The Molecular Mechanism Of SOSC1/2 Regulation Nuclear Factor ?B In Procine Macrophage Under Heat Stress

Heat stress is the most important stressful pathogenic factors of pig industry in southern China.In the long term,to select a anti-stress pig breed is an effective way to solve this problem.However,since the pathogenic mechanism of heat stress is still unclear and the exploration of anti-stress genetic material is not deep enough,the further development of breeding technology was greatly restricted.Suppressor of cytokine signaling(SOCS)is a negative regulator of cytokine signaling pathways,which can inhibit signal transmission of cytokines by negative feedback,and plays an important role in a variety of diseases and is speculated to be involved in the regulation of heat stress.In this study: we cloned the porcine SOCS1 and SOCS2 gene and predicted the potential molecular function.The real time quantitation polymerization chain reaction(qRT-PCR)methods were used to detect the expression of SOCS1/2 mRNA in the differental tissues of heat stressed pig.We also constructed eukaryotic expression vector pcDNA3.1-V5-SOCS?pcDNA3.1-V5-SOCS2 ? pcDNA3.1-V5-HSP70 and NF-?B reporter gene(in the pig macrophages transfected with HSP70 gene to construct porcine heat stress model in vitro),cotransfected all of them to 293 T cells and CRL-2845 cells,then analyze the influence of SOCS1/2 on the activity of NF-?B and downstream inflammatory factor expression;Furthermore,the activity of NF-?B were detected after cotransfection with a interference RNA vector(shRNA)of SOCS1/2 which silence SOCS1/2 gene.The results show that:(1)The porcine SOCS1 gene consists of 693 bp of ORF,encodes a protein precursor with 220 amino acids,which shares high amino acid sequence identity with that in pig(99.9%),Homo sapiens(97.1%),Bos Taurus(96.1%),Ovis aries(94.3%),and mus musculus(93.7%).The molecular weight of SOCS1 protein is 24.37 kDa,and PI is 10.98.It consisted of a central SH2 domain(Scr-homology 2),a variable length of N-terminal structural domain,a C-terminal 40-amino-acid SOCS box domain and a 12-amino-acid KIR domain.Meanwhile,we found that the expression of SOCS1 m RNA was increased significantly in spleen,intestine and thymus gland of heat stressed pigs by qRT-PCR(P ?0.05).Differently,the expression of SOCS1 in mesenteric lymph node were decreased atday 3,day 6 and day 9 after heat stress begin significantly(P?0.05).Porcine SOCS1 negatively regulates the activity of NF-?B which induced by overexpressing of HSP70 in both of 293 T cell line and CRL cell line.On the other hand,TNF-? expose amplicated the induce effect of HSP70 to NF-?B.We also showed that SOCS1 inhibited the phosphorylation of I?B-?and activated NF-?B via the degradation of Mal protein,a upstream molecular of NF-?B signal pathway.(2)The porcine SOCS2 gene consists of 597 bp of ORF,encodes a protein precursor with 198 amino acids,which shares high amino acid sequence identity with that in pig(99.9%),Bos Taurus(97.5%),Homo sapiens(96.0%)and mus musculus(94.4%).The molecular weight of SOCS2 protein is 22.25 kDa,and PI is 8.53.It consisted of a central SH2 domain(Scr-homology 2),a variable length of N-terminal structural domain,a C-terminal 40-amino-acid SOCS box domain.Meanwhile,the expression of SOCS2 were significantly decreased in spleen and mesenteric lymph node of heat stressed pigs(P <0.01),and with a time dependence manner.In contrast,we found a drastic increased of SOCS2 m RNA expression in small intestine and attained to peak at 6th day after the heat stress begin(P< 0.01).Porcine SOCS2 also negatively regulates the activity of NF-?B which induced by overexpressing of HSP70 in CRL cell line.We also showed that SOCS2 inhibited the phosphorylation of I?B-? and activated NF-?B via the degradation of Mal protein,a upstream molecular of NF-?B signal pathway.Our studies cloned the porcine SOCS1/2 cDNA successfully,and found a significant changes of SOCS1/2 mRNA expression in heat stressed pigs with a tissue and time dependence manner.Porcine SOCS1/2 negatively regulates the activity of NF-?B which induced by overexpressing of HSP70 in both of 293 T cell line and CRL cell line.TNF-?expose amplicated the induce effect of HSP70 to NF-?B.We also showed that SOCS1/2 inhibited the phosphorylation of I?B-? and activated NF-?B,in witch,SOCS1/2work via the degradation of Mal protein,a upstream molecular of NF-?B signal pathway.