Activation of αvβ3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner

Abstract

Purpose

Fibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of αvβ3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells.

Methods

TM-1 cells overexpressing wild-type β3 (WTβ3) or constitutively active β3 (CAβ3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. αvβ3 and α5β1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WTβ3 or CAβ3 integrin subunits were used to examine the role of αvβ3 integrin in HTM cells. The role of the canonical α5β1 integrin–mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the β1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632.

Results

Activation of αvβ3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on α5β1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CAβ3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched.

Conclusions

αvβ3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a β1 integrin/ROCK-independent mechanism. Thus, αvβ3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.