IKKβ Expression Induction-dependent And IKKβ Mediated NF-κB Activation By TFF3

IntroductionThe mammalian trefoil factor family has three members, TFF1, TFF2, and TFF3. In normal tissues, the predominant site for the expression of all three proteins is in the gastrointestinal tract.hTFF3 (hITF) acts as a small regulatory peptide to protect and restore the mucosal epithelia. Study showed that the elevated expression of TFF3 occurred in the tissues with inflammation or carcinoma. Besides the protection and reepithelial, TFF3 also involves in NF-κB signal transduction and regulates the development of varies of desease especially in relationship with inflammation.Nuclear factor kappa B (NF-κB), as a critical nuclear transcript factor regulates varieties of physiological and pathological process. Studies disclosed that NF-κB mediates the expression of cytokines, growth factors,and enzyme effectors, which response innate immunity and adaptive immunity with T cell receptor, B cell receptor, TNFR, CD40, BAFFR, LTβR and Toll/IL-1R. In vivo and vitro, varieties of stimulus may activate IKKs, the upstream IκB kinase complex, via diversity of receptors, receptor proximal molecules, and adaptors (such as TRAFs,RIPs), thus, resuting the phosphorylation and degradation of IκB activate and relocate NF-κB in cellular nucleus, which allow NF-κB to further activate downstream target genes. Specific activation of IKKs complex by different stimulatory effects on the innate immune and inflammatory responses may induce the activation of NF-κB hetrodimmer or homodimer, and their underlying cellular mechanisms are unknown.Studies on regulated activation in upstream NF-κB signaling by TFF3 may disclosed novel thearaputic targets to control the progression of diseases associated with inflammation, therefore, it is significant to clarify TFF3 involved signal transduction pathway. This study covers the modification for the activity of NF-κB and expression of upstream kinase complex, the results revealed a candidate mechanism of NF-κB activation by TFF3.Materials and Methods1. Construction of pEGFP-TFF3 vectorThe hTFF3 fragment was amplified by RT-PCR, after IEC RNA was extracted. After the purified hTFF3 and pEGFP vector were digested with the EcoRI and BgLII, they were ligated and transformated. Then, reconstructed vectors were verified by restriction endonuclease digesting and sequencing.2. Construction of pGEX-5x-1-IKKβ, Prokaryotic expression, and purificationAmplification of gene IKKβwas performaced based on M6P8 template. IKKP PCR product and pGEX-5x-1 vector were digested with restriction endonuclease BamH1 and EcoRI, recombinant Plasmid was developed by T4 DNA ligase,and transformed into E.coli BL21, expression of protein was induced by IPTG and detected by SDS-PAGE gel eleetrophoresis.3. The analysis of IKKβexpression modified by TFF3The modification of IKKβexpression was detected by RT-PCR, Western-blot after transfection of pEGFP-TFF3 plasmid.4. Luciferase reporter gene assay of NF-κB activityNF-κB promoter reporter gene expression vector was emploied and NF-κB promoter activity was detected. Results1. Construction of pEGFP-TFF3, pGEX-5x-1-IKKβvectorThe nucleotide sequencing of the gene encoding TFF3 in recombinant vector was performed, the sequence is identical with predicated one. The cloned gene was correctly inserted into the vectors as confirmed with restriction endonuclease and sequence analysis.2. Expression and purification of IKKβin prokaryotic cellsThe IKKβprotein was obstained after induction by IPTG from recombinant Ecoli.BL21. The fused GST-IKKβwith molecular mass around 73kD was identified by empolying SDS-PAGE gel electrophoresis.3. The expression of IKKβin transcription and translation levels modified by TFF3Modification of IKKβexpression was founded at both transcription and translation levels after transfection with pEGFP-TFF3.ConclusionTFF3 modified the expression of IKKβand activity of NF-κB. This discloses that IKKβmay involve in the upstream NF-κB transduction activated by TFF3. We constructed pGEX-5x-1-IKKβexpressing vector and purified the GST-IKKβprotein in prokaryotic cells, which may provide a candidate tool for further investigation of upstream NF-κB pathway activated by TFF3.