S1P1 and VEGFR-2 Form a Signaling Complex with Extracellularly Regulated Kinase 1/2 and Protein Kinase C-α Regulating ML-1 Thyroid Carcinoma Cell Migration

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor receptor 2 (VEGFR-2) signaling have been shown to integrate in many biological processes. The follicular thyroid carcinoma cell line ML-1 expresses VEGFR-2 and secretes substantial amounts of both vascular endothelial growth factor (VEGF)-A and VEGF-C. ML-1 cells also express S1P-receptors (S1P1–3,5). S1P is able to phosphorylate VEGFR-2, and inhibiting VEGFR-2 attenuates S1P-induced migration and down-regulates S1P1 expression in ML-1 cells. In the present study, we focused on the interactions between S1P1 and VEGFR-2. We show that S1P receptors form complexes with VEGFR-2 and that the S1P1/VEGFR-2 complex associates with protein kinase C (PKC)-α and ERK1/2. Furthermore, the complex evokes bidirectional signaling since the S1P-induced ERK1/2 phosphorylation is sensitive to VEGFR-2 kinase inhibition and VEGF-A-induced ERK1/2 phosphorylation is sensitive to pertussis toxin treatment as well as S1P1 small interfering RNA (siRNA) treatment. Both S1P- and VEGF-A-induced haptotaxis is sensitive to pertussis toxin treatment and S1P1 siRNA treatment. Phosphorylation of ERK1/2 evoked by both VEGF-A and the S1P1 agonist SEW-2871 is inhibited by PKC-α and PKC-βI siRNA. We hypothesize that VEGFR-2 forms a signaling complex with S1P1, evoking bidirectional signaling regulating both ERK1/2 phosphorylation and haptotaxis of ML-1 cells.