Study Of The Function And Molecular Mechanisms Of SMEK1 Gene In The Development Of T Cells And Adaptive Immune Response

T cells coordinate multiple aspects of adaptive immunity,including responses to pathogens,allergens,and tumors.The thymus is the main location for T cell development.The T cell precursors lacking CD4+and CD8+co-receptor expression undergo T cell receptor(TCR)rearrangement in the thymus,producing CD4+CD8+double positive(DP)thymocytes.DP cells undergo selection processes to produce CD4+or CD8+single positive(SP)thymocytes,which eventually develop into naive T cells and enter the peripheral lymphatic tissues.After naive T cells are in contact with antigen presenting cells(APC),a series of genes in the naive T cells undergo transcription and translation,and the cells proliferate and differentiate into effector T cells,mainly CD4+helper T cells and CD8+cytotoxic T cells,which provides immune protection for the organism.The development of T cells in thymus and the activation process in peripheral immune tissues are all subject to complex and precise regulation of signal molecules and transcription factors.The TCR binds to the MHC-antigen complex on the APC surface to generate the first signal.The costimulatory molecule CD28 interacts with the corresponding ligand B7 on APC to generate a second signal.The two signals work together to activate the TCR signaling pathway in the cell and regulate the activation process of T cells.SMEK1(Suppressor of MEK1)gene is located at 14q32.12 in human and was first reported by Michelle’s research group in 2005.Subsequent studies confirmed that SMEK1 participates in the formation of the regulatory subunits of Protein Phosphatase 4(PP4).Previous studies on PP4 have all knocked out its catalytic subunit PP4C.However,how SMEK1 functions as a regulatory subunit has not been clarified yet.Therefore,it is of great significance to study the specific regulatory mechanism of SMEK1.SMEK1 is the main regulator of many cellular physiological processes.Current studies have shown that SMEK1 can participate in the regulation of liver gluconeogenesis,inhibit the tumorigenesis and development of ovarian and cervical tumors,inhibit the proliferation of vascular endothelial cells,and regulate neurogenesis.Above studies indicates that SMEK1 is a widely expressed gene,suggesting that it may play a regulatory role in many tissues and organs.According to the database,SMEK1 is highly expressed in bone marrow,spleen,lymph node and peripheral T lymphocyte,suggesting that it may play an important role in T cells.Studies have shown that knocking out Smek1 in the whole mouse genome will affect the development of germ cells,thereby affecting the birth rate of mice.This makes it impossible for us to explore the function of Smek1 in T cells in the global knockout mice.Therefore,we constructed conditional knockout mice through the Cre-LoxP system,and got the conditional knockout of Smek1 in T cells.Phenotypic analysis showed that DP and CD4 SP cells in the thymus of conditional knockout mice showed a decreasing trend.Further analysis indicated that the expression of TCRβ and CD3 on the surface of DP cells was significantly down-regulated,but there were no significant changes in SP cells.Apoptosis level detection showed that the apoptotic levels of DP and SP cells of conditional knockout mice were up-regulated.We speculated that the impaired expression of TCRβ and CD3 in DP cells leads to the inability of cells to pass the positive selection process,which leads to cell apoptosis.The abnormal development of T cells in the thymus led to a decrease in peripheral T lymphocytes.In addition,deletion of Smek1 resulted in inhibited activation of peripheral CD4+T cells,impaired Th cell differentiation and reduced cytokine expression.We constructed a Jurkat cell line with low expression of SMEK1 and conducted preliminary investigations of the mechanism.We found that deletion of SMEK1 inhibited multiple TCR signaling pathways,such as MAPK,PI3K/Akt/NF-κB,and IL-6 receptor signaling pathways.The phosphorylation of protein molecules was suppressed,which led to impaired T cell activation.RNA sequencing results suggest that Smek1 plays a positive role in regulating gene expression during T cell activation.In summary,we found that SMEK1 is involved in maintaining the differentiation and development of T cells in the thymus and the activation process in peripheral immune tissues.The specific knockout of SMEK1 in T cells will cause abnormalities in these two processes.The results of this study provide inspiration for exploring the role of SMEK1 in the immune system.