The role of IPNV-induced Endoplasmic Reticulum (ER) stress and connects to mitochondrial-mediated necrotic cell death in CHSE-214 cells

• Main Authors: Hui-Ling Huang, 黃惠玲
• Other Authors: Jiann-Ruey Hong
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/71178556748557033567

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 97 === Infectious Pancreatic Necrosis Virus (IPNV) is one of the widespread fish pathogen and infects many economically important finfish and shellfish. It is a double-stranded, bi-segmented RNA genome virus (designated segments A and B) and within a medium-sized, non-enveloped single-shelled icosahedral particle. In previous study, IPNV-infected in CHSE-214 cells could induce cell death through apoptosis and post-apoptotic necrosis. The endoplasmic reticulum (ER) plays a vital role in a variety of cellular functions, including protein synthesis, folding, homeostasis of intracellular calcium, and apoptosis. ER stress is triggered when unfolded proteins accumulate in the ER due to increased input of proteins, such as virus infection, a large amount of viral proteins are synthesized in infected cells, which unfolded or misfolded proteins activate the ER stress responses. To cope with the stress, cells activate intracellular signaling pathway-the unfolded protein response (UPR) to provide adaptive responses for survival. In preliminary results, the ER stress markers either GRP78 or caspase-12 was up-regulated and activated with IPNV infection. Thus, we want to investigate IPNV-induced ER stress and this stress signaling how to correlate to induce mitochondrial disfunction in fish necrotic cells. We first found that IPNV infection can activate two components inositol-requiring 1 (IRE1) and activating transcription factor 6 (ATF6). We further observed that it also can activate phosphorylation of protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α), then induce the expression of transcription factor C/EBP homologous protein (CHOP) leading to down-regulate anti-apoptotic Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL. We further treated specific GRP78 inhibitor vomitoxin, which can’t affect PERK phosphorylation, but inhibit eIF2α phosphorylation to block the expression of CHOP and then increase anti-apoptotic Bcl-2 family proteins. Moreover, GRP78 inhibitor also can inhibit IPNV viral replication, which actives double-stranded RNA- dependent protein kinase (PKR) leading to increased eIF2α phosphorylation, and affect mitochondrial cytochrome c release. Furthermore, IPNV infection can introduce calcium releasing from ER, which calcium release blocked by its specific chelator BATPA that effectively rescues cell apoptosis, inhibits cytochrome c release and mitochondria membrane potential loss. Taken together, these results indicate that IPNV-induced ER stress signaling pathway connects with mitochondrial disfunction via down-regulation anti-apoptotic Bcl-2 family proteins in CHSE-214 cell, which may provide insight into IPNV pathogenesis.