The pathophysiology of pseudoexfoliation syndrome is affected by interaction of TGF-β1 and LOXL1
Abstract
Purpose
The cross-linking enzyme lysyl oxidase-like 1 (LOXL1) and profibrotic transforming growth factor (TGF)-ß1 play key roles in pathophysiology of pseudoexfoliation (PEX) syndrome/glaucoma. The purpose of this study was to investigate the interaction between LOXL1 and TGF-ß1 with respect to the PEX-specific disordered matrix metabolism.
Methods
Primary human Tenon's capsule fibroblasts (hTCF) obtained from patients were treated with TGF-ß1 (0-10 ng/ml) for 12-72 hours without or with preincubation with inhibitors of TGF-β signalling pathways. Expression of LOXL1 and PEX-specific extracellular matrix components was examined by using quantitative RT-PCR and Western immunoblot analysis. Direct binding of LOXL1 to TGF-ß1 was analyzed by blot overlay assay and solid phase ELISA using purified LOXL1, recombinant human TGF-ß1, TGFß1-LAP. The effect of LOXL1 on TGF-ß1 signaling was analyzed using TGF-ß receptor signaling real time PCR assays (BioRad) after transient transfection of hTCF with a full-length pCMV6-LOXL1 vector construct/with empty vector .
Results
TGFß1 significantly increased LOXL1 expression, secretion and enzymatic activity and correlated with enhanced expression of BMP-1, elastin, fibrillin-1, fibulin-4 and fibulin-5 with peak effects at 10 ng/ml for 48 hours. This induction was blocked by TGF-β receptor inhibitors and inhibitors of the canonical Smad and non-canonical signaling pathways. Direct binding between LOXL1 and TGFß1-LAP was demonstrated by Blot overlay assays and ELISA. LOXL1 overexpression temporarily upregulates different transcriptional regulators and some protein kinases of p38-MAPK signalling pathway after 12 to 24 hours post-transfection.
Conclusions
The results of this study indicate that the interaction of LOXL1 and TGF-ß1 plays an important role in the PEX-associated abnormal matrix metabolism and fibrosis.
