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.

Ceramide induces translocation of protein kinase C-alpha to the Golgi compartment of human embryonic kidney cells by interacting with the C2 domain.

Ceramide is a lipid second messenger produced by sphingolipid metabolism in cells exposed to a limited number of agonists and in turn triggers important cell responses including protein kinase C (PKC)-alpha activation. Using a fusion protein comprising bovine PKCalpha and the green fluorescent protein (GFP), we transfected human embryonic kidney (HEK) cells and investigated to which subcellular compartment ceramide triggers PKCalpha redistribution. Stimulation of HEK cells with exogenous C16-ceramide or bacterial sphingomyelinase (bSMase), which leads to increased endogenous ceramide formation, evokes a translocation of PKCalpha to the Golgi compartment. By using deletion mutants of PKCalpha lacking distinct domains in the regulatory region, it is shown that the Ca(2+)-dependent lipid binding C2 domain, but not one of the C1 domains is essentially required for the ceramide-triggered translocation of PKCalpha to the Golgi complex. In contrast, the C2 domain is not required for phorbol ester (TPA) binding and translocation of PKCalpha to the plasma membrane. In addition, evidence is provided that TPA requires only one of the two C1 subdomains to trigger translocation to the plasma membrane.In summary, our data provide evidence that ceramide either directly or indirectly interacts with the Ca(2+)-dependent lipid binding C2 domain of PKCalpha and thereby induces translocation of the enzyme to the Golgi compartment.

Identification of the LIM kinase-1 as a ceramide-regulated gene in renal mesangial cells.

Stimulation of rat renal mesangial cells with cell-permeable C6-ceramide for 6 and 24h induces the expression of several genes as analyzed by a RNA fingerprinting arbitrarily primed-PCR method. Sequencing of the differentially expressed bands identified the serine/threonine protein kinase LIM kinase-1 (LIMK-1), which is involved in the regulation of cytoskeletal organization, as a ceramide-induced gene. The ceramide-triggered upregulation of LIMK-1 was verified by semiquantitative reverse transcriptase-PCR. A detailed time course reveals a first detectable increase in RNA level after 2h of ceramide stimulation which reaches maximal levels after 6h of stimulation and remains elevated up to 24 h. This ceramide-induced gene transcription of LIMK-1 is accompanied by enhanced LIMK-1 protein levels with maximal protein expression seen after 6h of stimulation. Furthermore, cofilin, which is a specific substrate of LIMK-1, shows an increased phosphorylation at Ser-3 in mesangial cells exposed to C6-ceramide. Mechanistically, the ceramide-induced LIMK-1 expression is blocked by the Rho kinase inhibitor Y27632, but not by a farnesyl transferase inhibitor, suggesting the involvement of the small G protein Rho, but not Ras and Rac, in the expressional upregulation. Similar to exogenously added ceramide, also interleukin-1beta which is an established activator of the neutral sphingomyelinase that leads to endogenous ceramide formation upregulates LIMK-1 protein expression and activity. In summary, these data demonstrate for the first time that LIMK-1 is a ceramide-induced gene, thus suggesting that LIMK-1 may act as a link between stress-induced ceramide formation and reorganization of the actin cytoskeleton.