title: Effect of connective tissue growth factor on protein kinase expression and activity in human corneal fibroblasts.
abstract: OBJECTIVE
  To investigate signal transduction pathways for connective tissue growth factor (CTGF) in human corneal fibroblasts (HCF).
  
  
  METHODS
  Expression of 75 kinases in cultures of serum-starved (HCF) were investigated using protein kinase screens, and changes in levels of phosphorylation of 31 different phosphoproteins were determined at 0, 5, and 15 minutes after treatment with CTGF. Levels of phosphorylation of three signal transducing phosphoproteins (extracellular regulated kinase 1 [ERK1], extracellular regulated kinase 2 [ERK2] [MAPKs], and signal transducer and activator of transcription 3 [STAT3]) were measured at nine time points after exposure to CTGF using Western immunoblots. Inhibition of Ras, MEK1/2 (MAPKK), and ERK1/2, on CTGF-stimulated fibroblast proliferation and collagen gel contraction was assessed using selective inhibitors farnesylthiosalicylic acid, PD-98059, and SB203580, respectively.
  
  
  RESULTS
  Thirty two of the 75 kinases (43%) evaluated by the kinase screen were detected in extracts of quiescent HCF, suggesting these kinases are available to respond acutely to CTGF exposure. Addition of CTGF increased levels of phosphorylation of five phosphoproteins (ERK1 and 2, MEK1/2 [MAPKK], STAT3, and SAPK/JNK), and decreased levels of phosphorylation of 14 phosphoproteins (including protein kinases B and C) after 5 and 15 minutes. Further analysis of ERK1 and 2 and STAT3 phosphorylation showed rapid increases within 1 minute of CTGF exposure that peaked between 5 and 10 minutes then returned to pretreatment levels by 30 minutes. Treatment of HCF with selective inhibitors of Ras, MEK 1/2, and ERK1/2 individually blocked both CTGF induced cell proliferation, and collagen gel contraction.
  
  
  CONCLUSIONS
  Results from protein kinase screens and selective kinase inhibitors demonstrate Ras/MEK/ERK/STAT3 pathway is required for CTGF signaling in HCF.
authors.affiliation: Institute for Wound Research, University of Florida, Gainesville, Florida 32610-0294, USA.
authors.forename: Siva S
authors.initials: SS
authors.lastname: Radhakrishnan
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Blalock
authors.forename: Paulette M
authors.initials: PM
authors.lastname: Robinson
authors.forename: Genevieve
authors.initials: G
authors.lastname: Secker
authors.forename: Julie
authors.initials: J
authors.lastname: Daniels
authors.forename: Gary R
authors.initials: GR
authors.lastname: Grotendorst
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Schultz
mesh_terms: D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000494:pharmacology*; D060527:Corneal Keratocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D018631:ras Proteins / Q000378:metabolism
chemical_list: C526317:CCN2 protein, human; D010750:Phosphoproteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D055513:Connective Tissue Growth Factor; D011494:Protein Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins