Pharmacological agents currently in clinical trials for disorders in neurogastroenterology

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Esophageal, gastrointestinal, and colonic diseases resulting from disorders of the motor and sensory functions represent almost half the patients presenting to gastroenterologists. There have been significant advances in understanding the mechanisms of these disorders, through basic and translational research, and in targeting the receptors or mediators involved, through clinical trials involving biomarkers and patient responses. These advances have led to relief of patients’ symptoms and improved quality of life, although there are still significant unmet needs. This article reviews the pipeline of medications in development for esophageal sensorimotor disorders, gastroparesis, chronic diarrhea, chronic constipation (including opioid-induced constipation), and visceral pain.

Authors

Michael Camilleri

A conversation with Aaron Ciechanover

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Ushma S. Neill

Richard Scheller and Thomas Südhof receive the 2013 Albert Lasker Basic Medical Research Award

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Jillian H. Hurst

Hearing restoration: Graeme Clark, Ingeborg Hochmair, and Blake Wilson receive the 2013 Lasker~DeBakey Clinical Medical Research Award

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Corinne Williams

Bill and Melinda Gates honored with Lasker~Bloomberg Public Service Award

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Sarah Jackson

Emerging concepts in immunity to hepatitis C virus infection

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Since the discovery of hepatitis C virus (HCV) by molecular cloning almost a quarter of a century ago, unprecedented at the time because the virus had never been grown in cell culture or detected serologically, there have been impressive strides in many facets of our understanding of the natural history of the disease, the viral life cycle, the pathogenesis, and antiviral therapy. It is apparent that the virus has developed multiple strategies to evade immune surveillance and eradication. This Review covers what we currently understand of the temporal and spatial immunological changes within the human innate and adaptive host immune responses that ultimately determine the outcomes of HCV infection.

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Hugo R. Rosen

P-glycoprotein ABCB1: a major player in drug handling by mammals

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Mammalian P-glycoproteins are active drug efflux transporters located in the plasma membrane. In the early nineties, we generated knockouts of the three P-glycoprotein genes of mice, the Mdr1a, Mdr1b, and Mdr2 P-glycoproteins, now known as Abcb1a, Abcb1b, and Abcb4, respectively. In the JCI papers that are the subject of this Hindsight, we showed that loss of Mdr1a (Abcb1a) had a profound effect on the tissue distribution and especially the brain accumulation of a range of drugs frequently used in humans, including dexamethasone, digoxin, cyclosporin A, ondansetron, domperidone, and loperamide. All drugs were shown to be excellent substrates of the murine ABCB1A P-glycoprotein and its human counterpart, the MDR1 P-glycoprotein, ABCB1. We found that the ability of ABCB1 to prevent accumulation of some drugs in the brain is a prerequisite for their clinical use, as absence of the transporter led to severe toxicity or undesired CNS pharmacodynamic effects. Subsequent work has fully confirmed the profound effect of the drug-transporting ABCB1 P-glycoprotein on the pharmacokinetics of drugs in humans. In fact, every new drug is now screened for transport by ABCB1, as this limits oral availability and penetration into sanctuaries protected by ABCB1, such as the brain.

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Piet Borst, Alfred H. Schinkel

REEPing the benefits of an animal model of hereditary spastic paraplegia

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The hereditary spastic paraplegias (HSPs) are characterized by spasticity of the leg muscles due to axonal degeneration of corticospinal neurons. Beetz et al. report that the core motor phenotype and axonal pathology of HSPs are recapitulated in mice lacking the HSP-associated gene Reep1. REEP1 is shown to regulate ER structure in motor cortex neurons. The Reep1 knockout mouse should be a very useful model in which to study the mechanisms of progressive axon loss in HSPs and other disorders.

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Ariel Y. Deutch, Peter Hedera, Roger J. Colbran

Zinc, insulin, and the liver: a ménage à trois

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Insulin and Zn²⁺ enjoy a multivalent relationship. Zn²⁺ binds insulin in pancreatic β cells to form crystalline aggregates in dense core vesicles (DCVs), which are released in response to physiological signals such as increased blood glucose. This transition metal is an essential cofactor in insulin-degrading enzyme and several key Zn²⁺ finger transcription factors that are required for β cell development and insulin gene expression. Studies are increasingly revealing that fluctuations in Zn²⁺ concentration can mediate signaling events, including dynamic roles that extend beyond that of a static structural or catalytic cofactor. In this issue of the JCI, Tamaki et al. propose an additional function for Zn²⁺ in relation to insulin: regulation of insulin clearance from the bloodstream.

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Thomas V. O’Halloran, Melkam Kebede, Steven J. Philips, Alan D. Attie

Opening lines of communication in the distal nephron

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The distal nephron is composed of two main cell types: principal cells and intercalated cells. These cells have distinct morphologic features that allow them to be readily distinguished by light microscopy, as well as distinct suites of proteins that facilitate cell-specific transport properties. In this issue of the JCI, Gueutin and colleagues describe a new mechanism by which β-intercalated cells, via release of ATP and prostaglandin E₂ (PGE₂), influence the activity of transporters in principal cells.

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Thomas R. Kleyman, Lisa M. Satlin, Kenneth R. Hallows

Common variable immunodeficiency: two mutations are better than one

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B cells from common variable immunodeficiency (CVID) patients who have one mutant copy of the gene encoding the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) often display dysfunctional antibody production. Interestingly, some individuals with mutations in both TACI alleles do not present with CVID, suggesting that TACI mutations influence CVID pathogenesis via dominant interference or haploinsufficiency. In this issue of the JCI, Romberg and colleagues report how TACI mutations impact B cell activation, removal of autoreactive B cells, and the development of autoimmune disease in CVID.

