LKB1-AMPK Regulates NF-κB Signaling In T Cell Via BCL-10-MALT1

Background:The tumor suppressor LKB1(Liver kinase B1)is a serine/threonine protein kinase.Deletion or mutation of LKB1 in germline cells induces an autosomal dominant genetic disease,Peutz-Jeghers syndrome,which is characterized by gastrointestinal polyps,the formation of pigmented plaques in the skin and oral cavity,and a high incidence of cancer.A large number of clinicopathological studies have shown that gastrointestinal polyps in patients with Peutz-Jeghers syndrome have typical inflammatory symptoms,often accompanied by infiltration of inflammatory cells such as T lymphocytes.At the same time,using Cre/Lox P technology to establish T lymphocyte-specific LKB1 knockout transgenic heterozygous mice also formed inflammatory gastrointestinal polyps,suggesting that LKB1 in T lymphocytes has the function of regulating inflammatory response,but the molecular mechanism has not been studied.Long-term studies have found that when cells are in a state of nutrient/energy depletion,the AMP:ATP ratio is out of balance,and the protein kinase LKB1 inhibits protein synthesis,lipid synthesis and other energy-consuming pathways by activating the AMPK complex,and promotes glycolysis,glutamine decomposition,etc.The energy-producing pathway maintains the content of intracellular ATP and basic life activities.However,the regulatory effect and molecular mechanism of LKB1-AMPK on inflammatory signaling remains to be explored.The CARD11-BCL-10-MALT1(CBM)complex induced by T cell receptor(TCR)signaling activates the NF-κB inflammatory signaling pathway,initiates the transcription and expression of downstream target genes,and participates in T cell proliferation,differentiation and immune response,plays a key role in T cell immune activation and inflammatory response.For a long time,the research on LKB1-AMPK signaling in T cells has focused on the metabolic reprogramming under nutrient deprivation conditions or after activation of TCR signaling,and there is no research on the regulatory mechanism of inflammatory signaling,especially NF-κB signaling,under nutrient deprivation conditions.Therefore,exploring the effect of LKB1-AMPK on NF-κB signaling in T cells provides a new perspective and idea for understanding the regulation of nutrient metabolism on T cell function and inflammatory response.Methods:(1)LKB1 was reinfused in LKB1-deficient cell lines(He La,A549)or knocked down in a recombinant lentivirus-mediated sh RNA silent T cell line(Jurkat),and NF-κB signals were detected by Western Blot(WB)(IκB-α protein level),to investigate whether LKB1 can regulate NF-κB inflammatory signaling.(2)Jurkat cells were stimulated by glucose starvation or metformin treatment under two LKB1 activation conditions,and the protein level of IκB-α and the m RNA level of TNF-α were detected by WB and q PCR,respectively.(3)While using CD3 and CD28 monoclonal antibodies to activate the TCR signal of Jurkat cells,metformin was used to simultaneously stimulate the LKB1-AMPK signal of Jurkat cells,and the downstream molecules IL-2 and TNF-α regulated by NF-κB signal were detected by q PCR.Changes in m RNA levels.(4)Using glucose starvation or metformin to stimulate LKB1,BCL-10 or MALT1-silenced Jurkat cell lines,compare the differences in NF-κB signaling between these three lines and wild-type cells by WB,and analyze the role of these three molecules in nutrient/energy depletion.Regulation of NF-κB signaling in state.(5)Co-immunoprecipitation was used to detect the changes in the binding effect of BCL-10 and MALT1 under glucose starvation,and to investigate whether the formation of the complex between BCL-10 and MALT1 was regulated by nutritional signals;(6)The changes in the binding of BCL-10 and MALT1 were detected by coimmunoprecipitation in AMPK-α1/2 knockout MEF cells to investigate whether the binding between the two is regulated by AMPK.(7)In AMPK-α1/2 knockout MEF cells,wild-type AMPK-α1 or protein kinaseinactivating mutant AMPK-α1-K45 R was reinfused by recombinant retrovirus,and then BCL-10 was investigated by co-immunoprecipitation.Whether MALT1 binding is dependent on AMPK protein kinase activity.(8)Co-immunoprecipitation was used to verify whether each subunit of AMPK binds to BCL-10,and to explore the possibility of AMPK directly regulating BCL-10.(9)Phos-tag was used to study the phosphorylation level of BCL-10 during glucose starvation.(10)To explore the phosphorylation site and upstream protein kinase of BCL-10 by BCL-10 site-directed mutagenesis and in vitro protein kinase experiments.(11)The effect of phosphorylation at S138 of BCL-10 on binding to MALT1 was detected by co-immunoprecipitation.Results :(1)LKB1 gene inhibits NF-κB signaling in various cells: the expression level of LKB1 in He La,A549 and Jurkat is proportional to the protein level of IκB-α.(2)LKB1-AMPK activation inhibits NF-κB signaling in T cells: after glucose starvation or metformin treatment stimulates Jurkat,the protein level of IκB-α increases while the m RNA level of TNF-α decreases;the use of CD3,CD28 m Abs and metformin co-stimulates Jurkat cells,the m RNA levels of IL-2 and TNF-α were significantly inhibited.(3)Inhibition of NF-κB signaling in T cells under nutrient/energy deprivation depends on the presence of LKB1,BCL-10 and MALT1: LKB1,BCL-10 or MALT1 silencing Jurkat cell lines after glucose starvation or metformin treatment,IκB-α protein The level of growth was significantly reduced compared to wild type.(4)LKB1-AMPK signaling activated by nutrient signals regulates the formation of BCL-10-MALT1 complex: glucose starvation,AMPK-α1/2 knockout,and loss of AMPK-α1 kinase activity all inhibit the formation of BCL-10-MALT1 complex.(5)AMPK complex has direct binding with BCL-10: intracellular BCL-10 has binding effect with AMPK-α1,AMPK-β1 and AMPK-γ1.(6)BCL-10 was phosphorylated when LKB1-AMPK was activated: the phosphorylation level of BCL-10 increased during glucose starvation,and the phosphorylation band disappeared after 138S/A mutation.(7)Phosphorylation at the S138 site of BCL-10 regulates its complex formation with MALT1: After dephosphorylation of the BCL-10-138S/A mutation,the binding ability of BCL-10 to MALT1 is enhanced.Conclusions:Under the condition of nutrient/energy deprivation(glucose starvation or metformin treatment),T cells activate LKB1-AMPK signaling,inhibit the formation of BCL-10-MALT1 complex through AMPK phosphorylation modification,down-regulate the level of NF-κB signaling,and inhibit CD3,CD28-mediated TCR activating NF-κB signaling.