Sphingosine 1-phosphate, a novel signaling molecule, stimulates DNA binding activity of AP-1 in quiescent Swiss 3T3 fibroblasts.

Sphingosine and sphingosine 1-phosphate, metabolites of sphingolipids, stimulate cell proliferation in quiescent Swiss 3T3 fibroblasts and induce transient increases in intracellular free calcium (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). However, little is yet known of the nuclear events that follow the early responses induced by sphingolipid metabolites. Using a gel retardation assay, we found that specific DNA binding activity of activator protein-1 (AP-1) was markedly increased after treatment of quiescent Swiss 3T3 fibroblasts with sphingosine 1-phosphate and sphingosine. The DNA binding specificity of AP-1 was confirmed with competing probes containing consensus sequences of AP-1, AP-2, AP-3, SP-1, and NF1/CTF. The c-fos gene product was detected in the AP-1 complex using anti-c-Fos antibody. The dose response for stimulation of DNA binding activity of AP-1 by sphingosine 1-phosphate correlated closely with its effect on DNA synthesis. Furthermore, an inhibitor of sphingosine kinase, DL-threo-dihydrosphingosine, which inhibits sphingosine-induced DNA synthesis and the formation of sphingosine 1-phosphate, also inhibited sphingosine-stimulated AP-1 DNA binding activity. This result further supports our proposal that sphingosine 1-phosphate mediates the mitogenic effect of sphingosine. Our results indicate that sphingosine 1-phosphate-induced DNA synthesis and cell division may result from activation of AP-1 protein, linking signal transduction by sphingolipid metabolites to gene expression.