CCN Proteins: Methods and Protocols

593 Pages·2016·17.643 MB·English

by  Masaharu Takigawa

Methods in
Molecular Biology 1489
Masaharu Takigawa Editor
CCN Proteins
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB , UK
For further volumes:
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CCN Proteins
Methods and Protocols
Edited by
Masaharu Takigawa
Advanced Research Center for Oral and Craniofacial Sciences, Okayama University
Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
Editor
Masaharu Takigawa
Advanced Research Center for Oral and Craniofacial Sciences
Okayama University Dental School/Graduate School of Medicine
Dentistry and Pharmaceutical Sciences
Okayama, Japan
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6428-4 ISBN 978-1-4939-6430-7 (eBook)
DOI 10.1007/978-1-4939-6430-7
Library of Congress Control Number: 2016951254
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Prefa ce
CCN is an acronym that stands for C yr61 (Cysteine-rich 61)/CCN1, C TGF (connective
tissue growth factor)/CCN2, and N ov (nephroblastoma overexpressed)/CCN3, which
are the three founder members of this family. This family now comprises six distinctive
members with the addition of three more members, namely, WISP (Wnt-induced secreted
protein) 1-3/CCN4-6. They are all cysteine-rich secreted proteins and composed of four
distinct modules connected in tandem, i.e., IGF binding protein-like, von Willebrand type
C, thrombospondin type 1 repeat, and C-terminal modules, except for CCN5, which lacks
the CT module. They are known to play roles in fundamental biological processes by
serving as multifunctional growth and differentiation regulators that interact physically
with various cytokines, extracellular matrices, and cell membrane proteins in various micro-
environments. Abnormal regulation of these proteins is also involved in various diseases
such as fi brosis and malignancy.
This volume will be valuable for all those interested in CCN proteins and serve as a
valuable manual for cutting-edge methodologies and practical tips to overcome any obsta-
cles with experimentation pertaining to the chemistry, biology, physiology, pathology, and
pharmacology of CCN proteins in the context of basic, medical, and dental science’s. We
also believe that this comprehensive guide to methods and protocols for CCN research
utilizing both basic and state-of- the-art techniques will be a valuable resource for a wide
audience, ranging from the experienced CCN researchers looking for new approaches to
junior graduate students taking their fi rst steps into the fi eld of CCN research.
Okayama, Japan Masaharu Takigawa
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
1 T he CCN Proteins: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Masaharu Takigawa
PART I BASIC METHODS AND PROTOCOLS
2 G ene Expression Analysis of CCN Proteins: Whole-Mount
In Situ Hybridization of C cn2 in Developing Calcified Tissues . . . . . . . . . . . . 1 1
Tomoichiro Yamaai and Masaharu Takigawa
3 E xpression of CCN Genes and Proteins in Human Skin:
Methods and Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Yilei Cui , S arah Campbell , and Gary J. Fisher
4 A nalysis of Expression of CCN Family Genes
in Skeletal Tissue-Derived Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3
Harumi Kawaki , S atoshi Kubota , and Masaharu Takigawa
5 W estern Blotting Analysis of CCN Proteins
in Calcified Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3
Harumi Kawaki , S atoshi Kubota , and Masaharu Takigawa
6 I mmunohistochemical Analysis of CCN Proteins in Calcified Tissues. . . . . . . . 5 3
Harumi Kawaki , S atoshi Kubota , and Masaharu Takigawa
7 A nalysis of CCN Expression by Immunofluorescence on Skin Cells, Skin,
and Reconstructed Epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Muriel Cario-Andre
8 P roduction of Recombinant CCN2 Protein in E scherichia coli . . . . . . . . . . . . . 77
Eriko Aoyama , T akako Hattori , S atoshi Kubota , and Masaharu Takigawa
9 P roduction of Recombinant CCN Proteins by B revibacillus choshinensis . . . . . . 85
Hiroshi Hanagata and Makoto Mizukami
10 P roduction of Recombinant CCN2 Protein by Mammalian Cells. . . . . . . . . . . 95
Takashi Nishida , S atoshi Kubota , and Masaharu Takigawa
11 I n Vitro Transfection with and Expression of CCN Family of Genes . . . . . . . . 1 07
Danilo Janune and Masaharu Takigawa
12 P reparation of Module-Specific Antibodies Against CCN Family Members . . . 1 15
Satoshi Kubota and Masaharu Takigawa
1 3 E LISA of CCN Family Proteins in Body Fluids Including Serum and Plasma. . 1 27
Satoshi Kubota , H arumi Kawaki , and Masaharu Takigawa
vii
viii Contents
14 A nalysis of Signaling Pathways Activated by CCN Proteins . . . . . . . . . . . . . . . 1 39
Harumi Kawaki , S atoshi Kubota , and Masaharu Takigawa
15 P rotocols for Screening for Binding Partners of CCN Proteins:
