Tissue Transglutaminase Expression and Activity in Normal and Glaucomatous Trabecular Meshwork Cells

Abstract

Purpose:: Glaucoma is a leading cause of irreversible blindness in the world. A major risk factor for glaucoma is elevated intraocular pressure due to increased resistance of aqueous humor outflow through the trabecular meshwork (TM). In the glaucomatous TM there is an increased accumulation of extracellular matrix (ECM) material due to a disruption of the normal balance between ECM deposition and degradation. Tissue transglutaminase (tTgase) belongs to a family of calcium-dependent enzymes that catalyze the post-translational modification of the ECM by cross-linking proteins, thus making these proteins resistant to enzymatic and physical degradation. It is possible that the increase in ECM proteins seen in the glaucomatous TM is due to increased cross-linking activity of tTgase. The purpose of this study was to determine if there are differences in expression and activity of tTgase between normal and glaucoma TM cells.

Methods:: Normal (N=3 NTM) and glaucomatous (N=3 GTM) human TM cell lines were grown until confluent and cell lysates were collected using an extraction buffer. Western blot analysis was used to compare tTgase protein levels in NTM and GTM cells. tTgase enzyme activity between NTM and GTM cells was studied using a biotin cadaverine assay. In addition, immunohistochemistry was utilized to evaluate the expression of tTgase, fibronectin (FN) and N epsilon gamma glutamly lysine protein in NTM and GTM tissues.

Results:: Western blot analysis and immunohistochemistry demonstrated the presence of tTgase protein in both NTM and GTM cells. There was a significant increase in tTgase protein in GTM cells compared to NTM cells. In addition GTM cells demonstrated a significant increase in tTgase enzyme activity compared to NTM cells. Immunohistochemical results demonstrated increased expression of tTgase and FN in GTM tissues. Immunohistochemistry also demonstrated an increased co-localization of FN and N epsilon gamma glutamyl lysine protein indicating significant cross-linking of FN by tTgase in GTM tissues.

Conclusions:: This study demonstrated that both NTM and GTM cells express tTG. In addition, tTgase protein levels and enzyme activities are significantly elevated in GTM cells. There was a significant increase in co-localization of FN and N epsilon gamma glutamyl lysine protein in GTM tissues. These results indicate that tTgase may play an important role in the pathogenesis of glaucomatous by cross-linking ECM proteins such as FN and thus making the ECM more resistant to degradation.

Keywords: extracellular matrix • trabecular meshwork