title: Proteomic analysis of CTGF-activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration.
abstract: Connective tissue growth factor (CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF, we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using 2-dimensional gel electrophoresis followed by in-gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF-induced proteins, such as pro-alpha (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF-responsive molecules that may have important roles in ILD. Our study demonstrates that 1 of the novel CTGF-induced proteins, IQ motif containing GTPase activating protein (IQGAP) 1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF-induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF-induced cell migration and did not interfere with CTGF-induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and propose that CTGF-induced migration of lung fibroblasts to the damaged tissue is mediated via IQGAP1 and MAPK signaling pathways.
authors.affiliation: Medical University of South Carolina, Charleston, SC 29425, USA. bogatkev@musc.edu
authors.forename: Galina S
authors.initials: GS
authors.lastname: Bogatkevich
authors.forename: Anna
authors.initials: A
authors.lastname: Ludwicka-Bradley
authors.forename: C Beth
authors.initials: CB
authors.lastname: Singleton
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Bethard
authors.forename: Richard M
authors.initials: RM
authors.lastname: Silver
mesh_terms: D001344:Autopsy; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D055513:Connective Tissue Growth Factor; D015536:Down-Regulation; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D008168:Lung / Q000187:drug effects / Q000502:physiology*; D040901:Proteomics; D012595:Scleroderma, Systemic / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D015854:Up-Regulation; D014746:Vimentin / Q000235:genetics; D014945:Wound Healing
chemical_list: C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D014746:Vimentin; D055513:Connective Tissue Growth Factor