title: The Ras guanine nucleotide exchange factor RasGRF1 promotes matrix metalloproteinase-3 production in rheumatoid arthritis synovial tissue.
abstract: BACKGROUND
  Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS.
  
  
  METHODS
  Expression of RasGRF1, MMP-1, MMP-3, and IL-6 was detected in synovial tissue by immunohistochemistry and stained sections were evaluated by digital image analysis. Expression of RasGRF1 in FLS and synovial tissue was also assessed by immunoblotting. Double staining was performed to detect proteins in specific cell populations, and cells producing MMP-1 and MMP-3. RasGRF1 expression was manipulated in RA FLS by cDNA transfection and gene silencing, and effects on MMP-1, TIMP-1, MMP-3, IL-6, and IL-8 production measured by ELISA.
  
  
  RESULTS
  Expression of RasGRF1 was significantly enhanced in RA synovial tissue, and detected in FLS and synovial macrophages in situ. In cultured FLS and synovial biopsies, RasGRF1 was detected by immunoblotting as a truncated fragment lacking its negative regulatory domain. Production of MMP-1 and MMP-3 in RA but not non-RA synovial tissue positively correlated with expression of RasGRF1 and co-localized in cells expressing RasGRF1. RasGRF1 overexpression in FLS induced production of MMP-3, and RasGRF1 silencing inhibited spontaneous MMP-3 production.
  
  
  CONCLUSIONS
  Enhanced expression and post-translational modification of RasGRF1 contributes to MMP-3 production in RA synovial tissue and the semi-transformed phenotype of RA FLS.
authors.affiliation: Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands. j.r.abreu@amc.uva.nl
authors.forename: Joana R F
authors.initials: JR
authors.lastname: Abreu
authors.forename: Daphne
authors.initials: D
authors.lastname: de Launay
authors.forename: Marjolein E
authors.initials: ME
authors.lastname: Sanders
authors.forename: Aleksander M
authors.initials: AM
authors.lastname: Grabiec
authors.forename: Marleen G
authors.initials: MG
authors.lastname: van de Sande
authors.forename: Paul P
authors.initials: PP
authors.lastname: Tak
authors.forename: Kris A
authors.initials: KA
authors.lastname: Reedquist
mesh_terms: D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis*; D008875:Middle Aged; D013583:Synovial Membrane / Q000378:metabolism*; D014162:Transfection; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism*
chemical_list: C513039:RASGRF1 protein, human; D020666:ras-GRF1; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1
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