Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

CCN

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_559

Synonyms

Historical Background

In the early 1990s, three gene products with novel structural similarity were identified. The first member was discovered as a factor induced upon growth factor stimulation and was designated as cysteine-rich 61 (Cyr61), based on its structural characteristics. The second member was purified as a platelet growth factor-related molecule with a mitogenic effect on fibroblasts and thus, it was initially named connective tissue growth factor (CTGF). Subsequently, a gene with structural similarity to the above two genes was found to be overexpressed in nephroblastomas, which provided the name nephroblastoma-overexpressed (NOV) gene to this third member. Based on these findings, the acronym of the names of these three genes, CCN, was given for the first time as the family name (Perbal and Takigawa 2005). Thereafter, the other three members were identified as tumor suppressor-like gene products, or Wnt-inducible secreted proteins, in 1998; however, no CCN-related names were assigned to them (Perbal and Takigawa 2005). As such, a number of different names were confusingly given to the six genes encoding structurally quite similar proteins. In order to solve this problem, a unified nomenclature was proposed at the second International Workshop on the CCN Family of Genes and was approved by major researchers in this field in 2002. This proposal was published in 2003 to encourage comprehensive progress in the research on this novel family of genes and their products (Brigstock et al. 2003).

Structure

In the human genome, the six CCN family members are distributed on five chromosomes. Only ccn2 and ccn6 share the same chromosome, number 6, with a significant distance between them. The transcribed area of each gene consists of the first exon with a 5′-untranslated region (UTR) and a small portion encoding the signal peptide for secretion, a few intermediate exons corresponding to the amino-terminal and intermediate modules, and the last exon encoding the carboxy-terminal module with the 3′-UTR, together with the interspersed introns of diverse sizes. It should be noted that the exon-intron boundaries in these genes exactly correspond to those between the modules in their proteins, suggesting that these genes were established through exon shuffling during animal evolution (Chen and Lau 2009). The prototypic gene of the six members is anticipated to be either ccn1 or ccn2 since these genes are quite compact with short introns. The genetic promoter for ccn2 has been profoundly characterized, as described later on, whereas investigation of those promoters in the others is currently not (Leask and Abraham 2006).

Except for ccn6, the typical forms of the mRNAs of the CCN family members are characterized by the retention of the 3′-UTR of significant lengths, which suggests the functional significance of these 3′-UTRs. Indeed, the regulatory function of the 3′-UTR of ccn2 was exclusively analyzed, and its role in posttranscriptional gene regulation was uncovered (Kubota and Takigawa 2015). Splicing variants lacking 1 or 2 module-encoding exons were reported in the case of ccn4 and 6 (Perbal and Takigawa 2005).

The structures of the resultant proteins encoded by these genes are novel and distinctly analogous. All of the members have four conserved modules referred to as insulin-like growth-factor-binding protein-like (IGFBP), von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1), and C-terminal cystine knot (CT) modules, except for CCN5, which lacks the fourth one (Fig. 1). In addition, as many as 30 cysteine residues are present in each member, which also provides the structural basis for the functional property of CCN family proteins. Indeed, 38 cysteine residues are strictly conserved among mammalian CCN1, 2, 3, and 4. Internal VWC and TSP1 modules are connected by an amino acid stretch designated as the “hinge region” having a variety of lengths. This region is generally susceptible to cleavage by proteinases, which occasionally yield subfragments of the corresponding CCN family proteins. In silico prediction suggests remarkable similarities in the tertiary structures among the IGFBP modules of CCN1, 2, 3, and 6 and IGFBP4, among the VWC modules of all members and chordin, among the TSP1 modules of all members and thrombospondin, and among the CT modules of CCN1, 2, and 3 and BMP-7 (Holbourn et al. 2008).
CCN, Fig. 1

Gene and protein structure of the CCN family members. Abbreviations I, V, T, and C represent IGFBP, VWC, TSP1, and CT module-encoding regions, respectively

Molecular Action

As summarized above, CCN family proteins comprise four modules, which may be compared to “hands” since all four modules are highly interactive with other molecules. All they have are four hands; however, CCN family proteins can manipulate a number of biomolecules via multiple interactions with these hands. Therefore, CCN family proteins are able to play central roles in orchestrating the extracellular signaling network in a variety of microenvironments.

