Cell biology
Abstract
Cyclin D1 is an oncogene frequently overexpressed in human cancers that plays a dual function as cell cycle and transcriptional regulator, although the latter is widely unexplored. Here, we investigated the transcriptional role of cyclin D1 in lymphoid tumor cells with cyclin D1 oncogenic overexpression. Cyclin D1 showed widespread binding to the promoters of most actively transcribed genes and the promoter occupancy positively correlated with the transcriptional output of targeted genes. Despite this association, the overexpression of cyclin D1 in lymphoid cells led to a global transcriptional downmodulation that was proportional to cyclin D1 levels. This cyclin D1 dependent global transcriptional downregulation was associated with a reduced nascent transcription and an accumulation of promoter-proximal paused RNA Polymerase II (Pol II) that colocalized with cyclin D1. Concordantly, cyclin D1 overexpresion promoted an increment of the Poll II pausing index. This transcriptional impairment seems to be mediated by the interaction of cyclin D1 with the transcription machinery. In addition, cyclin D1 overexpression sensitized cells to transcription inhibitors revealing a synthetic lethality interaction that it was also observed in primary MCL cases. This global transcriptional dysregulation expands the oncogenic cyclin D1 functions and places the transcriptional machinery as a potential therapeutic target in cyclin D1 overexpressing tumors.
Authors
Robert Albero, Anna Enjuanes, Santiago Demajo, Giancarlo Castellano, Magda Pinyol, Noelia García, Cristina Capdevila, Guillem Clot, Helena Suárez-Cisneros, Mariko Shimada, Kennosuke Karube, Mónica López-Guerra, Dolors Colomer, Sílvia Beà, José Ignacio Martin-Subero, Elías Campo, Pedro Jares
Abstract
Red blood cells (RBCs) influence rheology, release ADP, ATP and nitric oxide suggesting a role for RBCs in hemostasis and thrombosis. Here we provide evidence for a significant contribution of RBCs to thrombus formation. Anemic mice showed enhanced occlusion times upon injury of the carotid artery. A small population of RBCs was located to platelet thrombi and enhanced platelet activation by a direct cell contact via the FasL-FasR (CD95) pathway known to induce apoptosis. Activation of platelets in the presence of RBCs led to platelet FasL exposure that activated FasR on RBCs responsible for externalization of phosphatidylserine (PS) on the RBC membrane. Inhibition or genetic deletion of either FasL or FasR resulted in reduced PS exposure of RBCs and platelets, decreased thrombin generation and reduced thrombus formation in vitro and protection against arterial thrombosis in vivo. Direct cell contacts of platelets and RBCs via FasL-FasR were shown after ligation of the inferior vena cava (IVC) and in surgical specimens of patients after thrombectomy. In a flow restriction model of the IVC, reduced thrombus formation was observed in FasL–/– mice. Taken together, our data reveal a significant contribution of RBCs to thrombosis by the FasL-FasR pathway.
Authors
Christoph Klatt, Irena Krüger, Saskia Zey, Kim-Jürgen Krott, Martina Spelleken, Nina Sarah Gowert, Alexander Oberhuber, Lena Pfaff, Wiebke Lückstädt, Kerstin Jurk, Martin Schaller, Hadi Al-Hasani, Jürgen Schrader, Steffen Massberg, Konstantin Stark, Hubert Schelzig, Malte Kelm, Margitta Elvers
Abstract
Jumonji D3 (JMJD3) histone demethylase epigenetically regulates development and differentiation, immunity, and tumorigenesis by demethylating a gene repression histone mark, H3K27-me3, but a role for JMJD3 in metabolic regulation has not been described. SIRT1 deacetylase maintains energy balance during fasting by directly activating both hepatic gluconeogenic and mitochondrial fatty acid β-oxidation genes, but the underlying epigenetic and gene-specific mechanisms remain unclear. In this study, JMJD3 was identified unexpectedly as a gene-specific transcriptional partner of SIRT1 and epigenetically activated mitochondrial β-oxidation, but not gluconeogenic, genes during fasting. Mechanistically, JMJD3, together with SIRT1 and the nuclear receptor PPARα, formed a positive autoregulatory loop upon fasting-activated PKA signaling and epigenetically activated β-oxidation–promoting genes, including Fgf21, Cpt1a, and Mcad. Liver-specific downregulation of JMJD3 resulted in intrinsic defects in β-oxidation, which contributed to hepatosteatosis as well as glucose and insulin intolerance. Remarkably, the lipid-lowering effects by JMJD3 or SIRT1 in diet-induced obese mice were mutually interdependent. JMJD3 histone demethylase may serve as an epigenetic drug target for obesity, hepatosteatosis, and type 2 diabetes that allows selective lowering of lipid levels without increasing glucose levels.
