bims-unfpre 2022-04-03 papers

Issue of 2022–04–03
six papers selected by
Susan Logue, University of Manitoba

Nat Commun. 2022 Apr 01. 13(1): 1748

Long-chain ceramides are cell non-autonomous signals linking lipotoxicity to endoplasmic reticulum stress in skeletal muscle.

Ben D McNally, Dean F Ashley, Lea Hänschke, Hélène N Daou, Nicole T Watt, Steven A Murfitt, Amanda D V MacCannell, Anna Whitehead, T Scott Bowen, Francis W B Sanders, Michele Vacca, Klaus K Witte, Graeme R Davies, Reinhard Bauer, Julian L Griffin, Lee D Roberts.

The endoplasmic reticulum (ER) regulates cellular protein and lipid biosynthesis. ER dysfunction leads to protein misfolding and the unfolded protein response (UPR), which limits protein synthesis to prevent cytotoxicity. Chronic ER stress in skeletal muscle is a unifying mechanism linking lipotoxicity to metabolic disease. Unidentified signals from cells undergoing ER stress propagate paracrine and systemic UPR activation. Here, we induce ER stress and lipotoxicity in myotubes. We observe ER stress-inducing lipid cell non-autonomous signal(s). Lipidomics identifies that palmitate-induced cell stress induces long-chain ceramide 40:1 and 42:1 secretion. Ceramide synthesis through the ceramide synthase 2 de novo pathway is regulated by UPR kinase Perk. Inactivation of CerS2 in mice reduces systemic and muscle ceramide signals and muscle UPR activation. The ceramides are packaged into extracellular vesicles, secreted and induce UPR activation in naïve myotubes through dihydroceramide accumulation. This study furthers our understanding of ER stress by identifying UPR-inducing cell non-autonomous signals.

DOI: https://doi.org/10.1038/s41467-022-29363-9

J Clin Invest. 2022 Mar 29. pii: e151591. [Epub ahead of print]

FOXK2 promotes ovarian cancer stemness by regulating the unfolded protein response pathway.

Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J Tanner, Mazhar Adli, Daniela Matei.

Understanding the regulatory programs enabling cancer stem cells (CSCs) to self-renew and drive tumorigenicity could identify new treatments. Through comparative chromatin state and gene expression analyses in ovarian CSCs vs. non-CSCs, we identified FOXK2 as a highly expressed stemness-specific transcription factor in ovarian cancer. Its genetic depletion diminished stemness features and reduced tumor initiation capacity. Our mechanistic studies highlight that FOXK2 directly regulated IRE1α (ERN1 gene) expression, a key sensor for the unfolded protein response (UPR). Chromatin immunoprecipitation-sequencing revealed that FOXK2 bound to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a catalytically inactive CRISPR/Cas9 (dCas9) diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 splicing and activation of stemness pathways, while genetic or pharmacological blockade of this sensor of the UPR inhibited ovarian CSCs. Collectively, these data establish a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in CSCs.

Keywords: Cancer; Cell Biology; Cell stress; Oncology; Transcription

DOI: https://doi.org/10.1172/JCI151591

Exp Hematol Oncol. 2022 Mar 31. 11(1): 18

RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation.

Dalia Quwaider, Luis A Corchete, Marta Martín-Izquierdo, Jesús M Hernández-Sánchez, Elizabeta A Rojas, Ignacio J Cardona-Benavides, Ramón García-Sanz, Ana B Herrero, Norma C Gutiérrez.

BACKGROUND: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells.
METHODS: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot.
RESULTS: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found.
CONCLUSION: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.

Keywords: ER stress; IRE1; Multiple myeloma; RIDD; UPR

DOI: https://doi.org/10.1186/s40164-022-00271-4

Gut. 2022 Apr 01. pii: gutjnl-2021-325013. [Epub ahead of print]

NFATc1 signaling drives chronic ER stress responses to promote NAFLD progression.

Muhammad Umair Latif, Geske Elisabeth Schmidt, Sercan Mercan, Raza Rahman, Christine Silvia Gibhardt, Ioana Stejerean-Todoran, Kristina Reutlinger, Elisabeth Hessmann, Shiv K Singh, Abdul Moeed, Abdul Rehman, Umer Javed Butt, Hanibal Bohnenberger, Philipp Stroebel, Sebastian Christopher Bremer, Albrecht Neesse, Ivan Bogeski, Volker Ellenrieder.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) can persist in the stage of simple hepatic steatosis or progress to steatohepatitis (NASH) with an increased risk for cirrhosis and cancer. We examined the mechanisms controlling the progression to severe NASH in order to develop future treatment strategies for this disease.
DESIGN: NFATc1 activation and regulation was examined in livers from patients with NAFLD, cultured and primary hepatocytes and in transgenic mice with differential hepatocyte-specific expression of the transcription factor (Alb-cre, NFATc1c.a . and NFATc1Δ/Δ ). Animals were fed with high-fat western diet (WD) alone or in combination with tauroursodeoxycholic acid (TUDCA), a candidate drug for NAFLD treatment. NFATc1-dependent ER stress-responses, NLRP3 inflammasome activation and disease progression were assessed both in vitro and in vivo.
RESULTS: NFATc1 expression was weak in healthy livers but strongly induced in advanced NAFLD stages, where it correlates with liver enzyme values as well as hepatic inflammation and fibrosis. Moreover, high-fat WD increased NFATc1 expression, nuclear localisation and activation to promote NAFLD progression, whereas hepatocyte-specific depletion of the transcription factor can prevent mice from disease acceleration. Mechanistically, NFATc1 drives liver cell damage and inflammation through ER stress sensing and activation of the PERK-CHOP unfolded protein response (UPR). Finally, NFATc1-induced disease progression towards NASH can be blocked by TUDCA administration.
CONCLUSION: NFATc1 stimulates NAFLD progression through chronic ER stress sensing and subsequent activation of terminal UPR signalling in hepatocytes. Interfering with ER stress-responses, for example, by TUDCA, protects fatty livers from progression towards manifest NASH.

