Effects Of Foxo1 Phosphorylation On Expression Of S1P1 In CD4~+ Thymocytes And Jurkat Cells

Background and ObjectiveT cells in the thymus undergoes CD4^-CD8^-double negative,CD4 ⁺CD8 ⁺ double positive and CD4 ⁺/CD8 ⁺ single positive stage and gradually develop mature,single-positive stage of thymocytes to express 1^-phosphate^-sphingosine receptor 1?Sphingosine-1-phosphate receptor 1,S1P1?and other molecules,under the action of the corresponding chemokines to leave the thymus to reach the peripheral settlement.S1P1 is a key molecule for T cells to move out of the thymus and its expression is regulated by FoxO1-KLF2-S1P1.FoxO1?forkhead transcription factors of the O class 1?is an important transcription factor that binds to Kruppel^-like factor 2?KLF2?expression in the nucleus in combination with homologous DNA sequences,then KLF2 affects the expression of CCR7,CD62 L and S1P1.The TCR activation signal phosphorylates FoxO1 in the nucleus through the PI3K^-AKt signaling pathway and transfers it to the cytoplasm [1],while losing the ability to promote the expression of downstream transcription factors KLF2 and downstream molecules S1P1,and thus affect thymocytes move out.We have reported that the expression of FoxO1,KLF2 and S1P1 in the thymus tissue of patients with myasthenia gravis?MG?was higher than that in the control group.To further elucidate the role of abnormal thymus and peripheral T cell subsets in the development of MG.In this paper,the expression of FoxO1 and its expression of KLF2,CCR7,CD69 and S1P1 in CD4 ⁺ thymocytes were studied.In this study,CD4 ⁺ thymocytes and Jurkat cell lines from MG patients were studied.The changes of KLF2 and S1P1 expression were observed by phosphorylation and dephosphorylation of FoxO1.Methods1.Subjects: Four normal thoracic tissues of non^-autoimmune congenital heart disease obtained from thoracotomy were treated aseptically and prepared with thymocytes.Jurkat cells were removed from the liquid nitrogen and resuscitated in a 37°water bath.The cells were then transferred to a 15 ml sterile centrifuge tube and a solution containing 10% fetal bovine serum 1640 was added dropwise.After centrifugation at 4? for 10 min,the supernatant was discarded and 5 ml of 10% fetal bovine serum 1640 was added thereto.The culture was incubated in a 37°5% CO₂ saturated humidity incubator.The cells were observed under the microscope every day.The And then select the logarithmic growth period of Jurkat cells were tested.2.CD4 ⁺ thymocytes were collected by immunomagnetic beads method: 10?l of single thymocyte cell suspension was counted and 80?l of sorted Buffer was added to 1 x 10⁷ cells?10?l?,20?l of CD4 monoclonal antibody was added and incubated at 4?15min,then add 2ml Buffer mix,300 g 4?centrifugal 10 min,discard the supernatant,add 500?l Buffer to repeat the cell to resuspend,and then MS column into the magnetic column,add 0.5ml PBE Run,Gravity flow under the natural flow,repeated washing after the separation column from the magnetic field,adding Buffer and collected,that is,to get CD4 ⁺ thymocytes.3.FoxO1 phosphorylation and dephosphorylation test: CD4 ⁺ SP thymocytes and Jurkat cells were divided into three groups,the first group of FoxO1 phosphorylation group.The 6^-well plates were coated with 5 ? g/ml CD3 monoclonal antibody,and the coating solution was discarded at 4? refrigerator.The concentration of 1?g/ ml CD28 monoclonal antibody and 10% fetal bovine serum was 2 × 10⁶/ml cell suspension 2ml;the second group of FoxO1 dephosphorylation group.The PI3K-AKt signal pathway specific inhibitor LY294002 12.5?g/ ml was added to the 2 ×10⁶ cells / ml cell suspension;the third group was the control group.Incubate with 10% fetal bovine serum 1640 medium and incubate at 37°5% CO₂ saturated humidity incubator for 48 h to collect cells.4.The mRNA expression of FoxO1,KLF2,S1P1 