CCN Proteins

The following sections are included:

• Contents

• Preface
  • REFERENCES

In this chapter, we introduce the general structures and functions of CCN proteins and briefly review major questions regarding the biological properties of this new family of signaling regulators.

CCN2/CTGF is a multifunctional growth factor for fibroblasts, chondrocytes, osteoblasts, and vascular endothelial cells. Depending on the type of cell with which it interacts, this factor promotes chemotaxis, migration, adhesion, proliferation, differentiation and/or extracellular matrix formation. Because of the very high level of gene expression of CCN2 in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor has been suggested to be the promotion of endochondral ossification. However, recent observations of its gene expression during embryonic development and wound healing suggest the involvement of this factor in growth and regeneration of various types of tissues including those of the skeletal and vascular systems. Also, the results of administration of recombinant CCN2 in vivo suggest the factor to be therapeutically useful for the regeneration and reconstruction of skeletal and vascular tissues. Moreover, CCN2 promotes at least chondrogenic differentiation of mesenchymal stem cells, suggesting that this factor is applicable to cell therapy. CCN2 is also known to be involved in fibrotic disorders, angiogenic diseases, and malignancies; but these pathological states may be due to uncontrolled overexpression of this growth factor. This chapter focuses on the role of CCN2 in the control of growth and regeneration, including that at the molecular level.

Known activities of CCN proteins in isolated cell systems can be attributed to their interaction with integrin receptors, with cell surface heparan sulfate proteoglycans (HSPGs) serving as coreceptors in some contexts. Recent studies have identified specific binding sites on CCN proteins for integrin α_vβ₃ and the α₆β₁-HSPG coreceptors, and mutations at these binding sites impair activities mediated through their cognate receptors. These findings provide compelling evidence that CCNs act through their binding to integrins and HSPGs, and provide a strategy for dissecting their integrin-mediated functions in vivo.

Odontogenesis is a complex, multi-stage and fundamental process that involves epithelial-mesenchymal interactions, polarized growth, morphogenesis and differentiation of ameloblasts and odontoblasts. Studies have shown that these complex developmental events require continuous and reiterative action by signaling molecules at successive stages and multiple sites. However, the exact nature of these factors, the steps controlled by them, and the means by which they exert their roles at multiple sites and times are not fully known. Our recent work described here shows for the first time that CCN2/connective tissue growth factor (CTGF), a member of the powerful CCN effector family, is involved in, and regulates, odontogenesis. CCN2 is first expressed at the onset of odontogenesis along with transforming growth factor β₁(TGF-β₁) and bone morphogenetic proteins 2 and 4 (BMP-2 and BMP-4), characterizes enamel knot and preameloblasts in conjunction with Sonic hedgehog (SHH) at subsequent stages, and then persists in differentiating and mature odontoblasts. CCN2 gene expression increases during differentiation of dental precursor cells in culture, and exogenous BMPs stimulate CCN2 expression in tooth germs explants. Exogenous recombinant CCN2 (rCCN2) stimulates proliferation in dental epithelium and mesenchyme. Interference with CCN2 action by neutralizing antibodies, however, inhibits cell proliferation and ameloblast and odontoblast differentiation. The results show that CCN2 is a powerful and developmentally controlled signaling molecules during tooth development, and has previously unsuspected roles such as stimulation of proliferation and promoting maturation in odontogenic cells. In this chapter, our previous studies are summarized and new data are presented. Taken together, our data demonstrate for the first time that CCN2 is expressed during, and regulates, odontogenesis. CCN2 expression is confined to specific sites and times in the developing tooth germ, is regulated by TGF-β and BMPs, and appears to be necessary for normal growth and cytodifferentiation of ameloblasts and odontoblasts.

Since their first description as CYR-61 (CCN1), CTGF (CCN2) and NOV (CCN3) proteins more than a decade ago, the number of publications and in turn our knowledge of the structure and function of these and other members of the CCN family of genes has increased in a logarithmic fashion. Their involvement has now been shown in multiple organ systems. One organ that has been the focus of a large number of these reports is the kidney. The majority of these renal studies have centered on, and have now documented, the role of CCN2 as an important pathogenic factor in fibrosis/sclerosis in a number of models of chronic kidney disease (CKD). This chapter will focus on these discoveries, the future direction, and the resulting possibilities for novel therapeutics and diagnostics, both targeting CCN. In addition, limited data indicates a possible role for CCN genes in embryonic development and in normal renal function. This area will also be briefly considered and the possible relationship to the pathological response discussed.

