The Role Of EGFR Phosphorylation In LPS Induced Endotoxemia

Background and ObjectionEGFR phosphorylation is reported to promote the inflammatory response of cells to LPS.However,the mechanism of how EGFR regulating the cell response to LPS is still remained unknown.We found LPS can increase the expression of EGFR and TLR4 on the cell surface of macrophage both in vitro and vivo.In LPS treatment,Rab5a mediated endocytosis of TLR4 and EGFR at early time is responsible for initiating the transporting of receptors onto cell surface at latter time.LPS trans-activated EGFR phosphorylation and its sub-stream signal molecule GRB2/EPS8/RIN1/RN-TRE coordinate to regulate the activity of Rab5a to ensure the proper endocytosis of TLR4 and EGFR.Therefore,inhibiting the phosphorylation of EGFR leads to the decrease of Rab5a activity and its mediated internalization of receptors.Therefore,less TLR4 is transported onto cell surface to recognize and bind with LPS.As a result,EGFR inhibitor such as PD168393 or erlotinib can effectively inhibit LPS induced expression of inflammatory cytokines(such as IL-1 ?,IL-6,IL-10 and TNF-?),activation of p38,ERK1/2 and I?B?,production of ROS and cell death(such as pyroptosis and necroptosis)in macrophage.IntroductionToll-like receptor 4(TLR4)being activated by lipopolysaccharide(LPS)from Gram-negative bacteria,is a key regulator of innate immunity and inflammation.The recognition between LPS and TLR4 needs several co-receptors for the successful and optimal LPS presentation and signal transduction.TLR4 receptor complex is comprised of TLR4 and myeloid differentiation factor 2(MD-2),which work together to activate a signaling cascade through the Toll/IL-1R(TIR)domain of its cytoplasmic tail.Two distinct signaling branches were triggered through the recruitment of the two TIR-containing adaptor proteins,myeloid differentiation primary response gene 88(MyD88)and TIR-domain-containing adapter-inducing interferon-?(TRIF),respectively.Like a double-edged sword,although it shows some extent of protective antimicrobial function,excessive or dysregulated TLR4 signaling can be detrimental to the host by causing bacterial sepsis.So,as the origin of LPS response;the amount of TLR4/MD-2 present on the cell surface is a tightly regulated process.Surface TLR4 amount is determined by the balance of receptor trafficking from Golgi apparatus to the cell membrane and internalization of cell surface receptor into endosomal compartments.However,compared with its subsequent signal transduction pathway after binding with LPS,the mechanism of how TLR4 is kept in balance on the cell surface is less well characterized.It has been reported that gp96,PRAT4A(protein associated with TLR4)and the interation of TLR4 and MD2 in the ER are all critical for proper processing of TLR4 in the endoplasmic reticulum(ER)and its secretion to the plasma membrane.In LPS treatment,TMED(transmembrane emp24 proteintransport domain containing)and Rab10 were reported to be responsible for transporting TLR4 from the Golgi to the plasma.On the other hand,LPS also induced TLR4 internalization into endosome with the help of CD 14,clathrin,rafts and Phospholipase C?-2.So up to now,how these two physiological processes of TLR4 coordinate with each other in the presence of LPS is barely studied.Epidermal growth factor receptor(EGFR,also known as ErbB1 or HER-1)is a transmembrane receptor which belongs to tyrosine kinase receptor family composed of an extracellular,a transmembrane and an intracellular domain.After binding to its ligands,such as epidermal growth factor(EGF),transforming growth factor a(TGF-a),amphiregulin(AR),epiregulin(EREG),betacellulin(BTC),heparin-binding EGF(HB-EGF)and epigen(EPGN),EGFR plays a fundamental role in development and normal physiology of cells,including stimulating cell proliferation,differentiation,and motility.Recently,more and more studies revealed that EGFR can be transactivated by LPS and plays an important regulatory role in the signal transduction process of LPS/TLR4 signaling.Both our previously study and some other researchers reported that EGFR phosphorylation promotes LPS induced TNF-? production and EGFR inhibitor erlotinib can effectively block LPS-induced cytokine expression in vivo and protects mice from LPS-mediated lethality.Although Chattopadhyay S et al.also found EGFR inhibitor protects mice from LPS-induced septic shock and death,but they stated that EGFR inhibitor have an effect on the production of TNF-? and it selectively blocking the IFN branch of TLR4 signaling but not the NF-?B pathway in RAW264.7 and primary bone marrow-derived dendritic cells(BMDCs).So far,there is no evidence to support EGFR can bind to TLR4 or other downstream signal molecules whatever in NF-?B pathway or IFN branch pathway.Therefore,the mechanism of how EGFR plays a role in LPS/TLR4 signal pathway is still remained unknown.In this study,we revealed that EGFR phosphorylation promotes the accumulation of both TLR4 and EGFR on the cell surface of macrophage in response to LPS.We also identified Rab5a mediated the endocytosis of TLR4 and EGFR at the early time of LPS treatment triggers the accumulation of TLR4 and EGFR on the cell surface of macrophage at the later time of LPS treatment.EGFR downstream molecules EPS8/RN-TRE and GRB2/RIN1 coordinate to keep the balance of GTP and GDP-bound form of Rab5a.Therefore,in LPS treatment,inhibiting the phosphorylation of EGFR can down-regulate the expression of TLR4 and EGFR on the cell surface.As a consequence,the activation of p38,ERK1/2 and I?B?,the generation of ros and cytokines(such as TNF-?,IL-1?,IL-10 and IL-6)and death(such as necroptosis and pyroptosis),in response to LPS,could all be inhibited by EGFR inhibitor.These results indicated that EGFR phosphorylation promotes the inflammatory process of LPS/TRL4 signal pathway at least in part through upregulating the amount of TLR4 on the cell surface.Methods1.LPS induced the phosphorylation of EGFR receptor,the phosphorylation of EGFR promotes both TLR4 and EGFR cell surface expression in response to LPS2.Rab5a mediated endocytosis initiates the accumulation of TLR4 and EGFR on the cell surface of macrophage in response to LPS3.Upregulated cell surface TLR4 expression sensitizes cell response to LPS4.EGFR phosporylation promotes LPS-induced macrophage pyroptosis and necrosis in macrophagesResults1.LPS induces the phosphorylation of EGFR with the help of TACE in macrophages2.EGFR phosphorylation promotes both TLR4 and EGFR cell surface expression in response to LPS3.Rab5a mediated endocytosis initiates the accumulation of TLR4 and EGFR on the cell surface of macrophage in response to LPS4.EPS8/GRB2/RN-TRE/RIN1 are involved in regulating EGFR phosphorylation induced Rab5a activity in LPS treatment5.Upregulated cell surface TLR4 expression sensitizes cell response to LPS6.EGFR phosporylation promotes LPS-induced macrophage death7.EGFR phosporylation promotes LPS induced cell death8.EGFR phosporylation promotes LPS induced pyroptosis and necrosis in macrophagesConclusionLPS can increase the expression of EGFR and TLR4 on the cell surface of macrophage both in vitro and vivo.In LPS treatment,Rab5a mediated ondocytosis of TLR4 and EGFR at early time is responsible for initiating the transporting of receptors onto cell surface at latter time.LPS trans-activated EGFR phosphorylation and its sub-stream signal molecule GRB2/EPS8/RIN1/RN-TRE coordinate to regulate the activity of Rab5a to ensure the proper endocytosis of TLR4 and EGFR.Therefore,inhibiting the phosphorylation of EGFR leads to the decrease of Rab5a activity and its mediated internalization of receptors.Therefore,less TLR4 is transported onto cell surface to recognize and bind with LPS.As a result,EGFR inhibitor such as PD 168393 or erlotinib can effectively inhibit LPS induced expression of inflammatory cytokines(such as IL-1 ?,IL-6,IL-10 and TNF-?),activation of p38,erkl/2 and I?B?,production of ROS and cell death(such as pyroptosis and necroptosis)in macrophage.