The known interacting counterparts of the classical CCN family members are illustrated in Fig. 2. As stated above, such multiple interactions indeed provide the basis of the molecular behavior and multiple effects of CCN family proteins. Owing to the interaction with extracellular matrix (ECM)-associated molecules, such as heparan sulfate proteoglycan (HSPG)s, fibronectin, vitronectin, and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) (Takigawa 2013; Chijiiwa et al. 2015) and its biological significance, CCN family proteins are frequently called “matricellular proteins.” It should be noted that interaction with integrins is a common property of all of the classical members, which eventually induces certain cellular responses, such as migration and adhesion through the adhesion signaling cascade under collaboration with cell-surface proteoglycans (Chen and Lau 2009; Jun and Lau 2011). Interaction with other receptor molecules on the cell surface, including low-density lipoprotein receptor-related proteins (LRP) s, FGF receptor (FGFR)s, receptor activator of NF-κB (RANK),  Notch, and TrkA receptor protein kinase, also promotes or modulates intracellular signal transduction (Katsube et al. 2009; Kubota and Takigawa 2013). Moreover, CCN family proteins are known to modify the signal emission/transduction by other growth factors and their inhibitors through direct binding. The effects of CCN2 on the action of transforming growth factor (TGF)-ß, bone morphogenetic protein (BMP)s, insulin-like growth factor (IGF)s, fibroblast growth factor (FGF)s, and vascular endothelial growth factor (VEGF) are classical examples, whereas interaction with other growth factors and its biological significance are currently being investigated as well (Kubota and Takigawa 2015). To date, the biological significance of the mutual interaction between CCN family members themselves has also been indicated (Perbal et al. 2010; Takigawa 2013; Kubota and Takigawa 2015). As a result of such interplay, a number of major intracellular signaling cascades, which include Wnt, JNK,  p38MAPK, ERK,  PI3K, ROS, AKT, and PKC-mediated pathways, are modulated by CCN family proteins (Chen and Lau 2009; Jun and Lau 2011).
CCN, Fig. 2

Interaction of CCN family member proteins with other biomolecules. Abbreviations are defined in the text except for Wnt inhibitory factor 1 (WIF1) and osteoprotegerin (OPG) that is a decoy of RANK

In other words, CCN family protein function is highly dependent upon the coexisting molecules in the microenvironment. Thus, the same CCN family member may exert apparently opposite biological effects under different local situations, as typically represented by the differential roles of CCN family proteins in the development of malignancies. On the other hand, it is widely recognized that CCN2 and CCN3 are functionally quite counteractive in skeletal development and renal fibrosis (Perbal et al. 2010). The fundamental difference between CCN2 and CCN3 functions may be established by the preference for their functional counterparts, which is determined by the property of their modules.

Physiological Function

Based on the molecular actions stated, several CCN family proteins, particularly three classical ones, are already known to mediate a variety of biological events occurring throughout tissue and organ development, especially in mesenchymal tissues. A major part of these functions, as represented by the angiogenic function common to all of the classical members, is thought to be the outcome of the common and prominent cell biological effects of promoting adhesion, migration, and proliferation of cells of mesenchymal origin, whose promotion is exerted by the integrin-mediated matricellular action of these proteins (Chen and Lau 2009). Interestingly, CCN5 is the only member that shows functionality opposite to that described above. Probably, this functional property of CCN5 can be ascribed to its structural property of lacking the CT module since binding sites for proteoglycans, such as heparan sulfate proteoglycan (HSPG)s, including perlecan, have been identified in the CT module in CCN2. Although CCN1, 2, and 3 are functionally quite similar in vitro, as stated below, their role in tissue/organ development significantly differs. These differences can be ascribed to their different temporospatial distribution during development, which is enabled by the proper action of their regulatory machinery for gene expression.

