CCN2 in Orofacial Tissue Development and Remodeling

Japanese Dental Science Review (2012) 48, 101—113

Available online at www.sciencedirect.com

jo urnal homepage: www.elsevier.com/locate/jdsr

Review article

CCN2 in orofacial tissue development and remodeling

Satoshi Kubota *

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry,

and Pharmaceutical Sciences, Okayama, Japan

Received 8 November 2011; received in revised form 13 January 2012; accepted 1 February 2012

KEYWORDS Summary CCN2 is one of the representative members of the CCN family, a group of proteins

CCN2; that orchestrate the extracellular signaling network. As anticipated by the original name,

CTGF; connective tissue growth factor, this molecule promotes the growth and development of

mesenchymal tissues, including bone and cartilage. Indeed, CCN2 is required for the proper

Tissue regeneration;

development of the orofacial region, which requirement is typically suggested by the frequent

Periodontal ﬁbrosis;

Bone; emergence of cleft palate in CCN2-null mice. The signiﬁcant contribution of CCN2 to mandibular

Cartilage morphogenesis and tooth germ development has also been indicated. Of note, CCN2 functions not

only during development, but also later in life, as it is a critical promoter of physiological and

pathological tissue remodeling, the latter of which denotes ﬁbrotic reconstruction of tissue. In

addition to its involvement in ﬁbrotic disorders in a variety of organs, CCN2 has been also reported

to be a mediator of periodontal ﬁbrosis caused by several factors including smoking. Based on

these cumulative ﬁndings, the utility of CCN2 to accelerate oral tissue regeneration by a

harmonized remodeling process is discussed herein, together with regulation of the gene

expression and molecular function of CCN2 as a therapeutic strategy against periodontal ﬁbrosis.

Contents

1. Introduction: the CCN family ...... 102

2. CCN2: one of the founders with multiple titles ...... 102

3. Roles of CCN2 in orofacial tissue development...... 104

3.1. Endochondral ossiﬁcation ...... 104

3.2. Intramembranous ossiﬁcation ...... 104

3.3. Articular and auricular cartilage formation/maintenance ...... 104

3.4. CCN2 in Meckel’s cartilage ...... 105

3.5. CCN2 and tooth development ...... 106

* Correspondence address: Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry

and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan. Tel.: +81 86 235 6646; fax: +81 86 235 6649.

E-mail address: [email protected].

102 S. Kubota

4. CCN2 as a mediator of tissue remodeling...... 106

4.1. CCN2 as a single coupling factor that coordinates efﬁcient bone remodeling ...... 106

4.2. Possible cartilage remodeling by CCN2 for maintaining tissue integrity...... 107

4.3. Tissue remodeling toward orofacial wound healing and ﬁbrosis by CCN2 ...... 107

5. CCN2 as a therapeutic tool or target in dental medicine ...... 108

5.1. Cartilage regeneration by CCN2 ...... 108

5.2. Bone regeneration by CCN2 ...... 108

5.3. Regulation of periodontal ﬁbrosis via CCN2 ...... 110

6. Conclusions for the future ...... 110

Acknowledgements ...... 111

References...... 111

interacts with multiple factors, the modules do not appear to

1. Introduction: the CCN family

collaborate to construct a speciﬁc functional domain with a

higher-ordered structure [11]. Thus, these proteins should be

Oral and maxillofacial tissues contain almost all types of the

categorized to a novel group of proteins deﬁned under a

hard tissues present in the human body. Not only the teeth,

paradigm distinct from that for regular signaling molecules.

which are found only in the oral cavity, but also bone and

In this context, CCN family proteins may be metaphorically

cartilage are indispensable components of the basic struc-

referred to as ‘‘4-handed conductors.’’ Orchestral conduc-

ture of this part of the body section that best represents the

tors do not actually play musical instruments to make sounds.

property of human being. Here, we should note that CCN2,

Instead, they manipulate or direct all of the players around

one of the classical members of the CCN family, is critically

them to manufacture an integrated world of music with a

involved in the development of these orofacial tissues as a

variety of sound sections. Fortunately, the CCN family pro-

unique director that orchestrates the extracellular signal

teins possess 4, or at least 3, hands to lead their colleagues in

trafﬁc in the microenvironment.

the microenvironment to organize the tissue.

The CCN family was founded by 3 classical members,

Among the 6 members, CCN2 has been standing out from

under the acronym of their original names [1]. The ﬁrst

the others in terms of its biological signiﬁcance. According to

member, CCN1, was identiﬁed as a cysteine-rich protein

the reports accumulated until today, critical involvement of

numbered 61 (Cyr61) induced immediately upon stimulation

CCN2 in skull and tooth development, as well as bone regen-

by growth factors. CCN2 was initially discovered as connec-

eration is indicated [1,5], whereas no comparable informa-

tive tissue growth factor (CTGF) with mitogenic activity

tion is available regarding the other members. Moreover,

toward ﬁbroblasts. Thereafter, another relevant molecule

CCN2 is the only member that mediates ﬁbrotic remodeling of

was found in a nephroblastoma overexpressed as a truncated

periodontal tissues. Here, the molecular behavior and func-

form, and was originally named NOV, and the CCN family was

tion of CCN2 in orofacial tissues are comprehensively

born in 1993 [2]. It took another 5 years until the other

described and discussed, based on such a scientiﬁc back-

members ﬁnally joined to establish a family with 6 members

ground. However, it should be also noted that contribution of

in mammals. Owing to the signiﬁcant impact of a single

the other members therein still remains to be explored at

report describing all of the 3 additional members as Wnt-

present.

inducible secreted proteins (WISP), these members have

been frequently referred to by this WISP terminology [1].

