Endocytosis is differentially regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) and phospholipase D (PLD). However, the relationship between HIF-1alpha and PLD in endocytosis is unknown. HIF-1alpha is degraded through the prolyl hydroxylase (PHD)/von Hippel-Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1alpha independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1alpha, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1alpha, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1alpha in the EGFR endocytosis pathway.
Animals ; Blood Proteins/chemistry/*metabolism ; Endocytosis ; Female ; HEK293 Cells ; HT29 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Mice, Nude ; Neoplasms/genetics/metabolism/pathology ; Phospholipase D/chemistry/*metabolism ; Phosphoproteins/chemistry/*metabolism ; Protein Structure, Tertiary ; Receptor, Epidermal Growth Factor/*metabolism ; Signal Transduction ; *Up-Regulation ; Vesicular Transport Proteins/*genetics/metabolism ; rab5 GTP-Binding Proteins/*metabolism

Amyotrophic Lateral Sclerosis ; genetics ; Animals ; Biological Transport, Active ; Bipolar Disorder ; genetics ; Glucose Transporter Type 4 ; metabolism ; Hepacivirus ; physiology ; Humans ; Neoplasm Metastasis ; Neoplasms ; metabolism ; Point Mutation ; Polymorphism, Single Nucleotide ; R-SNARE Proteins ; metabolism ; Tissue Distribution ; Transport Vesicles ; physiology ; Vesicular Transport Proteins ; chemistry ; genetics ; metabolism ; physiology ; Virus Replication

To study corneal lymphangiogenesis after corneal transplantation, corneal allogenic transplantation models were established in rats. 8 female Wister rats were used as donors, and 16 Sprague Dawley (SD) rats were used as recipients and 2 SD served as controls. Corneal lymphangiogenesis and hemangiogenesis was examined by electron microscopy 1 and 2 weeks after corneal penetrating transplantation, and the expression of lymphatic vessel endothelial receptor (LYVE-1) was examined 1, 3, 7, 14 days after the transplantation respectively. In addition, 19 allograft failed human corneas were examined by 5'-nase-alkaline phosphatase (5'-NA-ALP) double-enzyme-histochemistry staining to detect corneal lymphangiogenesis and hemangiogenesis. By immunohistochemistry for LYVE-1, it was found that blown lymphatics were localized in the stroma 3 days after the corneal transplantation. With electron microscopy, new lymphatic vessels and blood vessels were found 1 and 2 weeks after the corneal transplantation. By 5'-NA-ALP enzyme-histochemistry, corneal hemangiogenesis was found in all allograft failed human corneas and 5 of 19 (26.3 %) cases had developed corneal lymphangiogenesis. It is concluded that corneal lymphangiogenesis is present after corneal transplantation, which may play an important role in allograft rejection.
Cornea/*blood supply ; Cornea/chemistry ; Cornea/ultrastructure ; Corneal Neovascularization/etiology ; Corneal Neovascularization/metabolism ; Corneal Transplantation/adverse effects ; Corneal Transplantation/*methods ; Immunohistochemistry ; Lymphangiogenesis ; Microscopy, Electron ; Rats, Sprague-Dawley ; Rats, Wistar ; Vesicular Transport Proteins/biosynthesis

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.
Amino Acid Sequence ; Animals ; Exocytosis ; Female ; HEK293 Cells ; Humans ; Mice ; Molecular Sequence Data ; Mutant Proteins/metabolism ; Phosphorylation ; Phosphothreonine/metabolism ; Protein Binding ; Protein Interaction Mapping ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases/*metabolism ; Qa-SNARE Proteins/metabolism ; Rats ; Rats, Sprague-Dawley ; Synaptosomal-Associated Protein 25/*metabolism ; Synaptotagmins/metabolism ; Vesicle-Associated Membrane Protein 2/metabolism ; Vesicular Transport Proteins/chemistry/*metabolism

Animals ; COS Cells ; Cercopithecus aethiops ; Endoplasmic Reticulum ; GTP Phosphohydrolases ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; GTP-Binding Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Gene Expression ; Genetic Complementation Test ; HeLa Cells ; Humans ; Kinetics ; Membrane Fusion ; genetics ; Membrane Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Protein Multimerization ; RNA, Small Interfering ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Vesicular Transport Proteins ; genetics ; metabolism

In the present study, we investigated anti-inflammatory effect of Cardamine komarovii flower (CKF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We determined the effect of CKF methanolic extracts on LPS-induced pro-inflammatory mediators NO and prostaglandin E2 (PGE2), production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and related protein expression levels of MyD88/TRIF signaling pathways in peritoneal macrophages (PMs). Nuclear translocation of NF-κB-p65 was analyzed by immunofluorescence. For the in vivo experiments, an ALI model was established to detect the number of inflammatory cells and inflammatory factors (IL-1β, TNF-α, and IL-6) in bronchoalveolar lavage fluid (BALF) of mice. The pathological damage in lung tissues was evaluated through H&E staining. Our results showed that CKF can decrease the production of inflammatory mediators, such as NO and PGE2, by inhibiting their synthesis-related enzymes iNOS and COX-2 in LPS-induced PMs. In addition, CKF can downregulate the mRNA levels of IL-1β, TNF-α, and IL-6 to inhibit the production of inflammatory factors. Mechanism studies indicated that CKF possesses a fine anti-inflammatory effect by regulating MyD88/TRIF dependent signaling pathways. Immunocytochemistry staining showed that the CKF extract attenuates the LPS-induced translocation of NF-kB p65 subunit in the nucleus from the cytoplasm. In vivo experiments revealed that the number of inflammatory cells and IL-1β in BALF of mice decrease after CKF treatment. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In conclusion, our results suggest that CKF inhibits LPS-induced inflammatory response by inhibiting the MyD88/TRIF signaling pathways, thereby protecting mice from LPS-induced ALI.
Acute Lung Injury ; chemically induced ; drug therapy ; genetics ; metabolism ; Adaptor Proteins, Vesicular Transport ; genetics ; metabolism ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemistry ; Cardamine ; chemistry ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Flowers ; chemistry ; Humans ; Lipopolysaccharides ; adverse effects ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Plant Extracts ; administration & dosage ; chemistry ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo investigate the influences of Shensu Yin to RAW 264.7 on the expression of TLR3, TLR4 and the factors of the downstream in RAW 264. 7 cells.

METHODRAW 264.7 cell line was stimulated with Lipopolysaccharide and POLY I: C, respectively, and treated with the drug serum of Shensuyin simultaneously. 24 hours later, collected the supernatant and measured the inflammatory factors TNF-alpha and IFN-beta, extracted mRNA and measured the expression of TLR3, TLR4 and other correlated indexes of the downstream, analyzed and evaluated Shensu Yin's substance basis of pharmacodynamic actions.

RESULTShensu Yin drug serum depressed the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF mRNA, as a result, it decreased the amount of TNF-alpha and IFN-beta.

CONCLUSIONDepressing the expression of TLR3, MyD88, TRAM and TRIF mRNA may be the elementary basis of Shensu Yin to play heat-clearing and detoxicating effect.

Adaptor Proteins, Vesicular Transport ; genetics ; Animals ; Cell Line ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interferon-beta ; secretion ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; Plants, Medicinal ; chemistry ; Poly I-C ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Interleukin ; genetics ; Signal Transduction ; drug effects ; Toll-Like Receptor 3 ; genetics ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; secretion