Overlapping signaling pathways of sphingosine 1-phosphate and TGF-β in the murine Langerhans cell line XS52

TGF-beta 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-beta induces migration of the immature LC, XS52, a cell line expressing the signaling components, TGF-beta type I and II receptors and Smad2, 3, and 4 mRNA. TGF-beta stimulation induced transient Smad3/4 oligomerization and Smad3/DNA binding. Antisense oligonucleotides (ASO) targeting Smad3 abrogated TGF-beta-induced XS52 chemotaxis, proving the involvement of this Smad protein in the TGF-beta-dependent migration. In contrast, the typical CCR6-dependent chemotaxis of immature LC induced by CCL20/MIP-3alpha was not affected by Smad3 ASO. Most notably, we also identified the lysophospholipid sphingosine 1-phosphate (SIP) as a potent chemoattractant for immature LC, which expressed mRNA transcripts of lysophospholipid receptors SIP1-4. Additional experiments with specific ASO showed that the Galpha(i)-coupled receptors SIP1 and SIP3 were dominantly involved in the SIP-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-beta and SIP signal transduction pathways are indeed overlapping, as SIP augmented Smad activation and targeted DNA binding with kinetics comparable to TGF-beta. Finally, SIP failed to stimulate XS52 chemotaxis when Smad3 protein expression was abrogated. Thus, our data indicate a cross-communication between SIP and TGF-beta signaling that might be relevant for more than only migratory activities of immature LC.