| Translational medicine has become one of the newest concepts of international science and technology in the 21 st.It is aimed to break out the limitation between basic research and clinical medicine and focus on the patients to improve the whole medical level.With the coming of the aging society in china, translational medicine would take a significant role in the medical education and clinical application of the geriatric medicine.Cells are the major subjects to perform the organism structure and function. When extracellular signals transfer to the cell through the cell membrane,the cell will stimulate the corresponding biological chemical reaction based on the received signal to complete the signaling transduction. Notch signaling pathway is evolutionarily highly conserved pathway. Notch receptor and ligand are a single transmembrane receptor protein, which is involved in the regulation of development of multi-cellular organisms mainly through mediating the transmission of information between cells, resulted in a series of biological behaviors. Numerous studies have demonstrated that Notch signaling plays an important role in cell proliferation, differentiation and apoptosis. When Notch ligands on the cell surfaceactivate receptors of adjacent cells, intracellular domain of Notch(NICD) will be released into the nucleus, binding to the transcription factor RBP-J and recruiting coactivator complex, to initate the expression of downstream gene expression. Natural mutations and expression abnormalities of Notch pathway related genes often lead to a variety of human genetic diseases and cancers. Therefore, we assume that regulation of Notch pathway is useful for the clinical application..Studies overseas have showed that RBP-J, a key transcription molecule of Notch signaling pathway, can bind to FHL1C/Kyo T2 LIM protein and recruit a variety of transcriptional co-inhibitory molecules, thereby inhibiting RBP-J-mediated transcription of Notch signaling downstream genes. Our previous study revealed that Kyo T2 can participate in transcriptional regulation by recruiting RING1 and HPC2. However, FHL1C/Kyo T2 does not contain a nuclear localization signal. Its nuclear translocation is the most likely mediated by different modification or interaction with other nuclear proteins. Our previous protein profile data suggest that FHL1 C may interact with GNB2 protein, ? subunit of guanine nucleotide-binding protein. Therefore, to clarify the interaction between FHL1 C and GNB2 should be helpful to understand the molecular mechanisms of FHL1 C inhibited Notch signaling Macrophages are importantMacrophages are important phagocytosis and antigen-presenting cells. Macrophage types are related to tumor, metabolic diseases, brain injury and other diseases. The imbalance of macrophage polarization tends to cause a series of diseases. Thus, reasonable interventions of macrophage polarization have clinical values for disease therapy. Our previous study has demonstrated that Notch pathway activation can promote M1 polarization, whereas Notch signaling deficiency can promote M2 polarization in solid tumor. However, the underlying mechanism of Notch-mediated macrophage polarization remains unclear. In order to explore the mechanism of Notch signaling on macrophage polarization, we will examine the Kyo T2 function in Notch-mediated macrophage polarization, further to provide theoretical basement for establishing new strategy on targeting Notch signaling for clinical disease treatment.In order to completely explore biological function of Notch signaling inhibitors FHL1C/Kyo T2, We used co-immunoprecipitation and mammalian two-hybrid technology to verify the interaction between FHL1 C and GNB2. Using molecular biology methods and techniques, we observed the role of Kyo T2 on Notch mediated macrophage polarizationin, and further understanded the effects and mechanisms of Notch signaling on macrophage polarization, which will provide new ideas and new strategies on targeting Notch signaling pathway for clinical disease treatment.The detailed methods and results are as follows: 1. The verification of the interaction between FHL1 C and GNB2(1) Eukaryotic expression vectors of p CMV-VP16-GNB2 and p CMV-Flag-GNB2 were successfully constructed by molecular cloning, and their sequence were identifiedcorrect by enzyme digestion and DNA sequencing.Mealwhile, Amplification of eukaryotic expression vector p CMV-GAL4-DBD-FHL1 C and p CMV-myc-FHL1 C from our lab stock.(2) The mammalian two-hybrid assaywithtransient transfection and dual luciferase reporter assayshowed that FHL1 C and GNB2 cannot activate the expression of down stream report gene luciferase, suggesting the physicalinteraction was not presented between both of proteins Luciferase value changes were detected to verify the in vivo interaction between FHL1 C and GNB2.(3)Co-immunoprecipitationassay with Protein G microbead demonstrated that no specific band to GNB2 was detected using FHL1 C antibody, indicating that there was no the physiological interaction between FHL1 C and GNB2.2. The preliminary study on LIM structure domain protein Kyo T2 regulated macrophage polarization through Notch signal(1) Primary murine bone marrow were isolated and monocytes derived were induced. The polarization rate of CD11b/F4/80 positive macrophageswas identified by flow cytometry.. Futher,q RT-PCRshowed that M1 macrophages were successfully induced by LPS plus Interferon-?, and M2 macrophages by IL-4(P<0.01).(2) Kyo T2 expressionwas detected in polarized macrophages M1 and M2. Then,knocking down of Kyo T2 expression by si RNA resulted in the activation of Hes1 and Hey1 gene(P<0.05), down-stream of Notch signal,suggesting the activation of Notch signal..(3) Knockingdown of Kyo T2 expression by si RNA, the phenotype of M1 macrophagesdid not change significantly, wheres the phenotype of M2 macrophage was down-regulated(P<0.05), indicating that Kyo T2 promoted macrophage polarization to M2.Based on the above results, our study revealed the mechanism of LIM domain protein FHL1C/Kyo T2 regulated macrophage polarization by inhibiting Notch signaling. The following conclusions were drawn:1. There is no physiological interaction between LIM domain protein FHL1 C and GNB2, and the further study on translocationto nucleus of FHL1 C to inhibit Notch signal was needed.2. LIM domain protein Kyo T2 can promote macrophage polarization to M2 through Notch signaling, consistent to our previous study demonstrated by Notch knock-out or inhibition of Notch signal by GSI. These results suggest that Kyo T2 can be used as a candidate inhibitor of Notch signaling and macrophage polarization in preclinical studies. |
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