161 research outputs found
The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria
Components of the death receptors-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIPL), a well- known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca2+-release as well as ER-mitochondria tethering were decreased in c-FLIP-/- mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIPL and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIPL emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4
The Non-Steroidal FXR Agonist Cilofexor Improves Portal Hypertension and Reduces Hepatic Fibrosis in a Rat NASH Model
Background: The farnesoid X receptor (FXR) influences hepatic metabolism, inflammation
and liver fibrosis as key components of non-alcoholic steatohepatitis (NASH). We studied the effects
of the non-steroidal FXR agonist cilofexor (formerly GS-9674) on portal pressure and fibrosis in
experimental NASH. Methods: NASH was induced in Wistar rats using a choline-deficient high-fat
diet plus intraperitoneal sodium nitrite injections. First, a dose-finding study was performed with
10 mg/kg and 30 mg/kg of cilofexor, focusing on histological readouts. Liver fibrosis was assessed
by Picro-Sirius-Red, desmin staining and hepatic hydroxyproline content. Gene expression was
determined by RT-PCR. In a subsequent hemodynamic study, rats received 30 mg/kg cilofexor with
or without propranolol (25 mg/kg). Portal pressure, systemic hemodynamics and splanchnic blood
flow were measured. Results: Cilofexor dose-dependently induced FXR target genes shp, cyp7a1
and fgf15 in hepatic and ileal tissues, paralleled by a dose-dependent reduction in liver fibrosis
area (Picro-Sirius-Red) of β41% (10 mg/kg) and β69% (30 mg/kg), respectively. The 30 mg/kg
cilofexor dose significantly reduced hepatic hydroxyproline content (β41%), expression of col1a1
(β37%) and pdgfr-Ξ² (β36%), as well as desmin area (β42%) in NASH rats. Importantly, cilofexor
decreased portal pressure (11.9 Β± 2.1 vs. 8.9 Β± 2.2 mmHg; p = 0.020) without affecting splanchnic
blood-flow or systemic hemodynamics. The addition of propranolol to cilofexor additionally reduced
splanchnic inflow (β28%) but also mean arterial pressure (β25%) and heart rate (β37%). Conclusion:
The non-steroidal FXR agonist cilofexor decreased portal hypertension and reduced liver fibrosis
in NASH rats. While cilofexor seems to primarily decrease sinusoidal resistance in cirrhotic portal
hypertension, the combination with propranolol additionally reduced mesenteric hyperperfusion
Improving Interpretation of Cardiac Phenotypes and Enhancing Discovery With Expanded Knowledge in the Gene Ontology
This work was funded through grants from the British Heart Foundation (BHF, SP/07/007/23671, RG/13/5/30112) and the National Institute for Health Research University College London Hospitals Biomedical Research Centre; The Zebrafish Model Organism Database: National Human Genome Research Institute (NHGRI, HG002659, HG004838, HG004834); The Rat Genome Database: National Heart, Lung, and Blood Institute on behalf of the NIH (HL64541); The Mouse Genome Database: NGHRI (HG003300); FlyBase: UK Medical Research Council (G1000968); and Gene Ontology Consortium: NIH NHGRI (U41 HG002273) to Drs Blake, Cherry, Lewis, Sternberg, and Thomas. Professor Riley received BHF personal chair award (CH/11/1/28798). Professors Lambiase and Tinker received support from BHF and UK Medical Research Council. Professor Tinker received National Institute for Health Research Biomedical Research Centre at Barts and BHF grant (RG/15/15/31742). Dr Roncaglia received EMBL-EBI Core funds
Priorities and strategies for improving disabled women's access to maternity services when they are affected by domestic abuse:a multi-method study using concept maps
BACKGROUND: Domestic abuse is a significant public health issue. It occurs more frequently among disabled women than those without a disability and evidence suggests that a great deal of domestic abuse begins or worsens during pregnancy. All women and their infants are entitled to equal access to high quality maternity care. However, research has shown that disabled women who experience domestic abuse face numerous barriers to accessing care. The aim of the study was to identify the priority areas for improving access to maternity services for this group of women; develop strategies for improved access and utilisation; and explore the feasibility of implementing the identified strategies. METHODS: This multi-method study was the third and final part of a larger study conducted in the UK between 2012 and 2014. The study used a modified concept mapping approach and was theoretically underpinned by Andersenβs model of healthcare use. Seven focus group interviews were conducted with a range of maternity care professionals (nβ=β45), incorporating quantitative and qualitative components. Participants ranked perceived barriers to womenβs access and utilisation of maternity services in order of priority using a 5-point Likert scale. Quantitative data exploration used descriptive and non-parametric analyses. In the qualitative component of each focus group, participants discussed the barriers and identified potential improvement strategies (and feasibility of implementing these). Qualitative data were