2. CCN2: one of the founders with multiple

Furthermore, in addition to these popular names, a variety of

other names were given to most of the CCN family members, titles

which led to signiﬁcant scientiﬁc confusion [1,3—5]. There-

fore, a unified nomenclature was proposed in 2003 with the CCN2 was originally purified and identified as a platelet

consent of major researchers on the CCN family [6]. There- growth factor-associated protein [1]. Since this factor exerts

fore CCN2, rather than CTGF, is the name ofﬁcially recom- positive effects on the proliferation of a variety of the cells

mended by the International CCN Society. [1,3,4,8—10], this original name has been, and even is still

Structurally speaking, all CCN members are characterized being preferentially used among researchers in the ﬁeld.

by a commonly conserved modular structure. With the only However, a vast number of the studies revealed quite diverse

exception being CCN5 with 3 modules, CCN family members functionality of CCN2, which obviously represents the prop-

are composed of 4 distinct modules placed in tandem erty of this molecule as a CCN family member, as summarized

[1,3,4,7—10]. Following the signal peptide for secretion, in Table 1. Therefore, it is not surprising that nearly 10

insulin-like growth factor binding protein (IGFBP), von Will- different names have been assigned to this single molecule

ebrand factor type C repeat (VWC), thrombospondin type 1 during the progression of research on CCN2 [1,3—5].

repeat (TSP1) and carboxy-terminal cystine knot (CT) mod- Most critical biomolecules possess a dark side as well as a

ules are connected in this order. All of these modules are bright side of their functions. In this point of view, CCN2 is a

characterized by conserved cysteine residues and are highly typical double-edged sword in various microenvironments.

interactive. This novel structure comprising ‘‘sticky’’ mod- Indeed, CCN2 is involved in physiological events during the

ules provides the molecular background for the multifunc- development and growth of a variety of organ and tissues

tionality of the CCN family proteins. While each module itself [1,10], particularly of mesenchymal ones. Positive aspects of

CCN2 in orofacial tissue development and remodeling 103

Table 1 Diversity found in CCN2 terminology and function.

Proposed nomenclature CTGF FISP12 Hcs24 ßIG-M2

[1,5] HBGF-0.8 Ecogenin IGFBP8 IGFBP-rP2

Functiona l Cellular response Proliferation Differentiation Dedifferentiation

aspects [3,15 ] Adhesion Migration ECM production Apoptosis

Physiological roles End ochondral & Intramembranous ossification [5,14,75] Angiogenesis Lactogenic differentiation

Cardioprotection Wound healing

Pathological Fibrosis Liver Lung Heart Kidney

involvements Skin Pancreas Gingiva

[5,8,10,61,76] Tumors Breast cancer Skin cancer Pancreatic cancer

Colonolec tal cancer Melanoma Chondrosarcoma Oral cancer Bone metastasis Others Inflammation Biliary atresia Nephropathy

Atherosclerosis Alzheimer's disease

the functional properties of CCN2 are also represented by the malignant transformation and benign conversion under the

potential of this molecule to accelerate the regeneration of pathological state. Additionally, direct activation of intra-

damaged tissues, including bone and cartilage [12,13]. Of cellular signaling can be triggered through several cell-sur-

note, even a cardioprotective effect of CCN2 against myo- face receptors, such as integrins, LRP1, LRP6, and Trk A, to

cardial ischemia-reperfusion injury was reported [14]. Most which particular ligands other than CCN2 are already spe-

relevant reports describe the positive effects of CCN2 on cell ciﬁed [7,10,23,24]. Surprisingly, the direct interaction

growth and differentiation [9,10], save for one showing the between the intracellular estrogen receptor and CCN2

dedifferentiation of skeletal muscle cells induced by CCN2 and its functional signiﬁcance have also been recently

[15]. Contrarily, it is also widely recognized that CCN2 is a

major mediator of ﬁbrotic disorders and a critical determi-

nant of the phenotype of certain malignancies. Involvement

of CCN2 in ﬁbrosis of multiple organs was described in a

number of reports [4,7,16], conferring the title of proﬁbrotic

molecule on CCN2. As listed in Table 1, pathogenic involve-

ment of the CCN2 gene is frequently observed in a large

number of types of malignant tumors, including cancers that

originate in the epidermis [4,7,10,17]. These ﬁndings may

indicate the possible contribution of CCN2 to epidermal-

mesenchymal transition (EMT) events, because this factor

acts as a key player in mesenchymal tissue development.

However, the role of CCN2 in determining the malignant

phenotype is still controversial and complicated. It has been

solidly indicated that CCN2 promotes bone metastasis of

breast cancers [5,10], whereas its overexpression rather

shifts the phenotype of oral cancer cells toward a benign Figure 1 Molecular action of a CCN family member, CCN2.

one [18,19]. How can CCN2 action have such apparently CCN2 displays multiple functions via interactions with multiple

opposite outcomes? molecular counterparts, using 4 modules represented by the

As brieﬂy stated in the previous section, the multiple connected spheres in the center. Through the interaction with

biological outcomes effected by CCN2 are conferred by ECM molecules, this factor integrates ECM with the cells by

multiple interactions with a vast number of collaborative acting as a matricellular protein. By interacting with several

molecules (Fig. 1). In fact, CCN2 is reported to interact with growth factors, CCN2 either positively or negatively modulates

multiple molecular counterparts from different categories their activities. Finally, direct binding with cell-surface recep-

[7,10,20—23], which are summarized in Table 2. This inter- tors triggers cell adhesion, migration, and intracellular signal

action with a variety of collaborators enables CCN2 to emission to induce speciﬁc intracellular events. As a result, CCN2

execute apparently contradictory missions, i.e., cell growth manipulates a huge signaling network composed of numerous

and differentiation under the physiological state as well as biomolecules in the microenvironment.