Authors
Sunmi Seok, Young-Chae Kim, Sangwon Byun, Sunge Choi, Zhen Xiao, Naoki Iwamori, Yang Zhang, Chaochen Wang, Jian Ma, Kai Ge, Byron Kemper, Jongsook Kim Kemper
Abstract
T cells must migrate in order to encounter antigen-presenting cells (APCs) and to execute their varied functions in immune defense and inflammation. ATP release and autocrine signaling through purinergic receptors contribute to T cell activation at the immune synapse that T cells form with APCs. Here, we show that T cells also require ATP release and purinergic signaling for their migration to APCs. We found that the chemokine SDF-1α triggered mitochondrial ATP production, rapid bursts of ATP release, and increased migration of primary human CD4+ T cells. This process depended on pannexin-1 ATP release channels and autocrine stimulation of P2X4 receptors. SDF-1α stimulation caused localized accumulation of mitochondria with P2X4 receptors near the front of cells, resulting in a feed-forward signaling mechanism that promotes cellular Ca2+ influx and sustains mitochondrial ATP synthesis at levels needed for pseudopod protrusion, T cell polarization, and cell migration. Inhibition of P2X4 receptors blocked the activation and migration of T cells in vitro. In a mouse lung transplant model, P2X4 receptor antagonist treatment prevented the recruitment of T cells into allograft tissue and the rejection of lung transplants. Our findings suggest that P2X4 receptors are therapeutic targets for immunomodulation in transplantation and inflammatory diseases.
Authors
Carola Ledderose, Kaifeng Liu, Yutaka Kondo, Christian J. Slubowski, Thomas Dertnig, Sara Denicoló, Mona Arbab, Johannes Hubner, Kirstin Konrad, Mahtab Fakhari, James A. Lederer, Simon C. Robson, Gary A. Visner, Wolfgang G. Junger
Abstract
Medial vascular calcification, associated with enhanced mortality in chronic kidney disease (CKD), is fostered by osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Here, we describe that serum- and glucocorticoid-inducible kinase 1 (SGK1) was upregulated in VSMCs under calcifying conditions. In primary human aortic VSMCs, overexpression of constitutively active SGK1S422D, but not inactive SGK1K127N, upregulated osteo-/chondrogenic marker expression and activity, effects pointing to increased osteo-/chondrogenic transdifferentiation. SGK1S422D induced nuclear translocation and increased transcriptional activity of NF-κB. Silencing or pharmacological inhibition of IKK abrogated the osteoinductive effects of SGK1S422D. Genetic deficiency, silencing, and pharmacological inhibition of SGK1 dissipated phosphate-induced calcification and osteo-/chondrogenic transdifferentiation of VSMCs. Aortic calcification, stiffness, and osteo-/chondrogenic transdifferentiation in mice following cholecalciferol overload were strongly reduced by genetic knockout or pharmacological inhibition of Sgk1 by EMD638683. Similarly, Sgk1 deficiency blunted vascular calcification in apolipoprotein E–deficient mice after subtotal nephrectomy. Treatment of human aortic smooth muscle cells with serum from uremic patients induced osteo-/chondrogenic transdifferentiation, effects ameliorated by EMD638683. These observations identified SGK1 as a key regulator of vascular calcification. SGK1 promoted vascular calcification, at least partly, via NF-κB activation. Inhibition of SGK1 may, thus, reduce the burden of vascular calcification in CKD.
Authors
Jakob Voelkl, Trang T.D. Luong, Rashad Tuffaha, Katharina Musculus, Tilman Auer, Xiaoming Lian, Christoph Daniel, Daniel Zickler, Beate Boehme, Michael Sacherer, Bernhard Metzler, Dietmar Kuhl, Maik Gollasch, Kerstin Amann, Dominik N. Müller, Burkert Pieske, Florian Lang, Ioana Alesutan
Abstract
Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in β cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient-excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in β cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of β cell function and whole animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.
