Cell biology
Abstract
Maintaining cellular proteostasis is essential for oligodendrocyte viability and function; however, its underlying mechanisms remain unexplored. The UPR, comprising three parallel branches IRE1, PERK, and ATF6α, is a major mechanism that maintains cellular proteostasis by facilitating protein folding, attenuating protein translation, and enhancing autophagy and ERAD. Here we reported that impaired UPR in oligodendrocytes via deletion of PERK and ATF6α did not affect developmental myelination, but caused late-onset mature oligodendrocyte dysfunction and death in young adult mice. The detrimental effects of the impaired UPR on mature oligodendrocytes were accompanied by autophagy impairment and intracellular PLP accumulation, and were rescued by PLP deletion. Data indicate that PLP is degraded by autophagy and that intracellular PLP accumulation is cytotoxic to oligodendrocytes. Thus, these findings imply that the UPR is required for maintaining cellular proteostasis and the viability and function of mature oligodendrocytes in adults by regulating autophagy of PLP.
Authors
Sarrabeth Stone, Shuangchan Wu, Klaus-Armin Nave, Wensheng Lin
Abstract
The Notch signaling pathway mediates cell-cell communication regulating cell differentiation and proliferation and cell fate decisions in various tissues. In the urinary bladder, Notch acts as a tumor suppressor in mice, while mutations in Notch pathway components have been identified in human bladder cancer as well. Here we report that the genetic inactivation of Notch in mice leads to downregulation of cell-cell and cell-ECM interaction components, including proteins previously implicated in interstitial cystitis/bladder pain syndrome (IC/BPS), structural defects and mucosal sloughing, inflammation, and leaky urine-blood barrier. Molecular profiling of ailing mouse bladders showed similarities with IC/BPS patient tissue, which also presented low Notch pathway activity as indicated by reduced expression of canonical Notch targets. Urothelial integrity was reconstituted upon exogenous reactivation of the Notch pathway, implying a direct involvement of Notch. Despite damage and inflammation, urothelial cells failed to proliferate, uncovering a possible role for α4 integrin in urothelial homeostasis. Our data uncover a broad role for Notch in bladder homeostasis involving urothelial cell crosstalk with the microenvironment.
Authors
Varvara Paraskevopoulou, Vangelis Bonis, Vasilis S. Dionellis, Nikolaos Paschalidis, Pelagia Melissa, Evangelia Chavdoula, Eleni Vasilaki, Ioannis S. Pateras, Apostolos Klinakis
Abstract
Our integrative genomic and functional analysis identified transforming acidic coiled-coil–containing protein 2 (TACC2) as a chronic obstructive pulmonary disease (COPD) candidate gene. Here, we found that smokers with COPD exhibit a marked decrease in lung TACC2 protein levels relative to smokers without COPD. Single cell RNA sequencing reveals that TACC2 is expressed primarily in lung epithelial cells in normal human lungs. Furthermore, suppression of TACC2 expression impairs the efficiency of homologous recombination repair and augments spontaneous and cigarette smoke extract–induced (CSE-induced) DNA damage and cytotoxicity in immortalized human bronchial epithelial cells. By contrast, enforced expression of TACC2 attenuates the CSE effects. We also found that CSE enhances TACC2 degradation via the ubiquitin-proteasome system mediated by the ubiquitin E3 ligase subunit, F box L7. Furthermore, cellularly expressed TACC2 proteins harboring naturally occurring mutations exhibited altered protein lifespan coupled with modified DNA damage repair and cytotoxic responses. CS triggers emphysematous changes accompanied by accumulated DNA damage, apoptosis of alveolar epithelia, and lung inflammation in Tacc2–/– compared with Tacc2+/+ mice. Our results suggest that CS destabilizes TACC2 protein in lung epithelia by the ubiquitin proteasome system, leading to subsequent DNA damage, cytotoxicity, and emphysema.
Authors
Rama K. Mallampalli, Xiuying Li, Jun-Ho Jang, Tomasz Kaminski, Aki Hoji, Tiffany Coon, Divay Chandra, Starr Welty, Yaqun Teng, John Sembrat, Mauricio Rojas, Yutong Zhao, Robert Lafyatis, Chunbin Zou, Frank Sciurba, Prithu Sundd, Li Lan, Toru Nyunoya
Abstract
Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase-mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2f/f) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreERT2; Arpc2f/f). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting co-factors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell-extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.
Authors
Steven Tessier, Alexandra C Doolittle, Kimheak Sao, Jeremy D. Rotty, James E. Bear, Veronica Ulici, Richard F. Loeser, Irving M. Shapiro, Brian O. Diekman, Makarand V. Risbud
Abstract
Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.
Authors
Álvaro F. Fernández, Yang Liu, Vanessa Ginet, Mingjun Shi, Jihoon Nah, Zhongju Zou, Anwu Zhou, Bruce A. Posner, Guanghua Xiao, Marion Tanguy, Valérie Paradis, Junichi Sadoshima, Pierre-Emmanuel Rautou, Julien Puyal, Ming Chang Hu, Beth Levine
Abstract
Clinical and preclinical studies show tissue-specific differences in tumorigenesis. Tissue specificity is controlled by differential gene expression. We prioritized genes that encode secreted proteins according to their preferential expression in normal lungs to identify candidates associated with lung cancer. Indeed, most of the lung-enriched genes identified in our analysis have known or suspected roles in lung cancer. We focused on the gene encoding neuron-derived neurotrophic factor (NDNF), which had not yet been associated with lung cancer. We determined that NDNF was preferentially expressed in the normal adult lung and that its expression was decreased in human lung adenocarcinoma and a mouse model of this cancer. Higher expression of NDNF was associated with better clinical outcome of patients with lung adenocarcinoma. Purified NDNF inhibited proliferation of lung cancer cells, whereas silencing NDNF promoted tumor cell growth in culture and in xenograft models. We determined that NDNF is downregulated through DNA hypermethylation near CpG island shores in human lung adenocarcinoma. Furthermore, the lung cancer–related DNA hypermethylation sites corresponded to the methylation sites that occurred in tissues with low NDNF expression. Thus, by analyzing the tissue-specific secretome, we identified a tumor-suppressive factor, NDNF, which is associated with patient outcomes in lung adenocarcinoma.