104 S. Kubota

Table 2 Molecular counterparts of CCN2.

Category Name Outcome of interaction

Cell surface molecules [10,25] Integrins FAK activation, cell adhesion, migration

LRPs Modulation of Wnt, ERK and PKC signalings

Trk A Initiation of multiple signal transduction

Enhancement of cytokine production

Membrane HSPG Cell adhesion, differentiation, proliferation

Growth factors [10,25] BMPs Modiﬁcation of BMP signaling

TGF-ß Enhancement of TGF-ß signaling

FGF-2 Modulation of FGF signaling

VEGF Inhibition of VEGF function

ECM components [10,20,23] Fibronectin Cell adhesion

Aggrecan Enhancement of aggrecan production

Decorin Inhibition of CCN2 activity

Other [22] Estrogen receptor Suppression of transcriptional activation

Abbreviations: LRP, low density lipoprotein receptor-related protein; HSPG, heparan sulfate proteoglycan; BMP, bone morphogenetic

protein; FGF, ﬁbroblast growth factor; VEGF, vascular endothelial growth factor.

suggested [22]. As a result of these interactions, CCN2 both ECM production and vascular invasion at the growth

appears not only to participate in the generation of orofacial plate cartilage [26].

tissues during development, but also to promote remodeling

and regeneration thereafter.

3.2. Intramembranous ossiﬁcation

3. Roles of CCN2 in orofacial tissue Since the cranium is basically a fossil-like structure evolved

development from an ancient exoskeleton, a signiﬁcant number of the

facial bone components follow a distinct process entitled

intramembranous ossiﬁcation [27]. In this alternative path-

3.1. Endochondral ossiﬁcation

way of bone formation, mineralized bone is directly formed

by osteoblasts differentiated from mesenchymal stem cells,

The human skull consists of a number of elements of a variety

without forming any intermediate cartilaginous tissue.

of sizes and shapes. Upon delivery, the initial skull of a baby is

Recently, CCN2 was shown to be required for this bone-

composed of more than 40 elements, which eventually fuse

forming process, as well as endochondral ossiﬁcation in

into 22 bones to constitute the adult skull. Among them, the

mice. In normal osteoblasts isolated from the mouse cra-

major parts supporting the brain are formed through a

nium, CCN2 was found to be expressed at early stages of their

developmental pathway entitled endochondral ossiﬁcation

[3,25]. differentiation [28]; and exogenous CCN2 strongly enhanced

the production of bone ECM components and matrix calci-

Endochondral ossiﬁcation is a sophisticated biological

ﬁcation by osteoblasts [7,28,29]. Therefore, this molecule is

process established during vertebrate evolution. In this

thought to act on osteoblasts in an autocrine manner to

process, bones are primarily formed as cartilage anlagen

support the efﬁcient construction of the craniofacial bone

with comparable shapes. Along the course of development

elements. Consistent with these in vitro ﬁndings, CCN2 null

and growth, cartilaginous tissue grows and is gradually

mice display remarkably reduced levels of bone ECM produc-

replaced with mineralized bone (Fig. 2A). During this pro-

tion and mineral deposition in the cranial elements. In vitro

cess, CCN2 is vigorously produced at the early hypertrophic

analysis with CCN2 null osteoblasts further conﬁrmed a

(prehypertrophic) stage and inﬁltrates the surrounding

drastic reduction in osteogenic marker gene expression

extracellular matrix (ECM) toward target cells to accom-

and mineralization potential, which was efﬁciently compen-

plish multiple missions [3,25]. First, this molecule pro-

sated by the addition of CCN2 protein [28]. It should be noted

motes the proliferation and ECM deposition capability of

that the CCN2 null mice suffer from cleft palate [26], again

chondrocytes behind the producers. Second, it accumulates

supporting the biological signiﬁcance of CCN2 in orofacial

in hypertrophic chondrocytes to accelerate hypertrophy

bone development.

and apoptosis. Third, vascular invasion into the cartilage

is promoted by the angiogenic activity of CCN2. Fourth,

formation of osteoclasts/chondroclasts can be also pro- 3.3. Articular and auricular cartilage formation/

moted by CCN2. Fifth and ﬁnally, bone formation by osteo- maintenance

blasts is enhanced by the same factor, as described in the

next subsection. These profound roles of CCN2 in endo- In contrast to the cartilage anlagen that disappear after

chondral ossiﬁcation are represented by the phenotype bone morphogenesis and growth, several types of cartilage

observed in CCN2-null mice with retarded and abnormal persist there to execute their proper biological missions.

endochondral ossiﬁcation characterized by impairment of Among these permanent cartilages, the most typical and

CCN2 in orofacial tissue development and remodeling 105

other oral activities. Although the development and growth

scenario of the cartilage of the mandibular condyle are not

exactly the same as those of long bones [30], the roles and

biological signiﬁcance of CCN2 in both tissues are compar-

able. Thus, the ﬁndings described here on the CCN2 function

in relation to articular cartilage ought to apply also to the

TMJ cartilage.

