Abstract
Purpose.:
Choroideremia (CHM) is an X-linked progressive degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid caused by mutations in the CHM gene, which encodes Rab escort-protein-1 (REP-1). REP-1 enables posttranslational isoprenyl modification of Rab GTPases, proteins that control vesicle formation, movement, docking, and fusion. The aim of this study was to determine the effect of REP-1 depletion on vesicular trafficking in phagocytic and secretory pathways of human RPE.
Methods.:
In vitro, REP-1 expression was inhibited in human fetal RPE (hfRPE) cells by siRNA knockdown and its effects measured on the uptake of bovine photoreceptor outer segments (POS), proteolysis of POS rhodopsin, phagosomal pH, phagosome fusion with early and late endosomes/lysosomes, and polarized secretion of cytokines.
Results.:
Depletion of REP-1 in human RPE cells did not affect POS internalization but reduced phagosomal acidification and delayed POS protein clearance. REP-1 depletion also caused a decrease in the association of POS-containing phagosomes with late endosomal markers (Rab7, LAMP-1) and increases in the secretion of monocyte chemotactic protein (MCP-1) and interleukin (IL)-8 by hfRPE cells.
Conclusions.:
Lack of REP-1 protein expression in hfRPE cells leads to reduced degradation of POS most likely because of the inhibition of phagosome-lysosome fusion events and increased constitutive secretion of MCP-1 and IL-8. These observations may explain the accumulation of unprocessed outer segments within the phagolysosomes of RPE cells and the presence of inflammatory cells in the choroid of patients with CHM.
The mechanism of degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid in choroideremia (CHM) remains largely unknown. This X-linked, monogenic disorder is caused by mutations in the ubiquitously expressed
CHM gene, which encodes Rab escort-protein-1 (REP-1).
1,2 REP-1 participates in the geranylgeranylation of
ras-related GTPases, Rab proteins, that are integral to the trafficking of vesicles in endocytic and exocytic pathways.
3–6 Patients with CHM have a relative or complete lack of REP-1,
7–9 which conceivably cannot be compensated for in the eye by REP-2, a product of the CHM-like gene and the only other REP.
10 Alternately, the relative affinity of some Rab/REP-2 complexes for geranylgeranyl transferase may be less than that of Rab/REP-1.
11 The lack of REP-1 within cells of the eye has been hypothesized to affect the transport of opsin to the photoreceptor outer segment, the apical distribution of melanosomes in the RPE, and the phagocytosis of photoreceptor outer segments by the RPE.
12 For example, the RPE in the zebrafish
chm model fails to process outer segments within the phagolysosomes.
13
The RPE is a monolayer of polarized pigmented cells that lies between the neuroretina and the choroid and that plays a crucial role in the function and survival of the retina by performing a number of essential functions such as the phagocytosis of photoreceptor outer segments and the maintenance of immune privilege within the eye by polarized balanced secretion of anti-inflammatory and proinflammatory cytokines, among them interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1.
14,15 Various lines of evidence point to the RPE as having a central role in the pathogenesis of CHM. Rodrigues et al.,
16 studying the eye of a 19-year-old CHM affected male, identified a few pigment-filled macrophages within the retina that had attached outer segment structures, phagosomes, occasional melanin granules, and “curvilinear rod-like profiles.” The authors suggested the possibility of a defect in outer segment phagocytosis. Bonilha et al.,
17 reporting the pathology on a 91-year-old female CHM carrier, noted the absence of RPE apical microvilli and basal infoldings. Moreover, the RPE donor basal surface was dominated by the presence of banded fibers composed of clumps of widely spaced collagen. Bruch's membrane and the space between the basal membrane of the RPE contained many smooth and bristle-like-coated vesicles. RPE ultrastructural changes were consistent with cells that could not carry out several nurturing functions.
The aim of our study was to determine the effect of REP-1 depletion on cellular trafficking in endocytic and exocytic pathways in human RPE cells. Here we show that lack of REP-1 expression leads to reduced degradation of POS by RPE cells, most likely because of the inhibition of the phagosome-lysosome fusion events, and increased constitutive secretion of MCP-1 and IL-8 by RPE cells. These observations may explain the accumulation of unprocessed outer segments within the phagolysosomes of RPE cells and the finding of inflammatory cells in pathologic eye specimens from patients with CHM.
Confluent hfRPE cells were challenged with unlabeled POS for 2 hours (pulse) and chased up to 23 hours. Cells were lysed in RIPA buffer (Pierce, Rockford, IL) containing a cocktail of protease inhibitors (Complete; Roche, Indianapolis, IN). Degradation of phagocytosed POS opsin was determined by Western blot analysis of hfRPE cell protein extracts using a mouse monoclonal anti-rhodopsin antibody (Millipore/Chemicon, Temecula, CA). Integrated optical density (IOD) of the bands corresponding to the rhodopsin monomer were quantified, normalized to β-actin expression (IOD norm), and compared with the normalized IOD at 2-hour pulse/0-hour chase, which was set as 100%. Results were expressed as follows: % Degradation = 100% − IOD (norm) 23-hour chase/IOD (norm) 0-hour chase × 100%.
hfRPE cells plated in eight-well chamber slides (Laboratory-Tek; Nalge Nunc International, Naperville, IL) were continuously fed with POS over 6 hours, then were fixed in 4% paraformaldehyde-PBS, permeabilized, and blocked with normal goat serum in 0.05% Tween-20/PBS (PBST). The cells were double stained with antibodies to rhodopsin and LAMP-1 or Rab7A. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI; 1 μg/mL in PBS). Secondary antibodies used were conjugated with Alexa Fluor 488 or Alexa Fluor 568 (Invitrogen). Confocal microscopy was performed with a laser scanning confocal microscope (SP2, with TCS software version 11.04; Leica, Exton, PA) and 40× or 63× oil objectives. Magnifications varied, and scale bars are therefore digitally included in some of the pictures.