Authors

Antonio La Cava

PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells

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The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase–independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression — but not activity — of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase–independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1–positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

Authors

Paolo Neviani, Jason G. Harb, Joshua J. Oaks, Ramasamy Santhanam, Christopher J. Walker, Justin J. Ellis, Gregory Ferenchak, Adrienne M. Dorrance, Carolyn A. Paisie, Anna M. Eiring, Yihui Ma, Hsiaoyin C. Mao, Bin Zhang, Mark Wunderlich, Philippa C. May, Chaode Sun, Sahar A. Saddoughi, Jacek Bielawski, William Blum, Rebecca B. Klisovic, Janelle A. Solt, John C. Byrd, Stefano Volinia, Jorge Cortes, Claudia S. Huettner, Steffen Koschmieder, Tessa L. Holyoake, Steven Devine, Michael A. Caligiuri, Carlo M. Croce, Ramiro Garzon, Besim Ogretmen, Ralph B. Arlinghaus, Ching-Shih Chen, Robert Bittman, Peter Hokland, Denis-Claude Roy, Dragana Milojkovic, Jane Apperley, John M. Goldman, Alistair Reid, James C. Mulloy, Ravi Bhatia, Guido Marcucci, Danilo Perrotti

Accelerated neurodegeneration through chaperone-mediated oligomerization of tau

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Aggregation of tau protein in the brain is associated with a class of neurodegenerative diseases known as tauopathies. FK506 binding protein 51 kDa (FKBP51, encoded by FKBP5) forms a mature chaperone complex with Hsp90 that prevents tau degradation. In this study, we have shown that tau levels are reduced throughout the brains of Fkbp5^–/– mice. Recombinant FKBP51 and Hsp90 synergized to block tau clearance through the proteasome, resulting in tau oligomerization. Overexpression of FKBP51 in a tau transgenic mouse model revealed that FKBP51 preserved the species of tau that have been linked to Alzheimer’s disease (AD) pathogenesis, blocked amyloid formation, and decreased tangle load in the brain. Alterations in tau turnover and aggregate structure corresponded with enhanced neurotoxicity in mice. In human brains, FKBP51 levels increased relative to age and AD, corresponding with demethylation of the regulatory regions in the FKBP5 gene. We also found that higher FKBP51 levels were associated with AD progression. Our data support a model in which age-associated increases in FKBP51 levels and its interaction with Hsp90 promote neurotoxic tau accumulation. Strategies aimed at attenuating FKBP51 levels or its interaction with Hsp90 have the potential to be therapeutically relevant for AD and other tauopathies.

Authors

Laura J. Blair, Bryce A. Nordhues, Shannon E. Hill, K. Matthew Scaglione, John C. O’Leary III, Sarah N. Fontaine, Leonid Breydo, Bo Zhang, Pengfei Li, Li Wang, Carl Cotman, Henry L. Paulson, Martin Muschol, Vladimir N. Uversky, Torsten Klengel, Elisabeth B. Binder, Rakez Kayed, Todd E. Golde, Nicole Berchtold, Chad A. Dickey

Retinal angiogenesis suppression through small molecule activation of p53

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Neovascular age-related macular degeneration is a leading cause of irreversible vision loss in the Western world. Cytokine-targeted therapies (such as anti-vascular endothelial growth factor) are effective in treating pathologic ocular angiogenesis, but have not led to a durable effect and often require indefinite treatment. Here, we show that Nutlin-3, a small molecule antagonist of the E3 ubiquitin protein ligase MDM2, inhibited angiogenesis in several model systems. We found that a functional p53 pathway was essential for Nutlin-3–mediated retinal antiangiogenesis and disruption of the p53 transcriptional network abolished the antiangiogenic activity of Nutlin-3. Nutlin-3 did not inhibit established, mature blood vessels in the adult mouse retina, suggesting that only proliferating retinal vessels are sensitive to Nutlin-3. Furthermore, Nutlin-3 inhibited angiogenesis in nonretinal models such as the hind limb ischemia model. Our work demonstrates that Nutlin-3 functions through an antiproliferative pathway with conceivable advantages over existing cytokine-targeted antiangiogenesis therapies.

Authors

Sai H. Chavala, Younghee Kim, Laura Tudisco, Valeria Cicatiello, Till Milde, Nagaraj Kerur, Nidia Claros, Susan Yanni, Victor H. Guaiquil, William W. Hauswirth, John S. Penn, Shahin Rafii, Sandro De Falco, Thomas C. Lee, Jayakrishna Ambati

Lipotoxicity disrupts incretin-regulated human β cell connectivity

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Pancreatic β cell dysfunction is pathognomonic of type 2 diabetes mellitus (T2DM) and is driven by environmental and genetic factors. β cell responses to glucose and to incretins such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are altered in the disease state. While rodent β cells act as a coordinated syncytium to drive insulin release, this property is unexplored in human islets. In situ imaging approaches were therefore used to monitor in real time the islet dynamics underlying hormone release. We found that GLP-1 and GIP recruit a highly coordinated subnetwork of β cells that are targeted by lipotoxicity to suppress insulin secretion. Donor BMI was negatively correlated with subpopulation responses to GLP-1, suggesting that this action of incretin contributes to functional β cell mass in vivo. Conversely, exposure of mice to a high-fat diet unveiled a role for incretin in maintaining coordinated islet activity, supporting the existence of species-specific strategies to maintain normoglycemia. These findings demonstrate that β cell connectedness is an inherent property of human islets that is likely to influence incretin-potentiated insulin secretion and may be perturbed by diabetogenic insults to disrupt glucose homeostasis in humans.

Authors

David J. Hodson, Ryan K. Mitchell, Elisa A. Bellomo, Gao Sun, Laurent Vinet, Paolo Meda, Daliang Li, Wen-Hong Li, Marco Bugliani, Piero Marchetti, Domenico Bosco, Lorenzo Piemonti, Paul Johnson, Stephen J. Hughes, Guy A. Rutter

A SALL4/MLL/HOXA9 pathway in murine and human myeloid leukemogenesis

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The embryonic self-renewal factor SALL4 has been implicated in the development of human acute myeloid leukemia (AML). Transgenic mice expressing the human SALL4B allele develop AML, which indicates that this molecule contributes to leukemia development and maintenance. However, the underlying mechanism of SALL4-dependent AML progression is unknown. Using SALL4B transgenic mice, we observed that HoxA9 was significantly upregulated in SALL4B leukemic cells compared with wild-type controls. Downregulation of HoxA9 in SALL4B leukemic cells led to decreased replating capacity in vitro and delayed AML development in recipient mice. In primary human AML cells, downregulation of SALL4 led to decreased HOXA9 expression and enhanced apoptosis. We found that SALL4 bound a specific region of the HOXA9 promoter in leukemic cells. SALL4 overexpression led to enhanced binding of histone activation markers at the HOXA9 promoter region, as well as increased HOXA9 expression in these cells. Furthermore, we observed that SALL4 interacted with mixed-lineage leukemia (MLL) and co-occupied the HOXA9 promoter region with MLL in AML leukemic cells, which suggests that a SALL4/MLL pathway may control HOXA9 expression. In summary, our findings revealed a molecular mechanism for SALL4 function in leukemogenesis and suggest that targeting of the SALL4/MLL/HOXA9 pathway would be an innovative approach in treating AML.

