Cui-Luan Yao

The impact of acute temperature stress on hemocytes of invasive and native mussels (Mytilus galloprovincialis and Mytilus californianus): DNA damage, membrane integrity, apoptosis and signaling pathways

SUMMARY We investigated the effects of acute heat stress and cold stress on cell viability, lysosome membrane stability, double- and single-stranded DNA breakage, and signaling mechanisms involved in cellular homeostasis and apoptosis in hemocytes of native and invasive mussels, Mytilus californianus and Mytilus galloprovincialis, respectively. Both heat stress (28, 32°C) and cold stress (2, 6°C) led to significant double- and single-stranded breaks in DNA. The type and extent of DNA damage were temperature and time dependent, as was caspase-3 activation, an indicator of apoptosis, which may occur in response to DNA damage. Hemocyte viability and lysosomal membrane stability decreased significantly under heat stress. Western blot analyses of hemocyte extracts with antibodies for proteins associated with cell signaling and stress responses [including members of the phospho-specific mitogen-activated protein kinase (MAPK) family c-JUN NH2-terminal kinase (JNK) and p38-MAPK, and apoptosis executor caspase-3] revealed that heat and cold stress induced a time-dependent activation of JNK, p38-MAPK and caspase-3 and that these signaling and stress responses differed between species. The thermal limits for activation of cell signaling processes linked to the repair of stress-induced damage may help determine cellular thermal tolerance limits. Our results show similarities in responses to cold and heat stress and suggest causal linkages between levels of DNA damage at both extremes of temperature and downstream regulatory responses, including induction of apoptosis. Compared with M. californianus, M. galloprovincialis might have a wider temperature tolerance due to a lower amount of single- and double-stranded DNA damage, faster signaling activation and transduction, and stronger repair ability against temperature stress.

Thermal stress and cellular signaling processes in hemocytes of native (Mytilus californianus) and invasive (M. galloprovincialis) mussels: Cell cycle regulation and DNA repair

In a previous study using hemocytes from native and invasive congeners of Mytilus (Mytilus californianus and Mytilus galloprovincialis, respectively) we showed that DNA damage and cell signaling transduction processes related to the cellular stress response and apoptosis were induced by acute temperature stress. The present study extends this work by examining effects of acute heat- and cold stress on total hemocyte counts (THCs) and expression of key regulatory molecules involved in responding to stress: tumor suppressor factor (p53), cell cycle arrest activator (p21), and a DNA base excision repair enzyme (apurinic/apyrimidinic endonuclease (APE)). Hyperthermia (28 °C, 32 °C) led to significant decreases of THCs in both species. The extent of decrease in THC was temperature-, time-, and species-dependent; lower THC values were found in M. californianus, the more cold-adapted species. Western blot analyses of hemocyte extracts with antibodies specific for p53 protein, several site-specific phosphorylation states of p53, p21 protein, and APE indicated that heat- and cold (2 °C) stress induced a time-dependent activation of stress-related proteins in response to DNA damage; these stress-induced changes could govern cell cycle arrest or DNA damage repair. Our results show that the downstream regulatory response to temperature-induced cell damage may play an important role in deciding cellular fate following heat- and cold stress. Compared to M. californianus, the more warm-adapted M. galloprovincialis appears to have a higher temperature tolerance due to a lesser reduction in THC, faster signaling activation and transduction, and stronger DNA repair ability following heat stress.

Identification and expression profiles of ERK2 and ERK5 in large yellow croaker (Larimichthys crocea) after temperature stress and immune challenge

