Abstract
Purpose:
The profibrotic transforming growth factor (TGF)-ß1 and the cross-linking enzyme lysyl oxidase-like 1 (LOXL1) have been shown to play key roles in the pathophysiology of pseudoexfoliation (PEX) syndrome/glaucoma. In this study we investigated the interaction of both factors with respect to the PEX-specific disordered matrix metabolism.
Methods:
Primary human Tenon’s capsule fibroblasts (hTCF) obtained from PEX and control patients were treated with TGF-ß1 (0-10 ng/ml) for 12-72 hours without or with preincubation with inhibitors of Smad or non-Smad TGF-ß signalling pathways. Expression of LOXL1 and PEX-specific extracellular matrix components was examined by using quantitative RT-PCR and Western immunoblot analysis. Stable transfection of hTCF with a full-length LOXL1 vector construct or with empty vector as control served to analyze the effect of LOXL1 overexpression on TGF-ß1-induced gene expression by using a custom-made PCR array including 84 PEX-relevant genes related to extracellular matrix synthesis and remodelling. Differentially expressed genes were identified using the Qiagen RT2 Profiler Data Analysis Software V3.5 and verified by specific real-time PCR assays.
Results:
TGF-ß1 stimulated the expression, secretion, and enzymatic activity of LOXL1 in hTCF concomitant with an increase in expression levels of BMP-1, elastin, fibrillin-1, fibulin-4 and fibulin-5 with peak effects at 10 ng/ml and 48 hours. This induction was blocked by TGF-ß receptor inhibitors and by inhibitors of the canonical Smad and non-canonical p38 MAPK and JNK/AP-1 signaling pathways. Overexpression of LOXL1 in turn enhanced the ability of TGF-ß1 to induce elastic matrix synthesis and accumulation by increasing expression levels of elastic proteins (elastin, fibulins) and cross-linking enzymes (transglutaminase-2), and by decreasing expression levels of proteolytic enzymes (MMPs) compared with TGF-ß1-treated mock-transfected cells.
Conclusions:
These findings suggest that the concerted action of LOXL1 and TGF-ß1 plays an important role in the PEX-associated abnormal matrix metabolism and fibrosis. Further studies are warranted to address potential effects of the PEX-associated LOXL1 risk variants on TGF-ß1 action.