Authors

Ailing Li, Youyang Yang, Chong Gao, Jiayun Lu, Ha-Won Jeong, Bee H. Liu, Ping Tang, Xiaopan Yao, Donna Neuberg, Gang Huang, Daniel G. Tenen, Li Chai

Common genetic variation at the IL1RL1 locus regulates IL-33/ST2 signaling

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The suppression of tumorigenicity 2/IL-33 (ST2/IL-33) pathway has been implicated in several immune and inflammatory diseases. ST2 is produced as 2 isoforms. The membrane-bound isoform (ST2L) induces an immune response when bound to its ligand, IL-33. The other isoform is a soluble protein (sST2) that is thought to be a decoy receptor for IL-33 signaling. Elevated sST2 levels in serum are associated with an increased risk for cardiovascular disease. We investigated the determinants of sST2 plasma concentrations in 2,991 Framingham Offspring Cohort participants. While clinical and environmental factors explained some variation in sST2 levels, much of the variation in sST2 production was driven by genetic factors. In a genome-wide association study (GWAS), multiple SNPs within IL1RL1 (the gene encoding ST2) demonstrated associations with sST2 concentrations. Five missense variants of IL1RL1 correlated with higher sST2 levels in the GWAS and mapped to the intracellular domain of ST2, which is absent in sST2. In a cell culture model, IL1RL1 missense variants increased sST2 expression by inducing IL-33 expression and enhancing IL-33 responsiveness (via ST2L). Our data suggest that genetic variation in IL1RL1 can result in increased levels of sST2 and alter immune and inflammatory signaling through the ST2/IL-33 pathway.

Authors

Jennifer E. Ho, Wei-Yu Chen, Ming-Huei Chen, Martin G. Larson, Elizabeth L. McCabe, Susan Cheng, Anahita Ghorbani, Erin Coglianese, Valur Emilsson, Andrew D. Johnson, Stefan Walter, Nora Franceschini, Christopher J. O’Donnell, Abbas Dehghan, Chen Lu, Daniel Levy, Christopher Newton-Cheh, Honghuang Lin, Janine F. Felix, Eric R. Schreiter, Ramachandran S. Vasan, James L. Januzzi, Richard T. Lee, Thomas J. Wang

Renal β-intercalated cells maintain body fluid and electrolyte balance

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Inactivation of the B1 proton pump subunit (ATP6V1B1) in intercalated cells (ICs) leads to type I distal renal tubular acidosis (dRTA), a disease associated with salt- and potassium-losing nephropathy. Here we show that mice deficient in ATP6V1B1 (Atp6v1b1^–/– mice) displayed renal loss of NaCl, K⁺, and water, causing hypovolemia, hypokalemia, and polyuria. We demonstrated that NaCl loss originated from the cortical collecting duct, where activity of both the epithelial sodium channel (ENaC) and the pendrin/Na⁺-driven chloride/bicarbonate exchanger (pendrin/NDCBE) transport system was impaired. ENaC was appropriately increased in the medullary collecting duct, suggesting a localized inhibition in the cortex. We detected high urinary prostaglandin E₂ (PGE₂) and ATP levels in Atp6v1b1^–/– mice. Inhibition of PGE₂ synthesis in vivo restored ENaC protein levels specifically in the cortex. It also normalized protein levels of the large conductance calcium-activated potassium channel and the water channel aquaporin 2, and improved polyuria and hypokalemia in mutant mice. Furthermore, pharmacological inactivation of the proton pump in β-ICs induced release of PGE₂ through activation of calcium-coupled purinergic receptors. In the present study, we identified ATP-triggered PGE₂ paracrine signaling originating from β-ICs as a mechanism in the development of the hydroelectrolytic imbalance associated with dRTA. Our data indicate that in addition to principal cells, ICs are also critical in maintaining sodium balance and, hence, normal vascular volume and blood pressure.

Authors

Victor Gueutin, Marion Vallet, Maximilien Jayat, Janos Peti-Peterdi, Nicolas Cornière, Françoise Leviel, Fabien Sohet, Carsten A. Wagner, Dominique Eladari, Régine Chambrey

Kruppel-like factor 15 is critical for vascular inflammation

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Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle–specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.

Authors

Yuan Lu, Lisheng Zhang, Xudong Liao, Panjamaporn Sangwung, Domenick A. Prosdocimo, Guangjin Zhou, Alexander R. Votruba, Leigh Brian, Yuh Jung Han, Huiyun Gao, Yunmei Wang, Koichi Shimizu, Kaitlyn Weinert-Stein, Maria Khrestian, Daniel I. Simon, Neil J. Freedman, Mukesh K. Jain

Exclusive CX₃CR1 dependence of kidney DCs impacts glomerulonephritis progression

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DCs and macrophages both express the chemokine receptor CX₃CR1. Here we demonstrate that its ligand, CX₃CL1, is highly expressed in the murine kidney and intestine. CX₃CR1 deficiency markedly reduced DC numbers in the healthy and inflamed kidney cortex, and to a lesser degree in the kidney medulla and intestine, but not in other organs. CX₃CR1 also promoted influx of DC precursors in crescentic glomerulonephritis, a DC-dependent aggressive type of nephritis. Disease severity was strongly attenuated in CX₃CR1-deficient mice. Primarily CX₃CR1-dependent DCs in the kidney cortex processed antigen for the intrarenal stimulation of T helper cells, a function important for glomerulonephritis progression. In contrast, medullary DCs played a specialized role in inducing innate immunity against bacterial pyelonephritis by recruiting neutrophils through rapid chemokine production. CX₃CR1 deficiency had little effect on the immune defense against pyelonephritis, as medullary DCs were less CX₃CR1 dependent than cortical DCs and because recruited neutrophils produced chemokines to compensate for the DC paucity. These findings demonstrate that cortical and medullary DCs play specialized roles in their respective kidney compartments. We identify CX₃CR1 as a potential therapeutic target in glomerulonephritis that may involve fewer adverse side effects, such as impaired anti-infectious defense or compromised DC functions in other organs.

Authors

Katharina Hochheiser, Christoph Heuser, Torsten A. Krause, Simon Teteris, Anissa Ilias, Christina Weisheit, Florian Hoss, André P. Tittel, Percy A. Knolle, Ulf Panzer, Daniel R. Engel, Pierre-Louis Tharaux, Christian Kurts

ACTN3 genotype influences muscle performance through the regulation of calcineurin signaling

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α-Actinin-3 deficiency occurs in approximately 16% of the global population due to homozygosity for a common nonsense polymorphism in the ACTN3 gene. Loss of α-actinin-3 is associated with reduced power and enhanced endurance capacity in elite athletes and nonathletes due to “slowing” of the metabolic and physiological properties of fast fibers. Here, we have shown that α-actinin-3 deficiency results in increased calcineurin activity in mouse and human skeletal muscle and enhanced adaptive response to endurance training. α-Actinin-2, which is differentially expressed in α-actinin-3–deficient muscle, has higher binding affinity for calsarcin-2, a key inhibitor of calcineurin activation. We have further demonstrated that α-actinin-2 competes with calcineurin for binding to calsarcin-2, resulting in enhanced calcineurin signaling and reprogramming of the metabolic phenotype of fast muscle fibers. Our data provide a mechanistic explanation for the effects of the ACTN3 genotype on skeletal muscle performance in elite athletes and on adaptation to changing physical demands in the general population. In addition, we have demonstrated that the sarcomeric α-actinins play a role in the regulation of calcineurin signaling.

