretreatment, which blocks G(i/0) proteins, has no effect on Smad phosphorylation. 3.--Since TGF-beta is a potent profibrotic cytokine in mesangial cells and upregulates the connective tissue growth factor (CTGF) and collagen as important hallmarks in the fibrotic sequelae, we investigated whether FTY720 and phospho-FTY720 are able to mimic these effects of TGF-beta. Indeed, FTY720 and phospho-FTY720 markedly upregulate CTGF and collagen type IV protein expressions. In addition, the tissue inhibitor of metalloproteinase-1 is transcriptionally activated by FTY720, whereas cytokine-induced matrix metalloproteinase-9 is down-regulated by FTY720. 4.--Depletion of the TGF-beta receptor type II by the siRNA transfection technique blocks not only Smad phosphorylation but also CTGF upregulation. Similarly, Smad-4 depletion by siRNA transfection also abrogates CTGF upregulation induced by FTY720 and phospho-FTY720. 5.--In summary, our data show that FTY720 and phospho-FTY720 not only activate the Smad signalling cascade in mesangial cells, but also upregulate the expression of CTGF and collagen. These findings suggest that FTY720 may have additional effects besides the established immunomodulatory action and, importantly, a profibrotic activity has to be considered in future experimental approaches.

PPARalpha activation upregulates nephrin expression in human embryonic kidney epithelial cells and podocytes by a dual mechanism.

Nephrin is an important member of the glomerular ultrafiltration complex and changes in its expression are associated with severe proteinuria. In this study, we show that synthetic PPARalpha agonists, but not PPARgamma agonists, stimulate an increased nephrin mRNA and protein expression in cultures of human podocytes and A293 human embryonic kidney epithelial cells which are blocked by the PPARalpha antagonist Ru486. Furthermore, the PPARalpha agonists have an additive effect on the interleukin-1beta (IL-1beta)-induced nephrin upregulation. Luciferase-reporter assays reveal that human nephrin promoter activity is stimulated by the PPARalpha agonists. Neither IL-1beta nor TNFalpha alone has an effect on nephrin promoter activity suggesting that additional posttranscriptional regulatory events might be operative. The role of nephrin mRNA stability regulation was evaluated in cells treated with actinomycin D to stop further RNA transcription. In the presence of PPARalpha agonists, IL-1beta or TNFalpha, the decay of nephrin mRNA was drastically reduced thus arguing for an additional posttranscriptional mode of action. In summary, these data show that PPARalpha activation causes an increased nephrin expression by a dual action, on the one hand by stimulating nephrin promoter activity and on the other hand by reducing nephrin mRNA degradation. These findings may have importance for treatment strategies of renal diseases affecting the expression of nephrin and subsequently the proper action of the glomerular filtration apparatus.

Nitric oxide induces TIMP-1 expression by activating the transforming growth factor beta-Smad signaling pathway.

Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.

Heterologous desensitization of the sphingosine-1-phosphate receptors by purinoceptor activation in renal mesangial cells.

1 Sphingosine-1-phosphate (S1P) is considered a potent mitogen for mesangial cells and activates the classical mitogen-activated protein kinase (MAPK) cascade via S1P receptors. In this study, we show that S1P signalling is rapidly desensitized upon S1P receptor activation. A complete loss of S1P sensitivity occurs after 10 min of S1P pretreatment and remains for at least 8 h. A similar desensitization is also seen with the S1P mimetic FTY720-phosphate, but not with the nonphosphorylated FTY720, nor with sphingosine or ceramide. 2 Prestimulating the cells with extracellular ATP or UTP, which bind to and activate P2Y receptors on mesangial cells, a similar rapid desensitization of the S1P receptor occurs, suggesting a heterologous desensitization of S1P receptors by P2Y receptor activation. Furthermore, adenosine binding to P1 receptors triggers a similar desensitization. In contrast, two other growth factors, PDGF-BB and TGFbeta2, have no significant effect on S1P-induced MAPK activation. 3 S1P also triggers increased inositol trisphosphate (IP3) formation, which is completely abolished by S1P pretreatment but only partially by ATP pretreatment, suggesting that IP3 formation and MAPK activation stimulated by S1P involve different receptor subtypes. 4 Increasing intracellular cAMP levels by forskolin pretreatment has a similar effect on desensitization as adenosine. Moreover, a selective A3 adenosine receptor agonist, which couples to phospholipase C and increases IP3 formation, exerted a similar effect. 5 Pretreatment of cells with various protein kinase C (PKC) inhibitors prior to ATP prestimulation and subsequent S1P stimulation leads to a differential reversal of the ATP effect. Whereas the broad-spectrum protein kinase inhibitor staurosporine potently reverses the effect, the PKC-alpha inhibitor CGP41251, the PKC-delta inhibitor rottlerin and calphostin C show only a partial reversal at maximal concentrations. 6 Suramin, which is reported as a selective S1P3 receptor antagonist compared to the other S1P receptor subtypes, has no effect on the S1P-induced MAPK activation, thus excluding the involvement of S1P3 in this response. 7 In summary, these data document a rapid homologous and also heterologous desensitization of S1P signalling in mesangial cells, which is mechanistically triggered by PKC activation and eventually another staurosporine-sensitive protein kinase, as well as by increased cAMP formation.