CCN1, 2, and 3 are commonly characterized by their angiogenic functions. However, according to the phenotypic analysis of mutant mice, CCN1 appears to play the most critical part in the angiogenic developmental events. In fact, CCN1-null mice display severe defects in embryonic cardiovascular development. Even the disruption of a single ccn1 locus was reported to cause defects in the atrial septum of the mutant mice. Compared with that of CCN1, the phenotypic change induced by CCN2 or CCN3 deletion is relatively mild; but it should be noted that CCN3 deficiency induces cardiac septal defects in mice (Chen and Lau 2009).

Concerning the classical members, their involvement in skeletal development can be specified as another common property of these molecules, although the quality of contribution differs for each member. CCN1 is known to promote chondrogenesis, whereas CCN2 plays a central role in endochondral ossification by promoting all of the stages of this process. CCN2 is produced predominantly by growth-plate chondrocytes at late differentiation stages and is supplied and acts on the cells involved in a matricrine manner. The target cells include osteoblasts, preosteoclasts, and vascular endothelial cells in addition to chondrocytes; hence, intramembraneous ossification also requires CCN2 (Takigawa 2013; Kubota and Takigawa 2015). The regenerative effects of CCN2 on damaged articular cartilage were also confirmed in vivo. These findings are overall supported by the phenotype of CCN2-null mice (Perbal and Takigawa 2005). Such a profound and multiple functionality of CCN2 in osteogenesis is supposed to be based on the interaction of CCN2 with TGF-ß, BMPs, and other growth factors in the microenvironment. The contribution of CCN3 to skeletal development has been also indicated by previous studies on mutant mice. Overexpression of CCN3 in osteoblasts resulted in osteopenia; and, more interestingly, CCN3 deficiency causes severe joint malformation with appendicular skeletal defects. These findings suggest a critical role of CCN3 in joint formation and morphogenesis (Katsube et al. 2009; Chen and Lau 2009).

In addition to these functional characteristics shared by several members, the cell biological activities unique to each member have been revealed through recent investigations, as summarized in Table 1. The most outstanding of them is the action and effects of CCN3 on the hematopoetic system. It has been recently shown that CCN3 is produced by hematopoietic progenitors, regulates hematopoietic stem cell renewal, and induces clonal expansion of progenitors, in which the latter two actions are based on the interaction of CCN3 with Notch and BMPs (McCallum and Irvine 2009; Katsube et al. 2009). It should be noted that CCN1 plays critical roles in retinal development and placentation under the collaboration with another CCN family members (Kubota and Takigawa 2013; Chintala et al. 2015). In relation to this topic, cell biological function of CCN1 to promote cellular senescence in distinct types of the cells including trophoblasts is being indicated (Table 1), which also suggest the involvement of this CCN family member in the aging process. Additionally, involvement of CCN1 in the development of central nervous system is reported (Kubota and Takigawa 2013). Concerning CCN2, the researchers are clarifying novel roles of CCN2 in the development of a variety of tissues typically represented by olfactory bulb and pancreas, which are summarized in Table 2.
CCN, Table 1

Role of CCN family members in developmental processes

 

Cell biological activities

Relevant developmental processes

References

CCN1

Endothelial cells: adhesion (↑), migration (↑), proliferation (↑), survival (↑), tube formation (↑)

Angiogenesis

Aging

Cardiovascular development

Chondrogenesis

Hippocampal development

Placentation

Retinal development

Perbal and Takigawa (2005); Chen and Lau (2009); Du et al. (2014); Kubota and Takigawa (2013); Chintala et al. (2015); Kipkeew et al. (2016);

Chondrocytes: proliferation (↑), differentiation (↑),

Muscle progenitor cells: senescence (↑)

Fibroblasts: adhesion (↑), proliferation (↑)

Monocytes: adhesion (↑)

Platelets: adhesion (↑)

