See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/301541030 TGF-β Signaling Methods and Protocols Book in Methods in molecular biology (Clifton, N.J.) · June 2015 DOI: 10.1007/978-1-4939-2966-5 CITATION READS 1 386 3 authors, including: Pinglong Xu Xia Lin Zhejiang University Baylor College of Medicine 70 PUBLICATIONS 2,567 CITATIONS 96 PUBLICATIONS 5,287 CITATIONS SEE PROFILE All content following this page was uploaded by Pinglong Xu on 21 June 2017. The user has requested enhancement of the downloaded file. SEE PROFILE Methods in Molecular Biology 1344 Xin-Hua Feng Pinglong Xu Xia Lin Editors TGF-β Signaling Methods and Protocols METHODS IN MOLECULAR BIOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 [email protected] qwwwwwww [email protected] TGF-β Signaling Methods and Protocols Edited by Xin-Hua Feng Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang Province, China; Departments of Surgery, and Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas, USA Pinglong Xu Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang Proviince, China Xia Lin Department of Surgery, Baylor College of Medicine, Houston, USA [email protected] Editors Xin-Hua Feng Life Sciences Institute and Innovation Center for Cell Signaling Network Zhejiang University, Hangzhou Zhejiang Province, China Pinglong Xu Life Sciences Institute Zhejiang University Hangzhou, Zhejiang Proviince, China Xia Lin Department of Surgery Baylor College of Medicine Houston, USA Departments of Surgery and Molecular & Cellular Biology Baylor College of Medicine Houston, Texas, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2965-8 ISBN 978-1-4939-2966-5 (eBook) DOI 10.1007/978-1-4939-2966-5 Library of Congress Control Number: 2015951954 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) [email protected] Preface Cells that respond to environmental cues through the complex and dynamic network of signaling pathways maintain a critical balance between cellular proliferation, differentiation, and death. Since the original discovery of TGF-β about more than 30 years ago, the molecule only represents a prototype of a large family that consists of at least 33 members encoded by the human genome. The TGF-β superfamily members are secreted proteins, including TGF-β, activins, bone morphogenetic proteins, growth/differentiation factors, and Müllerian inhibiting substance, and can regulate many developmental processes in a range of organisms from worms to humans. At cellular level, they control a wide range of cellular functions such as proliferation, death, differentiation, and other functions in many cell types. Dysfunctions of the TGF-β family members often result in the pathogenesis of cancer, autoimmune diseases, diabetes, heart disease, hereditary hemorrhagic telangiectasia, Marfan syndrome, Vascular Ehlers-Danlos syndrome, Loeys–Dietz syndrome, and neurodegenerative diseases. The characterization of the TGF-β family underscores its importance in physiological and pathophysiological functions. The research on TGF-β biology, including its regulation, signaling, and physiological functions, has developed rapidly into a large field with thousands of publications per year. In the last 20 years, the components of the canonical signaling pathways—the TGF-β receptors and downstream intracellular effectors Smad proteins—have been identified, and the concept for context-dependent TGF-β actions has been well established. Many conventional methodologies or state-of-the-art technologies have been employed to elucidate how the TGF-β pathways are regulated and what functions they have in the context from single cells to complexed tissues to the whole organism. The rapidly evolving nature of TGF-β signaling research also necessitates a continuous updating of methods used. TGF-β Signaling: Methods and Protocols brings together a comprehensive collection of methods and techniques in TGF-β signaling research that are scientifically grounded within the cancer and development fields. Thus, this volume grows out of the necessity that a comprehensive method book covering biochemical, molecular, and biological description of TGF-β ligands, receptors, and intracellular events is needed for researchers who are already in the TGF-β field and for those who wish to enter the field. This volume provides the reader with up-to-date information in this continuing evolving field and attempts to take the reader into the exciting realm of TGF-β from the basic principles to the practical applications. All the chapters are provided by leading researchers in the TGF-β field. The first chapter by Budi and Derynck gives a basic introduction of TGF-β receptor signaling at the cell surface. Subsequent chapters are generally concerned with methods and techniques for the investigation of TGF-β signaling mechanism including receptors, intracellular kinases, microRNA, epigenetic regulation, post-translational regulations, non-Smad pathway; the physiological implications including those in epithelial-mesenchymal transition, endothelial cells, adipogenesis, Th differentiation, stem v [email protected] vi Preface cell, bone remodeling, ovary, zebrafish development, and frog animal capping; and the methodologies including metastasis imaging, 3D morphogenesis, membrane receptor quantification, conditional knockout, bone remodeling, kinase and phosphatase assays, BiFC interaction assays, and genome-wide siRNA screen. This book would not be possible were it not for all the contributors who devoted their precious time and considerable energy to bring this volume into reality and provide such clear and detailed accounts of their experimental protocols and useful hints. We are greatly indebted to them for their excellent contributions and for their patience in dealing with the editors. I also wish to thank Dr. P.J. Higgins and their overall help and patience in keeping the