The endoplasmic reticulum (ER) produces the vast majority of all proteins secreted into the extracellular space, including hormones and cytokines, as well as cell surface receptors and other proteins ...which interact with the environment. Accordingly, this organelle controls essentially all vital links to a cell's external milieu, responding to systemic metabolic, inflammatory, endocrine, and mechanical stimuli. The central role the ER plays in meeting protein synthetic and quality control requirements in the face of such demands is matched by an extensive and versatile ER stress response signaling network. ROS mediate several critical aspects of this response. Nox4, an ER resident capable of producing ROS, acts as a proximal signaling intermediate to transduce ER stress-related conditions to the unfolded protein response, a homeostatic corrective mechanism. However, chronic ER stress caused by unrelenting internal or external demands produces a secondary rise in ROS, generally resulting in cell death. Sorting out the involvement of ROS at different levels of the ER stress response in specific cell types is key to understanding the molecular basis for chronic diseases such as atherosclerosis, hypertension, and diabetes. Here, we provide an overview of ER stress signaling with an emphasis on the role of ROS.
Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a ...property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.
Although reactive oxidants have long been stigmatized as unwanted metabolic byproducts, the expression of oxidases specifically functioning to produce these same molecules in a regulated fashion is ...surprisingly pervasive throughout metazoan and plant evolution. Although the involvement of oxidants in many signaling pathways is well documented, the cellular strategies for conferring pathway specificity to such reactive molecules have remained more recondite. Recent studies now suggest that cells may spatially restrict oxidant production to allow microdomain-specific signaling.
RATIONALE:Autophagy is an essential survival mechanism during energy stress in the heart. Oxidative stress is activated by energy stress, but its role in mediating autophagy is poorly understood. ...NADPH oxidase (Nox) 4 is an enzyme that generates reactive oxygen species (ROS) at intracellular membranes. Whether Nox4 acts as a sensor of energy stress to mediate activation of autophagy is unknown.
OBJECTIVE:We investigated whether Nox4 is involved in the regulation of autophagy and cell survival during energy stress in cardiomyocytes.
METHODS AND RESULTS:Production of ROS in cardiomyocytes was increased during glucose deprivation (GD) in a Nox4-dependent manner. Protein levels and the ROS-producing activity of Nox4 were increased in the endoplasmic reticulum (ER), but not in mitochondria, in response to GD. Selective knockdown of Nox4, but not Nox2, or selective reduction of ROS in the ER with ER-targeted catalase, but not mitochondria-targeted perioxiredoxin 3, abrogated GD-induced autophagy. Nox4 promoted autophagy during GD through activation of the protein kinase RNA-activated-like ER kinase pathway by suppression of prolyl hydroxylase 4. The decrease in cell survival during GD in the presence of Nox4 knockdown was rescued by reactivation of autophagy by Atg7 overexpression, indicating that the effect of Nox4 on cell survival is critically mediated through regulation of autophagy. Nox4 was activated during fasting and prolonged ischemia in the mouse heart, where Nox4 is also required for autophagy activation and cardioprotection.
CONCLUSIONS:Nox4 critically mediates autophagy in response to energy stress in cardiomyocytes by eliciting ROS in the ER and stimulating the protein kinase RNA-activated-like ER kinase signaling pathway.
Tissue cells continually monitor anchorage conditions by gauging the physical properties of their underlying matrix and surrounding environment. The Rho and Ras GTPases are essential components of ...these mechanosensory pathways. These molecular switches control both cytoskeletal as well as cell fate responses to anchorage conditions and are thus critical to our understanding of how cells respond to their physical environment and, by extension, how malignant cells gainsay these regulatory pathways. Recent studies indicate that 2 proteins produced by the SHC1 gene, thought for the most part to functionally oppose each other, collaborate in their ability to respond to mechanical force by initiating respective Rho and Ras signals. In this review, we focus on the coupling of Shc and GTPases in the cellular response to mechanical anchorage signals, with emphasis on its relevance for cancer.
Acute exacerbations of fibrosing interstitial lung disease (ILD) occur in both idiopathic pulmonary fibrosis (IPF) as well as non-IPF ILDs. An expert consensus definition has allowed for more ...frequent reporting of IPF exacerbations. The same is lacking for non-IPF ILD exacerbations. The incidence of non-IPF ILD exacerbations is likely less than in IPF, but the two entities share similar risk factors, such as increased frequency as physiologic derangements advance. The radiologic and histopathologic spectrum of acute ILD exacerbations extends from organizing pneumonia (OP) to the more treatment-refractory diffuse alveolar damage (DAD) pattern. Indeed, responsiveness to various therapies may depend on the relative components of these entities, favoring OP over DAD. There are no proven therapies for acute ILD exacerbations. Corticosteroids are a mainstay in any regimen although clear evidence of benefit does not exist. A variety of immunosuppressant agents have purported success in historical cohort studies – cyclophosphamide, cyclosporine A, and tacrolimus most commonly. Only one randomized controlled trial has been published, studying recombinant thrombomodulin for IPF exacerbation, but the primary outcome of survivor proportion at 90 days was not met. Other novel therapies for ILD exacerbations are still under investigation. The short and long-term prognosis of acute exacerbations of ILD is poor, especially in patients with IPF. Transplant referral should be considered early for both IPF as well as fibrosing non-IPF ILDs, given the unpredictability of the exacerbation event.