Authors

Jane T. Seto, Kate G.R. Quinlan, Monkol Lek, Xi Fiona Zheng, Fleur Garton, Daniel G. MacArthur, Marshall W. Hogarth, Peter J. Houweling, Paul Gregorevic, Nigel Turner, Gregory J. Cooney, Nan Yang, Kathryn N. North

Maternal uterine NK cell–activating receptor KIR2DS1 enhances placentation

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Reduced trophoblast invasion and vascular conversion in decidua are thought to be the primary defect of common pregnancy disorders including preeclampsia and fetal growth restriction. Genetic studies suggest these conditions are linked to combinations of polymorphic killer cell Ig-like receptor (KIR) genes expressed by maternal decidual NK cells (dNK) and HLA-C genes expressed by fetal trophoblast. Inhibitory KIR2DL1 and activating KIR2DS1 both bind HLA-C2, but confer increased risk or protection from pregnancy disorders, respectively. The mechanisms underlying these genetic associations with opposing outcomes are unknown. We show that KIR2DS1 is highly expressed in dNK, stimulating strong activation of KIR2DS1⁺ dNK. We used microarrays to identify additional responses triggered by binding of KIR2DS1 or KIR2DL1 to HLA-C2 and found different responses in dNK coexpressing KIR2DS1 with KIR2DL1 compared with dNK only expressing KIR2DL1. Activation of KIR2DS1⁺ dNK by HLA-C2 stimulated production of soluble products including GM-CSF, detected by intracellular FACS and ELISA. We demonstrated that GM-CSF enhanced migration of primary trophoblast and JEG-3 trophoblast cells in vitro. These findings provide a molecular mechanism explaining how recognition of HLA class I molecules on fetal trophoblast by an activating KIR on maternal dNK may be beneficial for placentation.

Authors

Shiqiu Xiong, Andrew M. Sharkey, Philippa R. Kennedy, Lucy Gardner, Lydia E. Farrell, Olympe Chazara, Julien Bauer, Susan E. Hiby, Francesco Colucci, Ashley Moffett

A spastic paraplegia mouse model reveals REEP1-dependent ER shaping

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Axonopathies are a group of clinically diverse disorders characterized by the progressive degeneration of the axons of specific neurons. In hereditary spastic paraplegia (HSP), the axons of cortical motor neurons degenerate and cause a spastic movement disorder. HSP is linked to mutations in several loci known collectively as the spastic paraplegia genes (SPGs). We identified a heterozygous receptor accessory protein 1 (REEP1) exon 2 deletion in a patient suffering from the autosomal dominantly inherited HSP variant SPG31. We generated the corresponding mouse model to study the underlying cellular pathology. Mice with heterozygous deletion of exon 2 in Reep1 displayed a gait disorder closely resembling SPG31 in humans. Homozygous exon 2 deletion resulted in the complete loss of REEP1 and a more severe phenotype with earlier onset. At the molecular level, we demonstrated that REEP1 is a neuron-specific, membrane-binding, and membrane curvature–inducing protein that resides in the ER. We further show that Reep1 expression was prominent in cortical motor neurons. In REEP1-deficient mice, these neurons showed reduced complexity of the peripheral ER upon ultrastructural analysis. Our study connects proper neuronal ER architecture to long-term axon survival.

Authors

Christian Beetz, Nicole Koch, Mukhran Khundadze, Geraldine Zimmer, Sandor Nietzsche, Nicole Hertel, Antje-Kathrin Huebner, Rizwan Mumtaz, Michaela Schweizer, Elisabeth Dirren, Kathrin N. Karle, Andrey Irintchev, Victoria Alvarez, Christoph Redies, Martin Westermann, Ingo Kurth, Thomas Deufel, Michael M. Kessels, Britta Qualmann, Christian A. Hübner

CVID-associated TACI mutations affect autoreactive B cell selection and activation

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Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6–expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications.

Authors

Neil Romberg, Nicolas Chamberlain, David Saadoun, Maurizio Gentile, Tuure Kinnunen, Yen Shing Ng, Manmeet Virdee, Laurence Menard, Tineke Cantaert, Henner Morbach, Rima Rachid, Natalia Martinez-Pomar, Nuria Matamoros, Raif Geha, Bodo Grimbacher, Andrea Cerutti, Charlotte Cunningham-Rundles, Eric Meffre

Integrins protect cardiomyocytes from ischemia/reperfusion injury

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Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca²⁺ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca²⁺ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165–175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca²⁺ regulatory pathways and offers a means to protect the myocardium from ischemic injury.

Authors

Hideshi Okada, N. Chin Lai, Yoshitaka Kawaraguchi, Peter Liao, Jeffrey Copps, Yasuo Sugano, Sunaho Okada-Maeda, Indroneal Banerjee, Jan M. Schilling, Alexandre R. Gingras, Elizabeth K. Asfaw, Jorge Suarez, Seok-Min Kang, Guy A. Perkins, Carol G. Au, Sharon Israeli-Rosenberg, Ana Maria Manso, Zheng Liu, Derek J. Milner, Stephen J. Kaufman, Hemal H. Patel, David M. Roth, H. Kirk Hammond, Susan S. Taylor, Wolfgang H. Dillmann, Joshua I. Goldhaber, Robert S. Ross

2-Aminoadipic acid is a biomarker for diabetes risk

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Improvements in metabolite-profiling techniques are providing increased breadth of coverage of the human metabolome and may highlight biomarkers and pathways in common diseases such as diabetes. Using a metabolomics platform that analyzes intermediary organic acids, purines, pyrimidines, and other compounds, we performed a nested case-control study of 188 individuals who developed diabetes and 188 propensity-matched controls from 2,422 normoglycemic participants followed for 12 years in the Framingham Heart Study. The metabolite 2-aminoadipic acid (2-AAA) was most strongly associated with the risk of developing diabetes. Individuals with 2-AAA concentrations in the top quartile had greater than a 4-fold risk of developing diabetes. Levels of 2-AAA were not well correlated with other metabolite biomarkers of diabetes, such as branched chain amino acids and aromatic amino acids, suggesting they report on a distinct pathophysiological pathway. In experimental studies, administration of 2-AAA lowered fasting plasma glucose levels in mice fed both standard chow and high-fat diets. Further, 2-AAA treatment enhanced insulin secretion from a pancreatic β cell line as well as murine and human islets. These data highlight a metabolite not previously associated with diabetes risk that is increased up to 12 years before the onset of overt disease. Our findings suggest that 2-AAA is a marker of diabetes risk and a potential modulator of glucose homeostasis in humans.

