Nuclear factor E2-associated factor 2 and musculoaponeurotic fibrosarcoma K mediate regulation glutathione peroxidase of Cristaria plicata after microcystin-induced oxidative stress.

Nuclear factor E2-associated factor 2 (Nrf2)/Antioxidant Response Element (ARE) signaling pathway is an endogenous antioxidant pathway that protects cells from oxidative damage. This pathway is triggered when aquatic organisms are exposed to environmental toxicants. In this study, CpMafK (musculoaponeurotic fibrosarcoma K of Cristaria plicata) mRNA expression in hepatopancreas and gills were up regulated after Cristaria plicata (C. plicata) was exposed to microcystin (MC), which showed that CpMafK protected C. plicata from MC. After MC treatment and CpNrf2 (Nrf2 of Cristaria plicata) knockdown, the mRNA expression of CpMafK was down regulated. After MC treatment and CpMafK knockdown, the mRNA expression of CpNrf2 was down regulated. Indicating that the expression of CpNrf2 was positively correlated with CpMafK. CpGPx (GPx of Cristaria plicata) mRNA was also down regulated with the down regulation of CpMafK and CpNrf2. CpGPx promoter contains a variety of transcription factor binding sites, including Nrf2, ARE elements, etc. Gel blocking experiments showed that CpNrf2/CpMafK heterodimers were bound to CpGPx promoters in vitro. Dual luciferase reporter assay showed that CpNrf2/CpMafK heterodimer negatively regulated CpGPx promoter in cells. In conclusion, Nrf2 and MafK mediate regulation of GPx play a crucial role in protecting bivalves from MC.

GST-Mu of Cristaria plicata is regulated by Nrf2/Keap1 pathway in detoxification microcystin and has antioxidant function.

Glutathione S-transferase is a crucial phase II metabolic enzyme involved in detoxification and metabolism in aquatic organisms. This study aimed to investigate the regulation of Nrf2/Keap1 pathway on microcystin-induced CpGST-Mu expression and CpGST-Mu resistance to hydrogen peroxide. A mu class GST from Cristaria plicata (CpGST-Mu) was identified. The full-length cDNA was 1026 bp, with an open reading frame of 558 bp. Subcellular localization revealed that CpGST-Mu was localized in cytoplasm. The optimum pH and temperature for the catalytic activity of CpGST-Mu protein was pH 6 and 40 °C, respectively. The results of Real-time quantitative PCR showed that CpGST-Mu mRNA was constitutively expressed in tissues, with the highest expression level in hepatopancreas and the lowest expression level in gill. The mRNA level of CpGST-Mu was significantly increased under the stress of microcystins and hydrogen peroxide. CpGST-Mu had an antagonistic effect on hydrogen peroxide. In the knockdown experiments, the mRNA levels of CpGST-Mu exhibited corresponding changes while Nrf2 and Keap1 genes were individually knocked down. These findings indicated that GST-Mu exhibited antioxidant properties and its expression was regulated by Nrf2/Keap1 signaling pathway. The study provided new information on the function of GST-Mu and could contribute to future studies on how to excrete microcystins in molluscs.

Detoxification and antioxidant functions and regulatory mechanisms of two Delta-class GSTs in paddy crayfish (Procambarus clarkii) after imidacloprid stress.

