Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N^2,N^4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy

Effect of eplerenone on parathyroid hormone levels in patients with primary hyperparathyroidism: a randomized, double-blind, placebo-controlled trial

<p>Abstract</p> <p>Background</p> <p>Increasing evidence suggests the bidirectional interplay between parathyroid hormone and aldosterone as an important mechanism behind the increased risk of cardiovascular damage and bone disease observed in primary hyperparathyroidism. Our primary object is to assess the efficacy of the mineralocorticoid receptor-blocker eplerenone to reduce parathyroid hormone secretion in patients with parathyroid hormone excess.</p> <p>Methods/design</p> <p>Overall, 110 adult male and female patients with primary hyperparathyroidism will be randomly assigned to eplerenone (25 mg once daily for 4 weeks and 4 weeks with 50 mg once daily after dose titration] or placebo, over eight weeks. Each participant will undergo detailed clinical assessment, including anthropometric evaluation, 24-h ambulatory arterial blood pressure monitoring, echocardiography, kidney function and detailed laboratory determination of biomarkers of bone metabolism and cardiovascular disease.</p> <p>The study comprises the following exploratory endpoints: mean change from baseline to week eight in (1) parathyroid hormone(1–84) as the primary endpoint and (2) 24-h systolic and diastolic ambulatory blood pressure levels, NT-pro-BNP, biomarkers of bone metabolism, 24-h urinary protein/albumin excretion and echocardiographic parameters reflecting systolic and diastolic function as well as cardiac dimensions, as secondary endpoints.</p> <p>Discussion</p> <p>In view of the reciprocal interaction between aldosterone and parathyroid hormone and the potentially ensuing target organ damage, the EPATH trial is designed to determine whether eplerenone, compared to placebo, will effectively impact on parathyroid hormone secretion and improve cardiovascular, renal and bone health in patients with primary hyperparathyroidism.</p> <p>Trial registration</p> <p>ISRCTN33941607</p

Why are mineralocorticoid receptor antagonists cardioprotective?

Two clinical trials, the Randomized ALdosterone Evaluation Study (RALES) and the EPlerenone HEart failure and SUrvival Study (EPHESUS), have recently shown that mineralocorticoid receptor (MR) antagonists reduce mortality in patients with heart failure on top of ACE inhibition. This effect could not be attributed solely to blockade of the renal MR-mediated effects on blood pressure, and it has therefore been proposed that aldosterone, the endogenous MR agonist, also acts extrarenally, in particular in the heart. Indeed, MR are present in cardiac tissue, and possibly aldosterone synthesis occurs in the heart. This review critically addresses the following questions: (1) is aldosterone synthesized at cardiac tissue sites, (2) what agonist stimulates cardiac MR normally, and (3) what effects are mediated by aldosterone/MR in the heart that could explain the beneficial effects of MR blockade in heart failure? Conclusions are that most, if not all, of cardiac aldosterone originates in the circulation (i.e., is of adrenal origin), and that glucocorticoids, in addition to aldosterone, may serve as the endogenous agonist of cardiac MR. MR-mediated effects in the heart include effects on endothelial function, cardiac fibrosis and hypertrophy, oxidative stress, cardiac inotropy, coronary flow, and arrhythmias. Some of these effects occur via or in synergy with angiotensin II, and involve a non-MR-mediated mechanism. This raises the possibility that aldosterone synthase inhibitors might exert beneficial effects on top of MR blockade

Distinct Cytoplasmic and Nuclear Functions of the Stress Induced Protein DDIT3/CHOP/GADD153

DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Cell-autonomous cytotoxicity of type I interferon response via induction of endoplasmic reticulum stress

The interaction of IFN with specific membrane receptors that transduce death-inducing signals is considered to be the principle mechanism of IFN-induced cytotoxicity. In this study, the classic non–cell-autonomous cytotoxicity of IFN was augmented by cell-autonomous mechanisms that operated independently of the interaction of IFN with its receptors. Cells primed to produce IFN by 5-azacytidine (5-aza) underwent endoplasmic reticulum (ER) stress. The chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (4-PBA), as well as the iron chelator ciclopirox (CPX), which reduces ER stress, alleviated the cytotoxicity of 5-aza. Ablation of CCAAT-enhancer-binding protein homologous protein (CHOP), the major ER stress–associated proapoptotic transcription factor, protected fibroblasts from 5-aza only when the cytotoxicity was examined cell autonomously. In a medium-transfer experiment in which the cell-autonomous effects of 5-aza was dissociated, CHOP ablation was incapable of modulating cytotoxicity; however, neutralization of IFN receptor was highly effective. Also the levels of caspase activation showed a distinct profile between the cell-autonomous and the medium-transfer experiments. We suggest that besides the classic paracrine mechanism, cell-autonomous mechanisms that involve induction of ER stress also participate. These results have implications in the development of anti-IFN-based therapies and expand the class of pathologic states that are viewed as protein-misfolding diseases

Modulation of Pancreatic Islets&apos; Function and Survival During Aging Involves the Differential Regulation of Endoplasmic Reticulum Stress by p21 and CHOP

