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

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Abstract
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.