Yeast Two-Hybrid System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Mitsuhiro Hoshijima , T akako Hattori , and Masaharu Takigawa
16 P rotocols for Screening Peptide Motifs Binding to CCN Family Proteins . . . . 1 55
Satoshi Kubota , H arumi Kawaki , and Masaharu Takigawa
17 E valuation of Molecular Interaction between CCN2 Protein
and Its Binding Partners by Surface Plasmon Resonance (SPR). . . . . . . . . . . . 169
Eriko Aoyama and Masaharu Takigawa
18 P romoter Analyses of CCN Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Takanori Eguchi , S atoshi Kubota , and Masaharu Takigawa
19 A nalysis of Posttranscriptional Regulation of CCN Genes . . . . . . . . . . . . . . . . 1 87
S eiji Kondo , S atoshi Kubota , and Masaharu Takigawa
20 P rotein Imaging of CCN2 and CCN3 in Living Cells . . . . . . . . . . . . . . . . . . . 2 11
Takako Hattori , M itsuhiro Hoshijima , and Masaharu Takigawa
PART II FUNCTIONAL ANALYSIS
21 C ell Biological Assays for Measuring Chondrogenic Activities
of CCN2 Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Takashi Nishida , S atoshi Kubota , and Masaharu Takigawa
22 C ell Biological Assays for Measuring Angiogenic Activities
of CCN Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Tsuyoshi Shimo and Masaharu Takigawa
23 C ell Biological Assays for Measuring Odontogenic Activities
of CCN Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Koichiro Muromachi , H iroshi Sugiya , and Nobuyuki Tani-Ishii
24 S eparation and Enrichment of Hematopoietic Stem Cells for CCN Studies . . . 2 61
Lisa J. Crawford and Alexandra E. Irvine
25 I n Vivo Evaluation of Cartilage Regenerative Effects of CCN2 Protein . . . . . . 273
Takashi Nishida , S atoshi Kubota , and Masaharu Takigawa
26 G ene Expression Analysis of CCN Protein in Bone
Under Mechanical Stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Teruko Takano-Yamamoto , T omohiro Fukunaga , and Nobuo Takeshita
27 T he Bone Regeneration Model and Primary Osteoblastic Cell Culture
Used in the Analysis of C CN3 Transgenic and Knockout Mice . . . . . . . . . . . . 3 09
Kei Sakamoto , Y uki Matsushita , T okutaro Minamizato , Y uko Katsuki ,
Ken-ichi Katsube , and Akira Yamaguchi
28 D esign and Analysis of CCN Gene Activity Using CCN Knockout Mice
Containing LacZ Reporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Jie Jiang , Z hengshan Hu , and Karen M. Lyons
Contents ix
29 A nalysis of CCN4 Function in Osteogenic and Osteoclastic Cells
Using Gain and Loss of Function Approaches. . . . . . . . . . . . . . . . . . . . . . . . . 347
Azusa Maeda , M arian Young , and Mitsuaki Ono
30 C onstruction and Analysis of an Allelic Series of C cn1 Knockin Mice. . . . . . . . 361
Ricardo I. Monzon , K i-Hyun Kim , and Lester F. Lau
31 P roduction and Analysis of Conditional KO Mice of CCN2 in Kidney. . . . . . . 3 77
Naohiro Toda , H ideki Yokoi , K iyoshi Mori , and Masashi Mukoyama
32 G eneration and Analysis of Cartilage-Specific CCN2 Overexpression
in Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 91
Takako Hattori , S hinsuke Itoh , and Masaharu Takigawa
33 A nalysis of Transcytosis of CCN2 by Chondrocytes. . . . . . . . . . . . . . . . . . . . . 4 05
Kazumi Kawata , S atoshi K ubota , and Masaharu Takigawa
PART III PATHOPHYSIOLOGY
34 C CN2 in Skin Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 17
Andrew Leask
35 S tudying the CCN Proteins in Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 23
James Hutchenreuther , A ndrew Leask , and Katherine Thompson
36 A nalysis of Pathological Activities of CCN Proteins in Fibrotic Diseases:
Kidney Fibrosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Hideki Yokoi and Masashi Mukoyama
37 A nalysis of Pathological Activities of CCN Proteins in Fibrotic Diseases:
Liver Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 45
Li Chen and David R. Brigstock
38 C ellular or Exosomal microRNAs Associated with CCN Gene
Expression in Liver Fibrosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Li Chen and David R. Brigstock
39 T GF-β1- and CCN2-Stimulated Sirius Red Assay for Collagen
Accumulation in Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
Philip C. Trackman , D ebashree Saxena , and Manish V. Bais
40 C CN Detection of Cancer Tissues by Immunohistochemistry Staining . . . . . . 4 87
Cheng-Chi Chang , Y ue-Ju Li , and Min-Liang Kuo
41 D etection of CCN1 and CCN5 mRNA in Human Cancer Samples
Using a Modified In Situ Hybridization Technique. . . . . . . . . . . . . . . . . . . . . 495
Priyanka Ghosh , S nigdha Banerjee , G argi Maity , A rchana De ,
and Sushanta K. Banerjee
42 A nalysis of Pathological Activities of CCN Proteins in Bone Metastasis . . . . . . 5 05
Tsuyoshi Shimo , N orie Yoshioka , M asaharu Takigawa , and Akira Sasaki
43 A nalysis of Pathological Activities of CCN2/CTGF in Muscle Dystrophy . . . . 5 13
María José Acuña and Enrique Brandan