The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration

The NF-κB family of transcription factors is a well-established regulator of the immune and inflammatory responses and also plays a key role in other cellular processes, including cell death, proliferation, and migration. Conserved residues in the trans-activation domain of RelA, which can be posttranslationally modified, regulate divergent NF-κB functions in response to different cellular stimuli. Using rela(−/−) mouse embryonic fibroblasts reconstituted with RelA, we find that mutation of the threonine 505 (T505) phospho site to alanine has wide-ranging effects on NF-κB function. These include previously described effects on chemotherapeutic drug-induced apoptosis, as well as new roles for this modification in autophagy, cell proliferation, and migration. This last effect was associated with alterations in the actin cytoskeleton and expression of cellular migration–associated genes such as WAVE3 and α-actinin 4. We also define a new component of cisplatin-induced, RelA T505–dependent apoptosis, involving induction of NOXA gene expression, an effect explained at least in part through induction of the p53 homologue, p73. Therefore, in contrast to other RelA phosphorylation events, which positively regulate NF-κB function, we identified RelA T505 phosphorylation as a negative regulator of its ability to induce diverse cellular processes such as apoptosis, autophagy, proliferation, and migration

Mevalonate Cascade Regulation of Airway Mesenchymal Cell Autophagy and Apoptosis: A Dual Role for p53

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease

Lactic Acidosis Triggers Starvation Response with Paradoxical Induction of TXNIP through MondoA

Although lactic acidosis is a prominent feature of solid tumors, we still have limited understanding of the mechanisms by which lactic acidosis influences metabolic phenotypes of cancer cells. We compared global transcriptional responses of breast cancer cells in response to three distinct tumor microenvironmental stresses: lactic acidosis, glucose deprivation, and hypoxia. We found that lactic acidosis and glucose deprivation trigger highly similar transcriptional responses, each inducing features of starvation response. In contrast to their comparable effects on gene expression, lactic acidosis and glucose deprivation have opposing effects on glucose uptake. This divergence of metabolic responses in the context of highly similar transcriptional responses allows the identification of a small subset of genes that are regulated in opposite directions by these two conditions. Among these selected genes, TXNIP and its paralogue ARRDC4 are both induced under lactic acidosis and repressed with glucose deprivation. This induction of TXNIP under lactic acidosis is caused by the activation of the glucose-sensing helix-loop-helix transcriptional complex MondoA:Mlx, which is usually triggered upon glucose exposure. Therefore, the upregulation of TXNIP significantly contributes to inhibition of tumor glycolytic phenotypes under lactic acidosis. Expression levels of TXNIP and ARRDC4 in human cancers are also highly correlated with predicted lactic acidosis pathway activities and associated with favorable clinical outcomes. Lactic acidosis triggers features of starvation response while activating the glucose-sensing MondoA-TXNIP pathways and contributing to the “anti-Warburg” metabolic effects and anti-tumor properties of cancer cells. These results stem from integrative analysis of transcriptome and metabolic response data under various tumor microenvironmental stresses and open new paths to explore how these stresses influence phenotypic and metabolic adaptations in human cancers

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

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field