CCN proteins in health and disease (eBook)

Contents 5
Contributors 8
1 A Recent Breakthrough in the CCN Field: Functional Interactions Between CCN2 and CCN3 are Uncovered 13
1.1 Introduction 13
References 15
2 Report on the Fifth International Workshop on the CCN Family of Genes 17
References 24
3 Asking the Right Questions: What Can the Structure of the CCN Protein Domains Tell Us? 26
3.1 Introduction 26
3.2 The Building Blocks of the CCN Proteins 27
3.3 The IGFBP Domain 29
3.4 The von Willebrand Factor C Repeat Domain 33
3.5 The Thrombospondin Type 1 (TSP) Domain 37
3.6 The Cysteine Knot C-Terminal (CT) Domain 41
3.7 Conclusions 44
References 46
4 Nucleophosmin/B23: A Multifunctional Regulator that Determines the Fate of CCN2 mRNA 52
4.1 Post-transcriptional Regulation of Chicken ccn2 Via an RNA Element Interacting with Its Protein Counterpart 53
4.2 Identification of Nucleophosmin (NPM/B23) as a Counterpart of Element 3 -100/50 54
4.3 NPM, a Multifunctional Regulatory Molecule Shuttling Between the Nucleus and Cytoplasm 57
4.4 Novel Function of NPM as a Specific mRNA Destabilizer in Chondrocytes 58
4.5 Profound Role of the 3'-UTR-Mediated Post-transcriptional Regulation of ccn2 61
References 62
5 The CCN Genes as the Master Regulators of Angiogenesis, Vasculogenesis, Fibrogenesis and Cell Differentiation/Fate Specification in Mechanical Force-Driven Developmental Processes and Pathological Events 67
5.1 Introduction 68
5.2 Mechanical Forces as a Key Epigenetic Regulator of Physiological and Developmental Processes 69
5.3 Mechanical Forces as a Regulator of Pathological Processes 70
5.4 Adaptive Response and Mechanotransduction Mechanisms 71
5.5 Differential Expression of the CCN Proteins in Mechanical Stress-Related Diseases 73
5.6 Differential Expression of the CCN Proteins During Tissue Development 74
5.7 Role of CCN1 and CCN2 in Mechanotransduction 75
5.8 Regulatory Mechanisms of CCN1 and CCN2 Gene Expression by Mechanical Forces 78
5.9 Conclusion 80
References 81
6 A Monoclonal Antibody Approach to CCN5 Domain Analysis 87
6.1 Introduction 88
6.2 Materials and Methods 89
6.2.1 Cell Culture 89
6.2.2 Construction of CCN5 Expression Plasmids 90
6.2.3 Bacterial Expression of GST-Fusion Proteins 92
6.2.4 Production and Screening of Anti-CCN5 Positive Hybridomas 92
6.2.5 Plasmid Transfection and Immunofluorescence Microscopy 92
6.2.6 Purification of Monoclonal Antibody 93
6.2.7 Western Blot Analysis 93
6.2.8 Immunoprecipitation 94
6.2.9 Immunohistochemistry 94
6.3 Results 95
6.3.1 Production and Screening of Anti-CCN5 Monoclonal Antibodies 95
6.3.2 All Identified Monoclonal Antibodies Are Against the V-Domain of CCN5 96
6.3.3 Antibody 22H10 Characterization Demonstrates That It Recognizes the V-Domain 96
6.3.4 22H10 Can Immunoprecipitate Recombinant and Endogenous CCN5 Protein 97
6.3.5 22H10 Is Specific for Mouse and Rat CCN5 Without Cross-Reacting with Human CCN5 99
6.3.6 22H10 Detects Endogenous Mouse CCN5 in Tissues 101
6.4 Discussion 102
References 103
7 Matricellular Protein CCN2 Produced by Tubular Epithelial Cells Plays a Pivotal Role in Renal Fibrogenesis 106
7.1 Introduction 107
7.2 CCN2 Localization in the Fibrous Kidney 107
7.3 Roles of CCN2 Produced by Tubular Epithelial Cells 108
7.4 Molecular Regulation of CCN2 Gene Transcription in Tubular Epithelial Cells 109
7.5 Perspectives 110
References 111
8 Cooperative Regulation of Cell Proliferation and Differentiation by CCN2 and CCN3 113
8.1 Introduction 113
8.2 Effects of the CCN2 Deletion on the Differentiated Phenotype of Cartilage and Expression of CCN3 in Cartilage 114
8.3 Effect of Exogenous Recombinant CCN2 (rCCN2) and Recombinant CCN3 (rCCN3) on the Proliferation and Differentiation of ccn2-KO and Wild Type Chondrocytes 115
8.4 Effect of Exogenous Recombinant CCN2-3 on ccn2-3 Genes and Chondrocyte Differentiation-Associated Genes 115
