Hepatitis B X-interacting Protein HBXIP Negatively Regulates Toll-like Receptor Signaling

Objective:To investigate whether HBXIP is invoveled in LPS induced TLR4 pathway;To study the molecular mechanism whereby HBXIP modulated the process of TLR4 transport and degradation.Methods:1. Cloned the mouse homologue of HBXIP and identification.2. Raw264.7 cells were stimulated with LPS, the expression of HBXP was assayed by quantitative PCR and Western blot.3. Raw 264.7 cells were transfected with HBXIP shRNA or plasmids encoding HBXIP, and selected under Geneticin (G418). The effiency of silencing and overexpressing HBXIP were evaluated by Q-PCR and Western blot.4. Stable cell lines were stimulated with LPS(100ng/mL), the expression of inflammatory cytokines(IL-6, TNF-α, IL-1β, IFN-β) were assayed by quantitative PCR. The supernatants were then collected and the concentration of IL-6, Tnf-a, IL-1β evaluated by enzyme-linked immunoassay. The NF-κB signal pathways and MAPKs signal pathways were analyzed by Western blot(IKK α/β、 IκBα、p65、JNK、Erk、p38、IRF3).5. The transcription TLR4 expression was analyzed by quantitative PCR. The membrance TLR4 was analyzed by FACS. The total TLR4 expression was assayed by Western blot.6. mTOR signal pathway and LC3Ⅱ protein expression were assayed by Western blot.7. Lysosome quantity and pH were assayed by FACS according to the manufacturer’s instructions.8. The subcellular localization of TLR4, LAMP1 and LC3 were examined after immunostaining and indicated compartments markers under confocal microscopy.9. Nuclear localization of TFEB in HBXIP-overexpressed Raw264.7 cells was assayed by confocal microscopy. p-TFEB contained in lysates was examined by Western blot.10. Electron microscopic images of lysosome vacuoles in HBXIP overexpressing cell line.11. HBXIP overexpressing cell line was transfected with control siRNA, Rab siRNA or TFEB siRNA for 24 h, then stimulated with 100ng/mL LPS for indicated time, Western blotting assay TLR4 expression level in cells lysates. HBXIP overexpressing cell line was pre-treated with cycloheximide (CHX), Vinblastine and chloroquine (CQ) for 3h,2h and 3h, followed by LPS (100ng/mL) for indicated time, Western blot assay TLR4 expression level in cells lysates.Results:1. HBXIP expression is up-regulated upon TLR4 activation by LPS.2. Overexpression of HBXIP inhibits LPS-induced production of proinflammatory mediators (IL-6, TNF-a, IL-1β, IFN-β) and LPS-intiated signaling pathways in Raw264.7 cells.3. HBXIP negatively regulates the TLR4 expression.4. Overexpression HBXIP can promote LC3 Ⅱ, inhibiting mTOR signaling pathway related protein expression level, promote cell autophagy.5. HBXIP overexpression can reduce TFEB phosphorylation level, promote its nuclear transfer, thus affecting the number of lysosome and functions. After treated with autophagy-lysosome relevant inhibitors, the effect of HBXIP was largely aborogated.Conclusions:We demonstrate that HBXIP promotes the transcription factors TFEB activation, thereby improveing the lysosome and autophagy-lysosome activity. HBXIP affects the traffic and degradation of TLR4, in addition, HBXIP inhibits TLR4-induced IL-6, TNF-α production in macrophages. Therefore, we identify HBXIP as a novel negative regulator of TLR4 signaling.