Sphingosine 1-phosphate cross-activates the Smad signaling cascade and mimics transforming growth factor-beta-induced cell responses.

Exposure of renal mesangial cells to sphingosine 1-phosphate (S1P) leads to a rapid and transient activation of the mitogen- and stress-activated protein kinases but also the protein kinase B. Here, we show that S1P also induces phosphorylation of Smad proteins, which are members of the transforming growth factor-beta (TGF-beta) signaling device. However, Smad phosphorylation occurred more slowly with a maximal effect after 20-30 min of S1P stimulation when compared with the rapid activation of the MAPKs. Interestingly, Smad phosphorylation is increased by pertussis toxin, which is in contrast to the complete inhibition of S1P-induced MAPK phosphorylation by pertussis toxin. TGF-beta is a potent anti-inflammatory cytokine, which in mesangial cells attenuates the expression of (i) inducible nitricoxide synthase (iNOS) caused by interleukin (IL)-1beta, (ii) secreted phospholipase A(2) (sPLA(2)), and (iii) matrix metalloproteinase-9 (MMP-9). These gene products are also down-regulated by S1P in a concentration-dependent manner. Furthermore, the expression of connective tissue growth factor is enhanced by both TGF-beta(2) and S1P. These effects of S1P are not mediated by the MAPK cascade as neither pertussis toxin nor the MAPK cascade inhibitor U0126 are able to reverse this inhibition. Overexpression of the inhibitory Smad-7 or down-regulation of co-Smad-4 lead to a reversal of the blocking effect of S1P on IL-1beta-induced NO release. Moreover, down-regulating the TGF-beta receptor type II by the siRNA technique or antagonizing the S1P(3) receptor subtype with suramin abrogates S1P-stimulated Smad phosphorylation. In summary, our data show that S1P trans-activates the TGF-beta receptor and triggers activation of Smads followed by activation of connective tissue growth factor gene transcription and inhibition of IL-1beta-induced expression of iNOS, sPLA(2), and MMP-9.

Differential binding of ceramide to MEKK1 in glomerular endothelial and mesangial cells.

Previously, we have shown that ceramide is able to directly bind to and activate c-Raf and to trigger the downstream classical mitogen-activated protein kinase (MAPK/ERK) cascade in glomerular mesangial cells [Proc. Natl. Acad. Sci. USA 93 (1996) 6959]. In this study, we show that ceramide acts differently in glomerular endothelial cells in that treatment of endothelial cells with exogenous ceramide leads to a potent activation of the stress-activated protein kinase (SAPK/JNK) cascade but not to an activation of the classical ERK cascade. A similar effect was observed with the inflammatory cytokines TNFalpha and IL-1beta, which activate a sphingomyelinase and thereby increase intracellular ceramide levels. The activation of JNKs as shown by c-Jun phosphorylation assays was paralleled by increased phosphorylation of the two JNK isoforms, p45 and p54. In addition, also the activator of JNKs, SEK1, was found to be increasingly phosphorylated by exogenous ceramide as well as by TNFalpha. In contrast, dihydroceramide had no effect on JNK or SEK1 phosphorylation. To see whether ceramide directly binds to MEKK1, which is the c-Raf analog in the SAPK cascade, a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl]oxy]-carbonyl] propanoyl]-D-erythro-sphingosine) ([125I]TID-ceramide) was used. Stimulation of endothelial cells with this [125I]TID-ceramide for 5 min followed by a short photolysis defined MEKK1 as a direct target of ceramide. With the same method, protein kinase C-alpha (PKC-alpha) was identified as a ceramide target. In contrast, no binding to c-Raf or the MEKK1 activator p65-PAK could be detected. A direct binding of ceramide to MEKK1 was also confirmed by affinity chromatography using a ceramide-coupled sepharose column. Furthermore, the ceramide-activated SAPK/JNK cascade is clearly involved in the mechanism of apoptosis, since in the presence of a JNK inhibitor, ceramide-induced DNA fragmentation is significantly reduced. In summary, we have shown that ceramide potently activates the SAPK cascade but not the ERK cascade in endothelial cells, which contrasts to mesangial cells where ceramide activates the ERK pathway and has only a minor effect on the SAPK cascade. Regarding the direct target of ceramide binding and action in endothelial cells, we identified MEKK1 as a further member of the growing family of ceramide-activated protein kinases.

