Aberrant accumulation of fibulin-3 in the endoplasmic reticulum leads to activation of the unfolded protein response and VEGF expression
Aberrant accumulation of fibulin-3 in the endoplasmic reticulum leads to activation of the unfolded protein response and VEGF expression
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DOI：
10.1167/iovs.05-0070
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发表时间：
2005-11-01
影响因子：
4.4
通讯作者：
Abcouwer, SF
中科院分区：
文献类型：
--
作者：
Roybal, CN;Marmorstein, LY;Abcouwer, SF
目的。遗传性早发性黄斑退行性疾病，如leventinese malattia （ML）和Doyne蜂窝视网膜营养不良（DHRD）与导致突变纤维蛋白-3蛋白（R345W）产生的错义突变有关。R345W分泌不良，在ML/DHRD视网膜的RPE中积累。内质网（ER）内错误折叠蛋白的积累导致未折叠蛋白反应（UPR）信号的激活和内质网应激反应基因的表达，包括血管内皮生长因子（VEGF）。因此，我们假设R345W的表达激活了UPR，导致了VEGF的表达。利用腺病毒载体在ARPE-19细胞中过表达fibuin -3野生型（Wt）和R345W突变蛋白。采用Western blot和免疫细胞化学方法比较Wt和R345W的分泌和细胞内蓄积。通过测量葡萄糖调节蛋白78 （GRP78 [BiP]）的表达和编辑X-box结合蛋白（XBP-1） mRNA来评估UPR的激活。通过Northern blot分析、Western blot分析和使用含有8.2 kb人VEGF基因的新型VEGF启动子报告结构来测定VEGF的表达和VEGF启动子的转录激活。R345W在ARPE-19细胞中分泌不足，在内质网中积累，导致UPR激活，VEGF表达增加。与Wt突变蛋白相比，R345W的表达更有效地引起UPR激活，增加VEGF表达，并刺激VEGF启动子的转录。这些发现表明，突变的纤维蛋白-3的表达引起UPR激活和VEGF表达增加。突变纤维蛋白的表达可能通过引起内质网应激导致RPE功能障碍和VEGF表达增加而促进黄斑变性和脉络膜新生血管。
PURPOSE. The inherited early-onset macular degenerative disease known as malattia leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) have been linked to a missense mutation leading to production of a mutant fibulin-3 protein (R345W). R345W is poorly secreted and accumulates in the RPE of ML/DHRD retinas. Accumulation of misfolded proteins within the endoplasmic reticulum ( ER) causes activation of unfolded protein response (UPR) signaling and expression of ER stress - responsive genes, including vascular endothelial growth factor ( VEGF). Therefore, we hypothesized that the expression of R345W activates the UPR, leading to VEGF expression.METHODS. Adenoviral vectors were used to overexpress fibulin-3 wild-type (Wt) and R345W mutant proteins in ARPE-19 cells. Secretion and intracellular accumulation of Wt and R345W were compared by Western blot analysis and immunocytochemistry. Activation of the UPR was evaluated by measuring the expression of glucose-regulated protein 78 (GRP78 [BiP]) and editing of the X-box binding protein (XBP-1) mRNA. VEGF expression and transcriptional activation of the VEGF promoter were determined by Northern blot analysis, Western blot analysis, and use of a novel VEGF promoter-reporter construct containing 8.2 kb of the human VEGF gene.RESULTS. R345W was poorly secreted by ARPE-19 cells and accumulated in the ER, leading to UPR activation and increased VEGF expression. Compared with Wt mutant proteins, the expression of R345W was more effective at causing UPR activation, increasing VEGF expression, and stimulating transcription from the VEGF promoter.CONCLUSIONS. These findings demonstrated that the expression of mutated fibulin-3 caused UPR activation and increased VEGF expression. Expression of mutant fibulin proteins may contribute to macular degeneration and choroidal neovascularization by causing ER stress leading to RPE dysfunction and increased VEGF expression.