Cell biology
Abstract
The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of β1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.
Authors
Erin E. Hollander, Rosemary E. Flock, Jayne C. McDevitt, William P. Vostrejs, Sydney L. Campbell, Margo I. Orlen, Samantha B. Kemp, Benjamin M. Kahn, Kathryn E. Wellen, Il-Kyu Kim, Ben Z. Stanger
Abstract
Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress–related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins’ physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.
Authors
Yaqiong Zhang, Aizhen Yang, Zhenzhen Zhao, Fengwu Chen, Xiaofeng Yan, Yue Han, Depei Wu, Yi Wu
Abstract
Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that interleukin 13 (IL-13) activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibits viscoelastic liquid behavior and that mucus secreted by IL-13 activated HAECs exhibits solid-like behavior caused by mucin cross-linking. In addition, IL-13 activated HAECs show increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that is prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), is increased in IL-13 activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings is increased in asthma patients with high airway mucus plug scores. Together, our results show that IL-13 activated HAECs autonomously generate pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.
Authors
Maude A. Liegeois, Margaret Braunreuther, Annabelle R. Charbit, Wilfred W. Raymond, Monica Tang, Prescott G. Woodruff, Stephanie A. Christenson, Mario Castro, Serpil C. Erzurum, Elliot Israel, Nizar N. Jarjour, Bruce D. Levy, Wendy C. Moore, Sally E. Wenzel, Gerald G. Fuller, John V. Fahy
Abstract
The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.
Authors
Vipin Rawat, Patrick DeLear, Prarthana Prashanth, Mete Emir Ozgurses, Anteneh Tebeje, Philippa A. Burns, Kelly O. Conger, Christopher Solís, Yasir Hasnain, Anna Novikova, Jennifer E. Endress, Paloma González-Sánchez, Wentao Dong, Greg Stephanopoulos, Gina M. DeNicola, Isaac Harris, David Sept, Frank M. Mason, Jonathan L. Coloff
Abstract
The importance of the proper localization of most receptors at the cell surface is often underestimated, although this feature is essential for optimal receptor response. Endospanin 1 (Endo1) (also known as OBRGRP or LEPROT) is a protein generated from the same gene as the human leptin receptor and regulates the trafficking of proteins to the surface, including the leptin receptor. The systemic role of Endo1 on whole-body metabolism has not been studied so far. Here, we report that general Endo1-KO mice fed a high-fat diet develop metabolically healthy obesity with lipid repartitioning in organs and preferential accumulation of fat in adipose tissue, limited systematic inflammation, and better controlled glucose homeostasis. Mechanistically, Endo1 interacts with the lipid translocase CD36, thus regulating its surface abundance and lipid uptake in adipocytes. In humans, the level of Endo1 transcripts is increased in the adipose tissue of patients with obesity, but low levels rather correlate with a profile of metabolically healthy obesity. We suggest here that Endo1, most likely by controlling CD36 cell surface abundance and lipid uptake in adipocytes, dissociates obesity from diabetes and that its absence participates in metabolically healthy obesity.
Authors
Arturo Roca-Rivada, Marcio Do Cruzeiro, Raphaël G.P. Denis, Qiang Zhang, Christine Rouault, Yves Rouillé, Jean-Marie Launay, Céline Cruciani-Guglielmacci, Virginie Mattot, Karine Clément, Ralf Jockers, Julie Dam
Abstract
Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, the cellular source and expression regulation of COL4 in liver diseases is unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA-sequencing showed increased COL4 expression, which was further confirmed via immunofluorescence staining. scRNA-sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a tumor necrosis factor α–nuclear factor–κB dependent manner through an epigenetic mechanism in liver sinusoidal endothelial cells in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB-mediated epigenome editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.