analysed inductively using a framework analysis approach. RESULTS: The three most highly ranked barriers to womenβs access and utilisation of maternity services identified in the quantitative component were: 1) staff being unaware and not asking about domestic abuse and disability; 2) the impact of domestic abuse on women; 3) womenβs fear of disclosure. The top two priority strategies were: providing information about domestic abuse to all women and promoting non-judgemental staff attitude. These were also considered very feasible. The qualitative analysis identified a range of psychosocial and environmental barriers experienced by this group of women in accessing maternity care. Congruent with the quantitative results, the main themes were lack of awareness and fear of disclosure. Key strategies were identified as demystifying disclosure and creating physical spaces to facilitate disclosure. CONCLUSIONS: The study supports findings of previous research regarding the barriers that women face in accessing and utilising maternity services, particularly regarding the issue of disclosure. But the study provides new evidence on the perceived importance and feasibility of strategies to address such barriers. This is an important step in ensuring practice-based acceptability and ease with which improvement strategies might be implemented in maternity care settings
Endoplasmic Reticulum Stress Pathway-Mediated Apoptosis in Macrophages Contributes to the Survival of Mycobacterium tuberculosis
BACKGROUND: Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2Ξ± was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2Ξ± phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2Ξ±. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response. CONCLUSION/SIGNIFICANCE: These findings indicate that eIF2Ξ±/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria
p27 Deficiency Cooperates with Bcl-2 but Not Bax to Promote T-Cell Lymphoma
The effect of Bcl-2 on oncogenesis is complex and expression may either delay or accelerate oncogenesis. The pro-oncogenic activity is attributed to its well characterized anti-apoptotic function while the anti-oncogenic function has been attributed to its inhibition of cellular proliferation. Recent studies demonstrate that p27 may mediate the effects of Bcl-2 on cellular proliferation. We hypothesized that p27 may suppress tumor formation by Bcl-2 family members. To test this hypothesis, cell cycle inhibition and lymphoma development were examined in Lck-Bcl-2 and Lck-Bax38/1 transgenic mice deficient in p27. Strikingly, p27 deficiency synergistically cooperates with Bcl-2 to increase T cell hyperplasia and development of spontaneous T cell lymphomas. Within 1 year, >90% of these mice had developed thymic T cell lymphomas. This high penetrance contrasts with a one year incidence of <5% of thymic lymphoma in Lck-Bcl-2 or p27 β/β mice alone. In contrast, p27 deficiency had no effect on tumor formation in Lck-Bax38/1 transgenic mice, another model of T cell lymphoma. Histologically the lymphomas in p27 β/β Lck-Bcl-2 mice are lymphoblastic and frequently involve multiple organs suggesting an aggressive phenotype. Interestingly, in mature splenic T cells, Bcl-2 largely retains its anti-proliferative function even in the absence of p27. T cells from p27 β/β Lck-Bcl-2 mice show delayed kinetics of CDK2 Thr-160 phosphorylation. This delay is associated with a delay in the up regulation of both Cyclin D2 and D3. These data demonstrate a complex relationship between the Bcl-2 family, cellular proliferation, and oncogenesis and demonstrate that p27 up-regulation is not singularly important in the proliferative delay observed in T cells expressing Bcl-2 family members. Nonetheless, the results indicate that p27 is a critical tumor suppressor in the context of Bcl-2 expression
Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death
Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF
Abortive Autophagy Induces Endoplasmic Reticulum Stress and Cell Death in Cancer Cells
Autophagic cell death or abortive autophagy has been proposed to eliminate damaged as well as cancer cells, but there remains a critical gap in our knowledge in how this process is regulated. The goal of this study was to identify modulators of the autophagic cell death pathway and elucidate their effects on cellular signaling and function. The result of our siRNA library screenings show that an intact coatomer complex I (COPI) is obligatory for productive autophagy. Depletion of COPI complex members decreased cell survival and impaired productive autophagy which preceded endoplasmic reticulum stress. Further, abortive autophagy provoked by COPI depletion significantly altered growth factor signaling in multiple cancer cell lines. Finally, we show that COPI complex members are overexpressed in an array of cancer cell lines and several types of cancer tissues as compared to normal cell lines or tissues. In cancer tissues, overexpression of COPI members is associated with poor prognosis. Our results demonstrate that the coatomer complex is essential for productive autophagy and cellular survival, and thus inhibition of COPI members may promote cell death of cancer cells when apoptosis is compromised
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