The sphingolipid metabolite sphingosine-1-phosphate has emerged as a new bioactive molecule involved in the regulation of cell growth, differentiation, survival, and chemotaxis as well as angiogenesis and embryogenesis. These effects are mediated either via G-protein-coupled receptors or through intracellular actions. The most prominent sources of sphingosine-1-phosphate are human platelets suggesting its potential role in wound healing. In agreement with a positive function on reconstruction of wounded skin, we identified sphingosine-1-phosphate as a potent chemoattractant for keratinocytes as well as an activator of extracellular matrix production by fibroblasts. An unexpected finding is a strong cell growth arrest of keratinocytes after exposure to sphingosine-1-phosphate, as keratinocyte proliferation is critical for re-epithelialization of the wound. Most interestingly, the anti-proliferative effect of sphingosine-1-phosphate is not a result of cytotoxicity or apoptosis as sphingosine-1-phosphate even protects these cells from programmed cell death. Moreover, sphingosine-1-phosphate enhances differentiation of keratinocytes. To investigate further by which signaling pathway cell growth inhibition is mediated expression of the mRNA of all sphingosine-1-phosphate receptors (S1P1-5) was identified. 1 (Edg 1), 2 (Edg 5), 3 (Edg 3), 4 (Edg 6), and 5 (Edg 8) mRNA in keratinocytes was identified. As demonstrated in guanosine 5-[gamma-35S] triphosphate-gammaS binding assays, these G-protein-coupled receptors are functional at nanomolar concentrations. As the anti-proliferative effect of sphingosine-1-phosphate is only partially inhibited in the presence of pertussis toxin, it was investigated if intracellular actions are also involved. Microinjections of sphingosine-1-phosphate in keratinocytes also reduce proliferation suggesting that both sphingosine-1-phosphate receptors as well as intracellular actions mediate sphingosine-1-phosphate- induced cell growth arrest.

Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1alpha,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1alpha,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1alpha,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1alpha,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1alpha,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-alpha. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1alpha,25-dihydroxyvitamin D3. The effect of 1alpha,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1alpha,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1alpha,25-dihydroxyvitamin D3 in the presence of N,N-dimethylsphingosine. Taken together, here we report for the first time that 1alpha,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate.

The lysophospholipid sphingosine 1-phosphate and the cytokine-transforming growth factor ␤ are both released from degranulating platelets at wound sites, suggesting a broad spectrum of effects involved in wound healing. Interestingly, both of these molecules have been previously shown to induce chemotaxis but to strongly inhibit the growth of keratinocytes, while stimulating the proliferation of fibroblasts. In contrast to sphingosine 1-phosphate, the signaling cascade of the growth factor has been extensively examined. Specifically, Smad3 has been shown to be an essential mediator of transforming growth factor ␤-dependent chemotaxis of keratinocytes and mediates, in part, its growth-inhibitory effect. Here we show that sphingosine 1-phosphate, independently of transforming growth factor ␤ secretion, induces a rapid phosphorylation of Smad3 on its C-terminal serine motif and induces its partnering with Smad4 and the translocation of the complex into the nucleus. Moreover, sphingosine 1-phosphate fails to induce chemotaxis or inhibit the growth of Smad3-deficient keratinocytes, suggesting that Smad3 plays an unexpected functional role as a new target in sphingosine 1-phosphate signaling. Both sphingosine 1-phosphate receptors and the transforming growth factor ␤-type I receptor serine/threonine kinase are essential for activation of Smad3 by this lysophospholipid and the dependent biological responses, indicating a novel crosstalk between serine/threonine kinase receptors and G-protein coupled receptors.

Lysophosphatidic acid (LPA, 1-acyl-glycerol-3-phosphate) plays an important role in diverse biological responses including cell proliferation, differentiation, survival, migration, and tumor cell invasion. The most prominent source of LPA is platelets from which it is released after thrombin activation and is assumed to be an essential function of this lysophospholipid in cutaneous wound closure. Therefore, we examined the role of LPA on biological responses of keratinocytes. Although LPA potently enhances keratinocyte migration, it strongly induces growth arrest of proliferating epidermal cells. Thus, LPA possesses analogous actions to transforming growth factor-beta (TGF-beta), which is also released from degranulating platelets at wounded sites. In contrast to LPA, the intracellular signaling events of TGF-beta have been clearly identified and indicate that Smad3 is involved in chemotaxis and cell growth arrest of keratinocytes induced by this cytokine. Here we show that LPA, although it does not alter TGF-beta release is capable to activate Smad3 and results in a heteromerization with Smad4 and binding of the complex to its specific DNA-promoter elements. LPA completely fails to induce chemotaxis in Smad3-deficient cells, whereas growth inhibition is at least in part reduced. These findings indicate an essential role of Smad3 in diverse biological properties of LPA-stimulated keratinocytes.