Analysis of the stimulatory effect of sphingolipid, especially SPC, on the wound healing

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Sphingosylphosphorylcholine (SPC) is a sphingolipid metabolite known to stimulate healing by virtue of its potent mitogenic effect on various cells. To clarify the mechanism of SPC on the healing process, we examined the effect of SPC on wound contraction using an in vitro model and on the synthesis of fibronectin (FN), an extracellular matrix protein, in human dermal fibroblasts.(1) A mixture of human dermal fibroblasts and porcine type I collagen in a serum-free medium was gelled. The effect of SPC was analyzed by measuring the amount of contracted surface area.SPC stimulated collagen gel contraction dose dependently, maximal effect at 10 uM of SPC.The effect of SPC was not 'Inhibited by pre-treatment with anti-TGF-b or anti-PDGF-BB antibodies.Among the various signal transduction inhibitors, pertussis toxin, staurosporine and H7 were found to inhibit the action of SPC whereas genistein and tyrphostin A47 were not, suggesting that fibroblast contraction induced by SPC is mediated by a trimeric GTP-binding protein (G protein)-coupled receptor and protein kinase.(2) SPC was found dose-dependently to up-regulate the expression of protein and mRNA levels of FN.The effect of SPC on the expression of TGF-b, PDGF-BB, IL-4 and IL-6 in fibroblasts was then examined. Among the tested cytokines, the mRNA expression and protein level of only IL-6 was significantly up-regulated by the application of SPC in a dose dependent manner. Furthermore, northern blotting analysis revealed that the mRNA level of FN in fibroblasts was up-regulated by the addition of recombinant IL-6 and suppressed by the addition of neutralizing antibody against IL-6, suggesting that SPC promoted the synthesis of FN partially through IL-6 action.These results suggest that stimulatory effect of SPC on wound healing is exerted by the stimulation of wound contraction and extracellular matrix synthesis as well as the stimulation of reepithelialization.