Studys About Nf-κb Signaling Pathway In The Course Of The17β-Estradiol Regulating β-Defensin Gene1Expression In The Ovine Oviduct Epithelial Cells

Animals would be faced with the threats of infection by various bacteria in nature survival, and in the long process of evolution, the innate immune system in animals would against pathogen infection effectively. As endogenous cationic antimicrobial peptides(AMPs), defensins were rich in cysteine and widely distributed in various animal tissues and cells, and it was an important component of the innate immune system. Since defensins could against the infection by pathogens through their broad-spectrum resistance to bacteria and viruses, but their’s cytotoxicity would not beneficial to the cells growth, so the research of defensin expression regulation has an essential significance.Prior studies had shown that the estrogen changes in blood were positively associated with the expression of SBDl in reproduction organs during the estrus of sheep, and those indicated that estrogen may be involved in regulating the expression of SBD1in reproduction organs. Further studies showed that the expression of SBD1in oviduct mucosal epithelial cell surface, but not in lamina propria, muscle and connective tissue.The regulation mechanism of17p-estradiol has not been clearly established and we investigated the effects of E2on ovine oviduct epithelial cells in regard to SBD1expression.We treated ovine oviduct epithelial cells with different concentrations of E2and different times. The results showed that the optimum concentration of E2for the cells is10-8M and the greatest induction was observed around6h. Therefore, we treated the cells with E2(10-8M) to seek the time point of the greatest induction of SBD1expression in0to10.5h. The results shows that the greatest induction of SBD1expression was observed at3.5h and the induction of SBD1expression has significant differences from1.5to10.5h (P<0.05). We examined the expression of SBD1in differents time form the ovine oviduct epithelial cells preincubated with the high-affinity agonist for GPR30, inhibitor for ERs or inhibitor for the key proteins of PKA, PKC and NF-κB pathway.We found that in ovine oviduct epithelial cells E2-induced up-regulation of SBD1expression was GPR30-dependent during prophase and ERs-dependent during later-stage. Expression of SBD1included activation of the cAMP/PKA pathway, PKC pathway and NF-κB pathway via the Non Classical Genomic Mechanism, whereas the precondition that all three pathways were activated simultaneously was essential for this process. After the the treated cells with E2(10-8M), we examined the expression of NF-κB gene and the key proteins of NF-κB pathway in differents time using Western blot and RT-qPCR, and then detected the changes of the promoter binding SBD1ratio through ChIP. We discovered the mechanism of NF-κB pathway on E2-induced SBD1gene expression in ovine oviduct epithelial cells:the inactive complexes,which were formed from NF-κB and inhibiting protein IκB, free in the cell cytoplasm, while, the added E2as a external stimuli binded to GPR30and triggered a series of cascades; after0.5h, Iκ B was phosphorylated and generated p-I κB proteins, then the p-IκB was degraded by ubiquitination. Free NF-κB proteins were transported into the nucleus,1h later, the NF-κB proteins binded to the promoter of SBD1and activated the transcription of SBD1; with the lower I-κB protein in the cytoplasm, the new generated I-κB was phosphorylated by E2, but due to cytotoxicity from overexpression SBD1, the NF-κB pathway was stopped by negative feedback regulation. And then the cell returned to homeostasis through phosphorylation, dephosphorylation and ubiquitination.These results indicate that E2-induced up-regulation of SBD1expression was GPR30-dependent during prophase and ERs-dependent during later-stage. Expression of SBD1included activation of the cAMP/PKA pathway, PKC pathway and NF-κB pathway via the Non Classical Genomic Mechanism, whereas the precondition that all three pathways were activated simultaneously was essential for this process. Cells kept E2-induced SBD1gene expression via protein phosphorylation, ubiquitination, p65protein nuclear translocation and negative feedback regulation.