Capsid proteins of foot-and-mouth disease virus interact with TLR2 and CD14 to induce cytokine production.

The mechanism of recognition of the foot-and-mouth disease virus (FMDV) by host innate immune cells is not well-understood. In this study, we first found that binary ethylenimine inactivated-FMDV (BEI-FMDV) with structurally intact capsid activated TLR2, but not other TLRs, and this specific activation was blocked by anti-TLR2 Abs or knockout of TLR2. BEI-FMDV activated NF-κB to induce cytokines, notably interferon-ß and IL-6, in a TLR2 and MyD88-dependent manner. Coexpression of TLR6 and CD14 showed additive effects on BEI-FMDV/TLR2-mediated activation of NF-κB. Further studies demonstrated that recombinant capsid proteins rVP1 and rVP3 of FMDV but not rVP0 bound directly with CD14 and TLR2. The rVP1- and rVP3-mediated activation of TLR2 and NF-κB were enhanced by the coexpression of TLR1 or TLR6. Immunoprecipitation of either rVP1 or rVP3 with mouse macrophage cell extracts revealed that rVP1 or rVP3 associated with TLR2, CD14 and TLR6 suggesting that rVP1 and rVP3 interact with CD14, TLR2/TLR1, and TLR2/TLR6 heterodimer. Additional study confirmed that rVP1 and rVP3 interacted with the swine TLR2 signaling pathway to induce IL-6 in swine macrophages. Our results identify VP1 and VP3 of FMDV as novel TLR agonists whose recognition by CD14, TLR2/TLR1, and TLR2/TLR6 of host innate immune cells is critical for the induction of cytokine production.

A novel c-Kit/phospho-prohibitin axis enhances ovarian cancer stemness and chemoresistance via Notch3-PBX1 and ß-catenin-ABCG2 signaling.

BACKGROUND: The underlying mechanism involved in ovarian cancer stemness and chemoresistance remains largely unknown. Here, we explored whether the regulation of c-Kit and plasma membrane prohibitin (PHB) affects ovarian cancer stemness and chemotherapy resistance. METHODS: Mass spectrum analysis and an in vitro kinase assay were conducted to examine the phosphorylation of PHB at tyrosine 259 by c-Kit. The in vitro effects of c-Kit on membrane raft-PHB in ovarian cancer were determined using tissue microarray (TMA)-based immunofluorescence, western blotting, immunoprecipitation, colony and spheroid formation, cell migration and cell viability assays. In vivo tumor initiation and carboplatin treatment were conducted in nude mice. RESULTS: We found that c-Kit and PHB colocalized in the raft domain and were positively correlated in human ovarian serous carcinoma. c-Kit interacted with PHB and facilitated the phosphorylation of PHB at tyrosine 259 (phospho-PHBY259) in the membrane raft to enhance ovarian cancer cell motility. The generation of SKOV3GL-G4, a metastatic phenotype of SKOV3 green fluorescent protein and luciferase (GL) ovarian cancer cells, in xenograft murine ascites showed a correlation between metastatic potential and stem cell characteristics, as indicated by the expression of c-Kit, Notch3, Oct4, Nanog and SOX2. Further study revealed that after activation by c-Kit, raft-phospho-PHBY259 interacted with Notch3 to stabilize Notch3 and increase the downstream target PBX1. Downregulation of raft-phospho-PHBY259 increased the protein degradation of Notch3 through a lysosomal pathway and inhibited the ß-catenin-ABCG2 signaling pathway. Moreover, raft-phospho-PHBY259 played an important role in ovarian cancer stemness and tumorigenicity as well as resistance to platinum drug treatment in vitro and in vivo. CONCLUSIONS: These findings thus reveal a hitherto unreported interrelationship between c-Kit and PHB as well as the effects of raft-phospho-PHBY259 on ovarian cancer stemness and tumorigenicity mediated by the Notch3 and ß-catenin signaling pathways. Targeting the c-Kit/raft-phospho-PHBY259 axis may provide a new therapeutic strategy for treating patients with ovarian cancer.