Modulation of cathepsin D activity in retinal pigment epithelial cells

Abstract

This project used retinal pigment epithelial (RPE) cells to investigate the effects of up- and down-regulation of cathepsin D expression on the processing of cathepsin D and on the normal phagocytic and digestive function of these cells. RPE cells were transfected with a pHbetaApr-1-neo vector construct carrying the full-length sequence of the translated region of human cathepsin D in sense and antisense directions. Transfected cells were characterized for the presence and expression of the transgene by PCR amplification using transgene-specific primers. Total aspartic proteinase activity present in transformed RPE cells was measured by an enzyme assay using haemoglobin as substrate. Flow cytometry was used to quantify phagocytosis of fluorescein isothiocyanate-labelled rod outer segments (ROS), and lysosomal digestion of ROS was monitored by immunofluorescence. A 435 bp fragment was present in RPE cells carrying the cathepsin D transgene in sense and antisense orientations after PCR amplification. Expression of both 52 kDa procathepsin D and 34 kDa active cathepsin D was significantly up-regulated in sense cathepsin D-transfected RPE cells and down-regulated in RPE cells transfected with antisense cathepsin D. No other forms of cathepsin D were detected in the transfected cells, suggesting that, if pseudo-cathepsin D exists in RPE cells in vivo, it requires the presence of unknown specific regulatory elements. The up- and down-regulation of cathepsin D expression was further confirmed by enzyme assay. Transfected cells retained their phagocytosing ability after ROS challenge and maintained their ability to process ROS. The processing of ROS was significantly slower in RPE cells transfected with antisense than control vector or in sense-cathepsin D-transfected cells. These results demonstrate that cathepsin D is a major proteolytic enzyme participating in the lysosomal digestion of photoreceptor outer segments.

Melanoregulin (MREG) Modulates Lysosome Function in Pigment Epithelial Cells

“…proenzyme that is subsequently converted to a 46-kDa intermediate (54,55) and eventually an active 34-kDa enzyme during transport through the secretory pathway to lysosomes (56 -59). To determine whether loss of MREG alters Cat-D function in vivo, enzyme activity, expression levels, and distribution profiles were analyzed in RPE within retinas of Mreg Ϫ/Ϫ and Mreg ϩ/ϩ mice.…”

“…Ingested phagosomes are normally cleared from the apical RPE to the basal region as phagolysosomes are formed. Cat-D plays a major role in the degradation of opsin in phagolysosomes (30,53,54,60). In these next studies, we quantified the relative levels of opsin and Cat-D in phagosomes by double immunogold labeling of Mreg ϩ/ϩ and Mreg Ϫ/Ϫ retinas (Fig.…”

“…diminished lysosome maturation as reflected by decreased active Cat-D, is of particular significance when considered in the context of pathogenic changes of the retina. Upregulation of Cat-D results in the secretion of pro-Cat-D into the medium (54). Transgenic mice that overexpress mutant (inactive) Cat-D acquire features that resemble age-related retinal degenerative changes, including geographic atrophy, accelerated photoreceptor cell death, and presence of basal laminar deposits (82).…”

Macrophage and microglia ontogeny in the mouse visual system can be traced by the expression of Cathepsins B and D

“…However, strong cathepsin D but not cathepsin B expression was localized in the developing retinal pigment epithelium (RPE). In this regard cathepsin D is considered one of the most important lysosomal enzymes in RPE cells (Regan et al, 1980;Rakoczy et al, 1997), and plays a major role in degradation of the phagocytosed photoreceptors during their renewal process (el-Hifnawi, 1995). Several reports have described cathepsin D in the adult RPE of mammals (Wilcox, 1988;Ortego et al, 1997;Yamada et al, 1990;Rakoczy et al, 1999;Frohlich and Klessen, 2001;Wasselius et al, 2003;Ahuja et al, 2008) and, in concordance with our findings, Hayasaka and Shiono (1982) showed that, with the exception of cathepsin D displaying high levels of activity, most of the lysosomal enzyme activities detected in the rabbit RPE were low at birth.…”

The Phagocytosis of Rod Outer Segments is Inhibited by Selected Drugs in Retinal Pigment Epithelial Cell Cultures

“…There is evidence that tamoxifen, toremifene and chloroquine reduce the activities of cathepsin D and N-acetyl-b-glucosaminidase in retinal pigment epithelial cells (Toimela et al 1998). Cathepsin D is essential in rod outer segment digestion by human retinal pigment epithelial cells (Kennedy et al 1994;Rakoczy et al 1997). As a weakly basic compound, chloroquine accumulates in lysosomes, raising the lysosomal pH, which leads to inhibition of lysosomal enzymes (Ohkuma & Poole 1981;Toimela et al 1998;Schraermeyer et al 1999).…”