FHL-1 interacts with human RPE cells through the \u03b15\u03b21 integrin and confers protection against oxidative stress

Rawshan Choudhury,Simon J Clark,Martin J Humphries,Viranga Tilakaratna,Paul N Bishop,Maja Søberg Udsen,Selina Mcharg,Nadhim Bayatti,Janet A Askari,Ewa Szula,Richard Scharff

doi:10.1038/s41598-021-93708-5

Abstract

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

Highlights

The retinal pigment epithelium (RPE), a monolayer of cells in the retina, makes an essential contribution to the maintenance and support of the photoreceptor cells, and vision itself[1]
Despite the previously known important complement regulatory functions of FHL-17,18, the interaction described in this study demonstrates a non-canonical role of Factor H-like protein 1 (FHL-1) in protecting Retinal pigment epithelial (RPE) cells from oxidative-stress induced cell death
There is strong evidence implicating oxidative stress in the pathogenesis of AMD33–35, and the stability of RPE cells in vivo is dependent on their interactions, with one another, and their underlying extracellular matrix (ECM), Bruch’s membrane

Summary

Introduction

The retinal pigment epithelium (RPE), a monolayer of cells in the retina, makes an essential contribution to the maintenance and support of the photoreceptor cells, and vision itself[1]. RPE cells have a high metabolic turnover and are exposed to extreme levels of light-induced oxidative s tress[4]. RPE cells are separated from the choriocapillaris by an acellular barrier called Bruch’s membrane (Fig. 1a). This extracellular matrix (ECM) is comprised of five separate layers; the RPE basement membrane, inner collagenous layer, an elastin core, the outer collagenous layer, and the choriocapillaris basement membrane[6]. The attachment of RPE cells to their underlying Bruch’s membrane is required to maintain their homeostasis, and changes to this ECM affects RPE cell gene transcription, translation and protein s ecretion[9]. The α5β1 integrin recognises the conserved binding motif Arg-Gly-Asp (RGD) that is present in various ECM ligands including fibronectin (FN)[15]

Methods

Results

Conclusion