Trophoblasts: migration (↑), senescence (↑)

CCN2

Endothelial cells: adhesion (↑), migration (↑), proliferation (↑), survival (↑), tube formation (↑)

Adipogenesis

Angiogenesis

Astrogenesis

Endochondral ossification

Inner vertebral disc development

Hair follicle development

Intramembranous ossification

Osteoclastogenesis

Lactogenic differentiation

Odontogenesis

Olfactory bulb development

Pancreatic development

Periodontal ligament development

Perbal and Takigawa (2005); Kubota and Takigawa (2007); Chen and Lau (2009); Perbal et al. (2010); Takigawa (2013); Kubota and Takigawa (2015); Mendes et al. (2015); Song et al. (2015); Yuda et al. (2015)

Chondrocytes: proliferation (↑), differentiation (↑)

Osteoblasts: proliferation (↑), differentiation (↑)

Osteoclasts: differentiation (↑), fusion (↑)

Fibroblasts: adhesion (↑), proliferation (↑), ECM production (↑)

Monocytes: adhesion (↑)

Platelets: adhesion (↑)

Hepatic stellate cells: adhesion (↑)

Mesangial cells: apoptosis (↑)

Smooth muscle cells: apoptosis (↑)

Bone marrow stromal cells: adhesion (↑), migration (↑)

Dental epithelial cells: proliferation (↑), differentiation (↑)

Adipocytes: differentiation (↓)

Cementoblasts: differentiation (↑)

CCN3

Endothelial cells: adhesion (↑), migration (↑)

Angiogenesis

Skeletal morphogenesis

Hemopoietic stem cell renewal

Placentation

Perbal and Takigawa (2005); Chen and Lau (2009); Kipkeew et al. (2016)

Fibroblasts: adhesion (↑)

Clonal expansion of naïve cord blood progenitors (↑)

Trophoblasts: proliferation (↓), migration (↑)

CCN4

Bone marrow stromal cells: proliferation (↑), osteogenic differentiation (↑)

Chondrocytes: differentiation (↑)

Osteogenesis

Ono et al. (2011); Yoshioka et al. (2016)

CCN5

Smooth muscle cells: migration (↓), proliferation (↓)

(unknown)

Perbal and Takigawa (2005); Myers et al. (2014)

Osteoblastic cells: differentiation (↓)

CCN6

Mammary epithelial cells: milk protein synthesis (↑), proliferation (↑)

Mammary gland development

Jiang et al. (2015)

CCN, Table 2

Involvement of CCN family members in various diseases

 

Related disorders

Findings

References

CCN1

Restenosis

Promoter of neointimal hyperplasia

Chen and Lau (2009)

Nephropathy

Putative glomerular regenerator

Perbal and Takigawa (2005)

Sjögren’s syndrome

Pro-inflammatory factor

Li et al. (2016)

Pulmonary emphysema

Mediator of cigarette smoke-induced damage

Moon et al. (2014)

Atrial septal defect

Associated mutations

Perrot et al. (2015)

Lung fibrosis

Promoter of fibrosis

Kurundkar et al. (2016)

Psoriasis

Promoter of hyperplasia and inflammation

Sun et al. (2015)

Malignancies

Gastric cancer, ovarian cancer

Enhancer of tumorigenesis

Chen and Lau (2009); Jun and Lau (2011)

Melanoma, nonsmall cell lung cancer

Inhibitor of tumorigenesis

Glioma, glioblastoma, prostate cancer, breast cancer, pancreatic cancer, osteosarcoma, acute myeloid leukemia

Promoter of tumor growth

Jun and Lau (2011); Habel et al. (2015); Ishida et al. (2015); Niu et al. (2014); Haque et al. (2012)

Colon cancer

Expression associated with grade/stage

Jun and Lau (2011)

Myeloma, endometrial cancer, hepatocellular carcinoma,

Repressor of progression

Jun and Lau (2011); Johnson et al. (2014); Chen et al. (2016);

