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J. Biol. Chem., Vol. 283, Issue 7, 4439-4447, February 15, 2008

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Atrial Natriuretic Peptide-initiated cGMP Pathways Regulate Vasodilator-stimulated Phosphoprotein Phosphorylation and Angiogenesis in Vascular Endothelium^*

Hongjie Chen, Yehoshua C. Levine¹, David E. Golan^¶2, Thomas Michel³, and Alison J. Lin⁴

From the Cardiovascular Division and ^¶Hematology Division, Department of Medicine, Brigham and Women's Hospital and the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Nitric oxide (NO)- and atrial natriuretic peptide (ANP)-initiated cGMP signaling cascades are important in the maintenance of cardiovascular homeostasis. The molecular signaling mechanisms downstream of cGMP are not well understood, however. We have used small interfering RNA (siRNA) approaches to specifically knock down a series of signaling proteins in bovine aortic endothelial cells, and we have combined biochemical analyses with physiological assays to investigate cGMP-mediated signal transduction pathways. Activation of particulate guanylate cyclase (GC-A) by ANP leads to a substantial, dose-dependent, rapid, and sustained increase in intracellular cGMP. In contrast, stimulation of soluble guanylate cyclase by NO yields only a weak and transient increase in cGMP. ANP-induced cGMP production is selectively suppressed by siRNA-mediated knockdown of GC-A. ANP greatly enhances the phosphorylation at Ser-239 of the vasodilator-stimulated phosphoprotein (VASP), a major substrate of cGMP-dependent protein kinase (PKG) that significantly influences actin dynamics. Moreover, the ANP-induced phosphorylation of VASP at Ser-239 is accompanied by increased actin stress fiber formation and enhanced endothelial tube formation. siRNA-mediated knockdown of GC-A, VASP, or PKG abolishes ANP-induced VASP Ser-239 phosphorylation, stress fiber formation, and endothelial tube formation. We have demonstrated similar findings in human umbilical vein endothelial cells, where ANP substantially enhances intracellular cGMP content, phosphorylation of VASP at Ser-239, and endothelial tube formation. Taken together, our findings suggest that ANP-mediated cGMP signal transduction pathways regulate PKG phosphorylation of VASP Ser-239 in endothelial cells, resulting in reorganization of the actin cytoskeleton and enhancement of angiogenesis.

Received for publication, November 16, 2007

^* This work was supported in part by Grants HL46457, HL48743, GM36259 (to T. M.), HL32854, and HL70819 (to D. E. G.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Sarnoff Cardiovascular Research Foundation.

² To whom correspondence may be addressed: 250 Longwood Ave., SGMB 304, Boston, MA 02115. Tel.: 617-432-2256; E-mail: dgolan{at}hms.harvard.edu.

³ To whom correspondence may be addressed: 75 Francis St., Boston, MA 02115. Tel.: 617-732-7376; E-mail: tmichel{at}rics.bwh.harvard.edu.

⁴ To whom correspondence may be addressed: 250 Longwood Ave., SGMB 321, Boston, MA 02115. Tel.: 617-432-2257; E-mail: alison_lin{at}hms.harvard.edu.

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