CCN proteins regulate a broad spectrum of cellular functions including adhesion, migration, proliferation and survival. CCN family members are produced by various hepatic cells in response to injury or cellular transformation. Repair or regeneration following drug-induced hepatoxicity or hepatic resection is characterized by enhanced expression of CCN2 via pathways that are downstream of the actions of tumor necrosis factor-α and/or transforming growth factor-β. CCN2 and CCN3 levels are enhanced in primary and metastatic hepatocellular carcinoma, while CCN4 is over-produced in cholan-giocarcimonas. In hepatic fibrosis, CCN2 is produced by all major cell types, often downstream of transforming growth factor-β. CCN2 stimulates a pro-fibrotic phenotype in hepatic stellate cells, the principal fibrogenic cell type, through its regulation of proliferation, adhesion, migration, proliferation and survival as well as production of α-smooth muscle actin or collagen I. Binding of CCN2 to stellate cells occurs via cell surface integrin αvβ³ and low density lipoprotein receptor-related protein, both of which utilize heparan sulfate proteoglycans as a co-receptor. CCN proteins are a novel class of cell regulatory molecules that may represent new therapeutic targets for a variety of liver diseases, especially fibrosis and cirrhosis.

Gene expression patterns and numerous in vitro experiments suggest that CCNs may be required for multiple aspects of mammalian development. Roles for several CCN genes during mammalian development have been elucidated through genetic analysis in mice and humans. Mice lacking Ccn1 are characterized by insufficient placental vascularization and defective embryonic vasculature, confirming an essential and novel role for CCN1 in angiogenesis. Loss of Ccn2 in mice results in multiple defects in skeletal development, including impaired chondrocyte proliferation, decreased production of extracellular matrix components, and defective growth plate angiogenesis. Mutations in Ccn6 in humans are associated with defects in post-natal maintenance of articular cartilage. These genetic analyses have also shed light on signaling pathways utilized by CCNs and have revealed key molecular targets of CCN gene action.

The study of CCN genes in the development of the zebrafish, the frog Xenopus and the chick has only just begun. The main findings thus far complement the results obtained in better studied mammalian models. CCN1 in Xenopus embryos regulates gastrulation movements and can either antagonize or stimulate Wnt signaling, in a context dependent manner. In the same model system, CCN2 can also antagonize Wnt signaling, through interaction of its CT domain with the Wnt receptor complex. In Xenopus embryos CCN2 additionally antagonizes BMP pathway. The basis of this activity is binding of the VWC domain to BMP4. These activities likely contribute to the anteriorization of the nervous system and to the delay in neural differentiation seen in gain-of-function experiments.

The following sections are included:

• CCN3 EXPRESSION DURING EARLY STAGE DEVELOPMENT

• CCN3 EXPRESSION IN MIDDLE TO LATE STAGE DEVELOPMENT
  • Possible Role of CCN3 by its Analysis in Embryogenesis

• REFERENCES

CCN1/Cyr61 and CCN2/CTGF are highly expressed in several stress-related pathologies resulting from increased externally applied forces or from forces generated internally by the cellular components of tissue organs. In accordance with their in vivo distribution pattern, the CCN1/Cyr61 and CCN2/CTGF genes are strongly induced upon application of mechanical forces on cultured cells, or through manipulation of the cellular contractile apparatus. Mechanistically, alteration of the actin cytoskeleton tension and/or actin polymerization promoted by various components of the signaling machinery i.e. small Rho GTPases and MAP kinases, or by actin binding proteins seem to regulate the expression of the immediate early CCN genes. What could the significance be of enhanced CCN1/Cyr61 and CCN2/CTGF expression by mechanical stress? The role of these proteins in the cellular mechano-transduction process as response to environmental stress and their participation in long term modifications of the extracellular environment are issues of ongoing research.

The pathogenesis of systemic sclerosis(SSc) is not clarified yet, although some cytokines seemed to be closely related to the fibrotic process of this disorder. Transforming growth factor β(TGF-β) has received many attentions as an essential factor in the pathogenesis of various fibrotic disorders. In addition, we have shown that connective tissue growth factor (CCN2) is closely related to the pathogenesis of SSc as follows: (i) CCN2 mRNA expression was observed in the fibrotic lesions but not in the early non-fibrotic lesions or atrophic lesions; (ii) Serum CCN2 protein concentrations were significantly elevated, and this was correlated with skin sclerosis and lung fibrosis; and (iii) In our animal model, TGF-β induced subcutaneous fibrosis and subsequent CCN2 application caused persistent fibrosis, which was correlated with upregulation of procollagene mRNA promotor activities. Based on these data, we hypothesize that a two-step process of fibrosis occurs in SSc, that is TGF-β induces fibrosis in the early stage, and afterwards CCN2 acts to maintain tissue fibrosis.