Authors
Ya Zhang, Xuefeng Wu, Yan Kai, Chia-Han Lee, Fengdong Cheng, Yixuan Li, Yongbao Zhuang, Javid Ghaemmaghami, Kun-Han Chuang, Zhuo Liu, Yunxiao Meng, Meghana Keswani, Nancy R. Gough, Xiaojun Wu, Wenge Zhu, Alexandros Tzatsos, Weiqun Peng, Edward Seto, Eduardo M. Sotomayor, Xiaoyan Zheng
Abstract
Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor-dependent manner. Mass spectrometry analyses of human gigaxonin revealed nine candidate sites of O-GlcNAcylation, two of which – serine 272 and threonine 277 – are required for its ability to mediate IF turnover in novel gigaxonin-deficient human cell models that we created. Taken together, these results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the non-genetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.
Authors
Po-Han Chen, Jimin Hu, Jianli Wu, Duc T. Huynh, Timothy J. Smith, Samuel Pan, Brittany J. Bisnett, Alexander B. Smith, Annie Lu, Brett M. Condon, Jen-Tsan Chi, Michael Boyce
Abstract
Accumulation of senescent cells is associated with the progression of pulmonary fibrosis but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM) lipid extracts and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by anti-fibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR90) induced pro-fibrotic signaling in naïve fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl leukotriene in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cells removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time that senescent cells secrete functional LTs, significantly contributing to the LTs pool known to cause or exacerbate IPF.
Authors
Christopher D. Wiley, Alexis N. Brumwell, Sonnet S. Davis, Julia R. Jackson, Alexis Valdovinos, Cheresa Calhoun, Fatouma Alimirah, Carlos A. Castellanos, Richard Ruan, Ying Wei, Harold A. Chapman, Arvind Ramanathan, Judith Campisi, Claude Jourdan Le Saux
Abstract
Increased fibrosis is a characteristic remodeling response to biomechanical and neurohumoral stress and a determinant of cardiac mechanical and electrical dysfunction in disease. Stress-induced activation of cardiac fibroblasts (CF) is a critical step in the fibrotic response, although the precise sequence of events underlying activation of these critical cells in vivo remain unclear. Here, we test the hypothesis that a βIV-spectrin/STAT3 complex is essential for maintenance of a quiescent phenotype (basal non-activated state) in CFs. We report increased fibrosis, decreased cardiac function, and electrical impulse conduction defects in genetic and acquired mouse models of βIV-spectrin deficiency. Loss of betaIV-spectrin function promotes STAT3 nuclear accumulation and transcriptional activity, altered gene expression and CF activation. Furthermore, we demonstrate that a quiescent phenotype may be restored in βIV-spectrin deficient fibroblasts by expressing a βIV-spectrin fragment including the STAT3-binding domain or through pharmacological STAT3 inhibition. We find that in vivo STAT3 inhibition abrogates fibrosis and cardiac dysfunction in the setting of global βIV-spectrin deficiency. Finally, we demonstrate that fibroblast-specific deletion of βIV-spectrin is sufficient to induce fibrosis and decreased cardiac function. We propose that the βIV-spectrin/STAT3 complex is a determinant of fibroblast phenotype and fibrosis, with implications for remodeling response in cardiovascular disease.
Authors
Nehal J. Patel, Drew M. Nassal, Amara D. Greer-Short, Sathya D. Unudurthi, Benjamin W. Scandling, Daniel Gratz, Xianyao Xu, Anuradha Kalyanasundaram, Vadim V. Fedorov, Federica Accornero, Peter J. Mohler, Keith J. Gooch, Thomas J. Hund
Abstract
The roles of macrophages in orchestrating innate immunity through phagocytosis and T lymphocyte activation have been extensively investigated. Much less understood is the unexpected role of macrophages in direct tumor regression. Tumoricidal macrophages can indeed manifest cancer immunoediting activity in the absence of adaptive immunity. We investigated direct macrophage cytotoxicity in malignant pleural mesothelioma, a lethal cancer that develops from mesothelial cells of the pleural cavity after occupational asbestos exposure. In particular, we analyzed the cytotoxic activity of mouse RAW264.7 macrophages upon cell-cell contact with autologous AB1/AB12 mesothelioma cells. We show that macrophages killed mesothelioma cells by oxeiptosis via a mechanism involving enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27–specific (H3K27-specific) methyltransferase of the polycomb repressive complex 2 (PRC2). A selective inhibitor of EZH2 indeed impaired RAW264.7-directed cytotoxicity and concomitantly stimulated the PD-1 immune checkpoint. In the immunocompetent BALB/c model, RAW264.7 macrophages pretreated with the EZH2 inhibitor failed to control tumor growth of AB1 and AB12 mesothelioma cells. Blockade of PD-1 engagement restored macrophage-dependent antitumor activity. We conclude that macrophages can be directly cytotoxic for mesothelioma cells independent of phagocytosis. Inhibition of the PRC2 EZH2 methyltransferase reduces this activity because of PD-1 overexpression. Combination of PD-1 blockade and EZH2 inhibition restores macrophage cytotoxicity.
Authors
Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems
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