CCN2

Fibrosis

Skin

Inducer of sustained fibrosis

Perbal and Takigawa (2005); Chen and Lau (2009); Kubota and Takigawa (2013)

Liver, lung, muscle, gingiva

Mediator of fibrosis

Kidney

Inducer of fibrosis and nephropathy

Heart

Promoter and marker of fibrosis

Kubota and Takigawa (2015)

Malignancies

Esophageal and prostate cancers

Promoter of tumorigenesis

Chen and Lau (2009)

Pancreatic tumor

Promoter of tumor growth

Gallbladder cancer

Suppressor of tumor growth

Kubota and Takigawa (2015)

Breast cancer

Promoter of bone metastasis

Kubota and Takigawa (2007)

Inducer of apoptosis

Lung and colorectal cancers

Suppressor of metastasis

Chen and Lau (2009)

Acute lymphoblastic leukemia

Promoter of dissemination

Wells et al. (2016)

Glioma, ovarian cancer

Expression associated with grade/stage

Jun and Lau (2011)

Oral squamous cell carcinoma

Suppressor of malignant phenotype

Kubota and Takigawa (2007)

Osteoarthritis

Cartilage regenerator

Atherosclerosis

Overexpression observed

Myopia

Induction observed

Kubota and Takigawa (2015)

Alzheimer’s disease

Mediator of disease progression

Retinopathy

Mediator of neovascularization

CCN3

Marfan syndrome

Antagonist of fibrillogenesis

Lemaire et al. (2010)

Renal fibrosis

Blockade/reversion of fibrosis

Riser et al. (2014)

Obesity

Association with serum level

Pakradouni et al. (2013)

Malignancies

Melanoma

Repressor of proliferation

Perbal and Takigawa (2005); McCallum and Irvine (2009); Perbal et al. (2010); Kubota and Takigawa (2013); Jun and Lau (2011); Zhang et al. (2013)

Promoter of metastasis

Astrocytoma

Reduced expression observed

Prostate cancer

Promoter of neovascularization

Cervical cancer

Expression associated with grade/stage

Glioblastoma

Repressor of proliferation

Chronic myeloid leukemia

Association with drug sensitivity

Breast cancer

Association with tumor type

CCN4

Malignancies

Melanoma

Inhibitor of tumorigenesis

Chen and Lau (2009)

Lung cancer

Inhibitor of tumor invasion

 

Gastric cancer, prostate cancer

Promoter of metastasis

Perbal and Takigawa (2005); Ono et al. (2011)

Fibrosis

Liver, heart, lung

Inducer of fibrosis

Kubota and Takigawa (2013); Li et al. (2015)

Intimal thickening

Promoter of cell migration

Williams et al. (2016)

CCN5

Malignancies

Breast cancer

Inhibitor of tumorigenesis/progression

Jun and Lau (2011)

Pancreatic cancer

Inverse association with grade/stage

Jun and Lau (2011)

Fibrosis

Heart, Lung

Inhibition/reversion of fibrosis

Jeong et al. (2016)

CCN6

Pseudorheumatoid dysplasia

Genetic association

Perbal and Takigawa (2005)

Malignancies

Breast cancer

Suppressor of tumor growth

Chen and Lau (2009)

Gastric cancer

Promoter of proliferation and metastasis

Fang et al. (2014)

In contrast to those of the classical members, the distinct roles of the other three members in certain tissue/organ development are not yet certain, albeit their significant activities are observed in vitro. A few exceptions are the CCN4 in osteogenesis and the CCN6 in mammary grand, which were clarified within a few years until now, emphasizing the profound role of the CCN family in skeleton and mammary gland. A contribution of these CCN family members to aging is suggested as well (Du et al. 2014). According to a recent study (Perbal et al. 2010), the expression of ccn3, 4, 5, and 6 is elevated in human skin upon aging, suggesting their significant roles in biological processes long after development.