Authors

Thomas J. Wang, Debby Ngo, Nikolaos Psychogios, Andre Dejam, Martin G. Larson, Ramachandran S. Vasan, Anahita Ghorbani, John O’Sullivan, Susan Cheng, Eugene P. Rhee, Sumita Sinha, Elizabeth McCabe, Caroline S. Fox, Christopher J. O’Donnell, Jennifer E. Ho, Jose C. Florez, Martin Magnusson, Kerry A. Pierce, Amanda L. Souza, Yi Yu, Christian Carter, Peter E. Light, Olle Melander, Clary B. Clish, Robert E. Gerszten

Glucagon regulates gluconeogenesis through KAT2B- and WDR5-mediated epigenetic effects

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Abstract

Circulating pancreatic glucagon is increased during fasting and maintains glucose balance by stimulating hepatic gluconeogenesis. Glucagon triggering of the cAMP pathway upregulates the gluconeogenic program through the phosphorylation of cAMP response element–binding protein (CREB) and the dephosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic gene expression by promoting epigenetic changes that facilitate assembly of the transcriptional machinery. However, the nature of these modifications is unclear. Using mouse models and in vitro assays, we show that histone H3 acetylation at Lys 9 (H3K9Ac) was elevated over gluconeogenic genes and contributed to increased hepatic glucose production during fasting and in diabetes. Dephosphorylation of CRTC2 promoted increased H3K9Ac through recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat–containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle, whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Depletion of KAT2B or WDR5 decreased gluconeogenic gene expression, consequently breaking the cycle. Administration of a small-molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, suggesting that this enzyme may be a useful target for diabetes treatment.

Authors

Kim Ravnskjaer, Meghan F. Hogan, Denise Lackey, Laszlo Tora, Sharon Y.R. Dent, Jerrold Olefsky, Marc Montminy

ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

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Abstract

Aberrant regulation of the erythroblastosis oncogene B (ErbB) family of receptor tyrosine kinases (RTKs) and their ligands is common in human cancers. ErbB3 is required in luminal mammary epithelial cells (MECs) for growth and survival. Since breast cancer phenotypes may reflect biological traits of the MECs from which they originate, we tested the hypothesis that ErbB3 drives luminal breast cancer growth. We found higher ERBB3 expression and more frequent ERBB3 gene copy gains in luminal A/B breast cancers compared with other breast cancer subtypes. In cell culture, ErbB3 increased growth of luminal breast cancer cells. Targeted depletion of ErbB3 with an anti-ErbB3 antibody decreased 3D colony growth, increased apoptosis, and decreased tumor growth in vivo. Treatment of clinical breast tumors with the antiendocrine drug fulvestrant resulted in increased ErbB3 expression and PI3K/mTOR signaling. Depletion of ErbB3 in fulvestrant-treated tumor cells reduced PI3K/mTOR signaling, thus decreasing tumor cell survival and tumor growth. Fulvestrant treatment increased phosphorylation of all ErbB family RTKs; however, phospho-RTK upregulation was not seen in tumors treated with both fulvestrant and anti-ErbB3. These data indicate that upregulation of ErbB3 in luminal breast cancer cells promotes growth, survival, and resistance to fulvestrant, thus suggesting ErbB3 as a target for breast cancer treatment.

Authors

Meghan M. Morrison, Katherine Hutchinson, Michelle M. Williams, Jamie C. Stanford, Justin M. Balko, Christian Young, Maria G. Kuba, Violeta Sánchez, Andrew J. Williams, Donna J. Hicks, Carlos L. Arteaga, Aleix Prat, Charles M. Perou, H. Shelton Earp, Suleiman Massarweh, Rebecca S. Cook

Radiation-induced acid ceramidase confers prostate cancer resistance and tumor relapse

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Abstract

Escape of prostate cancer (PCa) cells from ionizing radiation–induced (IR-induced) killing leads to disease progression and cancer relapse. The influence of sphingolipids, such as ceramide and its metabolite sphingosine 1-phosphate, on signal transduction pathways under cell stress is important to survival adaptation responses. In this study, we demonstrate that ceramide-deacylating enzyme acid ceramidase (AC) was preferentially upregulated in irradiated PCa cells. Radiation-induced AC gene transactivation by activator protein 1 (AP-1) binding on the proximal promoter was sensitive to inhibition of de novo ceramide biosynthesis, as demonstrated by promoter reporter and ChIP-qPCR analyses. Our data indicate that a protective feedback mechanism mitigates the apoptotic effect of IR-induced ceramide generation. We found that deregulation of c-Jun induced marked radiosensitization in vivo and in vitro, which was rescued by ectopic AC overexpression. AC overexpression in PCa clonogens that survived a fractionated 80-Gy IR course was associated with increased radioresistance and proliferation, suggesting a role for AC in radiotherapy failure and relapse. Immunohistochemical analysis of human PCa tissues revealed higher levels of AC after radiotherapy failure than those in therapy-naive PCa, prostatic intraepithelial neoplasia, or benign tissues. Addition of an AC inhibitor to an animal model of xenograft irradiation produced radiosensitization and prevention of relapse. These data indicate that AC is a potentially tractable target for adjuvant radiotherapy.

Authors

Joseph C. Cheng, Aiping Bai, Thomas H. Beckham, S. Tucker Marrison, Caroline L. Yount, Katherine Young, Ping Lu, Anne M. Bartlett, Bill X. Wu, Barry J. Keane, Kent E. Armeson, David T. Marshall, Thomas E. Keane, Michael T. Smith, E. Ellen Jones, Richard R. Drake Jr., Alicja Bielawska, James S. Norris, Xiang Liu

Amelioration of ischemic brain damage by peritoneal dialysis

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Abstract

Ischemic stroke is a devastating condition, for which there is still no effective therapy. Acute ischemic stroke is associated with high concentrations of glutamate in the blood and interstitial brain fluid. The inability of the tissue to retain glutamate within the cells of the brain ultimately provokes neuronal death. Increased concentrations of interstitial glutamate exert further excitotoxic effects on healthy tissue surrounding the infarct zone. We developed a strategy based on peritoneal dialysis to reduce blood glutamate levels, thereby accelerating brain-to-blood glutamate clearance. In a rat model of stroke, this simple procedure reduced the transient increase in glutamate, consequently decreasing the size of the infarct area. Functional magnetic resonance imaging demonstrated that the rescued brain tissue remained functional. Moreover, in patients with kidney failure, peritoneal dialysis significantly decreased glutamate concentrations. Our results suggest that peritoneal dialysis may represent a simple and effective intervention for human stroke patients.

