TY - JOUR
T1 - FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress
AU - Choudhury, Rawshan
AU - Bayatti, Nadhim
AU - Scharff, Richard
AU - Szula, Ewa
AU - Tilakaratna, Viranga
AU - Udsen, Maja
AU - Mcharg, Selina
AU - Askari, Janet
AU - Humphries, Martin James
AU - Bishop, Paul
AU - Clark, Simon
N1 - Funding Information:
We thank Leo Zeef and Andy Hayes of the Bioinformatics and Genomic Technologies Core Facilities at the University of Manchester for providing support with regard to RNA-seq. This work was funded by a Fight for Sight research grant (1852/53), a Macular Society UK research grant, and an MRC Career development Fellowship (MR/K024418/1). SJC is funded by the Helmut Ecker Foundation, Germany.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/7/8
Y1 - 2021/7/8
N2 - 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.
AB - 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.
KW - Cell Communication
KW - Cell Death
KW - Cells, Cultured
KW - Extracellular Matrix/metabolism
KW - Gene Expression Regulation
KW - Humans
KW - Immobilized Proteins/metabolism
KW - Integrin alpha5beta1/metabolism
KW - Intracellular Signaling Peptides and Proteins/metabolism
KW - LIM Domain Proteins/metabolism
KW - Muscle Proteins/metabolism
KW - Oxidative Stress
KW - Protein Binding
KW - Retinal Pigment Epithelium/metabolism
KW - Telomerase/metabolism
U2 - 10.1038/s41598-021-93708-5
DO - 10.1038/s41598-021-93708-5
M3 - Article
C2 - 34239032
SN - 2045-2322
VL - 11
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 14175
ER -