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.

TGF-β has been defined as a key mediator for the induction and maintenance of immunological tolerance. Concomitantly, it is essential for homeostasis of specialized epithelial dendritic cells, namely, Langerhans cells (LC). Our data reveal that TGF-β induces migration of the immature LC, XS52, a cell line expressing the signaling components, TGF-β type I and II receptors and Smad2, 3, and 4 mRNA. TGF-β stimulation induced transient Smad3/4 oligomerization and Smad3/DNA binding. Antisense oligonucleotides (ASO) targeting Smad3 abrogated TGF-β-induced XS52 chemotaxis, proving the involvement of this Smad protein in the TGF-β-dependent migration. In contrast, the typical CCR6-dependent chemotaxis of immature LC induced by CCL20/MIP-3α was not affected by Smad3 ASO. Most notably, we also identified the lysophospholipid sphingosine 1-phosphate (S1P) as a potent chemoattractant for immature LC, which expressed mRNA transcripts of lysophospholipid receptors S1P1–4. Additional experiments with specific ASO showed that the Gαi-coupled receptors S1P1 and S1P3 were dominantly involved in the S1P-induced migration. In contrast, lysophosphatidic acid (LPA), also binding to members of the lysophospholipid receptor family, failed to induce XS52 migration. Intriguingly, we raised evidence that TGF-β and S1P signal transduction pathways are indeed overlapping, as S1P augmented Smad activation and targeted DNA binding with kinetics comparable to TGF-β. Finally, S1P failed to stimulate XS52 chemotaxis when Smad3 protein expression was abrogated. Thus, our data indicate a cross-communication between S1P and TGF-β signaling that might be relevant for more than only migratory activities of immature LC.

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.

1alpha,25-Dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] has been shown to induce cell growth arrest and to possess differentiation-inducing behaviour in both primary melanocytes and melanoma cell lines. Moreover, in several melanoma cell lines it has been demonstrated that the antiproliferative action is accompanied by an increase in apoptosis. In contrast, here we show that physiological concentrations of 1,25-(OH)(2)D(3) did not induce apoptosis in primary melanocytes despite a cell growth inhibitory effect. Furthermore, treatment with 1,25-(OH)(2)D(3) made melanocytes resistant to several inductors of programmed cell death, including tumour necrosis factor-alpha and ultraviolet radiation. The antiapoptotic effect of 1,25-(OH)(2)D(3) was completely abolished by the addition of N,N-dimethylsphingosine, which blocks the formation of the sphingolipid degradation product sphingosine 1-phosphate (S1P), suggesting a crucial role for this sphingolipid in 1,25-(OH)(2)D(3)-mediated cytoprotection. Indeed, stimulation of melanocytes with S1P also resulted in an antiapoptotic action. In addition, S1P induced cell growth arrest of human melanocytes. This was an unexpected finding, as S1P is generally known as a potent mitogenic molecule in a variety of cells, including fibroblasts. As both 1,25-(OH)(2)D(3) and S1P have been identified to modify the Bcl-2/Bax ratio in epithelial cells, we also measured the expressions of these proteins; however, treatment of melanocytes with either 1,25-(OH)(2)D(3) or S1P did not alter the Bcl-2/Bax ratio. In conclusion, 1,25-(OH)(2)D(3) was shown to protect human melanocytes from apoptosis by formation of S1P, which is opposite to its apoptotic action in diverse melanoma cell lines.