title: Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells.
abstract: The major structural change in the human trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG) is an increase in extracellular matrix (ECM) in the juxtacanalicular region of the TM. There is evidence that treatment with TGF-beta2 causes an induction of ECM deposition in cultured human TM cells and that TGF-beta2 is causatively involved in the JCT ECM increase in POAG. In the present study, we investigated the effects of connective tissue growth factor (CTGF) on the biology of cultured human TM cells. CTGF is a downstream mediator of TGF-beta2-signaling, which is expressed at high amounts in the human TM in situ. HEK293 cells were transfected with an eukaryotic expression plasmid containing the coding sequences of human CTGF. Secreted CTGF was isolated and purified by chromatography. Primary human TM cells were incubated for 24 h with CTGF at concentrations of 2.5-100 ng/ml. Following treatment with CTGF, the expression of various ECM components that are expressed in the JCT, matrix metalloproteinases (MMPs) and integrins was investigated by real-time RT-PCR and western blot analyses. In addition, the activity of MMPs was investigated by gelatine zymography. The effect of CTGF silencing on TGF-beta2-induced gene expression was investigated by transfection of immortalized HTM cells with CTGF-specific small interfering (si)RNA before TGF-beta2 treatment. CTGF-treated human TM cells showed an increase in the expression of fibronectin, collagen types I, III, IV and VI, as well as in the integrin subunits aV and beta1. Lower concentrations of CTGF caused an autoinduction of CTGF expression. No effects were observed on the expression and activity of MMP-2, MMP-9 and plasminogen activator inhibitor-1 (PAI-1). Transfection with CTGF-specific siRNA inhibited the TGF-beta2-induced upregulation of CTGF and fibronectin. Our results indicate that treatment of human TM cells with recombinant CTGF causes distinct changes in gene expression and that CTGF is a critical mediator of the effects of TGF-beta2 on ECM synthesis in human TM cells. An intriguing aspect supported by the data of the present work is that the pharmacologic modulation of CTGF might be a useful approach to develop novel therapeutic strategies to prevent or to reverse the structural changes that occur in the TM of eyes with POAG.
authors.affiliation: Institute of Human Anatomy and Embryology, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Junglas
authors.forename: Alice H L
authors.initials: AH
authors.lastname: Yu
authors.forename: Ulrich
authors.initials: U
authors.lastname: Welge-Lüssen
authors.forename: Ernst R
authors.initials: ER
authors.lastname: Tamm
authors.forename: Rudolf
authors.initials: R
authors.lastname: Fuchshofer
mesh_terms: D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005109:Extracellular Matrix / Q000187:drug effects* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008875:Middle Aged; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection
chemical_list: C526317:CCN2 protein, human; D016326:Extracellular Matrix Proteins; D016023:Integrins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D055513:Connective Tissue Growth Factor; D020782:Matrix Metalloproteinases