Interleukin-1beta inhibits ATP-induced protein kinase B activation in renal mesangial cells by two different mechanisms: the involvement of nitric oxide and ceramide.

1 Extracellular nucleotides, like ATP and UTP, have been shown to activate the protein kinase B (PKB) pathway in mesangial cells. In this study we report that the pro-inflammatory cytokine interleukin-1beta (IL-1beta) inhibits ATP-induced PKB activation. 2 Pretreatment of mesangial cells with IL-1beta leads to a time-dependent decrease of ATP-induced PKB phosphorylation. Maximal inhibition is seen after 6 h of pretreatment. Incubating cells with IL-1beta in the presence of the NO synthase inhibitor, N-monomethyl-L-arginine (L-NMMA), reversed the IL-1beta inhibition of PKB activity. A similar decrease in ATP-evoked PKB activation is obtained when cells were pretreated with the nitric oxide (NO) donor, (Z)-1-[2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (Deta-NO), but not with the cell-permeable cGMP analogue, 8-bromo-cGMP. 3 The NO- and IL-1beta-mediated delayed inhibition of PKB activity is completely reversed by the phosphatase inhibitor calyculin A, but not by ocadaic acid, suggesting that NO upregulates a protein phosphatase activity, which most probably belongs to the group of protein phosphatases type 1. 4 In addition, IL-1beta also triggers a short-term and transient inhibitory effect on ATP-induced PKB activation which is maximal after 2-5 min of pre-incubation with IL-1beta. This effect occurs independently of NO generation, because no NO synthase is expressed at that time, and consequently, L-NMMA does not reverse the effect. Rather an involvement of the sphingolipid ceramide is likely, since IL-1beta triggers rapid ceramide formation and incubation of cells with the cell-permeable C6-ceramide blocked ATP-induced PKB phosphorylation. 5 In summary, our data show that IL-1beta exerts both short-term and long-term inhibitory effects on ATP-stimulated PKB activation, the short-term effect probably involves ceramide formation, whereas the long-term effect is due to inducible NO synthase induction and subsequent NO formation. These results reveal a further facet in the mechanisms of ceramide- and NO-induced cell death, i.e. by turning off the survival signal elicited by PKB.

Interleukin-1 inhibits angiotensin II-stimulated protein kinase B pathway in renal mesangial cells via the inducible nitric oxide synthase.

Exposure of rat renal mesangial cells to angiotensin II and angiotensin III leads to a rapid phosphorylation and activation of the protein kinase B (PKB) pathway. The angiotensin II analogs angiotensin-(1-7), angiotensin-(1-6) and angiotensin-(3-8) were unable to activate PKB. The angiotensin II and III effects are mediated by the angiotensin type 1 receptor as documented by the inhibitory action of valsartan. Furthermore, angiotensin II-induced activation of PKB involves neither a pertussis toxin-sensitive pathway nor the small G proteins of the Rho/Rac/cdc42 family, but is completely blocked by inhibitors of the PI 3-kinase. Moreover, angiotensin II-stimulated PKB activation is inhibited by long-term pretreatment with interleukin-1 beta, an effect that is reversed by the NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). Similarly, the nitric oxide donor (Z)-1-[2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (Deta-NO) blocks the angiotensin II-induced PKB activation. The NO-mediated inhibition of PKB activation in turn is reversed by the phosphatase inhibitor calyculin A but not by ocadaic acid, implying the induction of a protein phosphatase 1 activity by NO.