Contribution of Calpain and Caspases to Cell Death in Cultured Monkey RPE Cells

Abstract

Purpose: AMD is the leading cause of human vision loss after 65 years of age. Several mechanisms have been proposed: (1) age-related failure of the choroidal vasculature leads to loss of RPE; (2) RPE dysfunctions due to accumulation of phagocytized, but unreleased A2E (N-retinylidene-N-retinylethanolamine); (3) zinc deficiency activation of calpain and caspase proteases, leading to cell death. The purpose of the present study is to compare activation of calpain and caspase in monkey RPE cells cultured under hypoxia or with A2E.

Methods: Monkey primary RPE cells were cultured under hypoxic conditions in a Gaspak pouch or cultured with synthetic A2E. Immunoblotting was used to detect activation of calpain and caspase. Calpain inhibitor, SNJ-1945, and pan-caspase inhibitor, z-VAD-fmk, were used to confirm activation of the proteases.

Results: (1) Hypoxia and A2E each decreased viability of RPE cells in a time-dependent manner. (2) Incubation under hypoxia alone induced activation of calpain, but not caspases. SNJ-1945 inhibited calpain activation, but z-VAD-fmk did not. (3) Incubation with A2E alone induced activation of calpain, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation. z-VAD-fmk inhibited caspase activation, suggesting no interaction between calpain and caspases.

Conclusions: Hypoxia activated the calpain pathway, while A2E activated both calpain and caspase pathways in monkey RPE cells. Such knowledge may be utilized in the treatment of AMD if inhibitor drugs against calpain and/or caspase are used to prevent RPE dysfunction caused by hypoxia or A2E.

Figure 1

Phase-contrast micrographs of monkey RPE cells under hypoxia. (A) initial, (B) 1-day hypoxia, (C) 1-day hypoxia/1-day reoxygenation, (D) 2-day hypoxia, and (E) 2-day hypoxia/1-day reoxygenation. These images were chosen from the most representative experiment in Figure 2 (n = 3).

Figure 2

Immunoblots of caspases, calpains, and their substrates in RPE cells cultured under hypoxia: (lane 1) initial, (lane 2) 1-day normal, (lane 3) 1-day hypoxia, (lane 4) 2-day normal, (lane 5) 1-day hypoxia/1-day reoxygenation, (lane 6) 2-day hypoxia, (lane 7) 3-day normal, and (lane 8) 2-day hypoxia/1-day reoxygenation. (A) caspase-3, (B) calpain 1, (C) calpain 2, (D) α-spectrin, a substrate for both capase-3 and calpain, and (E) β-actin (nonsubstrate gel-loading control). The bar graphs show the densities of bands for (F) α-spectrin fragments at 145-kDa (calpain-specific) normalized to β-actin and expressed as means ± SEM (n = 3). *P < 0.05 relative to the corresponding normal group (Dunnett's t-test).

Figure 3

Phase-contrast micrographs of RPE cells cultured under hypoxia with inhibitors. (A) 2-day normal, (B) 1-day hypoxia/1-day reoxygenation, (C) 1-day hypoxia/1-day reoxygenation + 100 μM SNJ-1945 (calpain inhibitor), (D) 1-day hypoxia/1-day reoxygenation + 100 μM z-VAD (pan-caspase inhibitor), and (E) 1-day hypoxia/1-day reoxygenation + 100 μM SNJ-1945 + 100 μM z-VAD. These images were chosen from the most representative experiment in Figure 4 (n = 5). (F) The bar graph showing the percentage of total area occupied by attached cells in each group compared to normal cells cultured for 2 days. Data are % ± SEM (n = 5 sets, each set was an average of 20 images). *P < 0.05 relative to the hypoxia/reoxygenation group (Dunnett's t-test).

