Adenovrial Mediated Expression Of SPK And Its Mutant Regulate The Migration Of Endothelial Cells

Endothelial cell migration is a very important initiating process for angiogenesis. Several angiogenic growth factors, such as, hepatocyte growth factor and vascular endothelialgrowth factor, regulate the migration of endothelial cells via multiple signals. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (S1P), which is a lipid messenger with strong angiogenic activity. S1P regulates the cell migration through intracellular as well as extracellular mechanisms. S1P is believed to serve both as an intracellular second messenger and as an extracellular ligand for a family of the G-protein-coupled receptors, EDGs. Extra cellular S1P binds EDG receptors in endothelial cells leading to activation of cell migration. So SPK is a key signal molecule in regulation of cell migration.Several cytokines active SPK via multiple signal pathways. Hepatocyte growth factor (HGF), also termed scatter factor, is a strong angiogenic factor, induced migration of endothelial cells. In our previous work, we have demonstrated that binding of HGF to c-Met by HGF actives SPK in endothelial cells. However, whether SPK activation is involved in the regulation of HGF-induced migration of endothelial cells remains unclear. In this study, we constructed the adenovirus carrying the wild type SPK gene and SPK mutant (SPKG82D) respectively, and evaluated the effect of the adenoviral mediated-expression of these genes on the HGF-induced migration of ECV304c cells. The SPK genes of interest were cut from PcDNA3-SPKWT and PcDNA3-SPKG82Dand then ligated them into a shuttle vector Pshuttle-cmv respectively. The recombinant plasmids Pshuttle-cmv-SPKWT (containing human wild type Sphingosine kinase, SPK ) and Pshuttle-cmv-SPKG82D(containing human mutation Sphingosine kinase, SPK) were constructed by the guideline of molecular cloning. Linearized by digesting with restriction endonuclease PmeI,Pshuttle-cmv-SPKWT and Pshuttle-cmv-SPKG82D was transformed respectively into electrocompetent E. coli BJ5183-AD-1 cells which contains adenoviral backbone3plasmid pAdEasy-1. Recombinant re-ad-SPKWT and re-ad- SPKG82Dwere confirmed by gene sequence and restriction endonuclease analysis. The linearized recombinant plasmid re-ad-SPKWT and re-ad-SPKG82D were transfected respectively into adenovirus packaging cell 293 cells. Finally, rAdenoviruses were amplified in 293 cells after plaque assay for purification.Human endothelial ECV 304 cells were chosen as a model system to investigate the role of SPK in the migration of endothelial cells induced by HGF, as ECV304 express both c-Met, the receptor for HGF, and the S1P. Recombinant HGF induces migration of ECV304 cells markedly. And binding of HGF to c-Met results in activation of SPK. We infected the ECV 304 cells with adenovirus carrying wide type SPK gene and SPK mutant. Overexpression of wild type SPK gene in ECV 304 cells increased the intracellular SPK activity and enhanced the HGF-induced migration. Whereas expression of a dominant negative SPK (SPKG82D) blocked the HGF-induced migration of ECV 304 cells. It is suggested that activation of SPK would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which SPK mediates signaling from HGF/c-Met to the endothelial cell migration.