Glutathione S-transferases (GSTs) are phase II metabolic detoxification enzymes, which are widely found in organisms, and play an important role in helping organisms to resist toxic compounds. In this study, the two Delta-class GSTs cDNA sequences were cloned from Procambarus clarkii (designated as PcGSTD1 and PcGSTD2). Tissue specific expression profile showed that PcGST1,2 were expressed in all 6 tissues, with the highest expression in hepatopancreas. Subcellular localization assay showed that PcGSTD1, 2 were mainly expressed in the cytoplasm of HEK-293 T cells. Recombinant PcGSTD1, 2 showed the highest catalytic activity to the GST model substrate 1-chloro-2,4-dinitrobenzene (CDNB) at 20 and 30 °C, pH 8 and 7, respectively. The mRNA expression of PcGSTD1, 2 and the GSTs activity varied with the time of imidacloprid challenge. The BL21(DE3) expressing PcGSTD1, 2 proteins could more resistant to H2O2. The dsRNA experiments showed that PcKeap1b, PcNrf1, and PcMafK affected the transcription levels of PcGSTD1, 2. The GST-Pulldown results revealed that PcbZIP and PcMafK recombinant proteins could bind to each other in vitro. The gel mobility shift assay demonstrated that PcMafK recombinant protein had affinity with the promoter of PcGSTD2. The Dual luciferase assays analyzed the activity of the promoters after different truncations, the core region of PcGSTD1 promoter was at -440 bp to +54 bp, and that of PcGSTD2 promoter was between -1609∼-1125 bp. These results suggested that PcGSTD1, 2 respond positively to imidacloprid stress in P. clarkii, and the transcriptional expressions of PcGSTD1, 2 were influenced by the factors of PcKeap1b/PcNrf1/PcMafK.

The thioredoxin expression of Cristaria plicata is regulated by Nrf2/ARE pathway under microcystin stimulation.

Thioredoxin plays an important role in inhibiting apoptosis and protecting cells from oxidative stress. This study was aimed to clarify how the expression of Trx from Cristaria plicata is regulated by Nrf2/ARE pathway. The expression of CpTrx mRNA was significantly up-regulated in gill and kidney tissues under microcystin stress. The Nrf2 gene of Cristaria plicata was identified to possess an auto active domain bit. While CpNrf2 was knocked down by specific small RNA, CpTrx mRNA expression was significantly down-regulated. The promoter of CpTrx gene had high transcriptional activity, and this basic transcriptional activity persisted after ARE element mutation. The region of promoter -206 to +217 bp was a core promoter region and had forward regulatory elements. Gel shift Assay exhibited that the CpTrx promoter could bind to the purified proteins CpNrf2 and CpMafK in vitro. The binding phenomenon disappeared after the ARE element mutation in promoter region. Subcellular localization experiments displayed that fluorescence overlap between CpNrf2 and Trx promoter increased under microcystin toxin stress. These results suggested that Trx expression was regulated by Nrf2/ARE pathway under oxidative stress.

MAPK/MAK/MRK overlapping kinase mediated apoptosis through caspase signaling pathway from Cristaria plicata.

MAPK/MAK/MRK Overlapping Kinase (MOK) belongs to MAP kinase superfamily, which plays an important role in regulating cell growth, division, and differentiation. Caspase-3, as the final executor of apoptosis, has an important position in the caspase-mediated apoptotic signaling pathway. The full-length cDNA of MOK and caspase-3 were cloned from Cristaria plicata (designated CpMOK and CpCaspase-3). The CpMOK gene was sequence with a full-length of 1413 bp, encoding a total of 470 amino acids, and containing an S_TKc structural domain. CpCaspase-3 has a sequence of 2425 bp, encoding 322 amino acids, containing a CASc domain. Real-time fluorescence quantitative PCR analysis showed that CpMOK and CpCaspase-3 distributed in various tissues of C. plicata, and the highest expression of CpMOK and CpCaspase-3 mRNA was in hepatopancreas. The expression of CpMOK was significantly changed in hepatopancreas, gills, and kidneys by the construction of wound model as well as stimulation of LPS, PGN, Poly I: C and Aeromonas hydrophila. Subcellular localization experiments confirmed that CpMOK was localized in the nucleus. Furthermore, the double-stranded RNA (dsRNA) of CpMOK was constructed for interference experiment, and the results showed that the mRNA expression of apoptotic gene signals caspase-1, caspase-3, caspase-7, caspase-8, and caspase-9 were increased. The expression of caspase-1, -3, -7, -9, cytochrome C (Cyt-c) and tumor necrosis factor-α (TNF-α) was detected by ELISA. Fluorescent staining of apoptotic cells using the Tunnel method revealed an increase in the number of apoptotic cells after interference. These results suggested that CpMOK knockdown could induce caspase-mediated apoptosis in C. plicata, and the phosphorylation of the kinase was disrupted during the process.