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PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

ABSTRACT: Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

SUBMITTER: Hiramatsu N

PROVIDER: S-EPMC6952592 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Hiramatsu Nobuhiko N Chiang Karen K Aivati Cathrine C Rodvold Jeffrey J JJ Lee Ji-Min JM Han Jaeseok J Chea Leon L Zanetti Maurizio M Koo Edward H EH Lin Jonathan H JH

The Journal of biological chemistry 20191202 1

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human ce ...[more]

PMID: 31792031

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Project description:UnlabelledInfluenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK) as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection.ImportanceInfluenza A viruses are responsible for annual epidemics and occasional pandemics with important consequences for human health and the economy. The unfolded protein response is a defense mechanism fired by cells when the demand of protein synthesis and folding is excessive, for instance, during an acute virus infection. In this report, we show that influenza virus downregulates the unfolded protein response mediated by the PERK sensor, while Montelukast, a drug used to treat asthma in humans, specifically stimulated this response and downregulated viral protein synthesis and multiplication. Accordingly, we show that PERK phosphorylation was reduced in virus-infected cells and increased in cells treated with Montelukast. Hence, our studies suggest that modulation of the PERK-mediated unfolded protein response is a target for influenza virus inhibition.

Project description:BackgroundAntioxidant enzymes play a fundamental role in counteracting oxidative stress induced by high glucose. Although mitochondrial superoxide dismutase (SOD2) is the principal defence against the toxicity of superoxide anions, the mechanism of its inactivation in diabetic subjects is still poorly understood. Recently, microRNA-21 has been associated with diabetes, although its function remains unclear. We sought to explore the mechanism underlying defective SOD2 antioxidant response in HUVECs during exposures to constant high glucose and oscillating glucose (as glucose variability model, GV) and the role of miR-21 in increasing the susceptibility to oxidative stress by disrupting reactive oxygen species (ROS) homeostasis.MethodsHUVECs exposed for 1 week to constant high glucose and GV were subjected to quantitative electron paramagnetic resonance for ROS measurements. Superoxide anions, SOD2 protein levels and mitochondrial membrane potential (??m) were also evaluated. Endogenous miR-21 and its putative ROS-homeostatic target genes (KRIT1, FoxO1, NFE2L2 and SOD2) were tested using mimic-miR-21 and quantified by qPCR. Luciferase assays were performed to test miR-21/3'-UTR-SOD2 binding.ResultsWe observed upregulation of microRNA-21, overproduction of superoxide anions and total ROS generation, depolarisation of the mitochondrial membrane potential (??m) and defective SOD2 antioxidant response in HUVECs subjected to constant high glucose and GV exposures. We also found that exogenous mimic-microRNA-21 targeted putative microRNA-21 ROS-homeostatic target genes, e.g., KRIT1, NRF2 and SOD2, which were significantly downregulated. All these effects were reverted by a microRNA-21 inhibitor, which improved SOD2 and KRIT1 expression, reduced the levels of ROS production and ameliorated ??m.ConclusionsOur data demonstrate the association of microRNA-21 with oscillating and high glucose and early mitochondrial dysfunction. We found that microRNA-21 may promote the suppression of homeostatic signalling that normally limits ROS damage. These data provide novel clues about the inhibition of microRNA-21 as a new therapeutic approach to protect against cellular oxidative injury in glucose variability and diabetes.

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PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

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PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

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