Phosphodiesterase 3A (PDE3A) Deletion Suppresses Proliferation of Cultured Murine Vascular Smooth Muscle Cells (VSMCs) via Inhibition of Mitogen-activated Protein Kinase (MAPK) Signaling and Alterations in Critical Cell Cycle Regulatory Proteins

Najma Begum,Steven Hockman,Vincent C. Manganiello

doi:10.1074/jbc.m110.214155

Abstract

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.

Highlights

Health through the Intramural Research Program of the NHLBI. □S The on-line version of this article contains supplemental Figs. 1– 6. 1 To whom correspondence should be addressed: National Institutes of
We examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation
Our results indicate that PDE3A depletion inhibited mitogen-induced VSMC proliferation via two complementary signaling pathways, i.e. protein kinase A (PKA)-catalyzed inhibitory phosphorylation of Raf-1, which interfered with activation of MAPK signaling, and PKA/cAMP response element-binding protein (CREB)-induced elevation of p21, leading to cell cycle arrest in G0/G1, as well as by increased accumulation of p53, which induces mitogen-activated protein kinase phosphatase 1 (MKP-1), p21, and WIP1, leading to inhibition of G1 to S cell cycle progression

Summary

Introduction

Health through the Intramural Research Program of the NHLBI. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1– 6. 1 To whom correspondence should be addressed: National Institutes of. Development of atherosclerosis may involve perturbation of the homeostatic balance between antiatherosclerotic signaling (nitric oxide (NO), atrial natriuretic peptide, and cyclic nucleotides) and proatherosclerotic signaling (tumor necrosis factor ␣ (TNF-␣) and angiotensin II) [5]. Both endothelial cells and VSMCs are critical targets for inflammatory molecules, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, that are increased during progression of atherosclerosis [6], and are capable of producing these molecules. PDE3A and PDE3B isoforms are expressed in VSMCs, but their exact role(s) is unclear in large part due to the lack of availability of specific inhibitors of individual PDE3 isoforms

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