Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2) Bcl-2 in the orange-spotted grouper (Epinephelus coioides)

Luo, Sheng‐Wei; Kang, Huan; Kong, Jing-Rong; Xie, Ren-Chong; Liu, Yuan; Wang, Weina; Xie, Fu-Xing; Sun, Zuoming

doi:10.1016/j.dci.2017.06.003

Cited by 33 publications

(12 citation statements)

References 98 publications

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“…Consistently, PO activity has been found to be significantly increased after LPS stimulation, as described by Zhou et al (38). Additionally, increased levels of BCL-2 gene expression have also been observed after V. parahaemolyticus challenge in the orange-spotted grouper Epinephelus coioides (39).…”

Section: Discussionmentioning

confidence: 97%

MITF Regulates Downstream Genes in Response to Vibrio parahaemolyticus Infection in the Clam Meretrix Petechialis

Zhang

Yue

et al. 2019

Front. Immunol.

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The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix-leucine zipper protein that plays a key role in cell proliferation, survival and immune defense through the direct transcriptional control of downstream genes. We have found that MITF participates in the immune response to Vibrio parahaemolyticus infection in the clam Meretrix petechialis . In this study, we focused on how MITF functions in immunity. First, PO, CTSK , and BCL-2 were identified as the target genes of MpMITF in the clam by RNAi. EMSAs showed direct binding between the MpMITF protein and the E-box of the MpPO, MpCTSK , and MpBCL-2 promoters. Yeast one-hybrid assays also suggested that MpMITF could activate the expression of these three downstream genes. These results demonstrated that the transcriptional expression of MpPO, MpCTSK , and MpBCL-2 is directly regulated by MpMITF. Second, we analyzed the roles of MpPO, MpCTSK , and MpBCL-2 in clam immunity. The mRNA expression of MpPO, MpCTSK , and MpBCL-2 increased significantly after V. parahaemolyticus challenge, which implied that these genes might take part in the immune defense against V. parahaemolyticus challenge in clams. The purified recombinant proteins, MpPO and MpCTSK, inhibited the growth of V. parahaemolyticus . Additionally, the apoptosis rate of clam haemocytes rose significantly when the activity of MpBCL-2 was suppressed. These results revealed that MpPO, MpCTSK , and MpBCL-2 are involved in the immune defense against V. parahaemolyticus . This study supports the idea that the MpMITF pathway plays a key role in immune defense through the direct regulation of the downstream genes MpPO, MpCTSK , and MpBCL-2 in the clam, M. petechialis .

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Section: Discussionmentioning

confidence: 97%

MITF Regulates Downstream Genes in Response to Vibrio parahaemolyticus Infection in the Clam Meretrix Petechialis

Zhang

Yue

et al. 2019

Front. Immunol.

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“…To measure the effect of TroBcl2 on the MMP, GPS cells were transfected with pTroBcl2 (or pCN3), and siTroBcl2 (or siTroBcl2-C) according to the transfection procedure described above. After transfection for 24 h, 2 μg/ml LPS was used to stimulate transfected cells for 24 h, as previously reported ( 4 , 40 ). After that, the MMP changes in transfected GPS cells were measured by an enhanced mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China) in accordance with the instructions.…”

Section: Methodsmentioning

confidence: 99%

“…The anti-apoptotic function of fish Bcl-2 seems to be conserved compared with that of mammalian Bcl-2. In orange-spotted grouper, overexpression of EcBcl-2 can inhibit lipopolysaccharide (LPS)-induced apoptosis of HeLa cells ( 4 ). Zebrafish Bcl-2 can block irradiation- and dexamethasone-induced apoptotic responses in lymphoid cells, increase the numbers of thymocytes and B cells in the kidney, and alter thymocyte homeostasis ( 35 ).…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process

Cao

Yang

et al. 2023

Front. Immunol.

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IntroductionB-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated.MethodsIn this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway.Results and discussionThe full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant “NWGR” motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1β. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus.

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“…However, the excess production of reactive oxygen species and the imbalance of antioxidant system activity may lead to oxidative stress and DNA, lipid and protein damage, resulting in a series of cellular dysfunctions [ 26 , 27 , 28 , 29 ]. Furthermore, oxidative stress could cause cellular apoptosis, a kind of programmed cell death which plays an important role in removing dying or infected cells [ 30 , 31 , 32 ]. To eliminate ROS and counteract the adverse effect of oxidative stress, cells have developed an antioxidant defense system, including low-molecular weight scavengers and enzyme defenses [ 33 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanism Based on Histopathology, Antioxidant System and Transcriptomic Profiles in Heat Stress Response in the Gills of Japanese Flounder

Yan

Qiao

et al. 2022

IJMS

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As an economically important flatfish in Asia, Japanese flounder is threatened by continuously rising temperatures due to global warming. To understand the molecular responses of this species to temperature stress, adult Japanese flounder individuals were treated with two kinds of heat stress—a gradual temperature rise (GTR) and an abrupt temperature rise (ATR)—in aquaria under experimental conditions. Changes in histopathology, programmed cell death levels and the oxidative stress status of gills were investigated. Histopathology showed that the damage caused by ATR stress was more serious. TUNEL signals confirmed this result, showing more programmed cell death in the ATR group. In addition, reactive oxygen species (ROS) levels and the 8-O-hDG contents of both the GTR and ATR groups increased significantly, and the total superoxide dismutase (T-SOD) activities and total antioxidant capacity (T-AOC) levels decreased in the two stressed groups, which showed damage to antioxidant systems. Meanwhile, RNA-seq was utilized to illustrate the molecular mechanisms underyling gill damage. Compared to the control group of 18 °C, 507 differentially expressed genes (DEGs) were screened in the GTR group; 341 were up-regulated and 166 were down-regulated, and pathway enrichment analysis indicated that they were involved in regulation and adaptation, including chaperone and folding catalyst pathways, the mitogen-activated protein kinase signaling (MAPK) pathway and DNA replication protein pathways. After ATR stress, 1070 DEGs were identified, 627 were up-regulated and 423 were down-regulated, and most DEGs were involved in chaperone and folding catalyst and DNA-related pathways, such as DNA replication proteins and nucleotide excision repair. The annotation of DEGs showed the great importance of heat shock proteins (HSPs) in protecting Japanese flounder from heat stress injury; 12 hsp genes were found after GTR, while 5 hsp genes were found after ATR. In summary, our study records gill dysfunction after heat stress, with different response patterns observed in the two experimental designs; chaperones were activated to defend heat stress after GTR, while replication was almost abandoned due to the severe damage consequent on ATR stress.

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Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2) Bcl-2 in the orange-spotted grouper (Epinephelus coioides)

Cited by 33 publications

References 98 publications

MITF Regulates Downstream Genes in Response to Vibrio parahaemolyticus Infection in the Clam Meretrix Petechialis

MITF Regulates Downstream Genes in Response to Vibrio parahaemolyticus Infection in the Clam Meretrix Petechialis

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process

Molecular Mechanism Based on Histopathology, Antioxidant System and Transcriptomic Profiles in Heat Stress Response in the Gills of Japanese Flounder

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