Authors
Can Gan, Usman Yaqoob, Jianwen Lu, Man Xie, Abid A. Anwar, Nidhi Jalan-Sakrikar, Sofia Jerez, Tejasav Sehrawat, Amaia Navarro-Corcuera, Enis Kostallari, Nawras W. Habash, Sheng Cao, Vijay H. Shah
Abstract
The roles of fibronectin leucine-rich transmembrane protein 2 (FLRT2) in physiological and pathological processes are not well known. Here, we identify a potentially novel function of FLRT2 in preventing endothelial cell senescence and vascular aging. We found that FLRT2 expression was lower in cultured senescent endothelial cells as well as in aged rat and human vascular tissues. FLRT2 mediated endothelial cell senescence via the mTOR complex 2, AKT, and p53 signaling pathway in human endothelial cells. We uncovered that FLRT2 directly associated with integrin subunit beta 4 (ITGB4) and thereby promoted ITGB4 phosphorylation, while inhibition of ITGB4 substantially mitigated the induction of senescence triggered by FLRT2 depletion. Importantly, FLRT2 silencing in mice promoted vascular aging, and overexpression of FLRT2 rescued a premature vascular aging phenotype. Therefore, we propose that FLRT2 could be targeted therapeutically to prevent senescence-associated vascular aging.
Authors
Hyun Jung Hwang, Donghee Kang, Jae-Ryong Kim, Joon Hyuk Choi, Ji-Kan Ryu, Allison B. Herman, Young-Gyu Ko, Heon Joo Park, Myriam Gorospe, Jae-Seon Lee
Abstract
Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.
Authors
Deirdre Nolfi-Donegan, Gowtham K. Annarapu, Claudette St. Croix, Michael Calderon, Cheryl A. Hillery, Sruti Shiva
Abstract
Myocardial ischemia/reperfusion (MI/R) injury is a major cause of adverse outcomes of revascularization following myocardial infarction. Anaerobic glycolysis during myocardial ischemia is well-studied, but the role of aerobic glycolysis during the early phase of reperfusion is incompletely understood. Lactylation of Histone H3 (H3) is an epigenetic indicator of the glycolytic switch. Heat shock protein A12A (HSPA12A) is an atypic member of the HSP70 family. In the present study, we report that during reperfusion following myocardial ischemia, HSPA12A was downregulated and aerobic glycolytic flux was decreased in cardiomyocytes. Notably, HSPA12A knockout in mice exacerbated MI/R-induced aerobic glycolysis decrease, cardiomyocyte death, and cardiac dysfunction. Gain- and loss-of-function studies demonstrated that HSPA12A was required to support cardiomyocyte survival upon hypoxia/reoxygenation (H/R) challenge, and that its protective effects were mediated by maintaining aerobic glycolytic homeostasis for H3 lactylation. Further analyses revealed that HSPA12A increased Smurf1-mediated Hif1α protein stability, thus increasing glycolytic gene expression to maintain appropriate aerobic glycolytic activity to sustain H3 lactylation during reperfusion, and ultimately improving cardiomyocyte survival to attenuate MI/R injury.
Authors
Wansu Yu, Qiuyue Kong, Surong Jiang, Yunfan Li, Zhaohe Wang, Qian Mao, Xiaojin Zhang, Qianhui Liu, Pengjun Zhang, Yuehua Li, Chuanfu Li, Zhengnian Ding, Li Liu
Abstract
In humans, Type 2 Diabetes Mellitus (T2DM) shows a higher prevalence in men compared to women, phenotype that has been attributed to a lower peripheral insulin sensitivity in men. Whether sex-specific differences in pancreatic β-cell function also contribute is largely unknown. Here we characterized the electrophysiological properties of β-cells in intact mouse male and female islets. Elevation of glucose concentration above 5 mM triggers an electrical activity with a similar glucose dependence in β-cells of both sexes. However, female β-cells have a more depolarized membrane potential and increased firing frequency compared to males. The higher membrane depolarization in female β-cells is caused by ~50% smaller Kv2.1 K+ currents compared to males but otherwise unchanged KATP, Ca2+-activated BK and SK, and background TASK1/TALK1 K+ current densities. In female β-cells the higher depolarization causes a membrane potential-dependent inactivation of the voltage-gated Ca2+ channels (CaV) resulting in reduced Ca2+ entry. Nevertheless, this reduced Ca2+ influx is offset by the higher action potential firing frequency. Since exocytosis of insulin granules does not show a sex-specific difference we conclude that the higher electrical activity promotes insulin release in females improving glucose tolerance.
Authors
Noelia Jacobo-Piqueras, Tamara Theiner, Stefanie M. Geisler, Petronel Tuluc
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