Aims: Although endoplasmic reticulum (ER) stress is recognized as a major mechanism causing pancreatic dysfunction in diabetes, little is known on how aging modulates the process. Here, we compared the response with ER stress, viability, and insulin release from pancreatic islets of young (6 weeks) or aged (14 months) mice. Results: Islets from aged mice were more sensitive to ER stress than their younger counterparts; they exhibited more pronounced unfolded protein response (UPR) and caspase activation and displayed compromised insulin release after high-glucose stimulation. Genetic ablation of p21 sensitized the islets to ER stress, especially in the aged group, whereas CHOP ablation was protective for islets from both aged and younger animals. Ciclopirox (CPX), an iron chelator that stimulates p21 expression, protected islets from glucotoxicity and mice from diet-induced diabetes, especially in the aged group in a manner that was both p21 and CHOP dependent. Innovation: For the first time, the study shows that age-dependent susceptibility to diet-induced diabetes is associated with the activity of p21 and CHOP in pancreatic islets and that CPX protects islets from glucotoxicity and mice from diabetes in an age-dependent manner. Conclusions: Our results identify ER stress as an age-dependent modifier of islet survival and function by mechanisms implicating enhancement of CHOP activity and inhibition of the protective activity of p21. These findings suggest that interventions restoring the homeostatic activity of ER stress, by agents such as CPX, may be particularly beneficial for the management of diabetes in the elderly. Antioxid. Redox Signal. 27, 185-200. © 2017, Mary Ann Liebert, Inc

Regulation of P21 during diabetes-associated stress of the endoplasmic reticulum

Endoplasmic reticulum (ER) stress plays a major role in the pathogenesis of diabetes by inducing β-cell apoptosis in the islets of Langerhans. In this study, we show that the transcription factor CHOP, which is instrumental for the induction of ER-stress-associated apoptosis and the pancreatic dysfunction in diabetes, regulates the expression of P21 (WAF1), a cell cycle regulator with anti-apoptotic activity that promotes cell survival. Deficiency of P21 sensitizes pancreatic β-cells to glucotoxicity, while in mice genetic ablation of P21 accelerates experimental diet-induced diabetes, results indicative of a protective role for P21 in the development of the disease. Conversely, pharmacological stimulation of P21 expression by nutlin-3a, an inhibitor of P53-MDM2 interaction, restores pancreatic function and facilitates glucose homeostasis. These findings indicate that P21 acts as an inhibitor of ER-stress-associated tissue damage and that stimulation of P21 activity can be beneficial for the management of diabetes and probably of other conditions in which ER-stress-associated death is undesirable. © 2015 Society for Endocrinology

CHOP-dependent regulation of p21/waf1 during ER stress

The transcription factor CHOP/GADD153 is induced during the unfolded protein response (UPR) and is associated to the induction of ER stress-related apoptosis. However, how the transition between the pro-survival and the pro-apoptotic role of ER stress is being orchestrated remains poorly understood. Here we show that tunicamycin, an antibiotic promoting ER stress, suppresses the expression of p21, a tumor suppressor that induces cell cycle arrest and inhibits apoptosis. This suppression of p21 levels was independent of p53 that is the major transcriptional regulator of p21, but could be reproduced by forced expression of CHOP. Consistently with these findings, siRNA-mediated inhibition of p21 levels restored the sensitivity of CHOP-deficient cells to tunicamycin. Our findings are consistent with a CHOP-dependent role for p21 in the shift from the pro-survival to the pro-apoptotic function of UPR. Copyright © 2010 S. Karger AG, Basel

Ciclopirox enhances pancreatic islet health by modulating the unfolded protein response in diabetes

Pancreatic dysfunction during diabetes is linked to the induction of endoplasmic reticulum (ER) stress on pancreatic beta (β) cells. Our laboratory recently discovered that p21 protects from diabetes by modifying the outcome of ER stress response. In the present study, we explored the antidiabetic activity of ciclopirox (CPX), an iron chelator and recently described activator of p21 expression. The effects of CPX in beta cell survival and function were assessed in cultured islets in vitro as well as in diabetic mice in vivo. The consequences of CPX in high glucose-induced insulin release and reactive oxygen species (ROS) production were also evaluated. Islet survival assays confirmed the significance of p21 in the regulation of glucotoxicity and suggested that CPX counteracts glucotoxicity in a manner that depends on p21. In vivo, administration of CPX in wild-type (WT) diabetic mice restored glucose homeostasis. In WT-cultured islets, CPX suppressed the expression of ER stress markers BiP, GRP94, and CHOP and reduced the levels of ROS during culture at high glucose. This reduction of ER stress may be associated with the ability of CPX to inhibit insulin release. Iron citrate stimulated insulin release, which was inhibited by CPX that functions as an iron chelator. It is conceivable that inhibition of insulin production constrains ER stress in islets promoting their survival and thus protecting from diabetes in vivo. This unfolded protein response (UPR)-antagonizing activity of CPX suggests application for the management not only of diabetes but also of other conditions related to ER stress. © 2016, Springer-Verlag Berlin Heidelberg