title: Role of MMP-9 in the breakdown of barrier integrity of the corneal endothelium in response to TNF-α.
abstract: TNF-α induces loss of barrier integrity of the corneal endothelium through mechanisms involving the activation of p38 MAP kinase. This study has investigated the role of matrix metalloproteinase-9 (MMP-9), known to be activated by mechanisms downstream of p38 MAP kinase, on the breakdown of the barrier integrity. Experiments were performed with primary cultures of bovine corneal endothelium. Changes in the trans-endothelial electrical resistance (TER), a measure of barrier integrity, were measured by electric cell-substrate impedance sensing. The integrity of the apical junctional assembly was imaged by immunolocalization of ZO-1. MMP-9 activity in the conditioned medium of cells treated with TNF-α was visualized by gelatin zymography. Transcriptional activation of MMP-9 was assessed by real-time RT-PCR. Exposure to TNF-α led to significant disruption of ZO-1 and also caused a continuous decline in TER for more than 20 h. These effects were opposed by cycloheximide (protein synthesis inhibitor), GM-6001 (broad spectrum inhibitor of MMPs), minocycline (MMP-2 and MMP-9 inhibitor), and MMP-9 inhibitor I (selective MMP-9 inhibitor). Cycloheximide, GM-6001, and MMP-9 inhibitor I also attenuated the increase in permeability to FITC-dextran (10 kDa). In addition, TNF-α led to an increased MMP-9 activity in the conditioned medium as well as a nearly 20-fold increase in mRNA for MMP-9 but not for MMP-2. The functional activity and increase in mRNA levels of MMP-9 were blocked by SB-203580 (selective p38 MAP kinase inhibitor) and cycloheximide. In conclusion, transcriptional and translational activation of MMP-9, downstream of p38 MAP kinase signaling, is involved in the (TNF-α)-induced loss of corneal endothelial barrier integrity.
authors.affiliation: Ophthalmology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Gangaraju
authors.initials: G
authors.lastname: Rajashekhar
authors.affiliation: School of Optometry, MCPHS University, Worcester, MA 01608, USA.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Shivanna
authors.affiliation: Pharmaceutical Sciences, University of Colorado, Aurora, CO 80045, USA.
authors.forename: Uday B
authors.initials: UB
authors.lastname: Kompella
authors.affiliation: School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405 USA.
authors.forename: Yueren
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405 USA. Electronic address: srinivas@indiana.edu.
authors.forename: Sangly P
authors.initials: SP
authors.lastname: Srinivas
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003911:Dextrans / Q000378:metabolism; D017097:Electric Impedance; D004728:Endothelium, Corneal / Q000187:drug effects* / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D020780:Matrix Metalloproteinase 9 / Q000502:physiology*; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D019108:Tight Junctions / Q000187:drug effects* / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D003911:Dextrans; D061965:Matrix Metalloproteinase Inhibitors; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; D062826:Zonula Occludens-1 Protein; C015219:fluorescein isothiocyanate dextran; D003513:Cycloheximide; D020780:Matrix Metalloproteinase 9; D016650:Fluorescein-5-isothiocyanate
keywords: TNF-α; corneal endothelium; matrix metalloproteinases; p38 MAP kinase; tight junctions