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-␤ (TGF-␤) 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-␤ is a potent anti-inflammatory cytokine, which in mesangial cells attenuates the expression of (i) inducible nitricoxide synthase (iNOS) caused by interleukin (IL)-1␤, (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-␤ 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-1␤-induced NO release. Moreover, down-regulating the TGF-␤ 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-␤ receptor and triggers activation of Smads followed by activation of connective tissue growth factor gene transcription and inhibition of IL-1␤-induced expression of iNOS, sPLA 2 , and MMP-9.

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.

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 TGFΒ- 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.

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-b (TGF-b) signalling device, thus suggesting a crosstalk between FTY720 and TGF-b 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-b 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-b. 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-b 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. British Journal of Pharmacology (2006Pharmacology ( ) 147, 164-174. doi:10.1038; published online 21 November 2005 Keywords: FTY720; mesangial cell; Smad; TGF-b 2 ; CTGF; collagen Abbreviations: DMEM, Dulbecco's modified Eagle medium; ECL, enhanced chemiluminescence; FTY720, 2-amino-2-[2-(4-octyl-phenyl)ethyl]-1,3-propanediol hydrochloride; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1b, interleukin-1b; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloproteinase-9; NO, nitric oxide; PKB, protein kinase B; SAPK/JNK, stress-activated protein kinase/Nterminal c-Jun kinase; S1P, sphingosine 1-phosphate; TGF-b, transforming growth factor-b; TIMP-1, tissue inhibitor of metalloproteinase-1