Activation of the mitochondrial caspase pathway and subsequent calpain activation in monkey RPE cells cultured under zinc depletion

Abstract

Purpose: Decreased zinc levels in the macula are reported in patients with age-related macular degeneration, and the zinc chelator N,N,N',N'-tetrakis (2- pyridylmethyl) ethylenediamine) (TPEN) causes death of human retinal pigment epithelial (RPE) cells. The purpose of the present study was to investigate signal transduction pathways during cell death initiated by TPEN, using monkey RPE cells.

Methods: RPE cells were cultured with TPEN. Activation of calpains and caspases, and proteolysis of their substrates were detected by immunoblotting. Incubation of calpain inhibitor SNJ-1945 or caspase inhibitor z-VAD-fmk was used to confirm activation of specific proteases.

Results: TPEN caused a time-dependent decrease in viable RPE cells. Cell death was accompanied by activation of calpain-1, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation and slightly inhibited caspase-9 activation. z-VAD-fmk inhibited caspases and calpain-1 activation. TPEN did not activate caspase-12.

Conclusions: Relative zinc deficiency in RPE cells causes activation of cytosolic calpain and mitochondrial caspase pathways without ER stress.

Figure 1

Growth curve for cultured monkey RPE cells. Data are expressed as means ±SEM (n=3–5).

Figure 2

Immunoblots of caspases, calpains, and their substrates: (lane 1) normal, (lane 2) 2-day 3 μM TPEN, (lane 3) 2-day 3 μM TPEN+100 μM SNJ-1945, (lane 4) 2-day 3 μM TPEN+10 μM SNJ-1945, (lane 5) 2-day 3 μM TPEN+100 μM z-VAD, and (lane 6) 2-day 3 μM TPEN+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) caspase-3 and calpain substrate, α-spectrin, (h) RPE cell marker—vimentin, (i) RPE cell marker—cytokeratin-18, and (j) β-actin. (g) The bar graph shows the density of the α-spectrin 145 kDa band (calpain-specific) and the 120 kDa band (caspase-3-specific) normalized to β-actin, and (i) the density of the cytokeratin-18 intact band and the 26 kDa band (caspase-3-specific) normalized to β-actin and expressed as means±SEM (n=3). **P<0.01 and *P<0.05, all relative to TPEN (Dunnett's t-test).

Figure 3

Phase-contrast micrographs of monkey RPE cells: (a) 2-day normal, (b) 3 μM TPEN at day 2, (c) 3 μM TPEN+100 μM SNJ-1945 (calpain inhibitor), (d) 3 μM TPEN+100 μM z-VAD (caspase inhibitor), and (e) 3 μM TPEN+100 μM SNJ-1945+100 μM z-VAD.

Figure 4

Immunoblots for ER caspase, caspase-12, calpains, α-spectrin, and ER stress markers: (lanes 1–3) ER stress marker treated with thapsigargin and (lanes 4–6) treated with TPEN. (Lane 1) 1-day normal, (lane 2) 1-day 10 μM thapsigargin, (lane 3) 1-day 10 μM thapsigargin+100 μM SNJ-1945, (lane 4) 2-day normal, (lane 5) 2-day 3 μM TPEN, and (lane 6) 2-day 3 μM TPEN+100 μM SNJ-1945. (a) Caspase-12, (b) calpain-1, (c) calpain-2, (d) caspase-3 and calpain substrate α-spectrin, (e) ER membrane marker calnexin, (f) ER stress marker CHOP, (g) ER stress marker Bip, and (h) β-actin.

Figure 5

Proposed pathways leading to TPEN-induced cell death in monkey RPE cells due to activation of caspases and calpains. Solid lines show the pathways confirmed in present study, green lines show the pathways reported in the literature,^{, ,} and dotted lines show the pathways not involved after TPEN treatment.