The modulation of Smac/DIABLO on mitochondrial apoptosis induced by LPS in Crassostrea gigas

• Published in: Fish & Shellfish Immunology
• Main Authors: Lv, Zhao, Song, Xiaorui, Xu, Jiachao, Jia, Zhihao, Yang, Bin, Jia, Yunke, Qiu, Limei, Wang, Lingling, Song, Linsheng
• Format: Report
• Language: English
• Published: ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD 2019
• Subjects: Mitochondrial pathway; Smac/DIABLO; LPS; Primary cultured hemocytes; C. gigas; Fisheries; Immunology; Marine & Freshwater Biology; Veterinary Sciences; Diablo; Crassostrea gigas
• Online Access: http://ir.qdio.ac.cn/handle/337002/160864; http://ir.qdio.ac.cn/handle/337002/160865; https://doi.org/10.1016/j.fsi.2018.10.035

The mitochondrial pathway of apoptosis is well studied as the major mechanism of physiological cell death in vertebrates. In the present study, a second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis-binding protein (IAP) with low pI protein (DIABLO) (designated as CgSmac) was identified from oyster Crassostrea gigas. The open reading frame of CgSmac was of 966 bp nucleotides encoding a predicted polypeptide of 321 amino acids with a conserved Smac/DIABLO domain containing a potential IAP-binding motif of VMPV. CgSmac proteins were distributed in hemocytes and co-localized with mitochondria. Western blotting analysis revealed that CgSmac proteins mainly existed in the dimer form in hemocytes, and the monomeric precursors and mature monomers were also detected. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSmac in hemocytes was significantly up-regulated and peaked at 6 h (12.26-fold, p < 0.05), and the protein level of its dimers was significantly up-regulated at 6 h, 12 h, 24 h, and 48 h, while that of CgSmac monomers was up-regulated at 6 h, 12 h and down-regulated at 24 h, 48 h. The decrease of mitochondrial membrane potential indicated that the occurrence of early stage of apoptosis in primary cultured hemocytes was induced by LPS, and RNA interference (RNAi) of CgSmac could not rescue this decrease. The caspase-3 activity in primary cultured hemocytes was significantly suppressed after RNAi of CgSmac. Correspondingly, the total apoptotic rate of primary cultured hemocytes was also significantly suppressed in dsCgSmac + LPS group (31.57%) compared to dsEGFP + LPS group (40.27%, p < 0.05), which in turn demonstrated the conserved pro-apoptotic function of CgSmac. Furthermore, the early apoptotic rate (10.4% vs. 8.5%, p < 0.05) was significantly higher in dsCgSmac + LPS group than that of dsEGFP + LPS group, while the necrosis (7.7% vs. 10.0%, p < 0.05) and late apoptotic rates (13.4% vs. 21.9%, p < 0.05) were lower in dsCgSmac + LPS group ...