title: CCN2 promotes keratinocyte adhesion and migration via integrin α5β1.
abstract: BACKGROUND
  CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.
  
  
  METHODS
  Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine-glycine-aspartic acid (RGD), 500 μM arginine-glycine-glutamate-serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.
  
  
  RESULTS
  CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.
  
  
  CONCLUSIONS
  Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.
authors.affiliation: Division of Plastic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 2115, USA; Department of Surgical Sciences, Plastic Surgery Unit, Uppsala University, Uppsala, Sweden.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kiwanuka
authors.forename: Lauren
authors.initials: L
authors.lastname: Andersson
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Caterson
authors.forename: Johan Pe
authors.initials: JP
authors.lastname: Junker
authors.forename: Bengt
authors.initials: B
authors.lastname: Gerdin
authors.forename: Elof
authors.initials: E
authors.lastname: Eriksson
mesh_terms: D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D039081:Integrin alpha5beta1 / Q000235:genetics / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
chemical_list: C526317:CCN2 protein, human; D005353:Fibronectins; D039081:Integrin alpha5beta1; D055513:Connective Tissue Growth Factor
keywords: CCN2; Cell adhesion; Cell migration; Fibronectin; Integrin; Keratinocyte