book on track. We hope this volume will prove valuable to all researchers, rookie or veteran, in the TGF-β signaling field and serve as a useful reference for many years to come. Hangzhou, China and Houston, TX, USA Hangzhou, China Houston, TX, USA [email protected] Xin-Hua Feng Pinglong Xu Xia Lin Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v ix 1 Regulation of TGF-β Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erine H. Budi, Jian Xu, and Rik Derynck 2 Determining TGF-β Receptor Levels in the Cell Membrane . . . . . . . . . . . . . . Long Zhang, Fangfang Zhou, Maarten van Dinther, and Peter ten Dijke 3 Posttranslational Modifications of TGF-β Receptors . . . . . . . . . . . . . . . . . . . . Xiaohua Yan and Ye-Guang Chen 4 Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-β Superfamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tao Huang and Andrew P. Hinck 5 Phosphorylation of Smads by Intracellular Kinases. . . . . . . . . . . . . . . . . . . . . . Fang Liu and Isao Matsuura 6 Analysis of Smad Phosphatase Activity In Vitro . . . . . . . . . . . . . . . . . . . . . . . . Tao Shen, Lan Qin, and Xia Lin 7 Three-dimensional Mammary Epithelial Cell Morphogenesis Model for Analysis of TGFß Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Juliet Rashidian and Kunxin Luo 8 TGF-β Signaling in Stem Cell Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wenlin Li, Wanguo Wei, and Sheng Ding 9 Analysis of Epithelial–Mesenchymal Transition Induced by Transforming Growth Factor β . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ulrich Valcourt, Jonathon Carthy, Yukari Okita, Lindsay Alcaraz, Mitsuyasu Kato, Sylvie Thuault, Laurent Bartholin, and Aristidis Moustakas 10 In Vitro Th Differentiation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takashi Sekiya and Akihiko Yoshimura 11 Interrogating TGF-β Function and Regulation in Endothelial Cells. . . . . . . . . J.A. Maring, L.A. van Meeteren, M.J. Goumans, and Peter ten Dijke 12 Isolation and Manipulation of Adipogenic Cells to Assess TGF-β Superfamily Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maria Namwanje, Juan C. Bournat, and Chester W. Brown 13 Imaging TGFβ Signaling in Mouse Models of Cancer Metastasis . . . . . . . . . . . Yibin Kang 14 Generation and Characterization of Smad7 Conditional Knockout Mice . . . . . Yi Pan and Yan Chen 15 Monitoring Smad Activity In Vivo Using the Xenopus Model System . . . . . . . Marco Montagner, Graziano Martello, and Stefano Piccolo 1 vii [email protected] 35 49 63 93 111 121 137 147 183 193 205 219 233 245 viii Contents 16 Animal Cap Assay for TGF-β Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chenbei Chang 17 Detection of Smad Signaling in Zebrafish Embryos . . . . . . . . . . . . . . . . . . . . . Xingfeng Liu, Qiang Wang, and Anming Meng 18 Role of TGF-β Signaling in Coupling Bone Remodeling . . . . . . . . . . . . . . . . . Janet L. Crane, Lingling Xian, and Xu Cao 19 Studying the Functions of TGF-β Signaling in the Ovary. . . . . . . . . . . . . . . . . Chao Yu, Jian-Jie Zhou, and Heng-Yu Fan 20 Quantitative Real-Time PCR Analysis of MicroRNAs and Their Precursors Regulated by TGF-β Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hara Kang and Akiko Hata 21 TGF-β-Regulated MicroRNAs and Their Function in Cancer Biology . . . . . . . Pengyuan Yang, Yun Zhang, Geoffrey J. Markowitz, Xing Guo, and Xiao-Fan Wang 22 Epigenomic Regulation of Smad1 Signaling During Cellular Senescence Induced by Ras Activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atsushi Kaneda, Aya Nonaka, Takanori Fujita, Ryota Yamanaka, Mai Fujimoto, Kohei Miyazono, and Hiroyuki Aburatani 23 The Role of Ubiquitination to Determine Non-Smad Signaling Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shyam Kumar Gudey and Marene Landström 24 Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xiaochu Chen and Lan Xu 25 Measuring TGF-β Ligand Dynamics in Culture Medium . . . . . . . . . . . . . . . . . Zipei Feng, Zhike Zi, and Xuedong Liu Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [email protected] 261 275 287 301 313 325 341 355 365 379 391 Contributors HIROYUKI ABURATANI • Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan LINDSAY ALCARAZ • Inserm U1052, Centre de Recherche en Cancérologie de Lyon, Lyon, France; CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, Lyon, France; Université de Lyon, Lyon, France; Université Lyon 1, Lyon, France; Centre Léon Bérard, Lyon, France LAURENT BARTHOLIN • Inserm U1052, Centre de Recherche en Cancérologie de Lyon, Lyon, France; CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, Lyon, France; Université de Lyon, Lyon, France; Université Lyon 1, Lyon, France; Centre Léon Bérard, Lyon, France JUAN C. BOURNAT • Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA CHESTER W. BROWN • Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA; Texas Children’s Hospital, Houston, TX, USA ERINE H. BUDI • Department of Cell and Tissue Biology, Broad Center, University of California, San Francisco, CA, USA XU CAO • Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA JONATHON CARTHY • Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden CHENBEI CHANG • Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA XIAOCHU CHEN • Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA YAN CHEN • Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China YE-GUANG CHEN • State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China JANET L. CRANE • Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA RIK DERYNCK • Department of Cell and Tissue Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Programs in Cell Biology, and Developmental and Stem Cell Biology, University of California, San Francisco, CA, USA PETER TEN DIJKE • Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands SHENG DING • Department of Pharmaceutical