The Molecular Mechanism Of Cbl-b In Suppressing The Activation Of CD4~+ T Cell

The proteins of Cbl family is a kind of E3 ubiquitin ligase containing RING-finger domain and are conserved in many species.Cbl-b functions as E3 ubiquitin ligase and multifunctional adaptor,can respond to a variety of internal and external stimulation,widely participates and regulates the signal transduction in cells.In T cells,early reports suggested that Cbl-b finely regulates antigen receptor signaling and immune response by ubiquitination of intracellular and extracellular receptors,receptor associated tyrosine kinases,and downstream signaling molecules.Therefore,Cbl-b,as the limited point of T cell activation,makes it possible to become a molecular target for autoimmune diseases and tumor therapy.However,the specific molecular mechanism of Cbl-b inhibiting the activation of T cells is still unclear.miRNA is a group of regulatory small molecule RNA which is evolutionary conservation and non-coding RNA,can regulate the various biological activities in cells by interfering target messenger RNA stability or translation.In recent years,through the identification of gene chip technology and deep sequencing of miRNAs in different stages of T cell development and activation,significant changes in miRNAs have been found in the process of T cell development and activation.Therefore,we guess whether the inhibitory effect of Cbl-b on CD4+T cell activation is achieved by influencing certain miRNA.Examined the CD4+ T cell activation by CCK8 experiment,we found that Cbl-b-/-CD4+ T cells could be activated by only anti-CD3 antibody stimulated,while WT CD4+T cells activation needed to be cositmulated with anti-CD3 and anti-CD28 antibody at the same time.The tumor inoculation experiments show that the lung and pancreatic cancer cell lines panc02 or LLC cells did not grow effectively in Cbl-b-/-mice,these results were similar with previous reported findings,indicating that Cbl-b inhibits the activation of T cells,and the lack of Cbl-b inhibits tumor growth.In order to verify the hypothesis:whether the inhibitory effect of Cbl-b on CD4+ T cell activation is achieved by influencing certain miRNA,we first used microarray to detect the expression profiles of miRNAs between CD4+ T lymphocytes from the spleen of WT and Cbl-b-/-mice purified by Magnetic absorption cell sorting(MACS)method,miR-99a and miR-125b were both higher expressed in Cbl-b-/-CD4+ T cells compared to WT CD4+ T cells,either in activated or non-activated CD4+ T cells.Then we pick out the most obvious difference miRNAs,miR-99a and miR-125b.we cloned miR-99a and miR-125b into the over-expression vector pMDHl-PGK-GFP and verified the over expression of the miRNAs in plasmid by qPCR.On this basis,we prepare miR-99a and miR-125b lentivirus to infect the EL4 to prepare stable cell strain.Examined by CCK8 experiment,we found that over-expression of miR-99a or miR-125b increase the cell proiferation,illustrating that Cbl-b may inhibit miR-99a/miR-125b expression to suppress the CD4+T cell activation.So,how does the Cbl-b regulate miR-99a/miR-125b expression?To investigate the molecular mechanisms by which Cbl-b regulates miRNA,thereby affecting the activation of CD4+ T cells.We consulted the literature to find the transcription factor HOXA10,which are important regulators of miR-99a and miR-125b.Therefore,we constructed luciferase report plasmids and applied luciferase assay system to verify the binding of 5'UTR with miR-99a and miR-125b,and the results showed that over-expression of HOXA10 promoted the expression of miR-99a and miR-125b.In addition,we detected the translocation of HOXA10 into the nucleus.We found HOXA10 translocated into the nucleus stimulated with the TCR/CD3 and CD28 signals in WT CD4+ T cells through immunofluorescence staining,whereas Cbl-b-/-CD4+ T cells were able to enter the nucleus when only stimulated with TCR/CD3 signal to promote the expression of miR-99a and miR-125b,then facilitated the CD4+ T cells activation.These results were consistent with the phenotype that CD4+ T cells are more susceptible to activation when deficiency of Cbl-b.This result gives us reason to speculate that cbl-b may regulate T cell activation through the HOXA10-mir-99a/mir-125b axis.To further investigate how Cbl-b regulates HOXA10 into the nucleus,we used STRING:functional protein association networks to predict the protein interaction with Cbl-b or HOXA10,cross analysis found that the related proteins SHP-1 and SHP-2,which may interacte with Cbl-b or HOXA10.The proteins were extracted from the activated CD4+ T lymphocytes from the spleen of WT and Cbl-b-/-mice isolated with MACS and,and submitted to western blot,the results show that the expression of SHP-2 in Cbl-b-/-mice increased significantly compared to WT mice,whereas SHP-1 has a little change.Then we cloned the genes of SHP-1 and SHP-2 in expressing vector.Subsequently,we co-expressed these genes with Cbl-b in 293 T cells.Western blot results showed that there were no any interaction between Cbl-b/SHP1 or Cbl-b/SHP2 when co-expressing with these genes in 293T cells.However,interaction between Cbl-b/SHP2 were obviously displayed when the WT CD4+ T cells were stimulated by anti-CD3 antibody alone.whereas CD28 co-stimulation decreased this interaction.To elucidate whether the reduction of SHP-2 is due to the E3 ubiquitin ligase Cbl-b.We isolated the CD4+T lymphocytes from the spleen of WT and Cbl-b-/-Amice and extracted protein after activation in vitro,the co-immunoprecipitation assay was employed to isolate SHP-2 protein complex and the ubiquitination of SHP-2 was identified.Upon TCR stimulation,the ubiquitination of SHP-2 was increased whereas CD28 co-stimulation reduced the ubiquitination level.We also validated the interaction between SHP-2 and HOXA10 and found that SHP-2 bound to HOXA10 whether activated or non-activated.Furthermore,we examined the protein tyrosine phosphatase activity of SHP-2 against HOXA10 in vitro,co-expressed SHP-2 and HOXA10 in 293T cells,immunoprecipitated against HOXA10,we find over-expression of SHP-2 inhibits the phosphorylation of HOXA10 and the phosphorylation of HOXA10 in Cbl-b-/-CD4+T cell is decreased,indicating that Cbl-b reduces the protein level of SHP-2 by ubiquitination to enhance the tyrosine phosphorylation of HOXA10,then prevents the HOXA10 translocate into nuclear,which inhibits the expression of miR-99a and miR-125b,and then increase the expression levels of PI3K/AKT to suppres the CD4 + T cell activation.