Proteins, separated by SDS-PAGE on 4% to 20% gradient mini-gels and transferred onto a 0.45-μm nitrocellulose membrane (Invitrogen), were probed with one of the following antibodies: rabbit anti-human LAMP-1, rabbit anti-human EEA-1, rabbit anti-Rab5A, rabbit anti-Rab 7A (all from Cell Signaling, Danvers, MA), or mouse anti-REP-1, clone 2F1 (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactivity was detected with a corresponding second anti-IgG antibody conjugated with horseradish peroxidase (Zymed Laboratory, Inc., San Francisco, CA) and imaged on x-ray film using chemiluminescent substrates (SuperSignal West Pico or SuperSignal West Dura; Pierce). Densitometric analysis was performed with ImageJ software. Results are presented as IOD normalized to β-actin expression.
Unless otherwise noted, all experiments were repeated at least three times, and data are presented as the mean ± SEM. Statistical comparisons were made using the Student's t-test (unpaired, two tailed, unless otherwise specified; Excel; Microsoft, Redmond, WA). Differences were considered to be significant if P < 0.05.
Phagocytosis of the POS consists of multiple processes such as POS recognition by RPE cells, binding, ingestion, phagosome maturation, and degradation of phagocytosed material. Interruption of any of these steps could lead to the accumulation of unprocessed material inside or outside RPE cells and eventually to retinal degeneration.
Here we show that lack of REP-1 expression results in altered degradation of POS by RPE cells or at least in the degradation of the major protein of rod outer segments, rhodopsin. Our data provide possible explanations for the observations of others studying clinical cases of CHM
16,17 and animal models
13 which have suggested a defect in phagocytosis.
To elucidate at what stage the process of phagocytosis is altered in REP-1-deficient cells, we first studied the internalization of POS labeled with rhodamine-based dye, which fluoresces only in an acidic environment. Depletion of REP-1 expression caused an average 20% reduction in fluorescence intensity of internalized rhodamine-based dye–POS particles compared with nontarget siRNA, likely as a result of the failure of POS-containing phagosomes to acidify properly. Quantitative analysis revealed that the differences in the numbers of rhodamine-based dye–POS ingested particles were not statistically significant compared with nontarget siRNA-treated cells. We found that the transfection of RPE cells with CHM siRNA resulted in an average 0.2 pH increase of phagosomal pH compared with nontarget siRNA-transfected cells. Several investigators have shown that acidification is a critical determinant in phagosome maturation.
26,27 In addition, most lysosomal proteases have an optimal proteolytic activity between pH 5.5 and 6.5,
28 and phagocytosed particles can begin to be degraded only when the phagosome reaches a sufficiently low pH. Deguchi et al.
21 demonstrated in vivo that bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, which increases intraphagosomal pH on average by 0.6 pH units, markedly inhibited the degradation of POS in the phagolysosomes.
Phagosomes are known to fuse first with early and late endosomes and then with lysosomes, thus progressively acquiring different proteins, such as Rab GTPases, the acidification machinery (V-type ATPase), and the lysosomal proteases from the endocytic pathway.
26,28,29
Rab GTPases have been implicated principally in the control of vesicle docking and fusion.
3,30 In one of the earliest known maturation events, Rab5 is recruited to phagosomes and fuses with early endosomes. Bridging between the two compartments is thought to be the role of early endosome antigen 1 (EEA1), the Rab5 effector.
26,31,32 Rab7 is essential for phagosome fusion with late endosomes or lysosomes.
30,33 Impairment of Rab7 function limits the degree of acidification of the phagosomal lumen. LAMP1 is a glycoprotein that is specifically localized to acidic lysosome structure and essential for the recruitment of Rab7 to the phagosome.
32
We hypothesize that under-prenylation of multiple Rab proteins resulting from REP-1 deficiency in the CHM knockdown human RPE cells could cause alteration in the fusion of phagosomes with endosomes and lysosomes and, as a result, the elevation of phagosomal pH and a deficiency in the degradation of POS.
In our experiments, silencing of CHM in human RPE cells resulted in a distribution of Rab5A and EEA1 consistent with delayed phagosomal maturation. The amount of Rab5A and EEA1 protein associated with latex bead–containing phagosomes was elevated in the first 6 hours and declined after 24 hours of phagocytosis compared with nontarget siRNA-transfected cells. At the same time, the association of late endosomal markers, LAMP-1 and Rab7A, with latex-containing and POS-containing phagosomes was diminished at 6 and 24 hours of phagocytosis, indicating reduced fusion of late endosomes and lysosomes with newly internalized targets.
Our observations suggest that a lack of REP-1 protein expression leads to a deficiency in the degradation of POS by RPE cells, most likely because of the failure of POS-containing phagosomes to acidify properly as a result of inhibition of the phagosome-lysosome fusion events. Decreased rates of POS degradation by RPE cells could lead to an accumulation of unprocessed material inside the cells and to gradual functional decline over many years. According to Burke,
34 even subtle modifications of cell shape or subcellular organization could have profound consequences for the ability of the RPE to support the survival of adjacent photoreceptors over time.
Effect of REP-1 Deficiency on Secretion of Chemokines and Growth Factors by hfRPE Cells