title: Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways.
abstract: BACKGROUND
  We have previously demonstrated that transforming growth factor-β (TGF-β) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-β-induced VSMC proliferation.
  
  
  METHODS
  Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with small interfering RNA (siRNA). For in vivo experiments, male Sprague-Dawley rats underwent carotid balloon injury, followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK, and proliferating cell nuclear antigen.
  
  
  RESULTS
  In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-β-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by proliferating cell nuclear antigen.
  
  
  CONCLUSIONS
  Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-β enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.
authors.affiliation: Division of Vascular Surgery, Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, Wisc., USA.
authors.forename: Pasithorn A
authors.initials: PA
authors.lastname: Suwanabol
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Seedial
authors.forename: Xudong
authors.initials: X
authors.lastname: Shi
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Dai
authors.initials: D
authors.lastname: Yamanouchi
authors.forename: Drew
authors.initials: D
authors.lastname: Roenneburg
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.forename: K Craig
authors.initials: KC
authors.lastname: Kent
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D049109:Cell Proliferation; D002470:Cell Survival; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D007150:Immunohistochemistry; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D051900:Smad3 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology
chemical_list: D051900:Smad3 Protein; C497787:Smad3 protein, rat; D016212:Transforming Growth Factor beta; D048049:Extracellular Signal-Regulated MAP Kinases