Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

K David Kennedy,Lakisha K Buie,Teresa Borrás,S A Anithachristy,Reiner Albert Veitia

doi:10.1371/journal.pone.0036301

Abstract

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma.

Highlights

The secreted glycoprotein, myocilin (MYOC), was identified in human trabecular meshwork (HTM) cells after prolonged exposure to dexamethasone (DEX) (Trabecular Meshwork Inducible protein, TIGR) [1]
We found that the myocilin mutants altered a high number of genes which had been previously associated with trabecular meshwork physiological and glaucomatous conditions
Prior to the generation of RNA to hybridize to the Affymetrix chips, the mutants vectors were tested on HTM cells for the overexpression of MYOC RNA and proteins using TaqMan PCR and WB as described above

Summary

Introduction

The secreted glycoprotein, myocilin (MYOC), was identified in human trabecular meshwork (HTM) cells after prolonged exposure to dexamethasone (DEX) (Trabecular Meshwork Inducible protein, TIGR) [1]. It was independently discovered in the ciliary body [2] and in the normal retina [3]. MYOC retained special properties in the trabecular meshwork and its induction by DEX is specific to this tissue [5]. POAG is the most common form of the disease, which in most cases, is triggered by an elevated intraocular pressure (IOP). Elevated IOP is the result of an increased resistance of the trabecular meshwork tissue to the aqueous humor outflow

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Results

Conclusion