Articular cartilage develops from the epiphysis of the

primordium, in which some of the chondrocytes are engaged

in constructing permanent cartilage, while the others

undergo ossiﬁcation around the secondary ossiﬁcation cen-

ters. CCN2 is believed to support both types of differentiation

process; whereas the fate of chondrocytes towards these 2

distinct missions is determined by another CCN family mem-

ber, CCN3 [31]. After the development of articular cartilage,

CCN2 gene expression in articular chondrocytes is not evi-

dently seen in vivo. Nevertheless, in vitro analyses showed

that the addition of CCN2 to cultures of articular chondro-

cytes isolated from adult cartilage promotes both prolifera-

tion and maturation of these cells [3,7]. Importantly, albeit

CCN2 promotes the calciﬁcation of chondrocytes following

the endochondral ossiﬁcation pathway, it never promotes the

calciﬁcation of articular chondrocytes leading to osteophyte

formation in osteoarthritis (OA). These ﬁndings indicate a

signiﬁcant role of CCN2 in the developmental processes of

articular cartilage as well.

In addition to TMJ cartilage, our cranium possesses other

cartilaginous components, which are of functional and par-

ticularly of esthetical importance. One of them is the auri-

cular cartilage constructing the ear. Concerning the role of

Figure 2 Production and roles of CCN2 in cartilage-directed CCN2 in this tissue, a single report has described the effect of

bone formation. CCN2 molecules are represented by marked CCN2 on it [32]. According to this study, CCN2 promotes the

stars. (A) Those acting in endochondral ossiﬁcation. In this proliferation and production of elastin ﬁbers, which is one of

process, bones are initially formed as cartilage anlagen (upper the typical characteristics of elastic cartilage cells. Of note,

left) and follow the development and growth pathway towards as in the case of articular chondrocytes, CCN2 does not

the completion of the adult form (upper right). Bone growth and induce either apoptosis or calciﬁcation in auricular chondro-

internal ossiﬁcation are carried out at the growth plates (bot- cytes. Therefore, CCN2 may also be a promoter of auricular

tom) located on the ossiﬁcation centers (upper center). Resting cartilage development.

chondrocytes (RC) ﬁrst proliferate (proliferative chondrocytes:

PC) and differentiate to support cartilage growth; and then these

3.4. CCN2 in Meckel’s cartilage

cells produce abundant CCN2 at the prehypertrophic stage

(PHC). CCN2 inﬁltrates around and accumulates in the hypertro-

Mandibular bone is recognized to be formed by intramem-

phic layer to enhance the activities of all of the cells involved.

branous ossiﬁcation. However in this process, Meckel’s

Along with growth, the cartilage matrix and hypertrophic chon-

cartilage, which is a transient cartilage developing from

drocytes (HC) in the hypertrophic zone are simultaneously

the ﬁrst branchial neural crest cells, mainly serves as a

replaced with bone tissue by chondroclasts (Cc) and osteoblasts

template of mandibular bone and contributes also as a

(Ob), along with vascular invasion by endothelial cells (VE). (B)

limited part after ossiﬁcation [33]. During mandibular

Comparable events occurring at the junction of Meckel’s carti-

development, CCN2 gene expression is observed to occur

lage and ossifying region at the late stage of development. Since

in 2 distinct stages [34]. At the ﬁrst stage, strong CCN2

Meckel’s cartilage does not grow to become mandibular bone in

gene expression is detected throughout the Meckel’s car-

mammals, the polarized growth plate structure is not evident;

tilage primordium before the cartilaginous structure

however, chondrocytes undergo hypertrophic-like differentia-

becomes evident, suggesting the contribution of CCN2 to

tion, while producing CCN2 along the ossiﬁcation site facing

Meckel’s cartilage formation itself. Indeed, CCN2 acceler-

the cartilage. Mandibular bone is formed attached to the ‘‘hy-

ates the adhesion and aggregation of the ﬁrst branchial

pertrophic’’ layer of Meckel’s cartilage with marginal bone

arch cells. Another study, one using TGF-b receptor 2

matrix invasion into the cartilage. CCN2 can be found also inside

knockout mice, indicated that CCN2 is a major signaling

the cartilage, suggesting its role in cartilage development.

molecule downstream of the TGF-b signal, which supports

ubiquitous one is the articular cartilage in joints. In the oral the chondrocyte proliferation required for the formation of

region, we ﬁnd the temporomandibular joints (TMJs) as the Meckel’s cartilage [35].

only joints, which collaborate together to realize the com- After the initial formation of Meckel’s cartilage, CCN2

plex movement needed for mastication, pronunciation, and production is then silenced in the chondrocytes. Then, it

106 S. Kubota

recurs upon the hypertrophic-like differentiation of these

cells [34]. Interestingly, along the emerging hypertrophic

chondrocyte-like cells, ossiﬁcation is initiated surrounding

Meckel’s cartilage, which is reminiscent of the events occur-

ring at the cartilage-bone boundary of the growth plate

during endochondral ossiﬁcation (Fig. 2B). This observation

suggests a critical role of CCN2 as a signaling molecule that is

released from Meckel’s cartilage and recruits the osteoblasts

from the developing mesenchyme. One may consider this

event distinct from endochondral ossiﬁcation, since ossiﬁca-

tion proceeds not from the inside, but from the outside of the

cartilage. Nevertheless, the fundamental role of CCN2 as a

messenger from chondrocytes to osteoblasts to guide the

ossiﬁcation is quite similar, well representing the functional

property of this molecule.