Roles in Pathological Conditions and Wound Healing

Up to the present, a vast number of malignant tumor tissues and cells from a variety of origins have shown increased expression of CCN family members. Particularly, cumulative findings indicate the involvement of classical CCN family members in multiple types of malignancies. Indeed, overexpression of ccn1 is observed in breast cancer, pancreatic tumors, and other tumors, as shown in Table 2. In the case of ccn2, basic and clinical investigations have revealed its overexpression in skin cancer, gallbladder cancer, chondrosarcoma, lymphoblastic leukemia, glioma, hepatocellular carcinoma, and melanoma, as well as in other cancers (Table 2), as based on functional evidence (Perbal and Takigawa 2005; Chen and Lau 2009). Also for ccn3, osteosarcoma, renal cell and prostate carcinomas, Ewing sarcoma, Wilms’ tumor, and rhabdomyosarcoma have been shown to overexpress this particular gene. Even 2, 3, or more CCN family members can be overexpressed simultaneously in the same tumor cells. These findings represent the close association of CCN family members with malignant tumors. However, whether such overexpression is a causative or inducing factor of malignant tumors, or an antitumorigenic response, is not easy to determine; and further experimentation is needed to clarify the situation. Moreover, even if specific effects were experimentally confirmed, the role of CCN1 and CCN2 in tumor development and metastasis still would remain quite controversial, for the forced expression of CCN1 or CCN2 in tumor cells occasionally yields apparently opposite outcome, depending upon the cell type. As discussed above, this functional variation may be conferred by the difference in the composition of the functional counterparts among those cells/tissues. Indeed, experimental evidence indicates the antitumorigenic functions of all of the members as well as tumorigenic ones (Table 2) (Perbal et al. 2010). It is also of great interest that CCN1 and CCN2 induce epithelial-mesenchymal transition (EMT), whereas the loss of CCN6 triggers EMT (Perbal and Takigawa 2005; Jun and Lau 2011). The structural basis to account for the difference in behaviors between CCN1/2 and CCN3–6 has not yet been established.

Probably the best-known CCN family-related disorder at present is fibrosis, in which CCN2 is believed to play a central role. Elevated CCN2 gene expression is observed in most of the fibrotic lesions of various organs. Unlike cancer cases, CCN2 always act positively on fibrosis, and thus the establishment of anti-CCN2 molecular therapy is desired. The utility of plasma CCN2 as a diagnostic marker for the fibrotic change in kidney and heart has also been suggested (Kubota and Takigawa 2007; Chen and Lau 2009; Kubota and Takigawa 2015). It is noteworthy that the actions of structurally and functionally similar CCN1 and CCN2 are contrastive in fibrotic kidney disease. CCN2 is known to be a promoter of diabetic nephropathy, whereas CCN1 expressed in podocytes enhances glomerular reconstruction by inhibiting the migration of mesangial cells and promoting neovascularization (Perbal and Takigawa 2005).

Fibrosis can be regarded as a failure in tissue repair after injury. Therefore, it is reasonable for CCN2 to participate in both fibrotic lesion formation and the wound healing process during which transient fibrosis occurs. Indeed, topical application of CCN2 that induces renal fibrosis improves wound healing in diabetes patients (Henshaw et al. 2015). Interestingly, CCN1 and CCN2 collaborate in wound healing process. Immediately after wounding, CCN2 is rapidly supplied by platelets; and ccn2 induction is also initiated in the cells around the injured tissue (Kubota and Takigawa 2007). During this initial step, both CCN1 and CCN2 support the adhesion of platelets and migration of monocytes (Chen and Lau 2009). After clotting, the migration of fibroblasts and granulation tissue formation are thought to be induced by the abundant CCN2. Moreover, involvement of CCN1, 3, and 5 in platelets was recently indicated (Hara et al. 2016), suggesting their collaborative action during clotting process. In this context, it is also noteworthy that CCN1 was found to mediate neutrophil efferocytosis in cutaneous wound healing (Jun et al. 2015).

