Published online before print August 6, 2009, doi: 10.1161/CIRCRESAHA.109.202333
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Submitted on June 4, 2009
Revised on July 23, 2009
Accepted on July 28, 2009
Heat Shock Protein 70 Enhances Vascular Bone Morphogenetic Protein-4 Signaling by Binding Matrix Gla Protein
Yucheng Yao ;
From the Division of Cardiology (Y.Y., A.D.W., S.J., K.I.B.), David Geffen School of Medicine; and Molecular Biology Institute (K.I.B.), University of California, Los Angeles.
* To whom correspondence should be addressed. E-mail: kbostrom{at}mednet.ucla.edu.
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.
Key words: heat shock protein 70 • matrix Gla protein • bone morphogenetic protein • protein interaction