3.5. CCN2 and tooth development

As observed in a number of organogenic processes including

heart, lung, and kidney development, odontogenesis fol-

lows multiple steps, in which epithelial—mesenchymal

interaction is critically required [36]. After the formation

of dental lamina, it penetrates into the underlying oral

mesenchyme to co-manufacture a tooth bud. The tooth

bud then develops into the enamel organ, dental papilla,

and dental follicle at the cap stage, and tooth morphogen-

esis is conducted during the following bell stage. At the ﬁnal

crown stage, cells localized in contact at the boundary

between the enamel organ and dental papilla rapidly dif-

ferentiate into ameloblasts and odontoblasts. Following the

initiation of dentin production by odonotoblasts, deposition

of enamel matrix is started by ameloblasts to accomplish the

tooth construction.

In this developmental pathway, CCN2 is recognized to play

Figure 3 Expression of the CCN2 gene during tooth germ

a signiﬁcant role as well. Throughout odontogenesis, CCN2 is

development. Dental epithelium and mesenchyme are repre-

produced under a distinct temporospatial regulation in mur-

sented by solid objects and stippled background, respectively.

ine tooth germs [1,37]. Expression of the CCN2 gene begins

Zones showing CCN2 gene expression are illustrated by shadows

immediately after the formation of dental lamina. At the cap

with arrowheads, according to the ﬁndings presented by Shimo

stage, prominent CCN2 expression is restricted to the enamel

et al. [41]. The area with a wavy pattern in the crown stage

knot located at the very center of the enamel organ-dental

denotes the nascent tooth enamel.

papilla attachment (Fig. 3). CCN2 gene expression persists in

the epithelial and mesenchymal cells along the epithelial—

mesenchymal boundary of the tooth germs throughout the

bell stage. However interestingly, the expression is shut down

Nevertheless, a recent report described that tooth germs

among ameloblasts at the crown stage. Detectable levels of

develop normally even in CCN2-null mice [38]. These appar-

CCN2 continue to be present in the dental follicle and outer

ently contradictory data suggest the functional redundancy

dental epithelium and even in the involuting dental lamina.

of the CCN family members. Indeed, both CCN1 and CCN2

It should be particularly noted that the induction of CCN2

have been reported to exert the same effects on the same

gene expression in the dental epithelium is dependent on the

cells, and both are strongly induced by the same factors [39].

signal provided by the direct contact between it and the

Therefore, it may be assumed that another CCN family

dental mesenchyme. Nevertheless, the molecules responsi-

member may be able to play a compensational role in

ble for this CCN2 induction have not been speciﬁed, though

odontogenesis in the absence of CCN2.

TGF-b and BMP-2 are capable of inducing CCN2 production in

the dental epithelium [37]. However, another report suggests

4. CCN2 as a mediator of tissue remodeling

that signals induced by CCN2 during odontogenesis may be

independent of TGF-b signalings [38].

In any case, studies with recombinant CCN2 and its 4.1. CCN2 as a single coupling factor that

neutralizing antibody indicate that CCN2 promotes not only coordinates efﬁcient bone remodeling

the proliferation but also the differentiation of both epithe-

lium and mesenchymal cells, which together form the tooth, As widely recognized, bone remodeling is continuously

thus indicating its solid contribution to odontogenesis. performed under the collaboration of osteoblasts and

CCN2 in orofacial tissue development and remodeling 107

4.2. Possible cartilage remodeling by CCN2 for

maintaining tissue integrity

Despite that cartilage evolved from a prototypic endoskele-

ton that supported the body, biological missions assigned to

the present permanent cartilage in the human body are quite

different from those of bone. Since uncalciﬁed cartilage does

not serve as a storage depot for calcium and phosphate, it is

not necessary for cartilage to be continuously remodeled to

support calcium homeostasis. Nevertheless, instead of being

major skeletal components of the face, our cartilage plays a

Figure 4 CCN2 as a single coupling factor for bone remodeling. critical role by enabling ﬂexible movement of our jaws,

In bone tissues, CCN2 is produced by immature osteoblasts and taking advantage of its elasticity and stiffness.

mononucleate osteoclast precursors, and initially stored in the The major role of articular cartilage is to soften the

ECM. CCN2 (a marked star) in the ECM acts on both cell types to friction and absorb the pressure between the bones upon

promote their maturation for the coupled resorption and syn- movement; hence, it is constantly subject to mechanical

thesis in a sustainable manner through matricrine actions. Ob, stress load and minor injury. Therefore, another mode of

osteoblast; Oc, osteoclast. tissue remodeling does occur in response to these stimuli, in

order to maintain the physical properties of articular carti-

lage. In this context, it is important to mention that CCN2

protein is induced in chondrocytes upon mechanical stress

osteoclasts, in order to maintain the integrity of bone tissue

load [45]. Furthermore, although CCN2 production by articu-

and calcium homeostasis of the whole body. Mineralized

lar chondrocytes is normally under the detection threshold,

bone is decalciﬁed and its matrix proteins are degraded by

marked CCN2 produced from clustering chondrocytes is

osteoclasts. In parallel with bone resorption, nascent bone

observed upon the development of OA [46]. This CCN2

is produced by osteoblasts to reconstruct the tissue. Not

induction seems to be mediated in part by macrophage-

only systemic factors, but also local factors and cell-surface

colony stimulating factor (M-CSF), also suggesting an unex-

molecules tightly regulate the behavior of osteoblasts and

pected anabolic functional aspect of this molecule in carti-

osteoclasts toward each other. These molecules that med-

lage [47]. According to the fact that CCN2 promotes the

iate the direct interaction between these cells are gener-

proliferation and ECM-molecule production of articular

ally designated as coupling factors. The best known pair of

chondrocytes in vitro, it is reasonable to consider that

coupling factors is the receptor activator of NF-kB (RANK)

