Regulation of cysteine cathepsin expression by oxidative stress in the retinal pigment epithelium/choroid of the mouse

Abstract

Cystatin C is the major inhibitor of the cysteine cathepsins. Polymorphisms in the cystatin C gene have recently been associated with the risk of developing Age-related Macular Degeneration (AMD). Oxidative stress is also thought to play a key role in the pathogenesis of AMD. We surveyed the retinal pigment epithelium (RPE) and choroid of the C57BL/6J mouse for the expression of the cysteine cathepsins under normoxic and hyperoxic (75% O(2)) conditions. Microarray analysis of RPE/choroid mRNA revealed the expression of cathepsins B and L, as well as cystatin C under all experimental conditions. The microarray results were confirmed by real-time quantitative polymerase chain reaction (PCR). Localization of the mRNA species for cystatin C and cathepsin B, as well as, localization of protein species for cystatin C, cathepsins B and L were performed to evaluate the tissue distribution of these species. Our results indicate that cystatin C is largely synthesized in the RPE and secreted from the basal side. Cathepsin B is the major cysteine protease in the RPE and choroid. The expression of all mRNAs and proteins was elevated by exposure to oxidative stress.

Fig. 1

Affymetrix GeneChip Expression Analysis of cathepsins B, L, S and cystatin C, in the RPE/choroid of C57BL/6J mice after 14 days of exposure to hyperoxic (75% oxygen) or normoxic conditions (21% oxygen). Gene expression of cathepsins B, L, S and cystatin C increased under oxidative stress (solid columns). Asterisks denotes a statistically significant difference between the normoxic and hyperoxic group (p<0.05).

Fig. 2

Cathepsin B, cathepsin L and cystatin C mRNA, measured using real-time quantitative TaqMan PCR on the RPE/choroid of C57BL/6J-Tyr^c-2J/J mice, after 14 days of exposure to hyperoxic or normoxic conditions. Each value represents an average of three measurements from three eyes. Cathepsin B, L and cystatin C mRNA levels were significantly elevated under oxidative stress (solid columns). Asterisks denotes a statistically significant difference between the normoxic and hyperoxic groups (p<0.005).

Fig. 3

Cathepsin B mRNA expression in the RPE/choroid of the C57BL/6J-Tyr^c-2J/J mouse, after 14 days of exposure to hyperoxic or normoxic conditions (control). (A) Staining of normoxic tissue with the sense riboprobe. (B) Staining of normoxic tissue with the anti-sense riboprobe. (C) Staining of hyperoxic tissue with the sense riboprobe. (D) Staining of hyperoxic tissue with the anti-sense riboprobe. Scale bar = 10 μM.

Fig. 4

Cystatin C mRNA expression in the RPE/choroid of the C57BL/6J-Tyr^c-2J/J mouse, after 14 days of exposure to hyperoxic or normoxic conditions (control). (A) Staining of normoxic tissue with the sense riboprobe. (B) Staining of normoxic tissue with the anti-sense riboprobe. (C) Staining of hyperoxic tissue with the sense riboprobe. (D) Staining of hyperoxic tissue with the anti-sense riboprobe. Scale bar = 10 μM.

Fig. 5

Cathepsin B immunoreactivity in the RPE/choroid of the C57BL/6J-Tyr^c-2J/J mouse, after 14 days of exposure to hyperoxic or normoxic conditions (control). (A) Staining of normoxic tissue with normal IgG. (B) Staining of normoxic tissue with anti-cathepsin B antibody. (C) Staining of hyperoxic tissue with normal IgG. (D) Staining of hyperoxic tissue with anti-cathepsin B antibody. Scale bar = 10 μM.

Fig. 6

Cathepsin L immunoreactivity in the RPE/choroid of the C57BL/6J-Tyr^c-2J/J mouse, after 14 days of exposure to hyperoxic or normoxic conditions (control). (A) Staining of normoxic tissue with normal IgG. (B) Staining of normoxic tissue with anti-cathepsin L antibody. (C) Staining of hyperoxic tissue with normal IgG. (D) Staining of hyperoxic tissue with anti-cathepsin L antibody. Scale bar = 10 μM.

Fig. 7

Cystatin C immunoreactivity in the RPE/choroid of the C57BL/6J-Tyr^c-2J/J mouse, after 14 days of exposure to hyperoxic or normoxic conditions (control). (A) Staining of normoxic tissue with normal IgG. (B) Staining of normoxic tissue with anti-cystatin C antibody. (C) Staining of hyperoxic tissue with normal IgG. Scale bar = 10 μM.