The pathophysiology of pseudoexfoliation syndrome is affected by interaction of TGF‐β1 and LOXL1

Abstract

PurposeThe 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.MethodsPrimary 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 .ResultsTGFß1 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.ConclusionsThe 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.