CCN2 is induced to reconstruct the damaged articular car-

on osteoclast progenitors and the RANK ligand (RANKL) on

tilage. Conversely, cartilaginous ECM degradation is directly

osteoblasts [40]. Additionally, another pair of cell-surface

carried out by proteases, such as MMPs and ADAM-TS5. It is

molecules was more recently nominated as novel coupling

known that mechanical injury induces the production of

factors of bone remodeling, i.e., the ephrin B2 ligand on

FGF-2 in articular cartilage, which subsequently provokes

osteoclasts and the EphB4 receptor tyrosine kinase on

the production of a number of MMPs including MMP-13. It is

osteoblasts [41]. Of interest, ephrin B2 has also been shown

important to note that a recent study revealed the mole-

to be produced by periodontal ligament cells in a strain-

cular interplay between CCN2 and FGF-2 in cartilage [48].

dependent manner, indicating its involvement in bone

Thus, CCN2 is believed to reorganize the articular cartilage

remodeling upon orthodontic tooth movement [42].

architecture against external stress to maintain its integrity

Here, another mode of coupling between these cells is

under collaboration with other signaling molecules such as

proposed (Fig. 4). As described in a previous section, CCN2 is FGF-2.

produced by osteoblasts at early stages of differentiation and

promotes their proliferation and osteoblastic differentiation

4.3. Tissue remodeling toward orofacial wound

[28]. Moreover, our recent study revealed that the same

healing and ﬁbrosis by CCN2

CCN2 molecule enhances osteoclastogenesis via interaction

with dendritic cell-speciﬁc transmembrane protein (DC-

STAMP), which is a cell-surface molecule playing a role in Upon oral tissue injury, the wounded areas are primarily

the cell fusion events that occur upon the maturation of ﬁlled with blood clot with polymerized ﬁbrin molecules

osteoclasts [43]. In that report, the production of CCN2 by formed through the activation of platelets and the coagula-

osteoclast progenitors was conﬁrmed as well. As such, this tion cascade. Subsequently, ﬁbroblasts migrate therein and

single molecule, CCN2, is indeed coupling osteoblasts and occasionally differentiate into myoﬁbroblasts to produce

osteoclasts and modulating the action of both cells; and thus ﬁbrotic ECM molecules such as type I collagen. This process

it deserves the title of single coupling factor of bone remo- may be followed by the contraction of the wounded area by

deling. Since CCN2 acts on both types of the cells positively, a-smooth muscle actin-positive myoﬁbroblasts. During this

this factor may be coordinating efﬁcient and balanced bone tissue regeneration process, CCN2 is supplied by multiple

remodeling. This functional property of CCN2 is well demon- sources and plays central roles in the regeneration.

strated by the regenerative effect of CCN2 on bone defects Although CCN2 is abundantly encapsulated in platelets

[13]. The fact that mechanical stimuli induce CCN2 produc- and released upon activation of them [25], it is also endo-

tion by osteocytes also indicates the property of CCN2 as a genously produced by the cells around the injured area. In

coordinator of bone remodeling [44]. fact, strong expression of the CCN2 gene is observed in the

108 S. Kubota

tooth extraction sockets [49], as well as in the wounded

5. CCN2 as a therapeutic tool or target in

dermal tissue [50]. Data obtained in vitro showed that CCN2

dental medicine

enhances the synthesis of type I collagen in ﬁbroblasts and

periodontal ligament cells [51,52], further supporting this

5.1. Cartilage regeneration by CCN2

notion.

Tissue ﬁbrosis, which is characterized by the abnormal

Flexible movement of synovial joints including the TMJ highly

accumulation of a vast amount of ﬁbrous ECM and functional

depends on the smooth, elastic, and lubricated surface

deﬁciency, can be regarded as an unsuccessful outcome of

properties of articular cartilage. Therefore, loss of integrity

continuous tissue remodeling toward reconstruction. Con-

of articular cartilage, which is usually caused by repetitive

sistently, CCN2 is known to be a key molecule that develops

mechanical stress load, results in OA with severe locomotive

ﬁbrotic disorders in a variety of organs. The target organs in

dysfunction. Because of the complicated movement required

humans include most tissues and organs including liver,

for their multiple activities, TMJ joints are highly susceptible

kidney, lungs, pancreas, and heart as well as orofacial

to OA-like damage. Indeed, TMJ dysfunction has been noted

tissues, save for neuronal and genital ones [1,4,7]. Over

as a common and major clinical complication in the area of

the past 10 years, CCN2 has been attracting the interest of

clinical dentistry for a long time, even before OA was recog-

dermatologists, since it mediates the pathological changes

nized as a serious issue in orthopedics.

