1] Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, OR, USA [2] Department of Integrative Biosciences, Oregon Health & Science University, Portland, OR, USA.
Department of Integrative Biosciences, Oregon Health & Science University, Portland, OR, USA.
Eye (Lond). 2014 Jan;28(1):85-92. doi: 10.1038/eye.2013.239. Epub 2013 Nov 8.
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.
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.
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.
Relative zinc deficiency in RPE cells causes activation of cytosolic calpain and mitochondrial caspase pathways without ER stress.
据报道，年龄相关性黄斑变性患者的黄斑区锌水平降低，锌螯合剂 N,N,N',N'-四（2-吡啶甲基）乙二胺（TPEN）可导致人视网膜色素上皮（RPE）细胞死亡。本研究旨在使用猴 RPE 细胞研究由 TPEN 引发的细胞死亡过程中的信号转导途径。
用 TPEN 培养 RPE 细胞。通过免疫印迹检测钙蛋白酶和半胱天冬酶的激活以及其底物的蛋白水解。通过孵育钙蛋白酶抑制剂 SNJ-1945 或半胱天冬酶抑制剂 z-VAD-fmk 来确认特定蛋白酶的激活。
TPEN 导致 RPE 细胞的存活率随时间呈依赖性下降。细胞死亡伴随着钙蛋白酶-1、半胱天冬酶-9 和半胱天冬酶-3 的激活。SNJ-1945 抑制钙蛋白酶的激活，略微抑制半胱天冬酶-9 的激活。z-VAD-fmk 抑制半胱天冬酶和钙蛋白酶-1 的激活。TPEN 未激活半胱天冬酶-12。
RPE 细胞中相对锌缺乏会导致细胞溶质钙蛋白酶和线粒体半胱天冬酶途径的激活，而不会导致内质网应激。