Authors

María del Carmen Godino, Victor G. Romera, José Antonio Sánchez-Tomero, Jesus Pacheco, Santiago Canals, Juan Lerma, José Vivancos, María Angeles Moro, Magdalena Torres, Ignacio Lizasoain, José Sánchez-Prieto

Dendritic epidermal T cells regulate skin antimicrobial barrier function

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Abstract

The epidermis, the outer layer of the skin, forms a physical and antimicrobial shield to protect the body from environmental threats. Skin injury severely compromises the epidermal barrier and requires immediate repair. Dendritic epidermal T cells (DETC) reside in the murine epidermis where they sense skin injury and serve as regulators and orchestrators of immune responses. Here, we determined that TCR stimulation and skin injury induces IL-17A production by a subset of DETC. This subset of IL-17A–producing DETC was distinct from IFN-γ producers, despite similar surface marker profiles. Functionally, blocking IL-17A or genetic deletion of IL-17A resulted in delayed wound closure in animals. Skin organ cultures from Tcrd^–/–, which lack DETC, and Il17a^–/– mice both exhibited wound-healing defects. Wound healing was fully restored by the addition of WT DETC, but only partially restored by IL-17A–deficient DETC, demonstrating the importance of IL-17A to wound healing. Following skin injury, DETC-derived IL-17A induced expression of multiple host-defense molecules in epidermal keratinocytes to promote healing. Together, these data provide a mechanistic link between IL-17A production by DETC, host-defense, and wound-healing responses in the skin. These findings establish a critical and unique role of IL-17A–producing DETC in epidermal barrier function and wound healing.

Authors

Amanda S. MacLeod, Saskia Hemmers, Olivia Garijo, Marianne Chabod, Kerri Mowen, Deborah A. Witherden, Wendy L. Havran

Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors

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Abstract

Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1^–/– background. We found that oncogenic Kras–induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.

Authors

Kwan-Hyuck Baek, Dongha Bhang, Alexander Zaslavsky, Liang-Chuan Wang, Anil Vachani, Carla F. Kim, Steven M. Albelda, Gerard I. Evan, Sandra Ryeom

Epithelial stem cell mutations that promote squamous cell carcinoma metastasis

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Abstract

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras^G12D activation and Smad4 deletion, to mouse keratin 15–expressing (K15⁺) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell–enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9–transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras^G12D activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

Authors

Ruth A. White, Jill M. Neiman, Anand Reddi, Gangwen Han, Stanca Birlea, Doyel Mitra, Laikuan Dionne, Pam Fernandez, Kazutoshi Murao, Li Bian, Stephen B. Keysar, Nathaniel B. Goldstein, Ningjing Song, Sophia Bornstein, Zheyi Han, Xian Lu, Joshua Wisell, Fulun Li, John Song, Shi-Long Lu, Antonio Jimeno, Dennis R. Roop, Xiao-Jing Wang

Human antibodies that neutralize respiratory droplet transmissible H5N1 influenza viruses

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Abstract

Recent studies described the experimental adaptation of influenza H5 HAs that confers respiratory droplet transmission (rdt) to influenza virus in ferrets. Acquisition of the ability to transmit via aerosol may lead to the development of a highly pathogenic pandemic H5 virus. Vaccines are predicted to play an important role in H5N1 control should the virus become readily transmissible between humans. We obtained PBMCs from patients who received an A/Vietnam/1203/2004 H5N1 subunit vaccine. Human hybridomas were then generated and characterized. We identified antibodies that bound the HA head domain and recognized both WT and rdt H5 HAs. We used a combination of structural techniques to define a mechanism of antibody recognition of an H5 HA receptor–binding site that neutralized H5N1 influenza viruses and pseudoviruses carrying the HA rdt variants that have mutations near the receptor-binding site. Incorporation or retention of this critical antigenic site should be considered in the design of novel H5 HA immunogens to protect against mammalian-adapted H5N1 mutants.

Authors

Natalie J. Thornburg, David P. Nannemann, David L. Blum, Jessica A. Belser, Terrence M. Tumpey, Shyam Deshpande, Gloria A. Fritz, Gopal Sapparapu, Jens C. Krause, Jeong Hyun Lee, Andrew B. Ward, David E. Lee, Sheng Li, Katie L. Winarski, Benjamin W. Spiller, Jens Meiler, James E. Crowe Jr.

Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis

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Abstract

The activating receptor NK cell group 2 member D (NKG2D) mediates antitumor immunity in experimental animal models. However, whether NKG2D ligands contribute to tumor suppression or progression clinically remains controversial. Here, we have described 2 novel lines of “humanized” bi-transgenic (bi-Tg) mice in which native human NKG2D ligand MHC class I polypeptide-related sequence B (MICB) or the engineered membrane-restricted MICB (MICB.A2) was expressed in the prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous carcinogenesis. Bi-Tg TRAMP/MICB mice exhibited a markedly increased incidence of progressed carcinomas and metastasis, whereas TRAMP/MICB.A2 mice enjoyed long-term tumor-free survival conferred by sustained NKG2D-mediated antitumor immunity. Mechanistically, we found that cancer progression in TRAMP/MICB mice was associated with loss of the peripheral NK cell pool owing to high serum levels of tumor-derived soluble MICB (sMICB). Prostate cancer patients also displayed reduction of peripheral NK cells and high sMIC levels. Our study has not only provided direct evidence in “humanized” mouse models that soluble and membrane-restricted NKG2D ligands pose opposite impacts on cancer progression, but also uncovered a mechanism of sMIC-induced impairment of NK cell antitumor immunity. Our findings suggest that the impact of soluble NKG2D ligands should be considered in NK cell–based cancer immunotherapy and that our unique mouse models should be valuable for therapy optimization.

Authors

Gang Liu, Shengjun Lu, Xuanjun Wang, Stephanie T. Page, Celestia S. Higano, Stephen R. Plymate, Norman M. Greenberg, Shaoli Sun, Zihai Li, Jennifer D. Wu

Proximal tubule H-ferritin mediates iron trafficking in acute kidney injury

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Abstract

Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule–specific FtH-knockout mice (FtH^PT–/– mice). FtH^PT–/– mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtH^PT–/– mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase–associated lipocalin, hemopexin, and transferrin were increased in FtH^PT–/– mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI.