Figure 4

Immunoblots of caspases, calpains, and their substrates in RPE cells cultured under hypoxia with inhibitors. (Lane 1) normal, (lane 2) 1-day hypoxia/1-day reoxygenation, (lane 3) 1-day hypoxia/1-day reoxygenation + 10 μM SNJ-1945, (lane 4) 1-day hypoxia/1-day reoxygenation + 100 μM SNJ-1945, (lane 5) 1-day hypoxia/1-day reoxygenation + 100 μM z-VAD, and (lane 6) 1-day hypoxia/1-day reoxygenation + 100 μM SNJ-1945 + 100 μM z-VAD. (A) Caspase-3, (B) caspase-7, (C) caspase-9, (D) calpain 1, (E) calpain 2, (F) PARP, (G) α-spectrin, (H) RPE cell marker cytokeratin-18, and (I) β-actin. The bar graphs show the densities of bands for the (J) α-spectrin 145-kDa fragment and the (K) intact band of CK-18 normalized to β-actin. The data are expressed as means ± SEM (n = 5). *P < 0.05, all relative to the 2-day hypoxia/reoxygenation group (Dunnett's t-test).

Figure 5

Phase-contrast micrographs of RPE cells cultured with A2E. (A) Initial, (B) plus 25 μM A2E at 12 hours, (C) 25 μM A2E at day 1, and (D) 25 μM A2E at day 2. These images were chosen from the most representative experiment in Figure 6 (n = 3).

Figure 6

Immunoblots of caspases, calpains, and their substrates in RPE cells cultured with A2E. (Lane 1) initial, (lane 2) 12-hour normal, (lane 3) 12-hour 25 μM A2E, (lane 4) 1-day normal, (lane 5) 1-day 25 μM A2E, (lane 6) 2-day normal, and (lane 7) 2-day 25 μM A2E. (A) Caspase-3, (B) calpain 1, (C) calpain 2, (D) α-spectrin, and (E) β-actin. Bar graphs show the densities of α-spectrin fragments at (F) 145 kDa (calpain-specific) and at (G) 120-kDa (caspase-3-specific) normalized to β-actin. Data are expressed as means ± SEM (n = 3). *P < 0.05 relative to the corresponding normal group (Dunnett's t-test).

Figure 7

Phase-contrast micrographs of RPE cells cultured with A2E and inhibitors. (A) 1-day normal, (B) plus 25 μM A2E at 1 day, (C) 25 μM A2E + 100 μM SNJ-1945, (D) 25 μM A2E + 100 μM z-VAD, and (E) 25 μM A2E + 100 μM SNJ-1945 + 100 μM z-VAD. These images were chosen from the most representative experiment in Figure 8 (n = 5).

Figure 8

Immunoblots for caspases, calpains, and their substrates in RPE cells cultured with A2E and inhibitors. (Lane 1) normal, (lane 2) 1-day 25 μM A2E, (lane 3) 25 μM A2E + 10 μM SNJ-1945, (lane 4) 25 μM A2E + 100 μM SNJ-1945, (lane 5) 25 μM A2E + 100 μM z-VAD, and (lane 6) 25 μM A2E + 100 μM SNJ-1945 + 100 μM z-VAD. (A) Caspase-3, (B) caspase-7, (C) caspase-9, (D) calpain 1, (E) calpain 2, (F) PARP, (G) α-spectrin, (H) intact cytokeratin-18 (CK-18) and CK-18 fragment at 26 kDa (caspase-3-specific), and (I) β-actin. The bar graphs show the densities of the bands for the (J) α-spectrin 145-kDa fragment, (K) α-spectrin 120-kDa fragment, (L) CK-18 intact band, and (M) the 26-kDa CK-18 fragment, all normalized to β-actin. Data are expressed as means ± SEM (n = 5). *P < 0.05 relative to A2E (Dunnett's t-test).

Figure 9

(A) Hypoxia-induced cell death in RPE cells is due only to activation of calpains. (B) Proposed pathway leading to A2E–induced cell death due to activation of caspase-3, -7, and -9, and calpains. Solid lines show the pathways confirmed in present study, blue lines show the pathways reported in the literature. Caspases-8 and -12 were not activated by either hypoxia or A2E.