Authors
Steven J. Millership, Gabriela da Silva Xavier, Agharul I. Choudhury, Sergio Bertazzo, Pauline Chabosseau, Silvia M.A. Pedroni, Elaine E. Irvine, Alex Montoya, Peter Faull, William R. Taylor, Julie Kerr-Conte, Francois Pattou, Jorge Ferrer, Mark Christian, Rosalind M. John, Mathieu Latreille, Ming Liu, Guy A. Rutter, James Scott, Dominic J. Withers
Abstract
Notch 1/2 genes play tumor-suppressing functions in squamous cell carcinoma (SCC), a very common malignancy in skin and internal organs. In contrast with Notch, we show that the transcription factor CSL (also known as RBP-Jκ), a key effector of canonical Notch signaling endowed with intrinsic transcription-repressive functions, plays a tumor-promoting function in SCC development. Expression of this gene decreased in upper epidermal layers and human keratinocytes (HKCs) undergoing differentiation, while it increased in premalignant and malignant SCC lesions from skin, head/neck, and lung. Increased CSL levels enhanced the proliferative potential of HKCs and SCC cells, while silencing of CSL induced growth arrest and apoptosis. In vivo, SCC cells with increased CSL levels gave rise to rapidly expanding tumors, while cells with silenced CSL formed smaller and more differentiated tumors with enhanced inflammatory infiltrate. Global transcriptomic analysis of HKCs and SCC cells with silenced CSL revealed major modulation of apoptotic, cell-cycle, and proinflammatory genes. We also show that the histone demethylase KDM6B is a direct CSL-negative target, with inverse roles of CSL in HKC and SCC proliferative capacity, tumorigenesis, and tumor-associated inflammatory reaction. CSL/KDM6B protein expression could be used as a biomarker of SCC development and indicator of cancer treatment.
Authors
Dania Al Labban, Seung-Hee Jo, Paola Ostano, Chiara Saglietti, Massimo Bongiovanni, Renato Panizzon, G. Paolo Dotto
Abstract
Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs plays an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed NGF-stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.
Authors
Ramesh Singh, Dileep Karri, Hong Shen, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Michael M. Ittmann, Bert W. O’Malley, Ping Yi
Abstract
Tyro3, Axl, Mer (TAM) receptor tyrosine kinases reduce inflammatory, innate immune responses. We demonstrate that tumor-secreted protein S (Pros1), a Mer/Tyro3 ligand, decreased macrophage M1 cytokine expression in vitro and in vivo. In contrast, tumor cells with CRISPR-based deletion of Pros1 failed to inhibit M1 polarization. Tumor cell–associated Pros1 action was abrogated in macrophages from Mer- and Tyro3- but not Axl-KO mice. In addition, several other murine and human tumor cell lines suppressed macrophage M1 cytokine expression induced by IFN-γ and LPS. Investigation of the suppressive pathway demonstrated a role for PTP1b complexing with Mer. Substantiating the role of PTP1b, M1 cytokine suppression was also lost in macrophages from PTP1b-KO mice. Mice bearing Pros1-deficient tumors showed increased innate and adaptive immune infiltration, as well as increased median survival. TAM activation can also inhibit TLR-mediated M1 polarization. Treatment with resiquimod, a TLR7/8 agonist, did not improve survival in mice bearing Pros1-secreting tumors but doubled survival for Pros1-deleted tumors. The tumor-derived Pros1 immune suppressive system, like PD-L1, was cytokine responsive, with IFN-γ inducing Pros1 transcription and secretion. Inhibition of Pros1/TAM interaction represents a potential novel strategy to block tumor-derived immune suppression.
Authors
Eric Ubil, Laura Caskey, Alisha Holtzhausen, Debra Hunter, Charlotte Story, H. Shelton Earp
Abstract
Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast–mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.
Authors
Neha Pincha, Edries Yousaf Hajam, Krithika Badarinath, Surya Prakash Rao Batta, Tafheem Masudi, Rakesh Dey, Peter Andreasen, Toshiaki Kawakami, Rekha Samuel, Renu George, Debashish Danda, Paul Mazhuvanchary Jacob, Colin Jamora
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Copyright © 2018 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)