Authors

Abolfazl Zarjou, Subhashini Bolisetty, Reny Joseph, Amie Traylor, Eugene O. Apostolov, Paolo Arosio, Jozsef Balla, Jill Verlander, Deepak Darshan, Lukas C. Kuhn, Anupam Agarwal

FGF18 as a prognostic and therapeutic biomarker in ovarian cancer

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Abstract

High-throughput genomic technologies have identified biomarkers and potential therapeutic targets for ovarian cancer. Comprehensive functional validation studies of the biological and clinical implications of these biomarkers are needed to advance them toward clinical use. Amplification of chromosomal region 5q31–5q35.3 has been used to predict poor prognosis in patients with advanced stage, high-grade serous ovarian cancer. In this study, we further dissected this large amplicon and identified the overexpression of FGF18 as an independent predictive marker for poor clinical outcome in this patient population. Using cell culture and xenograft models, we show that FGF18 signaling promoted tumor progression by modulating the ovarian tumor aggressiveness and microenvironment. FGF18 controlled migration, invasion, and tumorigenicity of ovarian cancer cells through NF-κB activation, which increased the production of oncogenic cytokines and chemokines. This resulted in a tumor microenvironment characterized by enhanced angiogenesis and augmented tumor-associated macrophage infiltration and M2 polarization. Tumors from ovarian cancer patients had increased FGF18 expression levels with microvessel density and M2 macrophage infiltration, confirming our in vitro results. These findings demonstrate that FGF18 is important for a subset of ovarian cancers and may serve as a therapeutic target.

Authors

Wei Wei, Samuel C. Mok, Esther Oliva, Sung-hoon Kim, Gayatry Mohapatra, Michael J. Birrer

Pak and Rac GTPases promote oncogenic KIT–induced neoplasms

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Abstract

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21–activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

Authors

Holly Martin, Raghuveer Singh Mali, Peilin Ma, Anindya Chatterjee, Baskar Ramdas, Emily Sims, Veerendra Munugalavadla, Joydeep Ghosh, Ray R. Mattingly, Valeria Visconte, Ramon V. Tiu, Cornelis P. Vlaar, Suranganie Dharmawardhane, Reuben Kapur

Myeloid-derived suppressor cell development is regulated by a STAT/IRF-8 axis

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Abstract

Myeloid-derived suppressor cells (MDSCs) comprise immature myeloid populations produced in diverse pathologies, including neoplasia. Because MDSCs can impair antitumor immunity, these cells have emerged as a significant barrier to cancer therapy. Although much research has focused on how MDSCs promote tumor progression, it remains unclear how MDSCs develop and why the MDSC response is heavily granulocytic. Given that MDSCs are a manifestation of aberrant myelopoiesis, we hypothesized that MDSCs arise from perturbations in the regulation of interferon regulatory factor–8 (IRF-8), an integral transcriptional component of myeloid differentiation and lineage commitment. Overall, we demonstrated that (a) Irf8-deficient mice generated myeloid populations highly homologous to tumor-induced MDSCs with respect to phenotype, function, and gene expression profiles; (b) IRF-8 overexpression in mice attenuated MDSC accumulation and enhanced immunotherapeutic efficacy; (c) the MDSC-inducing factors G-CSF and GM-CSF facilitated IRF-8 downregulation via STAT3- and STAT5-dependent pathways; and (d) IRF-8 levels in MDSCs of breast cancer patients declined with increasing MDSC frequency, implicating IRF-8 as a negative regulator in human MDSC biology. Together, our results reveal a previously unrecognized role for IRF-8 expression in MDSC subset development, which may provide new avenues to target MDSCs in neoplasia.

Authors

Jeremy D. Waight, Colleen Netherby, Mary L. Hensen, Austin Miller, Qiang Hu, Song Liu, Paul N. Bogner, Matthew R. Farren, Kelvin P. Lee, Kebin Liu, Scott I. Abrams

Inhibiting glycolytic metabolism enhances CD8⁺ T cell memory and antitumor function

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Abstract

Naive CD8⁺ T cells rely upon oxidation of fatty acids as a primary source of energy. After antigen encounter, T cells shift to a glycolytic metabolism to sustain effector function. It is unclear, however, whether changes in glucose metabolism ultimately influence the ability of activated T cells to become long-lived memory cells. We used a fluorescent glucose analog, 2-NBDG, to quantify glucose uptake in activated CD8⁺ T cells. We found that cells exhibiting limited glucose incorporation had a molecular profile characteristic of memory precursor cells and an increased capacity to enter the memory pool compared with cells taking up high amounts of glucose. Accordingly, enforcing glycolytic metabolism by overexpressing the glycolytic enzyme phosphoglycerate mutase-1 severely impaired the ability of CD8⁺ T cells to form long-term memory. Conversely, activation of CD8⁺ T cells in the presence of an inhibitor of glycolysis, 2-deoxyglucose, enhanced the generation of memory cells and antitumor functionality. Our data indicate that augmenting glycolytic flux drives CD8⁺ T cells toward a terminally differentiated state, while its inhibition preserves the formation of long-lived memory CD8⁺ T cells. These results have important implications for improving the efficacy of T cell–based therapies against chronic infectious diseases and cancer.

Authors

Madhusudhanan Sukumar, Jie Liu, Yun Ji, Murugan Subramanian, Joseph G. Crompton, Zhiya Yu, Rahul Roychoudhuri, Douglas C. Palmer, Pawel Muranski, Edward D. Karoly, Robert P. Mohney, Christopher A. Klebanoff, Ashish Lal, Toren Finkel, Nicholas P. Restifo, Luca Gattinoni

p16^INK4a protects against dysfunctional telomere–induced ATR-dependent DNA damage responses

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Abstract

Dysfunctional telomeres limit cellular proliferative capacity by activating the p53-p21– and p16^INK4a-Rb–dependent DNA damage responses (DDRs). The p16^INK4a tumor suppressor accumulates in aging tissues, is a biomarker for cellular senescence, and limits stem cell function in vivo. While the activation of a p53-dependent DDR by dysfunctional telomeres has been well documented in human cells and mouse models, the role for p16^INK4a in response to telomere dysfunction remains unclear. Here, we generated protection of telomeres 1b p16^–/– mice (Pot1b^Δ/Δ;p16^–/–) to address the function of p16^INK4a in the setting of telomere dysfunction in vivo. We found that deletion of p16^INK4a accelerated organ impairment and observed functional defects in highly proliferative organs, including the hematopoietic system, small intestine, and testes. Pot1b^Δ/Δ;p16^–/– hematopoietic cells exhibited increased telomere loss, increased chromosomal fusions, and telomere replication defects. p16^INK4a deletion enhanced the activation of the ATR-dependent DDR in Pot1b^Δ/Δ hematopoietic cells, leading to p53 stabilization, increased p21-dependent cell cycle arrest, and elevated p53-dependent apoptosis. In contrast to p16^INK4a, deletion of p21 did not activate ATR, rescued proliferative defects in Pot1b^Δ/Δ hematopoietic cells, and significantly increased organismal lifespan. Our results provide experimental evidence that p16^INK4a exerts protective functions in proliferative cells bearing dysfunctional telomeres.