Other specific and relevant association between CCN family members and corresponding disorders is also summarized in Table 2. The genetic linkage between a CCN6 missense mutation and pseudorheumatoid dysplasia in humans is clearly indicated; however, ccn6-null mice show no appreciable phenotypic changes. This discrepancy may represent a significant difference between primates and rodents (Chen and Lau 2009).

Gene Regulation

Since the biological outcome caused by CCN family members is highly dependent on the other molecules surrounding them in the microenvironment, when and where CCN family proteins are produced in vivo is a critical determinant of their function. Therefore, the molecular function of each CCN family protein is determined by its gene regulatory system as well as by its molecular architecture itself. Nevertheless, in general, the gene regulatory system of CCN family members has not been sufficiently investigated yet. In fact, little is known about the structure and function of the genetic promoters of ccn3, 4, 5, and 6.

In the case of ccn1, several reports have revealed particular regions in the ccn1 gene that are responsible for the transcriptional upregulation elicited by certain stimuli. A serum response element was identified within a <2 kb region upstream of the TATA-box in murine ccn1; and this element was shown to be essential for the transcriptional induction of ccn1 by serum and platelet-derived growth factor (PDGF) in fibroblasts (Chen and Lau 2009). Also, hypoxic induction of ccn1 is conducted by well-known mediators of hypoxic gene upregulation, such as hypoxia inducible factor (HIF)-1α (Chen and Lau 2009). This transcriptional regulation is mediated not by a few HIF-1α binding site-like sequences, but through a c-jun/AP-1 binding sequence located at approximately 620 bp upstream of the transcription initiation site in ccn1. This AP-1 binding site is also responsible for the ccn1 transcriptional induction by reactive oxygen species (ROS) (Qin et al. 2014). In addition, transcription of ccn1 is induced in response to mechanical stretch by the binding of early growth response (Egr)-2 factor to an element located within a 200 bp region upstream of the TATA-box. In terms of posttranscriptional regulation, it was recently reported that contact with mesenchymal stromal cells or CCN1 molecules promoted the splicing of ccn1 pre-mRNA, supporting the production of CCN1 from myeloma cells (Dotterweich et al. 2014). Regulation of ccn1 by miR-155 and miR-181 is indicated as well (Sumiyoshi et al. 2013; Yan et al. 2015).

Transcriptional and posttranscriptional regulatory systems of ccn2 have been extensively investigated (Fig. 3), and a number of valuable findings have been reported (Leask and Abraham 2006). According to these studies, the proximal promoter of ccn2 contains several functional cis-regulatory elements. Within the same region and in close proximity, 3 cis-elements are located in tandem, all of which mediate ccn2 induction by TGF-ß, either directly or indirectly. The one closest to the TATA-box and the third one accept the signal from TGF-ß via binding of Ets-1 and Smad transcription factors, respectively, whereas the second one in the middle mediates the signal from endothelin 1 (ET-1), a TGF-ß-inducible extracellular signaling molecule. It was recently revealed that Krupel-like factor 15 (KLF-15) negatively regulates ccn2 transcription by counteracting Smad 3 signaling (Perbal et al. 2010). This second element was initially referred to as the TGF-ß responsive element (TbRE) since it was characterized by TGF-ß-responsiveness and interaction with nuclear protein(s). Nowadays, the TGF-ß-responsiveness of this element is regarded as being indirect, which is mediated by the induction of ET-1 production; and so, an alternative name, basal control element-1 (BCE-1), has been given. Adjacent to them, another element with a novel protein counterpart has been built into the ccn2 promoter. This element, named transcriptional enhancer dominant in chondrocytes (TRENDIC), is a target of matrix metalloproteinase 3 (MMP-3), which is widely known to be an extracellular matrix-degrading enzyme. Surprisingly, MMP-3 is distributed also in the nuclei of chondrocytes and drives the transcription of ccn2, probably under the collaboration with chromatin remodeling molecules (Perbal et al. 2010). More recent studies uncovered the targets for myeloid zinc finger (MZF)1 (Piszczatowski et al. 2015) and SRY-box (SOX)9 (Oh et al. 2016) transcription factors in addition. Hypoxia is an effective stimulant to cause the induction of ccn2 as well as ccn1 at both transcriptional and posttranscriptional levels. Transcriptional induction of ccn2 upon hypoxia is mediated by two typical hypoxia responsive element (HRE)s, located relatively distant from the TATA-box in the proximal promoter; and the involvement of forkhead transcription factors, which are also mediators of ccn1 gene regulation, has been suggested (Chen and Lau 2009; Kubota and Takigawa 2013). Additionally, hypoxic signaling is also mediated by a posttranscriptional element, cis-acting element of structure-anchored repression (CAESAR) in the 3′-untranslated region of ccn2 mRNA.
CCN, Fig. 3