Chemistry, Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, CA, USA ix [email protected] x Contributors MAARTEN VAN DINTHER • Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands HENG-YU FAN • Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, China ZIPEI FENG • Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO, USA; Otto-Warburg Laboratory, Max Planck Institute for Molecular Genetics, Berlin, Germany MAI FUJIMOTO • Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan; Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan TAKANORI FUJITA • Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan M.J. GOUMANS • Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands SHYAM KUMAR GUDEY • Department of Medical Biosciences, Umeå University, Umeå, Sweden XING GUO • Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA AKIKO HATA • Cardiovascular Research Institute, University of California, San Francisco, CA, USA ANDREW P. HINCK • Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA TAO HUANG • Protein Chemistry, Novo Nordisk Research Center China, Beijing, China ATSUSHI KANEDA • Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan; Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan; CREST, Japan Science and Technology Agency, Saitama, Japan YIBIN KANG • Lewis Thomas Laboratory 255, Department of Molecular Biology, Princeton University, Princeton, NJ, USA HARA KANG • Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, Republic of Korea; Cardiovascular Research Institute, University of California, San Francisco, CA, USA MITSUYASU KATO • Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan MARENE LANDSTRÖM • Department of Medical Biosciences, Umeå University, Umeå, Sweden WENLIN LI • Department of Cell Biology, Second Military Medical University, Shanghai, China XIA LIN • Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA XUEDONG LIU • Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO, USA FANG LIU • Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway, NJ, USA; Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA; Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA [email protected] Contributors xi XINGFENG LIU • State-key Laboratory of Biomembrane and Membrane Engineering, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China KUNXIN LUO, PH.D. • Department of Molecular and Cell Biology (MCB), University of California, Berkeley, CA, USA J.A. MARING • Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands GEOFFREY J. MARKOWITZ • Department of Pharmacology and Cancer Biology, Duke University Medical Center, Duke University, Durham, NC, USA GRAZIANO MARTELLO • Department of Molecular Medicine, University of Padua School of Medicine, Padua, Italy ISAO MATSUURA • Division of Molecular Genomics and Medicine, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan L.A. VAN MEETEREN • Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands ANMING MENG • State-key Laboratory of Biomembrane and Membrane Engineering, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China; Institute of Zoology, Chinese Academy of Sciences, Beijing, China KOHEI MIYAZONO • Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan MARCO MONTAGNER • Department of Molecular Medicine, University of Padua School of Medicine, Padua, Italy ARISTIDIS MOUSTAKAS • Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden MARIA NAMWANJE • Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA AYA NONAKA • Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan YUKARI OKITA • Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan YI PAN • Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China STEFANO PICCOLO • Department of Molecular Medicine, University of Padua School of Medicine, Padua, Italy LAN QIN • Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA JULIET RASHIDIAN • Department of Molecular and Cell Biology (MCB), University of California, Berkeley, CA, USA TAKASHI SEKIYA • Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan TAO SHEN • Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA; Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA [email protected] xii Contributors SYLVIE THUAULT • Faculty of Pharmacy, INSERM UMR 911 CRO2, Marseille, France ULRICH VALCOURT • Inserm U1052, Centre de Recherche en Cancérologie de Lyon, Lyon, France; ; CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, France; Université de Lyon, Lyon, France; Université Lyon 1, Lyon, France; Centre Léon Bérard, Lyon, France QIANG WANG • Institute of Zoology, Chinese Academy of Sciences, Beijing, China XIAO-FAN WANG • Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA WANGUO WEI • Stem Cell and Regenerative Medicine Center, Chinese Academy of Science, Shanghai Advanced Research Institute, Shanghai, China LINGLING XIAN • Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA JIAN XU • Center for Craniofacial Molecular Biology, Ostrow School of Dentistry of USC, University of Southern California, Los Angeles, CA, USA LAN XU • Blueprint Medicines, Cambridge, MA, USA PINGLONG XU • Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, China RYOTA YAMANAKA • Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan XIAOHUA YAN • State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China PENGYUAN YANG • CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China AKIHIKO YOSHIMURA • Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan CHAO YU • Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, China LONG ZHANG • Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, China YUN ZHANG • Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA FANGFANG ZHOU • Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China JIAN-JIE ZHOU • Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, China ZHIKE ZI • Otto-Warburg Laboratory, Max Planck Institute for Molecular Genetics, Berlin, Germany [email protected] View publication stats