Induction of amyloid β accumulation by ER calcium disruption and resultant upregulation of angiogenic factors in ARPE19 cells
Induction of amyloid β accumulation by ER calcium disruption and resultant upregulation of angiogenic factors in ARPE19 cells
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DOI：
10.1167/iovs.07-1067
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发表时间：
2008-06-01
影响因子：
4.4
通讯作者：
Tohyama, Masaya
中科院分区：
文献类型：
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作者：
Koyama, Yoshihisa;Matsuzaki, Shinsuke;Tohyama, Masaya
目的。目的：探讨β淀粉样蛋白（A β）在人视网膜色素上皮细胞系arpe1中积累和血管生成的细胞内机制。作者使用了两种内质网（ER）应激诱导试剂，抑制肌浆/内质钙（Ca）(2+)- atp酶的thapsigargin （TG）和抑制n -链糖基化的tunicamycin （TM）。采用逆转录(RT)-PCR技术研究了A β前体蛋白（APP）剪接变异体的表达模式。通过实时RT-PCR和Western blot （VEGF）检测a β代谢相关因子和血管生成因子的细胞表达。采用实时RT-PCR和Western blot检测caspase-4的表达，评价内质网应激源的影响。通过钙离子成像实验评估TG引起的细胞内钙升高。二甲基亚砜作为非试剂对照，stauro-sporine作为线粒体诱导凋亡试剂，而不是内质网诱导凋亡试剂。tg处理的ARPE19细胞增加了诱导A β产生的APP剪接变体的mRNA表达，并降低了A β的分解代谢酶neprilysin的mRNA表达。tg处理的ARPE19细胞VEGF、tnf - α、TACE mRNA和VEGF蛋白表达增加，PEDF mRNA表达减少。tg处理的ARPE19细胞比tm处理的casepase-4更能诱导活性表达。仅tg处理的ARPE19细胞内Ca浓度升高。tg处理的ARPE19细胞显示A β积累诱导和血管生成因子mRNA表达模式。本研究提示内质网应激通过内质网钙破坏不仅可能诱导A β沉积促进因子的表达，还可能诱导视网膜色素上皮血管生成因子的表达。
PURPOSE. To investigate the intracellular mechanisms that induce amyloid beta (A beta) accumulation and angiogenesis in the human retinal pigment epithelial cell line ARPE19.METHODS. The authors used two endoplasmic reticulum (ER) stress-inducing reagents, thapsigargin (TG), which inhibits the sarcoplasmic/endoplasmic calcium (Ca)(2+)-ATPase, and tunicamycin (TM), which inhibits N-linked glycosylation. The expression pattern of A beta-precursor protein (APP) splice variants was investigated by reverse transcription (RT)-PCR. Cellular expressions of both a series of A beta metabolism-related factors and angiogenic factors were evaluated by real-time RT-PCR and Western blot (VEGF). Expression of caspase-4 was examined by real-time RT-PCR and Western blot to evaluate the effect of the ER stressor. Intracellular Ca elevation by TG was evaluated by Ca2+ imaging experiments. Dimethyl sulfoxide and stauro-sporine were used as a nonreagent control and as an apoptosis-inducing reagent through mitochondria not ER, respectively.RESULTS. TG-treated ARPE19 cells increased the mRNA expression of A beta production-inducing APP splice variants and reduced that of neprilysin, a catabolic enzyme for A beta. TG-treated ARPE19 cells produced increases in VEGF, TNF-alpha, TACE mRNA, and VEGF protein expressions and a decrease in PEDF mRNA expression. TG-treated ARPE19 cells induced the expression of active more than TM-treated casepase-4. The intracellular Ca concentration was elevated in only TG-treated ARPE19 cells.CONCLUSIONS. TG-treated ARPE19 cells showed both A beta accumulation-inducible and angiogenic factor mRNA expression patterns. This study suggests the possibility that ER stress through ER calcium disruption may induce the expression not only of A beta deposit-promoting factors but also angiogenic factors in the retinal pigment epithelium.