The following sections are included:

• INTRODUCTION

• SUMMARY OF DISCOVERIES AND INVESTIGATIONS OF CCN5
  • Studies of Vascular Smooth Muscle Cells
  • Uterine Smooth Muscle Cells
  • Studies of Rodent Embryonic Fibroblasts
  • Studies of Transformed Mammary Epithelial Cells and Human Colon Tumors
  • Studies of Human Bone Cells
  • Studies of Human Breast Cancer Cells

• CCN5 GENE, mRNA, AND PROTEIN STRUCTURE
  • Tools Developed for Studies of CCN5
    • Antibodies to CCN5
    • Viral and plasmid CCN5 expression constructs
    • Inhibitory RNA molecules for CCN5
    • Recombinant CCN5
  • CCN5 Gene Location, mRNA Structure, and Promoter
  • Analysis of the Molecular Size of the CCN5 Protein

• MODELS USED FOR THE STUDY OF CCN5 EXPRESSION
  • Tissue and Animal Models
  • Cultured Cell Models
  • Other Cells and Tissues Examined for CCN5 Expression

• BIOLOGICAL EFFECTS OFALTERED CCN5 EXPRESSION
  • Arrest of Cell Proliferation
  • Promotion of Cell Proliferation
  • Inhibition of Cell Motility
  • Cellular Invasiveness
  • Adhesion of Cells to Substrates
  • Induction of Apoptosis
  • Morphology
  • Tumorigenicity
  • Osteocalcin Production

• REGULATORY MOLECULES FOR CCN5 EXPRESSION (TABLE 7)
  • Heparin
  • Estrogen
  • Serum
  • Other CCN5 Regulatory Molecules

• SUMMARYAND FUTURE DIRECTIONS FOR CCN5 RESEARCH
  • Do General Pathophysiologic Roles Exist for CCN5?
  • Does the Data Support the Hypothesis that CCN5 Is the Anti-4-Domain CCN?
  • The Need for Genetically Tractable Animal Models for CCN5 Expression
  • CCN5—AVersatile ProteinWhose Function and Regulation Varies by Cell Type?

• REFERENCES

The CCN3 protein is one of the three founding members of the CCN family (see Chapter 1). As a prototypic CCN protein, it is constituted by the assembly of the IGFBP, VWC, TSP1, and CT modules. The N-terminal signal pep tide which is present in the neosynthesized protein allows efficient secretion of CCN3. Studies aimed at identifying the biological roles of CCN3 have been hampered by the low levels of CCN3 expressed in normal conditions, and by the lack of a simple biological system in which to study the properties of the normal CCN3 protein. Despite these limitations, the bulk of information that has been obtained over the past decade has permitted the identification of CCN3 as a key regulator of cell growth and differentiation. Recent results have indicated that CCN3 is a genuine signaling factor and that alterations in CCN3 expression constitute a marker for tumor typing. Furthermore, measurement of CCN3 amounts and inhibition or forced expression of CCN3 constitute potential therapeutic tools.

The following sections are included:

• INTRODUCTION

• CONNEXINS AND GAP JUNCTIONS

• GAP JUNCTIONS AND TUMOR SUPPRESSION

• CCN3 AND CONNEXIN INTERACTIONS

• CCN3 AND CONNEXIN IN GLIOMA CELLS

• CCN3 AND CONNEXIN IN CHORIOCARCINOMA CELLS

• SIGNIFICANCE OF INTERACTION BETWEEN CCN3 AND CONNEXIN43

• MODEL OF CCN3 INTERNALIZATION

• CONCLUSION

• ACKNOWLEDGMENTS

• REFERENCES

The following sections are included:

• INTRODUCTION

• CCN1 EXPRESSION IN CANCER
  • Increased Expression of CCN1 in Cancer
  • Reduced expression of CCN1 in cancer

• REGULATION OF CCN1 EXPRESSION IN CANCER

• EFFECT OF CCN1 ON TUMOR CELL LINES
  • Breast
  • Brain
  • Lung
  • Endometrium
  • Other

• CHEMORESISTANCE

• SUMMARY

• ACKNOWLEDGMENTS

• REFERENCES

Extracellular stimulation activates the particular receptors to transduce intra-cellular signals into the nucleus. It is well recognized that abnormal signal transduction occurs in cancer cells. Recent investigations revealed one of the major pathways implicated in gastrointestinal malignancies has been found to involve the Wnt-Wingless system. Indeed, we isolated a Wnt receptor gene designated Frizzled that represents a key signaling molecule for beta-catenin regulation in cancers. Among Wnt-inducible genes downstream of beta-catenin, it is noteworthy that three homologues of CCN family members has been identified as Wnt-inducible signaling pathway 1 (WISP1)/CCN4, WISP2/CCN5, and WISP3/CCN6. Recently, we isolated novel variants of WISP1/CCN4 and WISP3/CCN6 in human cancer tissues. This review focuses on Wnt in onco-genic signal transduction pathways and especially WISP/CCN variants, and emphasizes their regulation as an important concept that may influence future cancer treatments.

The following sections are included:

• INDEX