Transcriptional and posttranscriptional gene regulation of a CCN family member, CCN2. Abbreviations are defined in the Gene Regulation subsection in the text

CAESAR is the first identified posttranscriptional regulatory element in ccn2, which is located at the junction of the open reading frame and the 3′-UTR (Leask and Abraham 2006; Kubota and Takigawa 2007; Chen and Lau 2009). This structured RNA segment exerts dual functions: One is to retain the basal expression level by repressing translation, and the other is to alter the steady-state mRNA level by controlled RNA degradation in response to hypoxia. Subsequently, another element, 3′-100/50, was discovered in the 3′-UTR of chicken ccn2 mRNA as a regulatory element of mRNA stability in chondrocytes during endochondral ossification. Onto this element, nucleophosmin (NPM)/B23 specifically binds and accelerates the selective degradation of ccn2 mRNA in the cytoplasm (Perbal et al. 2010). Advance in miRNA research revealed a number of miRNAs negatively regulating ccn2 expression at posttranscriptional levels (Fig. 3) (Kubota and Takigawa 2015; Koga et al. 2015; Mu et al. 2016). Of interest, the miR-18a target is located quite close to the 3′-100/50 element, suggesting a mutual interaction between regulatory complexes.

Although genetic targets and precise regulatory mechanisms remain unclarified, a number of other extracellular/intracellular factors are known to regulate ccn family gene expression. The expression of the ccn1 gene is enhanced by angiotensin II, estrogen, the active form of vitamin D, mechanical stress, and UV light irradiation. CCN2 protein is induced by glucocorticoids, nicotine, inflammatory cytokines, mechanical stress, sphingosine 1-phosphate (S1P), rapamycin, radiation, and lysophosphatidic acid (LPA), occasionally in a cell-type-dependent manner (Kubota and Takigawa 2007; Chen and Lau 2009; Sar et al. 2015; Xu et al. 2015; Zhang et al. 2015; Kubota and Takigawa 2015; Yu et al. 2016). Repressive regulation of ccn2 by several miRNAs other than miR-18a has also been reported. It should be noted that TGF-ß enhances the transcription of as many as 4 CCN family members, ccn1, 2, 4, and 5, whereas it contrarily represses that of ccn3. As represented by the initial names given, ccn4 and 5 expression is induced by Wnt-1, which led to the discovery of these gene products (Perbal and Takigawa 2005).

Summary

The CCN family consists of six members in vertebrates, all of which are composed of 3–4 highly interactive protein modules. Using these modules as “hands,” CCN family proteins harmonize the extracellular signaling network via multiple interactions with molecular counterparts in the microenvironment. Therefore, the function of CCN family proteins is altered by the location and time point of gene expression, both of which are determined by their proper gene regulatory machinery. Physiologically, CCN family proteins enable harmonized development of the tissues involved and thus promote adequate tissue regeneration and wound repair. Conversely, aberrant expression of CCN family genes occasionally causes fibrotic disorders and frequently accompanies malignant transformation and invasion by tumor cells. Based on these unique properties, the medical utility of CCN family proteins and their derivatives is to be expected, and the development of anti-CCN family therapeutic strategy is currently being considered.

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular DentistryOkayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
  2. 2.Advanced Research Center for Oral and Craniofacial SciencesOkayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan