p38 MAPK involvement in the thermal stress response occurs via HSP27 and caspase3 in the large yellow croaker (Larimichthys crocea).

The p38 mitogen-activated protein kinase (p38 MAPK) is a multifunctional molecule that is involved in cellular response to various stressful stimuli. In the present study, the full-length cDNA sequence of p38 MAPK (Lcp38 MAPK) was identified from the large yellow croaker Larimichthys crocea, which encoded a polypeptide of 361 amino acid residues. The predicted Lcp38 MAPK protein contained a highly conserved Thr-Gly-Tyr (TGY) motif, a glutamate and aspartate (ED) site, a substrate binding site (Ala-Thr-Arg-Trp < ATRW>), and a serine/threonine kinase catalytic (S_TKc) domain characteristic of the MAPK family. The constitutive expression of Lcp38 MAPK was detected in most of the tissues examined with the strongest expression in intestine. Subcellular localization in LCK cells (kidney cell line from a L. crocea) revealed that Lcp38 MAPK existed in both the cytoplasm and cell nucleus. The expression of Lcp38 MAPK after temperature stress was tested in LCK cells. The results indicated that Lcp38 MAPK transcripts were significantly upregulated under both cold (10 °C) and heat stress (35 °C) (P < 0.05). Furthermore, the phosphorylation levels of p38 MAPK as well the transcriptional levels of heat shock protein 27 (HSP27) and caspase3 in LCK cells were significantly induced under thermal exposure (P < 0.05). However, the cold- and heat induced HSP27 and caspase3 expression was significantly suppressed by SB203580, a specific inhibitor of p38-MAPK (P < 0.05). These findings indicated that Lcp38 MAPK might be involved in the cellular stress response via HSP27 and caspase3 in large yellow croaker.

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.

Characterization and expression analysis of Toll-like receptor 2 gene in large yellow croaker, Larimichthys crocea.

Toll-like receptor 2 (TLR2) plays an important role in innate immune responses. Here we describe the isolation and characterization of the full-length cDNA sequence of toll-like receptor 2 in large yellow croaker Larimichthys crocea (LcTLR2). The LcTLR2 cDNA contains a 5'-terminal untranslated region (5'-UTR) of 135 bp, an open reading frame (ORF) of 2478 bp encoding a polypeptide of 825 amino acid residues and a 3'-UTR of 50 bp. Subcellular localization analysis suggested that the LcTLR2-pEGFP was mainly expressed in cytoplasm. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis revealed a broad expression of LcTLR2 in most examined tissues, with the most predominant expression in blood, followed by spleen, and the weakest expression in stomach. The expression levels of LcTLR2 after injection with Vibrio parahaemolyticus, Lipopolysaccharides (LPS) and poly inosinic:cytidylic (polyI:C) were investigated in spleen, head-kidney and liver. Our results showed that LcTLR2 transcripts increased significantly after all the three immune challenges (p < 0.05). However, compared with polyI:C and LPS, higher expression levels of LcTLR2 were induced in all examined tissues after V. parahaemolyticus stimulation. In addition, the expression levels of LcTLR2 after flagellin, polyI:C, peptidoglycan (PGN) and LPS challenge in LCK were investigated, our findings showed that high LcTLR2 transcripts were induced after flagellin and PGN stimulation, suggesting that LcTLR2 might play a vital role in fish defense against bacterial infection. Furthermore, compared with LPS, flagellin and peptidoglycan might play an important role in LcTLR2 induction in large yellow croaker.