Authors

Yang Wang, Norman Sharpless, Sandy Chang

Blood pressure homeostasis is maintained by a P311–TGF-β axis

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Abstract

P311 is an 8-kDa intracellular protein that is highly conserved across species and is expressed in the nervous system as well as in vascular and visceral smooth muscle cells. P311-null (P311^–/–) mice display learning and memory defects, but alterations in their vasculature have not been previously described. Here we report that P311^–/– mice are markedly hypotensive with accompanying defects in vascular tone and VSMC contractility. Functional abnormalities in P311^–/– mice resulted from decreased total and active levels of TGF-β1, TGF-β2, and TGF-β3 that arise as a specific consequence of decreased translation. Vascular hypofunctionality was fully rescued in vitro and in vivo by exogenous TGF-β1–TGF-β3. Conversely, P311-transgenic (P311^TG) mice had elevated levels of TGF-β1–TGF-β3 and subsequent hypertension. Consistent with findings attained in mouse models, arteries recovered from hypertensive human patients displayed increased P311 expression. Thus, we identified P311 as the first protein known to modulate TGF-β translation and the first pan-regulator of TGF-β expression under steady-state conditions. Together, our findings point to P311 as a critical blood pressure regulator and establish a potential link between P311 expression and the development of hypertensive disease.

Authors

Kameswara Rao Badri, Ming Yue, Oscar A. Carretero, Sree Latha Aramgam, Jun Cao, Stephen Sharkady, Gene H. Kim, Gregory A. Taylor, Kenneth L. Byron, Lucia Schuger

The diabetes-susceptible gene SLC30A8/ZnT8 regulates hepatic insulin clearance

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Abstract

Recent genome-wide association studies demonstrated that common variants of solute carrier family 30 member 8 gene (SLC30A8) increase susceptibility to type 2 diabetes. SLC30A8 encodes zinc transporter-8 (ZnT8), which delivers zinc ion from the cytoplasm into insulin granules. Although it is well known that insulin granules contain high amounts of zinc, the physiological role of secreted zinc remains elusive. In this study, we generated mice with β cell–specific Slc30a8 deficiency (ZnT8KO mice) and demonstrated an unexpected functional linkage between Slc30a8 deletion and hepatic insulin clearance. The ZnT8KO mice had low peripheral blood insulin levels, despite insulin hypersecretion from pancreatic β cells. We also demonstrated that a substantial amount of the hypersecreted insulin was degraded during its first passage through the liver. Consistent with these findings, ZnT8KO mice and human individuals carrying rs13266634, a major risk allele of SLC30A8, exhibited increased insulin clearance, as assessed by c-peptide/insulin ratio. Furthermore, we demonstrated that zinc secreted in concert with insulin suppressed hepatic insulin clearance by inhibiting clathrin-dependent insulin endocytosis. Our results indicate that SLC30A8 regulates hepatic insulin clearance and that genetic dysregulation of this system may play a role in the pathogenesis of type 2 diabetes.

Authors

Motoyuki Tamaki, Yoshio Fujitani, Akemi Hara, Toyoyoshi Uchida, Yoshifumi Tamura, Kageumi Takeno, Minako Kawaguchi, Takahiro Watanabe, Takeshi Ogihara, Ayako Fukunaka, Tomoaki Shimizu, Tomoya Mita, Akio Kanazawa, Mica O. Imaizumi, Takaya Abe, Hiroshi Kiyonari, Shintaro Hojyo, Toshiyuki Fukada, Takeshi Kawauchi, Shinya Nagamatsu, Toshio Hirano, Ryuzo Kawamori, Hirotaka Watada

Disruption of CEP290 microtubule/membrane-binding domains causes retinal degeneration

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Abstract

Mutations in the gene centrosomal protein 290 kDa (CEP290) cause an array of debilitating and phenotypically distinct human diseases, ranging from the devastating blinding disease Leber congenital amaurosis (LCA) to Senior-Løken syndrome, Joubert syndrome, and the lethal Meckel-Gruber syndrome. Despite its critical role in biology and disease, very little is known about CEP290’s function. Here, we have identified 4 functional domains of the protein. We found that CEP290 directly binds to cellular membranes through an N-terminal domain that includes a highly conserved amphipathic helix motif and to microtubules through a domain located within its myosin-tail homology domain. Furthermore, CEP290 activity was regulated by 2 autoinhibitory domains within its N and C termini, both of which were found to play critical roles in regulating ciliogenesis. Disruption of the microtubule-binding domain in a mouse model of LCA was sufficient to induce significant deficits in cilium formation, which led to retinal degeneration. These data implicate CEP290 as an integral structural and regulatory component of the cilium and provide insight into the pathological mechanisms of LCA and related ciliopathies. Further, these data illustrate that disruption of particular CEP290 functional domains may lead to particular disease phenotypes and suggest innovative strategies for therapeutic intervention.

Authors

Theodore G. Drivas, Erika L.F. Holzbaur, Jean Bennett

WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs

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Abstract

Authors

Ulrich Matt, Omar Sharif, Rui Martins, Tanja Furtner, Lorene Langeberg, Riem Gawish, Immanuel Elbau, Ana Zivkovic, Karin Lakovits, Olga Oskolkova, Bianca Doninger, Andreas Vychytil, Thomas Perkmann, Gernot Schabbauer, Christoph J. Binder, Valery N. Bochkov, John D. Scott, Sylvia Knapp

Metastasis-associated PRL-3 induces EGFR activation and addiction in cancer cells

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Abstract

Authors

Abdul Qader Omer Al-aidaroos, Hiu Fung Yuen, Ke Guo, Shu Dong Zhang, Tae-Hoon Chung, Wee Joo Chng, Qi Zeng

Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia

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Abstract

Authors

Yan Zhou, Matthew J. Gormley, Nathan M. Hunkapiller, Mirhan Kapidzic, Yana Stolyarov, Victoria Feng, Masakazu Nishida, Penelope M. Drake, Katherine Bianco, Fei Wang, Michael T. McMaster, Susan J. Fisher