Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fedcontrol mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH.

A recent study suggested that neuroinflammation plays a major role in the pathogenesis of a number of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Although the precise mechanism is obscure, dysregulation of the signaling transduction pathway in microglia may enhance inflammation, leading to synaptic dysfunction and ultimately to neuronal cell death. The expression and function of the P2X7 receptor (P2X7R), an ATP-gated ion channel abundantly expressed in microglia in the brain, is significantly up-regulated in the postmortem brain of Alzheimer's disease patients and various neurodegenerative disease animal models. This supports the role of the P2X7R pathway in the progression of neurodegeneration. Blocking P2X7R using brilliant blue G, a P2X7R antagonist that can cross the blood-brain barrier, has been shown to result in the amelioration of neuropathology in various animal models. Taken together, these results raise the possibility that the P2X7R signaling pathway could be a therapeutic target for treating various neurodegenerative diseases.

Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1β secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca2+-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space.

No abstract

No abstract

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group previously showed that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts. We further demonstrated that the former up-regulates Notch ligand delta-like 4 (Dll4) only in the presence of cell-cell contacts. Because Dll4/Notch signal restricts sprouting angiogenesis and promotes vascular stabilization, we investigated the mechanism of how the Ang1/Tie2 signal induces Dll4 expression to clarify the role of the Dll4/Notch signal in Ang1/Tie2 signal-mediated vascular quiescence. Under confluent endothelial cells, the basal Notch signal was observed. Ang1, moreover, induced Dll4 expression and production of the Notch intracellular domain (NICD). Ang1 stimulated transcriptional activity of ␤-catenin through phosphoinositide 3-kinase (PI3K)/AKTmediated phosphorylation of glycogen synthase kinase 3␤ (GSK3␤). Correspondingly, the GSK3␤ inhibitor up-regulated Dll4, whereas depletion of ␤-catenin by siRNA blocked Ang1-induced Dll4 expression, indicating the indispensability of ␤-catenin in Ang1-mediated up-regulation of Dll4. In addition, Dll4 expression by the GSK3␤ inhibitor was only observed in confluent cells, and was impeded by DAPT, a ␥-secretase inhibitor, implying requirement of the Notch signal in ␤-catenin-dependent Dll4 expression. Consistently, we found that either Ang1 or NICD up-regulates Dll4 through the RBP-J binding site within intron 3 of the DLL4 gene and that ␤-catenin forms a complex with NICD/RBP-J to enhance Dll4 expression. Ang1 induced the deposition of extracellular matrix that is preferable for basement membrane formation through Dll4/Notch signaling. Collectively, the Ang1/Tie2 signal potentiates basal Notch signal controlling vascular quiescence by up-regulating Dll4 through AKT-mediated activation of ␤-catenin.

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. in this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNP Sc ) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as Lc3-ii, ATG12-ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNP Sc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNP Sc in addition to attenuation of microgliosis and neuroprotection.

No abstract