Molecular Cloning Of P38 MAPK,c-fos,c-jun And Transcription In The Liver And Kidney Of Silver Carp Exposed To Microcystins

The mitogen-activated protein kinases(MAPKs) are intracellular important signal moleculars and they are involved in regulation of various physiological processes. p38 MAPK is an important member of MAPK pathway, which can be activated by various intracellular and extracellular stimuli, such as growth factors, cytokins, inflammation factors, UV irradiation, heat stress, inflammation factors, and pathogenic infections to regulate fundamental cellular signal transduction processes.The proto-oncogenes c-fos, c-jun and c-myc are members of the immediate early gene family(IEGs). Until now, more than a dozen of IEGs have been discovered in orginisms, among which c-fos and c-jun play an important role in regulating cellular metabolism and they also have been widely used as biomarkers for neurons activation after extracellular stimil.The present study aimed to clone full length of p38 MAPK, c-fos and c-jun cDNA from the liver of silver carp and to determine their expressions at mRNA level in the liver and kidney of silver carp following MCs exposure in order to elucidate the toxicological mechanism of MC hepatotoxicity in silver carp and to find new molicilar biomarker of MC hepatotoxicity and new drug target for liver cancer treatment. The results of RT-PCR and RACE reveal that the entire p38 MAPK cDNA is 2 418 bp and contains a 1 086 bp of long open reading frame(ORF) encoding a protein of 361 amino acids. The full-length c-fos cDNA is 1 719 bp, which includes a 1 059 bp ORF encoding 352 amino acids. As for c-jun, the entire cDNA is 1 931 bp that includes a 927 bp of ORF encoding a protein of 308 amino acids. Bioinformatics analysis indicates that the predicted relative molecular mass(Mr) of p38 MAPK is 41.64 kD and the theoretical isoelectric point(pI) is 5.54, suggesting that this protein may be unstable but strongly hydrophilic. The Mr of c-Fos is 38.24 kD and pI is 4.93. Simlar to p38 MAPK c-Fos is also unstable and hydrophilic. Finally, the Mr of c-Jun is 33.92 kD and pI is 9.08, which is predicted to be unstable and hydrophilic. Secondary structure prediction reveals that p38 MAPK contains 45.71% of α-helix, 13.02% of extended strand, 4.99% of β-turn and 36.29% of random coil. c-Fos contains 25.85% of α-helix, 12.78% of α-helix, 1.42% of β-turn and 59.94% of random coil. And c-Jun contains 32.14% of α-helix, 5.52% ofα-helix, 1.62% of β-turn and 60.71% of random coil.Structural domain analysis reveals that p38 MAPK has a MAPK functional domain(STKc_p38) between 35-309 aa while c-Fos and c-Jun has a BRLZ domain sequence between 108-172 aa and 227-291 aa, respectively. Phylogenetic trees of p38 MAPK, c-Fos and c-Jun in silver carp and other fish or animal species are constructed by Neighbor-Joining according to the amino acid sequence. The results indicate that the percent identity of deduced amino acid sequences of p38 MAPK is more than 96% with common carp, but c-Fos and c-Jun is more than 97% and 95% with grass carp, respectively. However, they only have a comparatively lower homology with mammals than that of fish.The results of the quantitative real-time PCR reveal that p38 MAPK, c-fos and c-jun constitutively express in all examined tissues of silver carp, such as liver, kidney, brain, and heart etc, however, in which the expression rate is different. The expression rate sequence of p38 MAPK is muscle >intestine>spleen >kidney >gill>liver >heart> brain. However, for c-fos and c-jun, the sequence is liver > spleen > kidney > intestine > muscle > gill > brain > heart and liver > intestine > heart > brain > spleen > kidney > gill > muscle, respectively.The result of acute toxicity test indicates that the 48 h LD50 of MCs on silver carp is 520 μg/kg determined by using the method of up and down procedure(UDP). According to the result of acute toxicity test, two dose of 50 and 200 μg·Kg-1 of MCs were adopt for intraperitoneal injection in silver carp, and then fish liver and kidney were taken after 1, 3, 8 and 12 h of exposure, respectively for transcription analysis of p38 MAPK, c-fos, c-jun, c-myc by using qPCR. The results showed that MCs siginificalty promoted the transcriptions of p38 MAPK, c-fos, c-jun, c-myc in the liver or kidney of MC-treated fish, suggesting that p38 MAPK, c-fos, c-jun, c-myc may be involved in the hepatotoxicity or renal toxicity of MCs in silver carp.Our study indicates that MCs can activate p38 MAPK pathway and proto-oncogenes c-fos, c-jun, c-myc in the liver or kidney of silver carp, suggesting that liver cancer induced by MC might be mediated by the activation of p38 MAPK pathway and over expression of oncogene c-fos, c-jun, c-myc. This result may be beneficil to elucidate the molecular mechanism of MC hepatotoxicity and to find new molecular biomarker of MC hepatotoxicity and new drug target for liver cancer treatment.