Interactions between sphingosine-1-phosphate and vascular endothelial growth factor signalling in ML-1 follicular...

Sphingosine-1-phosphate (S1P) induces migration of human ML-1 thyroid follicular cancer cells and inhibits migration of human FRO anaplastic thyroid cancer cells. As tumour cells often secrete vascular endothelial growth factor (VEGF), we investigated a possible interaction between S1P and VEGF signalling in the regulation of thyroid tumour cell migration. We found that both ML-1 and FRO cells secreted VEGF-A ( approximately 3.6 and <0.1 ng/10(6) cells/day respectively) and VEGF-C ( approximately 3.0 and 0.14 ng/10(6) cells/day respectively). S1P stimulated VEGF-A secretion in both cell lines, and blocking S1P receptors 1, 2 and 3 attenuated the S1P-evoked secretion of VEGF-A. Neither TSH nor insulin affected the amount of secreted VEGF-A or -C in ML-1 cells, while simultaneous stimulation with insulin and S1P increased VEGF-C secretion in FRO cells. Both cell lines expressed VEGF receptor 2 (VEGFR-2) mRNA and proteins. Serum-evoked migration of both ML-1 and FRO cells was attenuated when VEGFR-2 was inhibited. Moreover, inhibiting VEGFR-2 in ML-1 cells resulted in a rapid downregulation of S1P1 mRNA expression and S1P1 protein levels, suppression of S1P-induced migration and a decrease in S1P-induced Akt phosphorylation. A VEGF-neutralizing antibody also reduced S1P-induced migration. In ML-1 cells, S1P phosphorylated VEGFR-2. In addition, VEGFR-2 inhibition resulted in the upregulation of S1P3 mRNA within 24 h, but a significant increase in S1P3 protein levels was not observed. VEGFR-2 inhibition, but not a VEGF-neutralizing antibody, reduced ML-1 cell proliferation independently of S1P stimulation. The results indicate a complex interaction between S1P and VEGFR-2 in ML-1 cells, particularly in regulating migratory responses.