锌转运体ZnT和ZIP在人视网膜色素上皮中的表达及其受神经营养因子的调控

Expression of ZnT and ZIP zinc transporters in the human RPE and their regulation by neurotrophic factors.

作者信息

Leung Kar Wah, Liu Mugen, Xu Xuming, Seiler Magdalene J, Barnstable Colin J, Tombran-Tink Joyce

机构信息

Department of Neural and Behavioral Sciences, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.

出版信息

Invest Ophthalmol Vis Sci. 2008 Mar;49(3):1221-31. doi: 10.1167/iovs.07-0781.

DOI:10.1167/iovs.07-0781

PMID:18326752

Abstract

PURPOSE

Zinc is an essential cofactor for normal cell function. Altered expression and function of zinc transporters may contribute to the pathogenesis of neurodegenerative disorders including macular degeneration. The expression and regulation of zinc transporters in the RPE and the toxicity of zinc to these cells were examined.

METHODS

Zinc transporters were identified in a human RPE cell line, ARPE19, using a 28K human array, and their expression was confirmed by PCR, immunocytochemistry, and Western blot analysis in primary human RPE cultures and ARPE19. Zinc toxicity to ARPE19 was determined using monotetrazolium, propidium iodide, and TUNEL assays, and Zn(2+) uptake was visualized with Zinquin ethyl ester. The effect of various growth factors on zinc transporter expression also was examined.

RESULTS

Transcripts for 20 of 23 zinc transporters are expressed in fetal human RPE, 16 of 23 in adult human RPE, and 21 of 23 in ARPE19. Zn transporter proteins were also detected in ARPE19. ZnT5 expression was not observed, whereas ZnT6, ZIP1, and ZIP13 were the most abundantly expressed in all RPE samples. The addition of low concentrations of Zn(2+) to cultures resulted in a dose-dependent increase in intracellular Zn(2+) content in ARPE19, and >30 nM Zn(2+) induced necrosis with an LC(50) of 117.4 nM. Brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), and pigment epithelial-derived neurotrophic factor (PEDF) increased ZIP2 expression, GDNF and PEDF increased ZnT2 expression, and PEDF increased ZnT3 and ZnT8 expression. These neurotrophic factors also promoted Zn(2+) uptake in the RPE.

CONCLUSIONS

The array of zinc transporters expressed by the RPE may play a key role in zinc homeostasis in the retina and in ocular health and diseases.

摘要

目的

锌是正常细胞功能所必需的辅助因子。锌转运体表达和功能的改变可能导致包括黄斑变性在内的神经退行性疾病的发病机制。研究了视网膜色素上皮（RPE）中锌转运体的表达和调控以及锌对这些细胞的毒性。

方法

使用28K人类芯片在人RPE细胞系ARPE19中鉴定锌转运体，并通过PCR、免疫细胞化学和蛋白质印迹分析在原代人RPE培养物和ARPE19中确认其表达。使用单四氮唑盐、碘化丙啶和TUNEL试验确定锌对ARPE19的毒性，并用锌喹乙酯可视化Zn(2+)摄取。还研究了各种生长因子对锌转运体表达的影响。

结果

23种锌转运体中的20种转录本在胎儿人RPE中表达，23种中的16种在成人RPE中表达，23种中的21种在ARPE19中表达。在ARPE19中也检测到了锌转运体蛋白。未观察到ZnT5表达，而ZnT6、ZIP1和ZIP13在所有RPE样本中表达最为丰富。向培养物中添加低浓度的Zn(2+)导致ARPE19细胞内Zn(2+)含量呈剂量依赖性增加，>30 nM Zn(2+)诱导坏死，半数致死浓度（LC(50)）为117.4 nM。脑源性神经营养因子、睫状神经营养因子、胶质细胞源性神经营养因子（GDNF）和色素上皮源性神经营养因子（PEDF）增加ZIP2表达，GDNF和PEDF增加ZnT2表达，PEDF增加ZnT3和ZnT8表达。这些神经营养因子还促进了RPE中的Zn(2+)摄取。