8.5 Concluding Comments 116
References 116
9 The Role of CCN3 in Mesenchymal Stem Cells 118
9.1 Introduction 118
9.1.1 Chicken CCN3 Expression in Embryo 119
9.1.2 CCN3 and Mouse Bone Marrow Stromal Cells 120
9.1.3 CCN3 and Osteogenic Ability of Bone Marrow Derived Mesenchymal Stem Cells 121
9.1.4 Possible Role of CCN3 in Mesenchymal Stem Cells 123
References 125
10 Role of Connective Tissue Growth Factor in Cardiac Fibrosis 127
10.1 Introduction 127
10.2 CTGF (CCN2) in the Heart 128
10.3 Mechanism of Cardiac Fibrosis 128
10.4 Role of CTGF in Cardiac Remodeling 130
10.5 Novel Molecular Mechanisms Regulating CTGF 131
10.6 Future Directions 133
References 134
11 Gene Expression of CCN Family Members in Young and Aged Human Skin In Vivo 139
11.1 Introductions 140
11.2 Methods 140
11.2.1 Study Populations and Procurement of Human Tissue Samples 140
11.2.2 RNA Isolation and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) 141
11.2.3 Statistical Analysis 141
11.3 Results 141
11.3.1 Expression of CCN3-6 Genes in Human Skin In Vivo 141
11.3.2 Expression of CCN3-6 Genes in Young and Aged Human Skin In Vivo 142
11.4 Discussion 143
References 144
12 Global Expression Profiling Reveals a Role for CTGF/CCN2 in Lactogenic Differentiation of Mouse Mammary Epithelial Cells 147
12.1 Introduction 148
12.2 Materials and Methods 149
12.3 Results 152
12.4 Discussion 163
References 165
13 CCN3 (NOV): A Negative Regulator of CCN2 (CTGF) Activity and an Endogenous Inhibitor of Fibrosis in Experimental Diabetic Nephropathy 169
13.1 Introduction 170
13.2 Methods 171
13.2.1 Reagents 171
13.2.2 Cell Culture 171
13.2.3 Generation of a Stable Cell Line Over-Expressing Human CCN3 172
13.2.4 Transfection of the col1 Promoter Construct and Promoter Analysis by Luciferase Measurement 172
13.2.5 RNA Extraction, Reverse Transcription and PCR 172
13.2.6 ELISA 173
13.2.7 Western Blotting 174
13.2.8 Immunohistochemistry 174
13.2.9 Animal Experiments 174
13.2.10 Statistics 175
13.3 Results 175
13.3.1 Effect of CCN3 Treatment on TGF-ß Stimulated CCN2 and COL1 Production 175
13.3.2 CCN3 Expression in MC and Its Regulation by TGF-ß 177
13.3.3 Effect of Transfection with Over-Expression of the Human CCN3 179
13.3.4 Smad Signaling and CCN3 180
13.3.5 CCN3 Expression In Vivo in Diabetic Renal Fibrosis 180
13.4 Discussion 182
13.5 Competing Interests Statement 185
References 185
14 Inhibitors of Connective Tissue Growth Factor (CCN2)-Mediated Fibrogenesis: Underlying Mechanisms and Prospects for Anti-fibrotic Therapy 188
14.1 Introduction 189
14.2 Pharmacological Inhibitors 190
14.2.1 Tumor Necrosis Factor Alpha (TNF-a) 190
14.2.2 Prostaglandins (PG) 191
14.2.3 Peroxisome Proliferator-Activated Receptor-Gamma (PPAR-.) Agonists 192
14.2.4 Statins 192
14.3 Neutralizing Antibodies That Block CCN2 Action 193
14.4 In Vitro Use of CCN2 Antisense Oligonucleotides or siRNA 194
14.5 In Vivo Use of CCN2 Antisense Oligonucleotides or siRNA 195
14.6 New Data from the Authors Laboratory: Targeted CCN2 mRNA Silencing In Vivo 196
14.7 Conclusions 201
References 201
15 CCN3 Promotes Melanoma Progression by Regulating Integrin Expression, Adhesion and Apoptosis Induced by Cytotoxic Drugs 210
15.1 Introduction 211
15.2 CCN3 Protein Expression and Localizationin Melanoma Cells 212
15.3 Characterization of the Biological Activity of CCN3 in Melanoma Cells 213
15.4 CCN3 Promotes Visceral Metastatic Dissemination of Melanoma Cells 215
15.5 Conclusions 215
References 216
16 CCN3: A NOVel Growth Factor in Leukaemia 217
16.1 Introduction 217
16.2 BCR-ABL Expression Downregulates CCN3 in CML 219
16.3 CCN3 and Cell Signalling Pathways in CML 221
16.4 CCN3 and Normal Haematopoiesis 222
16.5 Conclusion 223
References 225
17 Prognostic Relevance of CCN3 in Bone Sarcomas 226
17.1 Introduction 226
17.2 Materials and Methods 228
