title: Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-beta responsiveness.
abstract: BACKGROUND
  The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-beta (TGF-beta) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-beta, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-beta. To explore this notion, we characterized TGF-beta-induced activation of fibroblasts from CCN2-null (CCN2(-/-)) mouse embryos.
  
  
  METHODS
  The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-beta signal transduction and regulation of collagen gene expression were examined in CCN2(-/-) MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays.
  
  
  RESULTS
  Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2(-/-) MEFs was markedly reduced compared to wild type MEFs, TGF-beta-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2(-/-) MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2.
  
  
  CONCLUSIONS
  Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-beta-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.
authors.affiliation: Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Yasuji
authors.initials: Y
authors.lastname: Mori
authors.forename: Monique
authors.initials: M
authors.lastname: Hinchcliff
authors.forename: Minghua
authors.initials: M
authors.lastname: Wu
authors.forename: Matthew
authors.initials: M
authors.lastname: Warner-Blankenship
authors.forename: Karen
authors.initials: K
authors.lastname: M Lyons
authors.forename: John
authors.initials: J
authors.lastname: Varga
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics; D024042:Collagen Type I; D055513:Connective Tissue Growth Factor; D004622:Embryo, Mammalian; D005109:Extracellular Matrix / Q000235:genetics; D005347:Fibroblasts; D005355:Fibrosis / Q000378:metabolism*; D015870:Gene Expression; D017874:Immediate-Early Proteins / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D051379:Mice; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000502:physiology*
chemical_list: C526318:CCN2 protein, mouse; D024042:Collagen Type I; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D003094:Collagen