The genomic response of the retinal pigment epithelium to light damage and retinal detachment

Abstract

The retinal pigment epithelium (RPE) plays an essential role in maintaining the health of the retina. The RPE is also the site of pathologic processes in a wide variety of retinal disorders including monogenic retinal dystrophies, age-related macular degeneration, and retinal detachment. Despite intense interest in the RPE, little is known about its molecular response to ocular damage or disease. We have conducted a comprehensive analysis of changes in transcript abundance (the "genomic response") in the murine RPE after light damage. Several dozen transcripts, many related to cell-cell signaling, show significant increases in abundance in response to bright light; transcripts encoding visual cycle proteins show a decrease in abundance. Similar changes are induced by retinal detachment. Environmental and genetic perturbations that modulate the RPE response to bright light suggest that this response is controlled by the retina. In contrast to the response to bright light, the RPE response to retinal detachment overrides these modulatory affects.

Figure 1.

Changes in RPE transcript levels in response to light damage. A, Scatterplot showing microarray hybridization intensity ratios and p values for three independent pairs of RPE samples from dark-adapted controls versus light-damaged BALB/c mice (20 eyes per sample). The scatterplot, including the subset of transcripts marked by red circles and blue squares (defined in the box in A), has not been corrected for redundancies in the Affymetrix M430 2.0 microarray annotation; thus, there are more data points than unique genes represented. B, RNA blot hybridization demonstrates increased or decreased abundances of different RPE transcripts 24 h after light damage. Left column, Transcripts induced by light damage, some of which are also induced in the retina. Right two columns, Transcripts encoding visual cycle proteins and RPE-specific G-protein-coupled receptors decrease in abundance after light damage. Gapdh and Rho transcripts serve, respectively, as (1) an internal control for equal loading of RNA and (2) retina contamination of the RPE sample. The Rpe65 blot demonstrates minimal contamination of the retina sample with RPE. C, Cellular localization of transcripts with altered abundances after light damage, as determined by in situ hybridization. Edn2, a transcript shown previously to accumulate in photoreceptors in response to bright light and other forms of damage, serves as a positive control. Vertical bars adjacent to each tissue section demarcate (from top to bottom) the RPE, outer nuclear layer, inner nuclear layer, and ganglion cell layer. ISH, In situ hybridization.

Figure 2.

Retinal detachment produces localized changes in transcript abundance in the retina and RPE. A–H, In situ hybridization 4 d after localized retinal detachment in albino BALB/c mice. Arrows indicate the lateral extent of the detachment. Within the detached region, there are localized increases in RPE transcripts (A, C, E), localized decreases in RPE transcripts (D, H), localized increases in retina transcripts (B, D, E), and localized decreases in retina transcripts (F). I–M, In situ hybridization to Serpin a3n transcripts 4 d after localized retinal detachment in a pigmented C57BL/6 × 129 hybrid eye. The detachment is seen on the left side of the eye in I. The melanin was partially bleached by overnight incubation with 3% hydrogen peroxide (see Materials and Methods). The left and right boxed zones in I are enlarged in J and L, respectively, and the boxed zones within J and L are further enlarged in K and M, respectively. Vertical black bars to the right of K and M demarcate the RPE. In the detached RPE, the purple in situ hybridization signal is seen superimposed on the light brown melanin.

Figure 3.

OSMR accumulation in the apical microvilli of the RPE after light damage. A, Specificity of OSMR-binding reagents. Left, Anti-OSMR immunoblot of control 293 cells (−) or 293 cells transfected with OSMR cDNA (+). Right, Anti-OSMR immunostaining (top) or AP-OSM binding followed by AP histochemistry (bottom) of control COS cells (−) or COS cells transfected with OSMR cDNA (+). B, Immunolocalization of OSMR (top) and binding of AP-OSM (bottom) in dark-adapted control (left) and light-damaged (right) BALB/c retinas. OSMR immunoreactivity and AP-OSM binding sites are induced 24 h after exposure to 1.5 h of bright light, and they localize to the apical face of the RPE and adjacent outer segment region. C, Immunoblots showing the time course of OSMR accumulation in the retina (top) and the RPE (bottom) after light damage to BALB/c mice. tub, Anti-β-tubulin control for sample loading; DAPI, 4′,6′-diamidino-2-phenylindole dihydrochloride; INL, inner nuclear layer; GCL, ganglion cell layer; ONL, outer nuclear layer; OS, outer segments; IS, inner segments.