17.2.1 Clinical Data 228
17.2.2 Cell Lines 229
17.2.3 Immunohistochemistry 229
17.2.4 Western Blotting 230
17.2.5 RNA Extraction and cDNA Synthesis 230
17.2.6 Microarray Analysis 231
17.2.7 Osteoblastic Differentiation in Human Mesenchymal Stem Cells and Osteosarcoma Cell Lines 231
17.2.8 Statistics 231
17.3 Results 231
17.3.1 Expression of CCN3 During Osteoblastic Differentiation 231
17.3.2 CCN3 Protein Expression in Ewing's Sarcoma Cell Lines 235
17.3.3 Detection of CCN3 Protein in EWS Tissue Arrays 236
17.3.4 Prognostic Value of CCN3 in Bone Sarcomas 237
17.3.5 Osteosarcomas 237
17.3.6 Ewing's Tumors 239
17.4 Discussion 240
References 243
18 CCN6 Regulates Breast Cancer Growth and Invasion Through Modulation of IGF Signaling and Epithelial to Mesenchymal Transition 247
18.1 Introduction 248
18.2 CCN6 Structure 248
18.3 CCN6 Regulates Breast Cancer Growth by Interfering with Extracellular Growth Factors 249
18.4 CCN6 Influences Breast Cancer Invasion by Regulating EMT 251
References 254
19 Novel Transcriptional Regulation of CCN2/CTGF by Nuclear Translocation of MMP3 256
19.1 Introduction 257
19.2 Nuclear Localization and DNA-Binding of MMP3 259
19.3 Transcription Factor-Like Function of MMP3 for CTGF/CCN2 Gene Expression 260
19.4 The Structural Basis of MMP3 for CCN2 Regulation 260
19.5 Nuclear Localization Signals (NLS) and Nuclear Translocation of MMP3 260
19.6 MMP: Matrix-Making Potential Via CCN2/CTGF Regulation 261
References 262
Fifth International Workshop on the CCN Family of Genes: Abstracts and Posters October 1822, 2008 266
Matricellular Proteins Regulate Cell Function: Studies of Thrombospondins 1 and 2 266
The CCN Family of Proteins: Structure–Function Relationships 267
Nucleophosmin/B23: A Multifunctional Regulator That Determines the Fate of CCN2 mRNA 268
Regulation of Chondrocytic Phenotype by micro RNA 18A: Involvement of CCN/CTGF as a Major Target Gene 269
CTGF/CCN-2 Expression Induced by TGF-b Is Modulated Through a Mechanism Dependent of Decorin and LRP-1 270
Identification of SOX9 Binding Site in CCN2 (CTGF) Gene by Use of ChIP on Chip Analysis 271
Mechanical Regulation of the CYR61/CCN1 Gene Requires the Combined Activity of the Myocardin-Related Transcription Factor (MRTF) – A and P300/CBP in Smooth Muscle Cells 272
Periostin Is Expressed in Human Skin and Is Regulated by Mechanical Strain 273
Functional and Biochemical Assessment of CCN5 Targeted to the Nucleus 274
Poly (ADP-Ribose) Polymerase-1 (PARP-1) and PARP-1 Binding Element (PBE) Enhance Murine CCN2 Gene Transcription in Proximal Tubular Epithelial Cells (PTEC) 275
Roles of CCN2 in Skeletal Growth and Regeneration – Requirement for Both Endochondral and Intramembranous Bone Formation 276
Skeletal Phenotype in Transgenic Mice Over-expressing CTGF in Cells of the Osteoblast Lineage 278
CCN1 and CCN2 Are Essential for Chondrogenesis 279
CCN3 and Notch/BMP Signals 280
FAK/SRC Suppresses Early Chondrogenesis: Central Role of CCN2 281
CTGF/CCN-2 is Produced by Non-chondrodystrophic Canine Intervertebral Disc-Derived Notochordal Cells and Upregulate Nucleus Pulposus Aggrecan Gene Expression 282
CCN2 Is Required for Vascular Remodeling In Vivo 283
KLF15 Regulates the Cardiac Response to Stress 284
The Role of CTGF in Paediatric Acute Lymphoblastic Leukaemia 285
Expression of CCN Proteins in Normal Human Skin In Vivo and After Wound Healing 286
Connective Tissue Growth Factor Promoter Activity in Normal and Wounded Skin 287
Ultraviolet Irradiation Induces CYR61/CCN1, a Novel Mediator of Collagen Homeostasis, Via Activation of Transcription Factor AP-1 in Human Skin Fibroblasts 288
The Interaction of ß-Catenin with Extra-cellular Matrix Components in Dermal Fibroblasts During Wound Healing 289
The Role of the Matricellular Protein DEL1 in Bone Fracture Healing 290
