Vitamin K-dependent Gas6 activates ERK kinase and stimulates growth of cardiac fibroblasts

The protein product of growth arrest specific gene 6 (Gas6), is the biological ligand for the Axl subfamily of receptor tyrosine kinases. We investigated the effects of exogenous Gas6 on growth of cardiac fibroblasts isolated from genetically Gas6-deficient mice. Recombinant Gas6, containing N terminal γ-carboxyglutamic acid residues formed from a vitamin K-dependent reaction, stimulated both DNA synthesis and proliferation of cardiac fibroblasts under serum-free conditions. Gas6 also markedly enhanced survival of cells during prolonged serum starvation. Gas6 stimulated tyrosine phosphorylation of Axl as well as phosphorylation of ERK kinase. The mitogenic effects of Gas6 were inhibited by neutralising anti-Gas6 antibodies and by a soluble Axl ectodomain fusion protein. In contrast, recombinant Gas6 from cells treated with warfarin, which prevents the γ-carboxylation reaction, neither stimulated fibroblast proliferation nor activated Axl tyrosine phosphorylation. Gas6-induced cell proliferation was additive to the effects of epidermal growth factor, suggesting activation of discrete signalling pathways. In conclusion, Gas6 appears to be a unique growth factor for fibroblasts and post-translational γ-carboxylation is necessary for its biological activity. These findings implicate vitamin K-dependent biochemical reactions in growth processes in development and in disease.