Fish is highly affected by many environmental stresses such as temperature and invasive infection. The extracellular signal-regulated kinase (ERK) pathway, part of the mitogen-activated protein kinase (MAPK) family, is found to act as crucial mediators for cell differentiation, proliferation and cell response to various stresses. In the present study, ERK2 (LcERK2) and ERK5 (LcERK2) were cloned and characterized from large yellow croaker, Larimichthys crocea. The full length cDNA sequence of LcERK2 was of 1910 bp, including an ORF of 1110bp encoding a polypeptide of 369 amino acids. The full length cDNA sequence of LcERK5 was of 3720bp, including an ORF of 3375bp encoding a polypeptide of 1124 amino acids. Multiple alignments showed that both LcERK2 and LcERK5 contained highly conserved TEY motif and S_TKc domain in MAPK family and the unique catalytic and active structures of ERK2 and ERK5. Subcellular localization revealed that both LcERK2 and LcERK5 expressed in the cytoplasm and cell nucleus. The expression of LcERK2 and LcERK5 were detected in most tissues of large yellow croaker, with the most predominant expression of LcERK2 in brain and LcERK5 in gill, and the weakest expression of LcERK2 in liver and LcERK5 in intestine, respectively. The expression levels of LcERK2 and LcERK5 after temperature stress and poly I:C and flagellin challenge were investigated in LCK (L. crocea kidney) cells. After temperature stress, significant down-regulations of LcERK2 transcripts were detected after 10 °C stress (p < 0.05) whereas LcERK2 transcripts increased significantly after 35 °C stress (p < 0.05). However, significant down-regulations of LcERK5 expression were detected at most time points after both cold and heat stress (p < 0.05). However, significant up-regulations of LcERK2 and LcERK5 transcripts were found after immune challenge (p < 0.05). Our results showed that LcERK2 transcripts enhanced after heat stress and both LcERK2 and LcERK5 transcripts could be induced by immune challenge. These findings indicated that LcERK2 might be more important in fish response to high temperature stress and both LcERK2 and LcERK5 might play an important role in fish immune response.

Subcellular Localization of Large Yellow Croaker (Larimichthys crocea) TLR21 and Expression Profiling of Its Gene in Immune Response

Toll-like receptor 21 (TLR21) is a non-mammalian type TLR, and plays an important role in innate immune response in fish. In this paper, the full-length cDNA sequence of TLR21 gene was identified and characterized from large yellow croaker, Larimichthys crocea and was termed as LcTLR21. It consists of 3365 bp, including a 5’-terminal untranslated region (UTR) of 97 bp, a 3’-terminal UTR of 331 bp, and an open reading frame (ORF) of 2937 bp encoding a polypeptide of 978 amino acid residues. The deduced LcTLR21 contains a signal peptide domain at N-terminal, 12 leucine-rich repeats (LRRs) at the extracellular region, a transmembrane domain and a cytoplasmic toll-interleukin-1 receptor (TIR) domain at the C-terminal. Subcellular localization analysis revealed that the LcTLR21-GFP was constitutively expressed in cytoplasm. Tissue expression analysis indicated that LcTLR21 gene broadly expressed in most of the examined tissues, with the most predominant abundance in spleen, followed by head-kidney and liver, while the weakest expression was detected in brain. The expression level of LcTLR21 after LPS, poly I:C and Vibrio parahaemolyticus challenges was investigated in spleen, head-kidney and liver. LcTLR21 gene transcripts increased significantly in all examined tissues after the challenges, and the highest expression level was detected in liver at 24 h after poly I:C stimulation (P < 0.05), suggesting that LcTLR21 might play a crucial role in fish resistance to viral and bacterial infections.

The interaction of TAK1 and TAB1 enhances LPS-induced cytokine release via modulating NF-κB activation (Larimichthys crocea)

