Carbon monoxide inhibits Fas activating antibody-induced apoptosis in endothelial cells

<p>Abstract</p> <p>Background</p> <p>The extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand, interacts with its cell surface receptor (<it>ie</it>., Fas/CD95), forming a death-inducing signaling complex (DISC). The Fas-dependent apoptotic pathway has been implicated in several models of lung or vascular injury. Carbon monoxide, an enzymatic product of heme oxygenase-1, exerts antiapoptotic effects at low concentration <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>Using mouse lung endothelial cells (MLEC), we examined the antiapoptotic potential of carbon monoxide against apoptosis induced by the Fas/CD95-activating antibody (Jo2). Carbon monoxide was applied to cell cultures <it>in vitro</it>. The expression and/or activation of apoptosis-related proteins and signaling intermediates were determined using Western Immunoblot and co-immunoprecipitation assays. Cell death was monitored by lactate dehydrogenase (LDH) release assays. Statistical significance was determined by student T-test and a value of <it>P </it>< 0.05 was considered significant.</p> <p>Results</p> <p>Treatment of MLEC with Fas-activating antibody (Jo2) induced cell death associated with the formation of the DISC, and activation of caspases (-8, -9, and -3), as well as the pro-apoptotic Bcl-2 family protein Bax. Exposure of MLEC to carbon monoxide inhibited Jo2-induced cell death, which correlated with the inhibition of DISC formation, cleavage of caspases-8, -9, and -3, and Bax activation. Carbon monoxide inhibited the phosphorylation of the Fas-associated death domain-containing protein, as well as its association with the DISC. Furthermore, carbon monoxide induced the expression of the antiapoptotic protein FLIP and increased its association with the DISC.</p> <p>CO-dependent cytoprotection against Fas mediated apoptosis in MLEC depended in part on activation of ERK1/2-dependent signaling.</p> <p>Conclusions</p> <p>Carbon monoxide has been proposed as a potential therapy for lung and other diseases based in part on its antiapoptotic effects in endothelial cells. In vitro, carbon monoxide may inhibit both Fas/caspase-8 and Bax-dependent apoptotic signaling pathways induced by Fas-activating antibody in endothelial cells. Strategies to block Fas-dependent apoptotic pathways may be useful in development of therapies for lung or vascular disorders.</p

The bone morphogenetic protein antagonist gremlin 1 is overexpressed in human cancers and interacts with YWHAH protein

BACKGROUND: Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of our study was to identify an unique gene that shows cancer-associated expression, and characterizes its function related to human carcinogenesis. METHODS: We used the differential display (DD) RT-PCR method using normal cervical, cervical cancer, metastatic cervical tissues, and cervical cancer cell lines to identify genes overexpressed in cervical cancers and identified gremlin 1 which was overexpressed in cervical cancers. We determined expression levels of gremlin 1 using Northern blot analysis and immunohistochemical study in various types of human normal and cancer tissues. To understand the tumorigenesis pathway of identified gremlin 1 protein, we performed a yeast two-hybrid screen, GST pull down assay, and immunoprecipitation to identify gremlin 1 interacting proteins. RESULTS: DDRT-PCR analysis revealed that gremlin 1 was overexpressed in uterine cervical cancer. We also identified a human gremlin 1 that was overexpressed in various human tumors including carcinomas of the lung, ovary, kidney, breast, colon, pancreas, and sarcoma. PIG-2-transfected HEK 293 cells exhibited growth stimulation and increased telomerase activity. Gremlin 1 interacted with homo sapiens tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide (14-3-3 eta; YWHAH). YWHAH protein binding site for gremlin 1 was located between residues 61–80 and gremlin 1 binding site for YWHAH was found to be located between residues 1 to 67. CONCLUSION: Gremlin 1 may play an oncogenic role especially in carcinomas of the uterine cervix, lung, ovary, kidney, breast, colon, pancreas, and sarcoma. Over-expressed gremlin 1 functions by interaction with YWHAH. Therefore, Gremlin 1 and its binding protein YWHAH could be good targets for developing diagnostic and therapeutic strategies against human cancers

Phosphorylation of Nicastrin by SGK1 Leads to Its Degradation through Lysosomal and Proteasomal Pathways

The gamma-secretase complex is involved in the intramembranous proteolysis of a variety of substrates, including the amyloid precursor protein and the Notch receptor. Nicastrin (NCT) is an essential component of the gamma-secretase complex and functions as a receptor for gamma-secretase substrates. In this study, we determined that serum- and glucocorticoid-induced protein kinase 1 (SGK1) markedly reduced the protein stability of NCT. The SGK1 kinase activity was decisive for NCT degradation and endogenous SGK1 inhibited gamma-secretase activity. SGK1 downregulates NCT protein levels via proteasomal and lysosomal pathways. Furthermore, SGK1 directly bound to and phosphorylated NCT on Ser437, thereby promoting protein degradation. Collectively, our findings indicate that SGK1 is a gamma-secretase regulator presumably effective through phosphorylation and degradation of NCT

Early structural and functional defects in synapses and myelinated axons in stratum lacunosum moleculare in two preclinical models for tauopaty

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer’s disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3β with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer’s disease

Novel benzidine and diaminofluorene prolinamide derivatives as potent hepatitis C virus NS5A inhibitors

Our study describes the discovery of a series of highly potent hepatitis C virus (HCV) NS5A inhibitors based on symmetrical prolinamide derivatives of benzidine and diaminofluorene. Through modification of benzidine, L-proline, and diaminofluorene derivatives, we developed novel inhibitor structures, which allowed us to establish a library of potent HCV NS5A inhibitors. After optimizing the benzidine prolinamide backbone, we identified inhibitors embedding meta-substituted benzidine core structures that exhibited the most potent anti-HCV activities. Furthermore, through a battery of studies including hERG ligand binding assay, CYP450 binding assay, rat plasma stability test, human liver microsomal stability test, and pharmacokinetic studies, the identified compounds 24, 26, 27, 42, and 43 are found to be nontoxic, and are expected to be effective therapeutic anti-HCV agents. (C) 2015 Elsevier Masson SAS. All rights reserved.1143sciescopu