Role of protein kinase C in endothelial cell migration and morphogenesis stimulated by sphingosine 1-phosphate

Protein kinase C (PKC)-mediated signal transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the role of various PKC isoforms in sphingosine 1-phosphate (SIP)-stimulated endothelial cells is not well understood. We show that S1P-stimulated human umbilical vein endothelial cells (HUVEC) activate PKCα and PKCϵ isoforms in a time-dependent manner. Immunoprecipitation and membrane association assays in CHO transfected cells show S1P1-specific activation of PKCϵ and S1P3-specific activation of PKCα in a similar time-dependent manner as in HUVEC. PKCϵ activation is inhibited by the addition of pertussis toxin. PKCϵ translocates to the nucleus following S1P stimulation. Infection of endothelial cells with dominant negative PKCα adenovirus reduces cell migration and greatly inhibits morphogenesis in cells stimulated with S1P. PKC-specific chemical inhibitor GF109203X also inhibits these two responses. Infection of endothelial cells with dominant negative PKCα reduces S1P-induced calcium rise. This maximal rise requires calcium uptake, but it does not require enzymatic activity of the kinase. Pretreatment of these cells with the PKC-specific inhibitor GF109203X does not inhibit S1P-induced calcium rise. S1P-induced morphogenesis but not cell migration is critically dependent on extracellular calcium. Pretreatment of endothelial cells with phorbol 12-myristate 13-acetate for five minutes abolishes S1P-stimulated rise in calcium but had little or no effect on migration. The PMA-inhibited calcium rise can be prevented by PKC inhibitor or infection with dominant negative PKCα. These data indicate PKC is a signaling molecule following the stimulation of specific GPCR with S1P.