title: Myristoylated Alanine-Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation.
abstract: BACKGROUND
  Transcription of the myristoylated alanine-rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS-mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo.
  
  
  RESULTS
  MARCKS knockdown blocked platelet-derived growth factor (PDGF)-induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP2, restored membrane-associated PIP2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation.
  
  
  CONCLUSIONS
  MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP2 at the plasma membrane. This study delineates a novel mechanism for MARCKS-mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.
authors.affiliation: Department of Surgery, Veterans Affairs Medical Center, Baltimore, MD (D.Y., T.S.M.) Department of Surgery, University of Maryland School of Medicine, Baltimore, MD (D.Y., G.M., D.K.S., R.S., T.S.M.) Center for Vascular and Inflammatory Disease, University of Maryland School of Medicine, Baltimore, MD (D.Y., D.K.S., R.S., T.S.M.).
authors.forename: Dan
authors.initials: D
authors.lastname: Yu
authors.affiliation: Department of Surgery, University of Maryland School of Medicine, Baltimore, MD (D.Y., G.M., D.K.S., R.S., T.S.M.).
authors.forename: George
authors.initials: G
authors.lastname: Makkar
authors.affiliation: Department of Surgery, University of Maryland School of Medicine, Baltimore, MD (D.Y., G.M., D.K.S., R.S., T.S.M.) Department of Physiology, University of Maryland School of Medicine, Baltimore, MD (D.K.S., T.A.B., R.S.) Center for Vascular and Inflammatory Disease, University of Maryland School of Medicine, Baltimore, MD (D.Y., D.K.S., R.S., T.S.M.).
authors.forename: Dudley K
authors.initials: DK
authors.lastname: Strickland
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD (D.K.S., T.A.B., R.S.).
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Blanpied
authors.affiliation: The Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC (D.J.S., P.J.B.).
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Stumpo
authors.affiliation: The Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC (D.J.S., P.J.B.).
authors.forename: Perry J
authors.initials: PJ
authors.lastname: Blackshear
authors.affiliation: Department of Surgery, University of Maryland School of Medicine, Baltimore, MD (D.Y., G.M., D.K.S., R.S., T.S.M.) Department of Physiology, University of Maryland School of Medicine, Baltimore, MD (D.K.S., T.A.B., R.S.) Center for Vascular and Inflammatory Disease, University of Maryland School of Medicine, Baltimore, MD (D.Y., D.K.S., R.S., T.S.M.).
authors.forename: Rajabrata
authors.initials: R
authors.lastname: Sarkar
authors.affiliation: Department of Surgery, Veterans Affairs Medical Center, Baltimore, MD (D.Y., T.S.M.) Department of Surgery, University of Maryland School of Medicine, Baltimore, MD (D.Y., G.M., D.K.S., R.S., T.S.M.) Center for Vascular and Inflammatory Disease, University of Maryland School of Medicine, Baltimore, MD (D.Y., D.K.S., R.S., T.S.M.).
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Monahan
mesh_terms: D000818:Animals; D020212:Carotid Artery Injuries / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D002465:Cell Movement; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004789:Enzyme Activation; D006801:Humans; D006965:Hyperplasia; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000201:enzymology* / Q000473:pathology; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D058426:Neointima; D009479:Neuropeptides / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D011554:Pseudopodia / Q000201:enzymology / Q000473:pathology; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C581598:Cdc42 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D009479:Neuropeptides; D018129:Phosphatidylinositol Phosphates; C107953:RAC1 protein, human; C484602:Rac1 protein, mouse; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein
keywords: Rac1; membrane lipids; migration; restenosis; vascular smooth muscle