Published online before print January 25, 2007, doi: 10.1161/01.RES.0000258854.03388.02
© 2007 American Heart Association, Inc.
Molecular Medicine |
Axl/Phosphatidylinositol 3-Kinase Signaling Inhibits Mineral Deposition by Vascular Smooth Muscle Cells
From the Wellcome Trust Centre for Cell-Matrix Research (G.D.M.C., A.P.S., J.P.K., A.P.G., A.E.C.), Faculty of Life Sciences; and Division of Cardiovascular & Endocrine Sciences (G.D.M.C., M.Y.A., A.E.C.), Faculty of Medical & Human Sciences, University of Manchester, UK.
Correspondence to Dr Ann Canfield, University of Manchester, Michael Smith Building, Oxford Rd, Manchester M13 9PT, UK. E-mail ann.canfield{at}manchester.ac.uk
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 Axl is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Axl 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 Axl 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 Axl, using recombinant adenoviruses, enhances Axl phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Axl significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and 45Ca 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, Axl-mediated signaling is not enhanced by overexpression of kinase-dead Axl and mineralization is accelerated, although ß-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD.fmk attenuates the increased mineralization induced by kinase-dead Axl, suggesting that kinase-dead Axl stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Axl. 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.
Key Words: Axl receptor tyrosine kinase • calcification • differentiation • signaling pathways • vascular smooth muscle cells
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