Sanders Esmond J, Parker Eve, Harvey Steve
Department of Physiology, University of Alberta, 755 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada.
Exp Eye Res. 2006 Nov;83(5):1205-14. doi: 10.1016/j.exer.2006.06.009. Epub 2006 Aug 7.
Several variants of growth hormone (GH) are found in the retina and vitreous of the chick embryo, where they appear to act as cell survival factors, having neuroprotective effects on retinal ganglion cells (RGCs). Here, we investigate the molecular mechanisms of the anti-apoptotic effect of GH in cultured RGCs. GH treatment increased Akt phosphorylation in these cells, which is an anti-apoptotic event. Whereas unphosphorylated Akt was detected in both nucleus and cytoplasm of RGCs by immunocytochemistry, the phosphorylated form of Akt (Akt-phos) was located primarily in the cytoplasm of both normal and apoptotic cells, although levels were markedly lower in the latter. It was found that GH treatment of RGCs reduced Akt levels, while concomitantly raising Akt-phos levels, consistent with a role for Akt signaling pathways in GH neuroprotective action. This was substantiated using Wortmannin, which, like GH antiserum, inhibited Akt phosphorylation and initiated apoptosis. The addition of Wortmannin to RGC cultures simultaneously with GH significantly reduced the anti-apoptotic effect of GH. The induction of apoptosis by GH antiserum was clearly accompanied by an increase in caspase-3 activation and PARP-1 cleavage, both of which were significantly reduced in the presence of the broad spectrum caspase inhibitor, Q-VD-OPh, which itself had a dramatic neuroprotective effect on cultured RGCs. Calpain activation appeared to be a major caspase-independent pathway to PARP-1 cleavage and apoptosis in these cells. Calpain inhibitor III (MDL 28170) was able to reduce PARP-1 cleavage and abrogate the apoptogenic effect of GH antiserum. The results support the view that caspase and calpain inhibitors are major neuroprotective agents for RGCs, and that pathways that activate both caspases and calpains are important for the anti-apoptotic actions of GH in these cells.
在鸡胚的视网膜和玻璃体中发现了几种生长激素（GH）变体，它们似乎作为细胞存活因子发挥作用，对视网膜神经节细胞（RGCs）具有神经保护作用。在此，我们研究了GH在培养的RGCs中抗凋亡作用的分子机制。GH处理增加了这些细胞中Akt的磷酸化，这是一个抗凋亡事件。通过免疫细胞化学在RGCs的细胞核和细胞质中均检测到未磷酸化的Akt，而磷酸化形式的Akt（Akt-phos）主要位于正常细胞和凋亡细胞的细胞质中，尽管后者的水平明显较低。研究发现，GH处理RGCs会降低Akt水平，同时提高Akt-phos水平，这与Akt信号通路在GH神经保护作用中的作用一致。使用渥曼青霉素证实了这一点，渥曼青霉素与GH抗血清一样，抑制Akt磷酸化并引发凋亡。在RGC培养物中同时添加渥曼青霉素和GH可显著降低GH的抗凋亡作用。GH抗血清诱导的凋亡明显伴随着caspase-3激活和PARP-1裂解的增加，在存在广谱caspase抑制剂Q-VD-OPh的情况下，这两者均显著降低，而Q-VD-OPh本身对培养的RGCs具有显著的神经保护作用。钙蛋白酶激活似乎是这些细胞中PARP-1裂解和凋亡的主要非caspase依赖性途径。钙蛋白酶抑制剂III（MDL 28170）能够减少PARP-1裂解并消除GH抗血清的凋亡诱导作用。这些结果支持以下观点：caspase和钙蛋白酶抑制剂是RGCs的主要神经保护剂，激活caspase和钙蛋白酶的途径对于GH在这些细胞中的抗凋亡作用很重要。
