Heat Shock Protein 70 Enhances Vascular Bone Morphogenetic Protein-4 Signaling by Binding Matrix Gla Protein

Abstract

Rationale: Matrix Gla protein (MGP) is a calcification inhibitor, which binds and inhibits bone morphogenetic protein (BMP)-2 and -4. Objective: The objective was to determine whether MGP also binds other proteins, which could interfere with its function. Methods and Results: We transfected bovine aortic endothelial cells with N-terminally FLAG-tagged MGP and used immunoprecipitation and liquid chromatographic–tandem mass spectrometric analysis to identify MGP-binding proteins. Heat shock protein (HSP)70, a stress-induced protein expressed in atherosclerotic lesions and soluble in serum, was identified as a novel MGP-binding protein. The interaction between MGP and HSP70 was confirmed by coimmunoprecipitation and chemical crosslinking, and blocked the interaction between MGP and BMP-4. In endothelial cells, HSP70 enhanced BMP-4–induced proliferation and tube formation, and in calcifying vascular cells, HSP70 enhanced BMP-induced calcium deposition. In addition, HSP70 mediated the procalcific effect of interleukin-6 on calcifying vascular cells. In apolipoprotein E–null mice, a model for atherosclerosis, levels of BMP-4, HSP70, MGP, and interleukin-6 were elevated in the aortic wall. Levels of BMP-4, HSP70, and interleukin-6 were also elevated in serum, and anti-HSP70 antibodies diminished its procalcific effect on calcifying vascular cells. Conclusion: HSP70 binds MGP and enhances BMP activity, thereby functioning as a potential link between cellular stress, inflammation, and BMP signaling.