and CCR7,CD69 Total RNA was extracted from each group.FoxO1,KLF2,S1P1,CD69,CCR7,CCR9,KLF2 mRNA expression level,using 2-??Ct method analysis FoxO1 phosphorylation group,FoxO1 dephosphorylation group and the control group compared the relative expression of different genes.5.The expression of FoxO1,KLF2,S1P1 and P-FoxO1 protein: The total protein of each group was extracted and the protein expression of FoxO1,KLF2,S1P1 and P^-FoxO1 were detected by Western blot.6.Statistical analysis Data statistics were analyzed using mean ± standard deviation,using SPSS 21.0 statistical software for analysis of data.The comparison between the two groups was analyzed by t test,and the data were analyzed by GraphPad Prism 5.0.The results were considered statistically significant at P <0.05.Results1.The effects of CD3 and CD28 monoclonal antibody on the expression of FoxO1,KLF2,S1P1,CCR7 and CD69 in CD4 ⁺ SP thymocytes: The changes of FoxO1,KLF2,S1P1,CCR7 and CD69 mRNA were detected by real-Time PCR.The results showed that FoxO1,KLF2,S1P1,CCR7 mRNA levels in FoxO1 phosphorylation group were significantly lower than those in blank control group?P <0.05?.The CD69 mRNA level was significantly higher than that of the blank control group?P <0.05?.2.Effects of PI3K-AKt signal pathway specific inhibitor LY294002 on the expression of FoxO1,KLF2,S1P1,CCR7 and CD69 in CD4 ⁺ SP thymocytes: The Real-Time PCR was used to detect the changes of FoxO1,KLF2,S1P1,CCR7 and CD69 mRNA levels in each group.The results showed that FoxO1 mRNA level in FoxO1 dephosphorylation group was significantly higher than that in blank control group?P<0.05?,while KLF2 and S1P1 mRNA levels were significantly lower than those in blank control group?P<0.05?CCR7 and CD69 mRNA levels were significantly decreased compared with the blank control group?P> 0.05?3.Effects of CD3 and CD28 on the expression of FoxO1,KLF2,S1P1,CCR7 and CD69 in Jurkat cells: Real-Time PCR was used to detect the expression of FoxO1,KLF2,S1P1 and CCR7,CD69 mRNA in 3h,6h,12 h,24h and 48 h Variety.The results of CD3 and CD28 treatment showed that FoxO1,KLF2,S1P1 and CCR7 mRNA levels in FoxO1 phosphorylation group were significantly lower than those in blank control group?P <0.05?.CD69 mRNA level in the blank control group was significantly higher than that in the control group?P <0.05?.4.Effects of PI3K-AKt signal pathway specific inhibitor LY294002 on the expression of FoxO1,KLF2,S1P1,CCR7 and CD69 in Jurkat cells: Real-Time PCR was used to detect FoxO1,KLF2,S1P1 in 3h,6h,12 h,CD69 mRNA levels.The results of LY294002 treatment showed that FoxO1,KLF2 and S1P1 mRNA levels in FoxO1 dephosphorylation group were significantly higher than those in blank control group?P <0.05?while while CD69 mRNA level was significantly higher than that of blank control group?P>0.05?,CCR7 mRNA level was significantly lower than that of blank control group?P> 0.05?.5.Effects of FoxO1 phosphorylation and FoxO1 dephosphorylation on the expression of P-FoxO1,FoxO1,KLF2 and S1P1 proteins in Jurkat cells: The expression levels of FoxO1,KLF2 and S1P1 protein in FoxO1 phosphorylation group after CD3 and CD28 treatment were compared with those of blank control group?P<0.05?.The expression of P^-FoxO1 protein was significantly increased compared with the blank control group?P<0.05?.The expression level of FoxO1,KLF2 and S1P1 protein in FoxO1 dephosphorylation group was significantly higher than that in blank control group?P <0.05?,and the expression of P-FoxO1 protein was significantly higher than that of blank control group?P<0.05?,and the level of FoxO1,KLF2 and S1P1 protein in FoxO1 dephosphorylation group treated with PI3K-AKt signal pathway specific inhibitor LY294002?P> 0.05?.ConclusionsFoxO1 phosphorylation and dephosphorylation are important factors in reg ulating the status of immature T cells.