observed in progressive systemic sclerosis or scleroderma

OA is one of the major complications frequently found in

[4,53,54]. In the oral cavity, the most prominent ﬁndings

aged persons in advanced countries [63]. Since OA greatly

indicating the relevance of the CCN2 molecule to ﬁbrotic

affects the quality of life of these patients, exploring a novel

disorders have come from investigations on drug-induced

and fundamental therapeutic strategy is earnestly desired by

gingival overgrowth [55,56]. This symptom is characterized

societies worldwide. In OA cartilage, the articular cartilage

by an overwhelming growth of gingiva composed of a highly

has biologically and physically disintegrated mainly due to

ﬁbrous tissue with elevated CCN2 production, which is

long-term mechanical overload, in spite of the attempt of

induced by an anti-convulsant, phenytoin, or a calcium-

articular chondrocytes to proliferate and to produce CCN2 for

channel blocker, nifedipine. Similar changes may be induced

repair [46]. Therefore, it would be quite reasonable to use

under certain oral conditions; for example, occlusal dys-

CCN2 as a supplement to overcome the damage to the

function and mechanical tension on individual cells have

cartilage in OA. Indeed, exogenous application of CCN2

been implicated in the induction of CCN2 in gingiva [57,58].

remarkably ameliorates the OA-like changes in an experi-

Mechanical stress load-induced CCN2 would supposedly

mental rat model [12], and forced expression of CCN2 in

mediate periodontal tissue remodeling both physiologically

cartilage protects the articular cartilage from OA-like

and pathologically.

damage in mice [64]. These ﬁndings indicate a solid utility

Apart from these factors, there is another major risk

of CCN2 for the treatment of OA and TMJ dysfunction (Fig. 5).

factor that causes periodontal ﬁbrosis. Clinically, gingival

The regeneration potential of CCN2 is more clearly seen

ﬁbrosis has been frequently observed among smokers,

from the results of experiments with another rat model. For

although the mechanism of the onset has not yet been

obtaining severest damage to articular cartilage, Nishida

clariﬁed. However, it is now clear from a recent study that

et al. prepared rats with a full-thickness defect in their knee

nicotine in the smoke is the major agent that induces period-

cartilage [12]. Exogenous application of CCN2 into the car-

ontal ﬁbrosis [59]. In cultures of normal human gingival

tilage defect, in combination with gelatin hydrogel for gra-

ﬁbroblasts and periodontal ligament cells, nicotine promotes

dual release, efﬁciently regenerated articular cartilage

CCN2 production and type I collagen deposition that can be

therein, without causing overgrowth.

neutralized by anti-CCN2 antibody. It is already known that

As stated in a previous section, CCN2 is also capable of

these periodontal cells produce CCN2 in response to TGF-b by

enhancing the regeneration of auricular cartilage [32].

transcriptional activation of the CCN2 gene [60,61], whereas

Indeed, the application of CCN2 onto the cultured auricular

no transcriptional activation is observed upon nicotine treat-

chondrocyte pellets enhances their growth in vivo as sub-

ment. Thus, a TGF-b-independent, post-transcriptional reg-

cutaneous explants. Although no reports have described the

ulation is driving the expression of CCN2 mRNA in this event

regeneration of damaged pinna in vivo, CCN2 may be useful

occurring in a smoker’s oral cavity.

in esthetic reconstruction of ears, if an appropriate drug

Since plaque control and other management of period-

delivery system can be established as actually done in the

ontal disease under a ﬁbrous gum are difﬁcult, the period-

case of articular cartilage.

ontal disease that occurs in these patients is generally

resistant to dental therapeutics. Therefore, molecular ther-

apy targeting CCN2 can be considered for the gingival ﬁbrosis 5.2. Bone regeneration by CCN2

mentioned above, even though quitting smoking is the best

choice for the last case. Finally, CCN2 is also known to play a In addition to the in vitro ﬁndings that CCN2 promotes the

signiﬁcant role in the maintenance of the integrity of oral proliferation and differentiation of osteoblasts, it was also

mucosa other than the gingiva. According to a recent report, shown that CCN2 is produced at the site of regeneration after

CCN2 is suggested to be responsible for the submucous a bone fracture [3]. Since the endochondral ossiﬁcation-like

ﬁbrosis induced by arecoline, an active ingredient of Areca process recurs during the repair of a bone fracture, it may be

nuts traditionally consumed in certain Asian countries, on the supposed that a sustainable supply of CCN2 is made possible

buccal oral mucosa [62]. Both nicotine and arecoline are from platelets for immediate discharge and from prehyper-

known as carcinogens, supporting the ﬁndings that CCN2 is trophic chondrocytes for delayed production after differen-

involved in malignant tumors of various organs as well. tiation. By mimicking this process, we are able to accelerate

CCN2 in orofacial tissue development and remodeling 109

the regeneration of intractable bone defects. In a rat model, promotes the bone regeneration up to the exactly desired

application of CCN2 adsorbed onto gelatin hydrogel for dur- level without inducing any overgrowth. In these cases, the

able and gradual release efﬁciently accelerated the regen- boundary of the original cortical tissue and the regenerated

eration of intractable bone defects [13]. This is not of a great one at the defect site is almost invisible by histological

impact per se; for other factors, such as BMP-2 and FGF-2, observation [13]. The property of CCN2 to enhance the

have been indicated to be useful in promoting bone repair. healing potential of bone tissue in a harmonized manner is