•Exacerbations of IPF and non-IPF ILDs are a devastating complication with significant morbidity and mortality.•Exacerbations of non-IPF ILDs are less reported and studied than exacerbations of IPF.•Incidence of ILD exacerbations increases as disease progresses.•Histopathology of exacerbations occurs along a spectrum of manifestations that often overlap and/or occur simultaneously.•Response to treatment favors organizing pneumonia over diffuse alveolar damage during the exacerbation episode.
Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second ...messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.
In the advanced stages of cancer, autophagy is thought to promote tumor progression through its ability to mitigate various cellular stresses. However, the details of how autophagy is homeostatically ...regulated in such tumors are unknown. Here, we report that NUPR1 (nuclear protein 1, transcriptional regulator), a transcriptional coregulator, is aberrantly expressed in a subset of cancer cells and predicts low overall survival rates for lung cancer patients. NUPR1 regulates the late stages of autolysosome processing through the induction of the SNARE protein SNAP25, which forms a complex with the lysosomal SNARE-associated protein VAMP8. NUPR1 depletion deregulates autophagic flux and impairs autolysosomal clearance, inducing massive cytoplasmic vacuolization and premature senescence in vitro and tumor suppression in vivo. Collectively, our data show that NUPR1 is a potent regulator of autolysosomal dynamics and is required for the progression of some epithelial cancers.
OBJECTIVES—In response to endoplasmic reticulum (ER) stress, endothelial cells initiate corrective pathways such as the unfolded protein response. Recent studies suggest that reactive oxygen species ...produced on the ER may participate in homeostatic signaling through Ras in response to ER stress. We sought to identify mechanisms responsible for this focal signaling pathway.
APPROACH AND RESULTS—In endothelial cells, we found that ER stress induced by tunicamycin activates the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 focally on the ER surface but not on the plasma membrane. Ras activation is also restricted to the ER, occurs downstream of Nox4, and is required for activation of the unfolded protein response. In contrast, treatment with the growth factor VEGF (vascular endothelial growth factor) results in Ras activation and reactive oxygen species production confined instead to the plasma membrane and not to the ER, demonstrating local coupling of reactive oxygen species and Ras signals. We further identify the calcium-responsive, ER-resident guanyl exchange factors RasGRF1 and RasGRF2 as novel upstream mediators linking Nox4 with Ras activation in response to ER stress. Oxidation of the sarcoendoplasmic reticulum calcium ATPase and increases in cytosolic calcium caused by ER stress are blocked by Nox4 knockdown, and reduction in cytosolic free calcium prevents both Ras activation and the unfolded protein response.
CONCLUSIONS—ER stress triggers a localized signaling module on the ER surface involving Nox4-dependent calcium mobilization, which directs local Ras activation through ER-associated, calcium-responsive RasGRF.
In the antiphospholipid syndrome (APS), antiphospholipid antibody (aPL) recognition of β2 glycoprotein I promotes thrombosis, and preclinical studies indicate that this is due to endothelial nitric ...oxide synthase (eNOS) antagonism via apolipoprotein E receptor 2 (apoER2)-dependent processes. How apoER2 molecularly links these events is unknown. Here, we show that, in endothelial cells, the apoER2 cytoplasmic tail serves as a scaffold for aPL-induced assembly and activation of the heterotrimeric protein phosphatase 2A (PP2A). Disabled-2 (Dab2) recruitment to the apoER2 NPXY motif promotes the activating L309 methylation of the PP2A catalytic subunit by leucine methyl transferase-1. Concurrently, Src homology domain-containing transforming protein 1 (SHC1) recruits the PP2A scaffolding subunit to the proline-rich apoER2 C terminus along with 2 distinct regulatory PP2A subunits that mediate inhibitory dephosphorylation of Akt and eNOS. In mice, the coupling of these processes in endothelium is demonstrated to underlie aPL-invoked thrombosis. By elucidating these intricacies in the pathogenesis of APS-related thrombosis, numerous potential new therapeutic targets have been identified.
•The activation of PP2A in endothelium underlies thrombus formation induced by aPL in mice.•Endothelial apoER2 serves as a scaffold for aPL-induced assembly of a Dab2 and SHC1- containing complex that assembles and activates PP2A.
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