Keywords: fatty liver; hepatic fibrosis; inflammation; nonalcoholic steatohepatitis

DOI: https://doi.org/10.1136/gutjnl-2021-325013

Cell Mol Life Sci. 2022 Mar 28. 79(4): 213

ER-mitochondria communication is involved in NLRP3 inflammasome activation under stress conditions in the innate immune system.

Ana Catarina Pereira, Jessica De Pascale, Rosa Resende, Susana Cardoso, Isabel Ferreira, Bruno Miguel Neves, Mylène A Carrascal, Mónica Zuzarte, Nuno Madeira, Sofia Morais, António Macedo, Anália do Carmo, Paula I Moreira, Maria Teresa Cruz, Cláudia F Pereira.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are key events in the initiation and/or progression of several diseases, are correlated with alterations at ER-mitochondria contact sites, the so-called "Mitochondria-Associated Membranes" (MAMs). These intracellular structures are also implicated in NLRP3 inflammasome activation which is an important driver of sterile inflammation, however, the underlying molecular basis remains unclear. This work aimed to investigate the role of ER-mitochondria communication during ER stress-induced NLRP3 inflammasome activation in both peripheral and central innate immune systems, by using THP-1 human monocytes and BV2 microglia cells, respectively, as in vitro models. Markers of ER stress, mitochondrial dynamics and mass, as well as NLRP3 inflammasome activation were evaluated by Western Blot, IL-1β secretion was measured by ELISA, and ER-mitochondria contacts were quantified by transmission electron microscopy. Mitochondrial Ca2+ uptake and polarization were analyzed with fluorescent probes, and measurement of aconitase and SOD2 activities monitored mitochondrial ROS accumulation. ER stress was demonstrated to activate the NLRP3 inflammasome in both peripheral and central immune cells. Studies in monocytes indicate that ER stress-induced NLRP3 inflammasome activation occurs by a Ca2+-dependent and ROS-independent mechanism, which is coupled with upregulation of MAMs-resident chaperones, closer ER-mitochondria contacts, as well as mitochondrial depolarization and impaired dynamics. Moreover, enhanced ER stress-induced NLRP3 inflammasome activation in the immune system was found associated with pathological conditions since it was observed in monocytes derived from bipolar disorder (BD) patients, supporting a pro-inflammatory status in BD. In conclusion, by demonstrating that ER-mitochondria communication plays a key role in the response of the innate immune cells to ER stress, this work contributes to elucidate the molecular mechanisms underlying NLRP3 inflammasome activation under stress conditions, and to disclose novel potential therapeutic targets for diseases associated with sterile inflammation.

Keywords: Bipolar disorder (BD); Calcium; Endoplasmic reticulum (ER) stress; Mitochondria; Sterile inflammation; Unfolded protein response

DOI: https://doi.org/10.1007/s00018-022-04211-7

Cell Death Discov. 2022 Mar 28. 8(1): 133

GRP78 blockade overcomes intrinsic resistance to UBA1 inhibitor TAK-243 in glioblastoma.

Xu Zhang, Runqiu Wu, Cong Tian, Wanzhou Wang, Lingni Zhou, Tongxuan Guo, Jiefeng Yu, Changyong Wu, Yang Shen, Xuejiao Liu, Rutong Yu.

Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor of the central nervous system. Despite continuous progression in treatment options for GBM like surgery, radiotherapy, and chemotherapy, this disease still has a high rate of recurrence. The endoplasmic reticulum (ER) stress pathway is associated with chemotherapeutic drug resistance. The UBA1 inhibitor TAK-243 can induce strong ER stress. However, the sensitivity of TAK-243 varies greatly in different tumor cells. This study evaluated the antitumor effects of the GRP78 inhibitor, HA15, combined with TAK-243 on GBM in the preclinical models. HA15 synergistically enhanced the sensitivity of GBM cells to TAK-243. When compared with TAK-243 monotherapy, HA15 combined with TAK-243 significantly inhibited GBM cell proliferation. It also induced G2/M-phase arrest in the cell cycle. In vivo studies showed that HA15 combined with TAK-243 significantly inhibited the growth of intracranial GBM and prolonged survival of the tumor-bearing mice. Mechanistically, HA15 and TAK-243 synergistically activated the PERK/ATF4 and IRE1α/XBP1 signaling axes, thereby eventually activating PARP and the Caspase families, which induced cell apoptosis. Our data provided a new strategy for improving the sensitivity of GBM to TAK-243 treatment and experimental basis for further clinical trials to evaluate this combination therapy.

DOI: https://doi.org/10.1038/s41420-022-00950-5