Connective Tissue Growth Factor (CTGF/CCN2) as a Regulator of ß1 Integrin-Mediated Cell Adhesion and Survival During Lactogenic Differentiation of Mouse Mammary Epithelial Cells 291
CCN2/CTGF – Cardioprotective Factor in Myocardial Ischemia/Reperfusion Injury and Heart Failure 292
High Glucose and Free Fatty Acid Adverse Effects on Cardiac Myocytes Are Mediated by CCN2/Connective Tissue Growth Factor (CTGF) Through TrkA 293
Balancing CCN-2 (CTGF) and BMP(S) in Complicationsof Diabetes Mellitus 295
CCN2/CTGF Is Transactivated Through Its Enhancer Elementby SOX9 in Fibroblasts: Possible Roles in Fibrosis 296
In Vivo Effects of CCN5 Overexpression in Fibroids 297
Microvascular Pericytes Express CCN2 in Tissue Fibrosis 298
Epithelial and Connective Tissue Cell CTGF/CCN2 Expressionin Gingival Fibrosis: Role of Epithelial-Mesenchymal Transition 299
Fibrosis in Duchenne Muscular Dystrophy Role of CCN2 and Its Potential Inhibition by Decorin
Tissue-Specific Regulation of CCN2/CTGF in Gingival Fibroblasts and Its Relationship to Gingival Fibrosis 301
CCN3 (NOV) Is a Negative Regulator of CCN2 (CTGF) and a Novel Endogenous Inhibitor of Fibrosis in Experimental Nephropathy 302
Targeted CCN2 Gene Therapy in Experimental Liver Fibrosis 303
Cell Type-Specific Regulation of Connective Tissue Growth Factor by Hypoxia 304
Elastin Receptor Subunits Facilitate Elastic Fiber Assembly and Modulate Cellular Proliferation 305
Abnormal Extracellular Matrices in Rare and CommonDisorders 306
Cell and Gene Therapy Preserves Matrix Homeostasis: A NovelParacrine Mechanism 307
MT1-MMP and Contractility Promote Invasive Behaviourby Ovarian Cancer Cells 309
CCN3 Promotes Melanoma Progression by Regulating IntegrinExpression, Adhesion and Chemoresistance 310
CCN3 Restores Growth Regulation in Chronic MyeloidLeukaemia 311
Prognostic Role of CCN3 in Ostesarcoma and Ewing’s Sarcoma 312
CCN1/CYR61 Enhances the Metastatic Potential of HumanOsteosarcoma Cell Lines and Activates the AKT Pathway 313
CCN3 Suppresses Growth and Induces Actin CytoskeletalReorganization in Breast Cancer Cells 314
CCN1: A New Target for Chemotherapeutical Drugs in BreastCancer Treatment 315
Suppression of Invasive Front by CCN5/WISP-2 in BreastCancer Is Mediated Through Silencing of microRNA-10B 316
Identification of CCN Proteins as Substrates forKallikrein-Related Peptidases 317
The CCN Family Member CCN6: Inhibition of CCN6 RegulatesE-Cadherin Expression in the Breast Epithelium ThroughUp-Regulation of Snail and ZEB1 318
The CCN1/a6ß1 Connection and the Hormonal Responsein Breast Cancer 319
Novel Transcriptional Regulation of CCN2/CTGF by NuclearTranslocated MMP3 320
Integrin-Mediated Matrix Signaling in Cell Death and Survival 321
Induction of CTGF by TGF-ß1 in Osteoblasts: Independent Effects of SRC and ERK on Smad Signaling 322
Cross-Talk Between CTGF and TGF-ß1 in Mesenchymal Stem Cell Condensation 323
TGF-b Receptor I Kinase Activity Is Required for CTGF/CCN-2 Expression Induced by TGF-b and LPA 325
CTGF/CCN-2 Inhibits BMP-7 Signaling Activity in Diabetic Nephropathy 326
Plasma CCN-2/CTGF Is an Independent Predictor of End-Stage Renal Disease and Mortality in Type 1 Diabetic Nephropathy 327
Nuclear Localization of CCN5, a Classic Matricellular Protein 328
Early Embryonic Expression of CCN5 in Mouse Embryos 329
A Three Dimensional Cell Culture Model to Study the Function of CCN6 in Breast Tumorigenesis 330
Involvement of CYR61 in Growth, Migration, and Metastasis of Prostate Cancer Cells 331
Urinary CCN2 and CCL-2 (MCP-1) Are Predictors of Initiation and Progression of Diabetic Nephropathy 332
CCN3/NOV Is Recruited, Trafficked and Modified During All-Trans Retinoic Acid Induced Neuronal Differentiation on a 3D Matrix: Analysis Using Domain Specific Antibodies 334
Sponsors of the Fifth InternationalWorkshop on the CCN Family of Genes 335
Index 336

