Pleiotropic Influence of TNF- on the Barrier Integrity of Corneal Endothelium: Role of MMP-9

Abstract

Purpose: : TNF-α, which is implicated in corneal allograft rejection, is known to break down the barrier integrity of corneal endothelium through mechanisms involving activation of p38 MAP kinase, microtubule disassembly, and disruption of the peri-junctional actomyosin ring (PAMR). In this study, we have characterized the role of MMP-9 as the downstream target of p38 MAP kinase signaling in monolayers of cultured bovine corneal endothelial cells.

Methods: : Changes in the barrier integrity were assessed in terms of trans-endothelial electrical resistance (TER) obtained from electrical cell-substrate impedance sensing (ECIS^TM, Applied Biophysics, Inc, NY). Apical junctional assembly was visualized by immunolocalization of ZO-1 and cadherins (with a pan-cadherin a/b). The changes in organization of PAMR were ascertained by staining for f actin. MMP-9 activity in the conditioned media from cells treated with TNF-α was visualized by gelatin zymography. Transcriptional activation was assessed by real-time RT-PCR.

Results: : Exposure to TNF-α (20 ng/ml; 6 hr) induced dispersion of ZO-1 and cadherins, and also led to disruption of PAMR. TNF-α also caused a decline in the TER, which sustained for more than 20 hr. These effects were opposed by pre-treatment with a (MMP-9)-selective inhibitor as well as GM-6001 (50 µM; 1 hr), a broad spectrum MMP inhibitor. An increase in MMP-9 activity, but not that of MMP-2, in the conditioned media was observed upon exposure to the cytokine for 20 hr. Similarly, the transcriptional levels of MMP-9 but not MMP-2 demonstrated > 20-fold increase in the mRNA. All responses to cytokine were blocked by a selective p38-MAP kinase inhibitor (SB-203580; 10 µM; 1 hr) and the protein synthesis inhibitor, cycloheximide (10 µg/ml; 1 hr).

Conclusions: : Transcriptional and translational activation of MMP-9, downstream of p38 MAP kinase signaling, are involved in the (TNF-α)-induced loss of barrier integrity of the corneal endothelium.

Keywords: cornea: endothelium • cytokines/chemokines • pump/barrier function