Hsp90 Mediates Notch Signaling To Regulate Th22 Cell Differentiation

Background and Objective:Th22 cells are one of the subtypes of CD4~+T cells,which mainly participate in occurrence and development of diseases or pathological processes,such as inflammatory responses,autoimmune diseases and cancer through the secretion of cytokine IL-22.Therefore,it is important to understand the mechanism by which Th22 differentiation is regulated.It has been reported that activation of the Notch signaling pathway is closely related to the development and differentiation of T cells,and also involved in the regulation of differentiation of Th1,Th2,Th17 and Treg cells.Our previous study demonstrated that high expression of the transcription factor Hes-1 in the Notch signaling pathway can inhibit the differentiation of Th22 cells.Hsp90 can play a role in stabilizing the intracellular segment of the Notch molecule(NICD)in T lymphocytic leukemia,and promote the occurrence and development of leukemia,which may be related to the stimulation of T cell proliferation.When Hsp90 is inhibited,ubiquitination degradation of NICD occurrs,and expressions of Hes-1 and c-myc are decreased,suggesting that the signaling of the Notch signaling pathway is affected by Hsp90.Whether does Hsp90mediate Notch signaling to regulate the differentiation of naive CD4~+T cells into Th22 cells?So far,no relevant reports have been found.Therefore,to explore roles of Hsp90 in the regulation of Th22 differentiation by Notch signaling pathway may enrich understanding of Th22 differentiation and lay a theoretical foundation for treatment of related diseases.Methods:Mouse lymph nodes were taken out,single cell suspension was prepared,and naive CD4~+T cells were isolated by immunomagnetic beads.The cells were induced by anti-CD3,anti-CD28,IL-6 and TNF-α.At the same time,Jagged-1was used to treat naive CD4~+T cells.The phenotype of Th22 cells was detected by flow cytometry.The expression of Hes-1 and Hsp90 was detected by qPCR and Western Blot.Then,Hsp90 inhibitor SNX2112 and Hsp90-siRNA were used to block or interfere with expression of Hsp90,and phenotypic changes of Th22 cells were observed.Total RNA was extracted by a Trizol method.RT-PCR and qPCR were used to detect changes of il-22,Hes-1,ahr and arnt at their mRNA levels.Western Bolt analysis was performed for IL-22,Hes-1,AhR and ARNT expressions.ELISA was utilized to test IL-4,IL-9,IL-10,IL-18,IL-21,IL-22,TNF-α,IFN-γ,TGF-βand IL-17A expression levels in the cell culture supernatants.In vivo immunofluorescence staining was performed for the expression relationship between Hsp90 and IL-22,Hes-1 and AhR,Hes-1 and IL-22,AhR and CCR6 in CD4~+T cells.Afterwards,Hsp90agonist GGA(geranylgeranylacetone)and Hsp90 overexpression vector were further used to stimulate the cells,and changes of Th22 cell phenotype,signaling molecules,transcription factors and cytokines were detected and analyzed in the same manner.The CO-IP was then done for interaction between Hsp90 and NICD.Finally,it was determined that Hsp90 could exert the same effect by in in vivo transfecting Hsp90overexpression vector or siRNA into mice.Results:IL-6 and TNF-αsignificantly induced phenotypic transformation of na?ve CD4~+T cells into Th22 cells in vitro.Jagged-1 activation of Notch signaling pathway inhibited the phenotypic transformation of naive CD4~+T cells to Th22 cells,which could be reversed by anti-Jagged-1 and DAPT.SNX2112 or knockdown of Hsp90 can promote the phenotypic transformation of naive CD4~+T cells to Th22 cells,up-regulation of il-22,ahr,arnt expressions,and significant down-regulation of the Notch signaling pathway-targeted transcription factor Hes-1.Compared with the control group,SNX2112 or Hsp90-siRNA promoted IL-22 production by CD4~+T cells,while Jagged-1 inhibited IL-22 production.In contrast,GGA or overexpression of Hsp90 could inhibit the production of IL-22 by CD4~+T cells.Different concentrations of Jagged-1 activate Notch signaling or DAPT inhibits Notch signaling,resulting in down-regulation or up-regulation of Th22 cell phenotype,but no change was observed in Hsp90 mRNA and protein levels.Inhibition or promotion of Hsp90expression may reduce or increase Hes-1 Level.CO-IP showed that Hsp90 interacted with the intracellular segment NICD of the Notch signal.The in vivo overexpression of Hsp90 could suppress deviation of Th22 cells,which were reversed by the in vivo knockdown of hsp90 gene。Conclusions:The results indicate that the activation of Jagged-1-Notch signal pathway can inhibit the differentiation of naive CD4~+T cells into Th22 cells,and that there is no obvious correlation with Hsp90.Activation of Hsp90 molecule also inhibits the differentiation of Th22 cells,which is involved in the activation of the Notch signaling pathway,and Hsp90 interacts with the intracellular segment of the Notch receptor.The results suggest that Hsp90 can mediate Jagged-1-Notch signaling to inhibit Th22 cell differentiation through binding to NICD.