"Chapter 2 Report on the Fifth InternationalWorkshop on the CCN Family of Genes (p. 5-6)

A. E. Irvine, B. Perbal, and H. Yeger

Abstract The Fifth International Workshop on the CCN Family of Genes was held in October 2008. This bi-annual meeting provides a unique forum for researchers in the CCN field to present and exchange ideas. The CCN family of regulatory proteins play key roles in both normal cell development and a wide range of pathologies. This was reflected in the breadth of basic cell biology and translational studies presented at the conference. Exciting new in vitro and in vivo model systems are providing new insights into the functional complexity of the CCN family and promise much more to come.

Keywords CCN1 · CCN2 · CCN3 · CCN4 · CCN5 · CCN6 · Cyr61 · CTGF · NOV · Wisp-1 · Wisp-2 · Wisp-3

The Fifth InternationalWorkshop on the CCN Family of Genes was held in Toronto, October 18–22, 2008. The meeting was organised by Herman Yeger, Bernard and Annick Perbal and provided an excellent environment for discussion and interaction amongst a diverse and enthusiastic group of researchers. The Journal of Communication and Cell Signalling has now been launched as the official journal of the International CCN Society.

The publishers, Springer Science & Business Media, sponsored the opening plenary session of the conference and also three scholarships for young investigators to attend the meeting. The first Springer Award for outstanding research in this field was presented by Peter Butler (Springer) to Professor Paul Bornstein by for his work on matricellular proteins. Professor Bornstein gave a stimulating talk focussed on the concept of ‘dynamic reciprocity’ and presented his studies using thrombospondin (TSP-1 and TSP-2) null mice. Opening remarks by Professor Perbal coupled with Professor Bornstein’s lecture set the stage nicely for the meeting to follow. The first session of the meeting on CCN Structure/Function and Expression opened with a presentation by Dr. Ravi Acharya (Bath, England).

Dr. Acharya examined the protein structure of the CCN family and discussed how this might contribute to the functional differences between family members. By modelling the three dimensional structure of the domains he provided insight as to how this might influence interaction with other key molecules. This was followed by a series of presentations on CCN2 gene regulation lead by Dr. Satoshi Kubota (Okayama, Japan) who described the characterisation of Nucleophosmin/B23 as a regulator of CCN2 in chicken chondrocytes. Nucleophosmin has the ability to shuttle between the nucleus and the cytoplasm and Satoshi presented extensive experiments demonstrating both transcriptional (nuclear) and post-transcriptional (cytoplasmic) regulation of CCN2.

Further work from this group, presented by Dr. Ogawara, demonstrated that mi-RNA 18a acts on CCN2 via the 3-UTR and regulates human chondrocytic differentiation. This work now introduces a new level of CCN regulation that should get increasing attention. In a change of cellular context, Dr. Cabello-Verrugio (Santiago, Chile) presented work on the regulation of CCN2 by TGF-b and LPA in skeletal muscle cells in parallel with other studies from his lab suggesting dependency on decorin and involvement of the endocytic receptor LRP-1. He used the C2C12 myoblast cell line to show a dose-dependent induction of CCN2 where the induction is mediated classically through TGFbR1 and SMAD2/3."