title: MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.
abstract: BACKGROUND
  Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.
  
  
  RESULTS
  siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.
  
  
  CONCLUSIONS
  MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.
authors.affiliation: Department of Surgery, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, United States of America; Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Dan
authors.initials: D
authors.lastname: Yu
authors.affiliation: Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: George
authors.initials: G
authors.lastname: Makkar
authors.affiliation: Department of Surgery, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, United States of America.
authors.forename: Tuo
authors.initials: T
authors.lastname: Dong
authors.affiliation: Department of Surgery, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, United States of America; Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Dudley K
authors.initials: DK
authors.lastname: Strickland
authors.affiliation: Department of Surgery, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, United States of America; Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Rajabrata
authors.initials: R
authors.lastname: Sarkar
authors.affiliation: Department of Surgery, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, United States of America; Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Thomas Stacey
authors.initials: TS
authors.lastname: Monahan
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000502:physiology*; D019938:Cyclin D1 / Q000096:biosynthesis / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000201:enzymology*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D044786:S-Phase Kinase-Associated Proteins / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C530104:CCND1 protein, human; C436291:CDKN1B protein, human; C530105:Ccnd1 protein, mouse; C493961:Cdkn1b protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D044786:S-Phase Kinase-Associated Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27