Figure 4.

The response of the retina and RPE to light damage is modified by constant light preconditioning. A, RNA blots showing Serpin a3n and Osmr transcript accumulation in BALB/c mouse retina either preconditioned for 5 d in constant light (CL; ∼100 lux) or maintained in darkness (DA; dark adapted) and then exposed for 1.5 h to 6000 lux with pupil dilation, followed by a 24 h recovery in darkness. B, Immunoblot showing OSMR accumulation in the retina and RPE of BALB/c mice treated as described in A. C, In situ hybridization to Serpin a3n, Cebpd, and Mmp3 transcripts in the RPE and/or retina 24 h after light damage to BALB/c mice that were either dark adapted or constant light preconditioned. Light-damage induction of RPE transcripts is suppressed by preconditioning. D, In situ hybridization to Serpin a3n and Osmr transcripts in constant light-preconditioned mice after retinal detachment. Induction of these transcripts in retina and RPE is not suppressed by preconditioning. Vertical bars to the right of each tissue section are described in Figure 1.

Figure 5.

The stress responses of the retina and RPE are modulated by genetically based retinopathies. A–C, Immunoblots showing OSMR levels in the retina and RPE of control and mutant albino mice that were dark adapted for 7 d and then exposed to 1.5 h of 6000 lux white fluorescent light, followed by a 24 h recovery in darkness. Comparisons are shown for Gnat1 ^+/− and Gnat1 ^−/− (A), RPE65 ^+/− and RPE65 ^−/− (B), and rd1 ^+/− and rd1 ^−/− (C). D, In situ hybridization to Serpin a3n and Osmr transcripts 24 h after light damage of dark-adapted albino Gnat1 ^−/− and RPE65 ^−/− mice. Light-damage induction of RPE transcripts is not affected in the Gnat1 ^−/− retina and RPE, but is suppressed in the RPE65 ^−/− retina and RPE. E, In situ hybridization showing Serpin a3n and Osmr transcript accumulation in the retina and RPE of albino RPE65 ^−/− mice 4 d after a local retinal detachment. Regions that are unaffected (left) or detached (right) are from the same eye. Retinal detachment leads to the accumulation of both Serpin a3n and Osmr transcripts in the RPE, and to an increase in the already elevated level of these transcripts in the retina. Vertical bars adjacent to each tissue section are described in Figure 1.

Figure 6.

The RPE damage response is maintained in vitro in an eyecup explant. A, Transmission electron microscopy of the RPE and adjacent choroid from a C57BL/6 mouse after 6 d in vitro reveals relatively normal RPE morphology including apical microvilli. CC, Choriocapillaris; BM, Bruch's membrane; MV, microvilli. Size bar, 5 μm. B, RPE cells in an eyecup explant maintain transcriptional and translational competence. An eyecup from a BALB/c mouse was infected in vitro with a recombinant adenovirus vector carrying a β-galactosidase coding region under the control of a cytomegalovirus enhancer/promoter, incubated for 2 d, and then fixed and stained with X-gal. The blue X-gal reaction product is localized to the RPE. C, OSMR expression by RPE cells in eyecup explants prepared at different times after 1.5 h of light damage in vivo. Dark-adapted BALB/c mice were exposed to toxic light, RPE explants were prepared at the indicated times, and after 1 d in vitro the eyecups were immunostained for OSMR.