Axl/phosphatidylinositol 3-kinase signaling inhibits mineral deposition by vascular smooth muscle cells

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. The receptor tyrosine kinase Ax1 is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Ax1 prevents the deposition of a calcified matrix by vascular smooth muscle cells (VSMCs) and that this occurs via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. First, we demonstrate that Ax1 is expressed and phosphorylated in confluent VSMCs and that its expression is markedly downregulated as these cells calcify their matrix. Second, we demonstrate that overexpression of wild-type Ax1, using recombinant adenoviruses, enhances Ax1 phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Ax1 significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and Ca-45 accumulation. Third, the addition of a PI3K inhibitor, wortmannin, negates the inhibition of mineralization by overexpression of wild-type Axl, suggesting that activation of downstream signaling via PI3K is crucial for its inhibitory activity. In contrast, Ax1-mediated signaling is not enhanced by overexpression of kinase-dead Axl1 and mineralization is accelerated, although beta-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD. fmk attenuates the increased mineralization induced by kinase-dead Ax1, suggesting that kinase-dead Ax1 stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Ax1. Together, these results demonstrate that signaling through Axl inhibits vascular calcification in vitro and suggest that therapeutics targeting this receptor may open up new avenues for the prevention of vascular calcification in vivo.