Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

AbstractCeramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains

Two different B-type creatine kinase subunits dimerize in a tissue-specific manner

AbstractBrain-type creatine kinase B-CK (EC 2.7.3.2) was purified from several chicken tissues, e.g. cardiac muscle, brain, gizzard and retina. Two major monomeric chicken B-CK subunits, designated Bb (basic) and Ba (acidic), which differ in isoelectric point, were separated by chromatofocussing in the presence of 8 M urea on a MonoP column. The two subunits were shown by peptide mapping, amino acid analysis and partial sequencing, as well as by immunological criteria, to be distinct B-CK polypeptides. The N-tenninal sequence of 30 amino acid residues of Bb correspond entirely to data derived from a B-CK c-DNA clone termed H4 [(1986) Nucleic Acids Res. 14, 1449-1463], whereas the N-terminus of the acidic Ba species was blocked. Native dimeric B-CK isoenzymes obtained from these tissues were separated by ion exchange chromatography on a MonoQ column yielding two B-CK dimer populations, type-I and type-II B-CK, varying in relative proportions. Quantitation of the CK activity peak ratios of these two populations revealed the existence of a tissue-specific, post-translational mechanism regulating B-CK dimerization in neural tissues. Tissue-specific dimerization of the two distinct B-CK monomer species may represent a means of specifying the intracellular distribution of the dimeric B-CK subspecies

Caveolin-1-mediated post-transcriptional regulation of inducible nitric oxide synthase in human colon carcinoma cells

Reactive oxygen species are now widely recognized as important players contributing both to cell homeostasis and the development of disease. In this respect nitric oxide (NO) is no exception. The discussion here will center on regulation of the inducible form of nitric oxide synthase (iNOS) for two reasons. First, only iNOS produces micromolar NO concentrations, amounts that are high by comparison with the picomolar to nanomolar concentrations resulting from Ca2+-controlled NO production by endothelial eNOS or neuronal nNOS. Second, iNOS is not constitutively expressed in cells and regulation of this isoenzyme, in contrast to endothelial eNOS or neuronal nNOS, is widely considered to occur at the transcriptional level only. In particular, we were interested in the possibility that caveolin-1, a protein that functions as a tumor suppressor in colon carcinoma cells (Bender et al., 2002; this issue), might regulate iNOS activity. Our results provide evidence for the existence of a post-transcriptional mechanism controlling iNOS protein levels that involves caveolin-1-dependent sequestration of iNOS within a detergent-insoluble compartment. Interestingly, despite the high degree of conservation of the caveolin-1 scaffolding domain binding motif within all NOS enzymes, the interaction detected between caveolin-1 and iNOS in vitro is crucially dependent on presence of a caveolin-1 sequence element immediately adjacent to the scaffolding domain. A model is presented summarizing the salient aspects of these results. These observations are important in the context of tumor biology, since down-regulation of caveolin-1 is predicted to promote uncontrolled iNOS activity, genotoxic damage and thereby facilitate tumor development in human

A Novel Role for Helicobacter pylori Gamma-Glutamyltranspeptidase in Regulating Autophagy and Bacterial Internalization in Human Gastric Cells

The risk of developing gastric cancer is strongly linked to Helicobacter pylori (H. pylori) infection. Alternatively, autophagy is a conserved response that is important in cellular homeostasis and provides protection against bacterial infections. Although H. pylori is typically considered an extracellular bacterium, several reports indicate that it internalizes, possibly to avoid exposure to antibiotics. Mechanisms by which H. pylori manipulates host cell autophagic processes remain unclear and, importantly, none of the available studies consider a role for the secreted H. pylori virulence factor gamma-glutamyltranspeptidase (HpGGT) in this context. Here, we identify HpGGT as a novel autophagy inhibitor in gastric cells. Our experiments revealed that deletion of HpGGT increased autophagic flux following H. pylori infection of AGS and GES-1 gastric cells. In AGS cells, HpGGT disrupted the late stages of autophagy by preventing degradation in lysosomes without affecting lysosomal acidification. Specifically, HpGGT impaired autophagic flux by disrupting lysosomal membrane integrity, which leads to a decrease in lysosomal cathepsin B activity. Moreover, HpGGT was necessary for efficient internalization of the bacteria into gastric cells. This important role of HpGGT in internalization together with the ability to inhibit autophagy posits HpGGT as a key virulence factor in the development of gastric cancer

Signaling triggered by Thy-1 interaction with ß3 integrin on astrocytes is an essential step towards unraveling neuronal Thy-1 function

Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Recently described evidence that Thy-1 interacts with ß3 integrin on astrocytes will be discussed. Thy-1 binding to ß3 integrin triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment and spreading. Thy-1 has been reported to modulate neurite outgrowth by triggering a cellular response in neurons. However, our data indicate that Thy-1 can also initiate signaling events that promote adhesion of adjacent astrocytes to the underlying surface. Preliminary results suggest that morphological changes observed in the actin cytoskeleton of astrocytes as a consequence of Thy-1 binding is mediated by small GTPases from the Rho family. Our findings argue that Thy-1 functions in a bimodal fashion, as a receptor on neuronal cells and as a ligand for ß3 integrin receptor on astrocytes. Since Thy-1 is implicated in the inhibition of neurite outgrowth, signaling events in astrocytes are likely to play an important role in this proces

Metformin prevents nerve growth factor-dependent proliferative and proangiogenic effects in epithelial ovarian cancer cells and endothelial cells

Background: Epithelial ovarian cancer (EOC) is characterized by exacerbated angiogenesis regulated by proangiogenic and growth factors. Nerve growth factor (NGF) is overexpressed in EOC where it promotes proliferation as well as survival and is considered a proangiogenic factor. Metformin, a drug commonly used in the treatment of diabetes, is attributed to antineoplastic effects, but the underlying mechanisms remain unknown. Given that current therapies yield modest results in EOC patients, the aim of this study was to determine the effects of metformin on NGF-enhanced proliferation of EOC cells and the angiogenic potential of endothelial cells. Methods: A2780 (EOC), HOSE (human ovarian surface epithelial) and EA.hy926 (endothelial) cells were treated with NGF and metformin. Cell viability, cell proliferation and cell cycle were evaluated in all three cell lines, and the angiogenic potential in endothelial EA.hy926 cells. Results: NGF enhanced cell proliferation in A2780, HOSE and EA.hy926 cells ( p < 0.05), while metformin treatment decreased cell proliferation in A2780 and EA.hy926 cells ( p < 0.05). Moreover, the NGF-enhanced angiogenic score in EA.hy926 cells was prevented by metformin ( p < 0.05). Conclusions: Given that NGF plays a significant role in EOC progression, our current findings suggest that metformin holds considerable promise as an adjuvant treatment in ovarian cancer

The 5’ untranslated region of the anti-apoptotic protein Survivin contains an inhibitory upstream AUG codon

Survivin (BIRC5) is an anti-apoptotic protein that is important in cancer. Mechanisms responsible for controlling Survivin levels in cells include transcriptional regulation and modulation of protein stability via post-translational modifications; however to date, translational control has been poorly studied. Here, we focused particularly on the primary control elements present in the Survivin 5' untranslated region (5'UTR). Bioinformatic analysis of ribosome occupancy on the Survivin 5'UTR revealed the presence of elongating ribosomes upstream of the canonical initiator AUG, suggesting an alternative upstream initiator AUG (uAUG) might exist. This uAUG was found out-of-frame at position -71 and appeared as a conserved element in mammals. RACE analysis revealed different transcriptional start sites for BIRC5, which indicated that translational control by this uAUG is restricted to longer 5'UTR variants. We studied the activity of the uAUG in different cell types by cloning the Survivin 5'UTR DNA sequence (wild-type and mutated variants) upstream of renilla luciferase (RLuc) into a pcDNA3 plasmid. Changes in RLuc activity were determined by luminescence assays and Western blotting. Results showed that when this uAUG was mutated to AUU or AGG in the cloned Survivin 5'UTR, RLuc activity was significantly increased. Similar results were obtained when uAUG was positioned inframe with the RLuc initiator AUG. Immunodetection of Renilla (35 kDa) by Western blotting revealed the presence of a second band (37 kDa approximately) in cells transfected with the Inframe reporter constructs, indicating that the uAUG was functional in our experimental conditions. In conclusion, our experimental data demonstrate the presence of an alternative and inhibitory initiator uAUG in the Survivin 5' UTR. This inhibitory uAUG may help understanding how Survivin expression is downregulated under physiological or pathological conditions.Comisión Nacional de Investigación Cientifica y Tecnológica of Chile (Conicyt) Comisión Nacional de Investigación Cientifica y Tecnológica (CONICYT) CONICYT FONDECYT 1171615 1170925 Comisión Nacional de Investigación Cientifica y Tecnológica (CONICYT) CONICYT FONDAP 1513001