However, CCN2 has a precious advantage that is not found in of course ascribable to the molecular action of this protein as

the case of these other bone-generating factors. In concor- a CCN family member. In clinical dentistry, alveolar bone

dance with the effect on damaged articular cartilage, CCN2 regeneration is a critical issue for the efﬁcient support of

Figure 5 Experimental prevention and treatment of OA with CCN2. Under normal conditions, adequate mechanical stress causes

minimal degradation and balanced synthesis of the ECM to maintain the integrity of articular cartilage, which is supported by the CCN2

(marked stars) stored therein (upper left panel). Mechanical or another extensive stress that induces massive ECM degradation (‘‘ECM’’

in white) also harms the matricrine retention of CCN2 as well as the physical properties of the cartilage. In response, chondrocytes

proliferate, forming clusters, and produce ECM (‘‘ECM’’ in black) components and CCN2, which, however, end up being in insufﬁcient

quantity for regeneration of a duly organized ECM (upper right panel). In the presence of excessive CCN2 storage prepared in advance,

chondrocytes are ready to produce sufﬁcient amounts of ECM molecules to maintain the articular cartilage, even when it incurs

extensive damage (lower left panel). Even after the onset of OA, external and durable administration of CCN2 compensates the loss of

matricrine stimulation by CCN2, resulting in the efﬁcient assistance of articular chondrocytes to regenerate the disintegrated cartilage

tissue. Dotted, solid, and open arrows denote molecule production by, effect on, and proliferation of the articular chondrocytes, respectively.

110 S. Kubota

dentures and stable ﬁxation of dental implants for rigid However, selective activation of Smad7 inside the cells by

prosthesis. With BMP-2, for example, nowadays it is not an extracellular approach appears difﬁcult at present.

difﬁcult to manufacture bone on the alveolar arch or at Another report has described that nifedipine-induced gingi-

the bottom of a sinus. Nevertheless, clinicians sometimes val ﬁbrosis that is mediated by CCN2 in concert with androgen

face difﬁculty in the maintenance of the added bone against receptors can be inhibited by ﬂutamide, a non-steroidal

subsequent resorption [65]. Considering its molecular nat- receptor antagonist of androgens [67]. Regulation of CCN2

ure, CCN2 may be more effective in keeping the engineered gene expression by direct administration of antisense oligo-

bone in a desired form as a coordinator of bone remodeling. nucleotides is being attempted as well in vivo. According to a

recent study, injection of an anti-CCN2 modiﬁed oligonucleo-

tide into skin wounds represses scar formation without

5.3. Regulation of periodontal ﬁbrosis via CCN2

affecting the proper wound healing process [68]. As such,

development of novel strategies to down-regulate CCN2 is

Since CCN2 is a central mediator of ﬁbrotic disorders, ther-

desired and is actually underway to control gingival ﬁbrosis.

apeutic approach targeting CCN2 in controlling ﬁbrosis in

every relevant organ ought to be explored in the future. In

the tissues present deep inside of the body, such as the heart, 6. Conclusions for the future

kidneys and liver, a highly sophisticated drug delivery system

is indispensable to speciﬁcally target CCN2 in these organs According to the scientiﬁc evidence that has accumulated to

with agents that regulate its gene expression or modify its date, it is obvious that CCN2 plays a key role in the devel-

molecular behavior. In contrast, the oral cavity and skin are opment of orofacial tissues. Consistent with this notion, the

relatively easy to access without speciﬁc device for targeted medical utility of CCN2 in regenerating cartilage, bone, and

delivery. Therefore, the anti-CCN2 strategy against skin other connective tissues has been established. Moreover, the

dermal and gingival ﬁbrosis is currently actively explored. functional property of CCN2 as a critical mediator of ﬁbro-

Molecular therapy targeting CCN2 is being developed genesis is expected to provide a clue for the development of

against drug-induced gingival ﬁbrosis, since removal of etio- anti-ﬁbrotic therapeutics targeting this molecule. However,

logical factors is difﬁcult in those cases [56]. A recent report one should carefully consider the novel molecular action and

has indicated that activation of Smad7, a repressive mediator multiple functionality of CCN2 in exploring this anti-CCN2

of intracellular signal transduction, in gingival ﬁbroblasts is strategy in the future.

effective in preventing the CCN2 induction and ﬁbroblast/ As explained in this article, CCN2 function is exerted by

myoﬁbroblast transition that cause gingival ﬁbrosis [66]. the molecular interaction with a huge number of molecules

Figure 6 Regulation of the CCN2 gene and possible strategies for its therapeutic control. Factors affecting the expression of the CCN2

gene are shown in ellipses, whereas key factors for anti-CCN2 strategies are marked by rectangles. Solid and dotted arrows indicating

regulations represent direct and indirect effects of the corresponding molecules. Transcriptional and post-transcriptional regulations

are illustrated, respectively above and below the illustrated CCN2 gene structure. Thick and thin boxes indicate exons and the

promoter, whereas lines indicate introns in the gene structure. TGF-b utilizes both Smad- and Rac1/cdc42-mediated signaling

pathways for CCN2 transcriptional activation as well as indirect induction through endothelin-1 (ET-1). Coding exons comprising the

open reading frame (ORF) are also speciﬁed by solid boxes, whereas untranslated regions (UTRs) are in open boxes in the structures

representing the CCN2 gene and its mRNA. Other abbreviations: GAPDH, glyceraldehyde 3-dehydrogenase; NPM, nucleophosmin/B23.

CCN2 in orofacial tissue development and remodeling 111

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