Integrin αVβ5-mediated Removal of Apoptotic Cell Debris by the Eye Lens and Its Inhibition by UV Light Exposure

Daniel Chauss,Lisa A Brennan,Olga Bakina,Marc Kantorow

doi:10.1074/jbc.m115.688390

Daniel Chauss, Lisa A Brennan + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m115.688390

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Abstract

Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important for establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens, and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high throughput RNA sequencing and bioinformatics analysis, we establish that lens epithelial cells express members of the integrin αVβ5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVβ5 and with RAB7 and Rab-interacting lysosomal protein that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVβ5 receptor is required for lens epithelial cell phagocytosis and that UV light treatment of lens epithelial cells results in damage to the αVβ5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVβ5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.

Highlights

An excellent model system to identify these mechanisms is the transparent eye lens that functions to focus light onto the retina where visual information is transmitted to the brain [11]
The results suggest that UV light-induced damage to integrin ␣V␤5 and the consequential loss of lens phagocytosis could result in accumulation of toxic apoptotic cell debris, death of lens epithelial cells, and cataract formation
Lens Epithelial Cells Phagocytize Multiple Substrates—To evaluate whether lens cells were capable of phagocytosis, we first examined the ability of human SRA 01/04 lens epithelial cells to internalize fluorescently labeled polystyrene beads over a period of 16 h (Fig. 1A)

Summary

Experimental Procedures

Cell Culture and Transfection of Human Lens Epithelial Cells—Human lens epithelial cells (SRA 01/04) [44] were cultured in DMEM (Invitrogen) supplemented with 15% FBS (Invitrogen), gentamicin (50 units/ml; Invitrogen), penicillin/ streptomycin antibiotic mix (50 units/ml; Invitrogen), and fungizone (5 ␮l/ml; Invitrogen) at 37 °C in the presence of 5% CO2. Preparation of Chicken Primary Lens Epithelial Cells—Primary chicken lens epithelial cell cultures were prepared from the lenses of embryonic day 10 (E10) White Leghorn embryonated chicken eggs (Charles River Laboratories, Storrs, CT) using the method of Menko et al [45]. Primary lens cells were isolated from chicken lenses by trypsinization and agitation. Cells were plated onto glass bottom dishes coated with mouse laminin 23017015, Invitrogen) and cultured in Medium 199 Cells were plated onto glass bottom dishes coated with mouse laminin (catalog no. 23017015, Invitrogen) and cultured in Medium 199 (catalog no. 11150067, Invitrogen) supplemented with 10% FBS (catalog no. 10437028, Invitrogen) and

The abbreviations used are

Results

Discussion