ABSTRACT Transforming growth factor‐&bgr;‐activating kinase 1 (TAK1) is triggered by foreign pathogenic infection and involves in proinflammatory response through the activation of nuclear factor‐&kgr;B (NF‐&kgr;B), which is specifically regulated by TAK1‐binding protein 1 (TAB1). However, the expression and regulatory characterizations of TAK1 and TAB1 in fish immune response remain largely unknown. In the present study, the cDNA sequences of TAK1 (LcTAK1) and TAB1 (LcTAB1) were identified from large yellow croaker, Larimichthys crocea. The open reading frame (ORF) of LcTAK1 was 1725 bp in length, encoding 574 amino acids. The putative LcTAK1 protein contained a protein kinase domain and a C‐terminal coiled‐coil region. The ORF of LcTAB1 was 1518 bp encoding 505 amino acids. And a typical PP2Cc domain and a conserved sequence motif (PYVDFSQFYLLWGSDH) at C‐terminal were identified in the predicted LcTAB1 protein. Multiple alignments showed that LcTAK1 shared 74.0–97.9% and LcTAB1 shared 37.4–95.8% sequence identities with TAK1 and TAB1 proteins from other species, respectively. Quantitative PCR analysis indicated that both LcTAK1 and LcTAB1 were broadly expressed in all examined tissues, with the most predominant expression in brain and the weakest expression in muscle, respectively. Subcellular localization revealed that both LcTAK1 and LcTAB1 expressed in the cytoplasm. In addition, LcTAK1 transcripts increased significantly in LCK cells after flagellin, LPS and poly I:C stimulation while LcTAB1 enhanced greatly after LPS and poly I:C challenge. Furthermore, the roles of them in NF‐&kgr;B activation were investigated by overexpression of LcTAK1 and LcTAB1 in HEK293T cells. Our results revealed that NF‐&kgr;B luciferase promoter expression could not be induced by overexpression of LcTAK1 or LcTAB1 alone, however, it could be induced by co‐expression of LcTAK1 and LcTAB1 together. Moreover, the roles of LcTAK1 and LcTAB1 in immune response analysis showed that NF‐&kgr;B activation enhanced significantly in co‐overexpressed HEK293T cells following LPS and poly I:C stimulation. However, the expression levels of tumor necrosis factor (TNF)‐&agr;, Interleukin‐6 (IL‐6) and IL‐8 were induced only after LPS challenge (p<.05). These findings suggested that the TAK1‐TAB1 complex of large yellow croaker might play an important role in pro‐inflammatory cytokines and chemokine release after LPS stimulation via inducing NF‐&kgr;B activation. HIGHLIGHTSBoth TAK1 and TAB1 localized in cytoplasm.LcTAK1 increased after flagellin, LPS and poly I:C stimulation while LcTAB1 did not response to flagellin.Only co‐overexpression of LcTAK1 and LcTAB1 could induce NF‐&kgr;B activation and IL‐8 transcript.When target genes co‐overexpressed, NF‐&kgr;B enhanced after LPS and poly I:C stimulation.The secretion of TNF‐&agr;, IL‐6 and IL‐8 only induced by LPS stimulation in LcTAK1 and LcTAB1 co‐overexpressed cells.

The impact of acute thermal stress on green mussel Perna viridis : Oxidative damage and responses

Examining the physiological responses of mussels to thermal stress is crucial to evaluate their biogeographic distribution and ability to adapt to a changing climate. In the present study, we investigated the effects of acute cold (8 °C and 15 °C) and heat (35 °C and 42 °C) stress on the mortality rate, reactive oxygen species (ROS) production, malondialdehyde (MDA) content, mitochondrial membrane potential (MMP) and antioxdative responses in the gill tissue of the green mussel species Perna viridis. Our results showed that cold and heat stress induced a temperature-dependent increase in mortality rate. ROS production increased significantly (p < 0.01) after both cold and heat stress. However, the activities of antioxidant enzymes, including SOD, CAT and GSH-Px, were greatly enhanced only after heat stress. In addition, MDA content and MMP increased significantly under both cold and heat stress. The up-regulation of Hsp70 transcripts was only detected after acute stress at 35 °C. However, p38-MAPK phosphorylation levels increased after both cold and heat stress. In addition, a moderate activation of caspase-3 was found after mussels were exposed to 8 °C and 42 °C stress. Our results suggest that both extreme cold and heat stress could induce ROS production in the gill tissue of P. viridis, which might result in lipid peroxidation and mitochondria dysfunction. Antioxidative enzymes and Hsp70 might be important in the heat stress response of animals, whereas p38-MAPK might be crucial in the acute response to both cold and heat stress. However, caspase-3 activation might be very weak under both cold and heat stress.