Bettina Sauer

Overlapping Signaling Pathways of Sphingosine 1-Phosphate and TGF-β in the Murine Langerhans Cell Line XS52

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.

Glucocorticoids mediate differential anti-apoptotic effects in human fibroblasts and keratinocytes via sphingosine-1-phosphate formation†

Glucocorticoids are potent anti‐inflammatory and immunomodulatory drugs which also induce growth inhibition in a variety of cell types. For this reason long‐term treatment of inflammatory skin diseases may result in irreversible skin atrophy. To elucidate whether the antiproliferative action of glucocorticoids in fibroblasts is accompanied by induction of apoptosis we investigated the influence of dexamethasone (DEX) on both parameters. Interestingly, we revealed that growth inhibitory concentrations of this glucocorticoid did not induce fibroblast apoptosis. Moreover, DEX protected these cells from apoptosis induced by tumor necrosis factor α (TNFα)/actinomycin, UV‐irradiation, and cell permeable ceramides. These findings are in contrast to the lack of anti‐apoptotic effects detected in keratinocytes. Although DEX inhibited TNFα mediated nuclear factor‐kappa (NF‐κB) activity in fibroblasts, this mechanism was not involved in its cytoprotection as it was verified by specific NF‐κB inhibitors. Therefore, we looked for alternative intracellular mediators. Coincubation of fibroblasts with the sphingosine kinase inhibitor N,N‐dimethylsphingosine, which blocks formation of the sphingolipid degradation product sphingosine‐1‐phosphate (S1P), abrogated the protective glucocorticoid effect almost completely. As preincubation with S1P reduced the number of apoptotic cells after stimulation with TNFα/actinomycin and moreover DEX increased the intracellular S1P content a role of this sphingolipid in the cytoprotection by DEX is suggested.