title: Transforming growth factor-beta2 utilizes the canonical Smad-signaling pathway to regulate tissue transglutaminase expression in human trabecular meshwork cells.
abstract: Transforming growth factor-beta2 (TGF-β2) is elevated in the aqueous humor of patients with glaucoma. This growth factor is known to increase extracellular matrix (ECM) deposition in the trabecular meshwork (TM) as well as increase intraocular pressure (IOP) in perfused human cultured anterior eye segments. In addition overexpression of TGF-β2 in the mouse TM leads to elevated IOP. Exogenous TGF-β2 also increases tissue transglutaminase (TGM2) protein levels and enzyme activity in TM cells. TGM2 is a calcium-dependent enzyme that mediates cross-linking of ECM proteins, thus making ECM proteins resistant to enzymatic degradation and physical breakdown. We have investigated the signaling pathway by which TGF-β2 induces TGM2 in human TM cells. Primary cultures of human TM cells (N = 6) were treated for 48 h with TGF-β2 (0-10 ng/ml) in serum-free medium. TGM2 enzyme activity differences between non-treated and TGF-β2 treated TM cells were studied using a biotin cadaverine assay. Endogenous TGF-β2 protein levels were examined in normal trabecular meshwork (NTM) and glaucomatous trabecular meshwork (GTM) cell strains. Immunohistochemistry was used to evaluate the expression and co-localization of TGF-β2 and TGM2 in NTM and GTM tissues. Activation of Smad3 signaling pathway was evaluated by western immunoblot analysis using phospho-specific antibodies following exogenous TGF-β2 treatment. Pharmacological specific inhibitor of Smad3 (SIS3) and short interfering (si)RNAs were used to suppress Smad3 activity and CTGF gene expression respectively. Endogenous TGF-β2 levels were significantly elevated in cultured GTM cells (p < 0.05) when compared to NTM cells. Immunohistochemistry studies also demonstrated elevated expression and co-localization of both TGF-β2 and TGM2 in glaucoma human TM tissues. Exogenous TGF-β2 increased both TGM2 protein levels and enzyme activity in TM cells. Phosphorylation of Smad3 was stimulated in TM cell strains by exogenous TGF-β2. TGF-β2 induction of TGM2 was not inhibited with selective siRNA knockdown of CTGF. In contrast, a specific inhibitor of Smad3 (SIS3) and siRNA knockdown of Smad3 (p < 0.05) suppressed TGF-β2 induction of TGM2. This study demonstrated that TGF-β2 induction of TGM2 can be mediated via the canonical Smad-signaling pathway but does not appear to involve CTGF as a downstream mediator. Regulation of the Smad-signaling pathway in the TM may be useful in the therapy for glaucoma associated with aberrant TGF-β2 signaling.
authors: Department of Cell Biology and Anatomy, University of North Texas Health Science Center at Fort Worth, TX 76134, USA.
authors: Tara
authors: T
authors: Tovar-Vidales
authors: Abbot F
authors: AF
authors: Clark
authors: Robert J
authors: RJ
authors: Wordinger
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000502:physiology; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins; D005902:Glaucoma, Open-Angle / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D010766:Phosphorylation; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D053781:Transforming Growth Factor beta2 / Q000378:metabolism / Q000494:pharmacology*; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: C508504:6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride; C526317:CCN2 protein, human; D007546:Isoquinolines; D011725:Pyridines; D011758:Pyrroles; D034741:RNA, Small Interfering; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C000717608:TGM2 protein, human; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins