High mobility group box protein 1 (HMGB1) modulates the innate immune response when present in the extracellular compartment. Receptors for HMGB1 include TLR4, TLR2, and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce LPS tolerance. HMGB1 dose-response experiments were performed on IFN-gamma-differentiated human monocyte-like THP-1 cells. Treatment with 1 microg/ml HMGB1 18 h before exposure to LPS (1 microg/ml) decreased TNF release, NF-kappaB nuclear DNA-binding activity, phosphorylation, and degradation of IkappaBalpha. Preconditioning with HMGB1 alone and HMGB1 in the presence of polymyxin B decreased LPS-mediated, NF-kappaB-dependent luciferase reporter gene expression. The specificity of HMGB1 in tolerance induction was supported further by showing that boiled HMGB1 failed to induce tolerance, and antibodies against HMGB1 blocked the induction of LPS tolerance. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type mice became LPS-tolerant following HMGB1 exposure ex vivo, but macrophages derived from RAGE-deficient mice failed to develop tolerance and responded normally to LPS. Mice preconditioned with HMGB1 (20 microg) 1 h before LPS injection (10 mg/kg) had lower circulating TNF compared with control mice preconditioned with saline vehicle. Similarly, decreased nuclear DNA binding of hepatic NF-kappaB was observed in mice preconditioned with HMGB1. Taken together, these results suggest that extracellular HMGB1 induces LPS tolerance, and the RAGE receptor is required for this induction.

BackgroundThe receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated.Methodology/Principal FindingsC57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice.Conclusions/SignificanceLack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.

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Edited by Alex TokerUnlike other ErbB family members, HER2 levels are maintained on the cell surface when the receptor is activated, allowing prolonged signaling and contributing to its transforming ability. Interactions between HER2, HSP90, PMCA2, and NHERF1 within specialized plasma membrane domains contribute to the membrane retention of HER2. We hypothesized that the scaffolding protein ezrin, which has been shown to interact with NHERF1, might also help stabilize the HER2-PMCA2-NHERF1 complex at the plasma membrane. Therefore, we examined ezrin expression and its relationship with HER2, NHERF1, and PMCA2 levels in murine and human breast cancers. We also used genetic knockdown and/or pharmacologic inhibition of ezrin, HSP90, NHERF1, PMCA2, and HER2 to examine the functional relationships between these factors and membrane retention of HER2. We found ezrin to be expressed at low levels at the apical surface of normal mammary epithelial cells, but its expression is up-regulated and correlates with HER2 expression in hyperplasia and tumors in murine mammary tumor virus-Neu mice, in human HER2-positive breast cancer cell lines, and in ductal carcinoma in situ and invasive breast cancers from human patients. In breast cancer cells, ezrin co-localizes and interacts with HER2, NHERF1, PMCA2, and HSP90 in specialized membrane domains, and inhibiting ezrin disrupts interactions between HER2, PMCA2, NHERF1, and HSP90, inhibiting HER2 signaling and causing PKC␣-mediated internalization and degradation of HER2. Inhibition of ezrin synergizes with lapatinib in a PKC␣-dependent fashion to inhibit proliferation and promote apoptosis in HER2-positive breast cancer cells. We conclude that ezrin stabilizes a multiprotein complex that maintains active HER2 at the cell surface. . 2 The abbreviations used are: EGFR, epidermal growth factor receptor; ERM, ezrin/radixin/moesin/merlin; MMTV, murine mammary tumor virus; DCIS, ductal carcinoma in situ; PH, pleckstrin homology; EGF, epidermal growth factor; PLA, proximity ligation assay; IP, immunoprecipitation; shRNA, short hairpin RNA; KD, knockdown; PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin; CA, constitutively active; BrdU, bromodeoxyuridine; DAPI, 4Ј,6-diamidino-2-phenylindole.

Highlights The SARS-CoV-2 receptor ACE2 C-terminal PDZrecognition motif 802 QTSF 805 binds to NHERF1 NHERF1 and ACE2 interact directly in SARS-CoV-2-susceptible lung and intestine cells NHERF1 expression correlates with SARS-CoV-2 entry by regulating ACE2 membrane abundance b-Arrestins may cooperate with NHERF1 to promote ACE2-mediated SARS-CoV-2 cell entry