title: 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.
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.
authors.affiliation: Cardiovascular-Pulmonary Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. begumn@mail.nih.gov
authors.forename: Najma
authors.initials: N
authors.lastname: Begum
authors.forename: Steven
authors.initials: S
authors.lastname: Hockman
authors.forename: Vincent C
authors.initials: VC
authors.lastname: Manganiello
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000172:deficiency* / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D055786:Gene Knockout Techniques; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics*; D008297:Male; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D010982:Platelet-Derived Growth Factor; D034741:RNA, Small Interfering; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D054638:Dual Specificity Phosphatase 1; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3