title: Regulation of fibronectin-EDA through CTGF domain-specific interactions with TGFβ2 and its receptor TGFβRII.
abstract: OBJECTIVE
  To investigate the role of fibronectin containing extra domain A (FN-EDA) in the pathogenesis of proliferative vitreoretinopathy (PVR) and the regulation of FN-EDA by transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) in retinal pigment epithelial (RPE) cells.
  
  
  METHODS
  Expression of FN-EDA in normal human retinas and PVR membranes was evaluated by immunohistochemistry. The effects of TGFβ and CTGF on FN-EDA mRNA and protein expression in primary cultures of human RPE cells were analyzed at different time points by real-time PCR and Western blot, respectively. The interaction of CTGF with TGFβ2 or with its type II receptor TGFβRII was examined by ELISA, immunoprecipitation, and solid-phase binding assays.
  
  
  RESULTS
  FN-EDA was abundantly expressed in PVR membranes but absent from the RPE monolayer in normal human retinas. Treatment of RPE cells with TGFβ2 induced FN-EDA expression in a time- and dose-dependent manner, but CTGF alone had no effect. However, CTGF, through its N-terminal half fragment, augmented TGFβ2-induced expression of FN-EDA at the protein level. This effect was blocked by antibodies against TGFβ2 or TGFβRII. Interaction of TGFβ2 or TGFβRII with CTGF was dose dependent and specific. CTGF directly bound TGFβ2 and TGFβRII at its N- and C-terminal domains, respectively.
  
  
  CONCLUSIONS
  These findings suggest that CTGF promotes the profibrotic activities of TGFβ acting as a cofactor through direct protein interactions and complex regulatory mechanisms.
authors.affiliation: Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.
authors.forename: Rima
authors.initials: R
authors.lastname: Khankan
authors.forename: Noelynn
authors.initials: N
authors.lastname: Oliver
authors.forename: Shikun
authors.initials: S
authors.lastname: He
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Ryan
authors.forename: David R
authors.initials: DR
authors.lastname: Hinton
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D012160:Retina / Q000196:embryology / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053781:Transforming Growth Factor beta2 / Q000378:metabolism* / Q000494:pharmacology; D015854:Up-Regulation; D018630:Vitreoretinopathy, Proliferative / Q000378:metabolism
chemical_list: C526317:CCN2 protein, human; D005353:Fibronectins; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor; D012313:RNA; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II