The Functions of Runx Family Transcription Factors and Cbfb in Skeletal Development

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Oral Science International 12 (2015) 1–4

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Review

The functions of Runx family transcription factors and Cbfb in

skeletal development

∗

Toshihisa Komori

Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan

a r a

t i c l e i n f o b s t r a c t

Article history: Runx family transcription factors consist of Runx1, Runx2, and Runx3. Runx1 is involved in chondro-

Received 9 October 2014

cyte differentiation at an early stage. Runx2 is a major transcription factor for chondrocyte maturation,

Received in revised form 1 December 2014

and Runx3 has a redundant function with Runx2 and it is partly involved in chondrocyte maturation.

Accepted 4 December 2014

Runx2 directly regulates Ihh expression and enhances chondrocyte proliferation. Runx2 is an essential

transcription factor for osteoblast differentiation. Heterozygous mutation of RUNX2 causes cleidocranial

Keywords:

dysplasia, which is characterized by hypoplastic clavicles, open fontanelles, and supernumerary teeth.

Runx2

Runx2 directly regulates Sp7 expression, and Runx2 together with Sp7 and canonical Wnt signaling com-

Cbfb

Chondrocytes pletes determination of the lineage of mesenchymal cells into osteoblasts. Runx2 expression in osteoblasts

Osteoblasts is regulated by a 343-bp enhancer. Dlx5/6 and Mef2 directly bind this enhancer; form an enhanceosome

Enhancer with Tcf7, ␤-catenin, Sox5/6, Smad1, and Sp7; and activate this enhancer. Msx2 inhibits this enhancer,

and switching of the binding of Msx2 to Dlx5 is important for this activation. Core binding factor ␤ (Cbfb)

forms a heterodimer with Runx family proteins and enhances their DNA-binding capacity. In Cbfb con-

ditional knockout mice in osteoblast and chondrocyte lineages, the differentiation of chondrocytes and

osteoblasts is inhibited. All of the Runx family proteins are reduced in the cartilaginous limb skeletons

and calvariae in Cbfb conditional knockout mice, although the reduction of Runx2 protein in calvariae is

much milder than that in cartilaginous limb skeletons. Therefore, Cbfb regulates skeletal development

by stabilizing Runx family proteins, and Runx2 protein stability is less dependent on Cbfb in calvariae

than in cartilaginous limb skeletons.

Contents

1. Introduction ...... 1

2. Runx2 and chondrocyte differentiation ...... 2

3. Runx2 and osteoblast differentiation ...... 2

4. Transcriptional regulation of Runx2 gene for osteoblast differentiation ...... 3

5. Cbfb and Runx family transcription factors ...... 3

6. Conclusions ...... 4

References ...... 4

1. Introduction then replaced with bone by osteoblasts and osteoclasts through

the process of endochondral ossiﬁcation. After mesenchymal

The vertebrate skeleton is composed of cartilage and bone. cell condensation, pluripotent mesenchymal cells differentiate

Bone is formed through either intramembranous or endochondral into immature chondrocytes, which produce Col2a1 and proteo-

ossiﬁcation. Osteoblasts directly form intramembranous bones, glycan. The immature chondrocytes mature into hypertrophic

while chondrocytes ﬁrst form a cartilaginous skeleton, which is chondrocytes, which express Col10a1, and ﬁnally become ter-

minal hypertrophic chondrocytes, which express Spp1 (secreted

phosphoprotein 1/osteopontin), Ibsp (integrin-binding sialopro-

∗ tein/bone sialoprotein II), Mmp13 (matrix metallopeptidase 13),

Tel.: +81 95 819 7630; fax: +81 95 819 7633.

and Vegf (vascular endothelial growth factor) [1–3].

E-mail address: [email protected]

http://dx.doi.org/10.1016/S1348-8643(14)00032-9

2 T. Komori / Oral Science International 12 (2015) 1–4

Runx family transcription factors consist of Runx1, Runx2, and

Runx3. Runx2 is an essential transcription factor for osteoblast

differentiation [4,5]. Heterozygous mutation of RUNX2 causes clei-

docranial dysplasia (CCD), which is characterized by hypoplastic

+/−

clavicles, open fontanelles, and supernumerary teeth, and Runx2

mice also show hypoplastic clavicles and open fontanelles [6]. Fur-

thermore, Runx2 regulates chondrocyte maturation and Runx3 is

partly involved in it [2,7–12]. Runx2 is also required for vascular

invasion into the mineralized cartilage, which is composed of ter-

minal hypertrophic chondrocytes [13,14]. Runx1 is also involved in

chondrocyte differentiation, at least in the development of some of

the endochondral bones [15,16]. Core binding factor ␤ (Cbfb) is a

co-transcription factor, which forms a heterodimer with Runx fam-

ily transcription factors [17]. In this review, the major functions

of Runx2 in osteoblasts and chondrocytes, the functions of Cbfb

in skeletal development, and involvement of Runx1 and Runx3 in Fig. 2. Regulation of osteoblast differentiation by Runx2, Tcf/Lef, Ctnnb1, and Sp7.

Runx2, Tcf/Lef, Ctnnb1, and Sp7 are required for the differentiation of pluripotent

endochondral ossiﬁcation are described.

mesenchymal cells to osteoblasts. Osteoblast-speciﬁc expression of Runx2 is reg-

ulated by Dlx5/6, Mef2, Tcf7, Ctnnb1, Smad1/5, Sp7, and Sox5/6, which activate

2. Runx2 and chondrocyte differentiation the 343-bp osteoblast-speciﬁc enhancer. Runx2 regulates Sp7 and Tcf7 expression.

Therefore, there are reciprocal regulations between Runx2 and Sp7 and between

Runx2 and Tcf7 [29].

Runx2 is weakly expressed in resting and proliferating chon-

drocytes and upregulated in prehypertrophic chondrocytes, and

maturation and vascular invasion into the cartilage (Fig. 1). How-

the high expression is maintained in hypertrophic and terminal

−/− ever, these ﬁndings also indicate that some transcription factors

hypertrophic chondrocytes [2]. Runx2 mice die at birth due to

have a redundant function with Runx2 in chondrocyte maturation.

the failure of breathing caused by the lack of ossiﬁcation in the

−/− Runx3 is also expressed in prehypertrophic and hypertrophic

thoracic cage. In the skeleton of Runx2 newborn mice, all endo-

chondrocytes. Chondrocyte maturation is mildly retarded in

chondral bones are cartilaginous, and mineralization is virtually

−/−

Runx3 mice at E15.5 but not at E18.5. Hypertrophic chondro-

absent throughout the skeleton, except the tibia, ﬁbula, radius,

cytes are completely absent, and the expression of Ihh and Pth1r

and ulna [4]. When the chondrocytes terminally differentiate, the

−/− −/−

is undetectable in the whole skeleton in Runx2 Runx3 mice,

matrix in the terminal hypertrophic chondrocyte layer is miner-

−/− indicating that chondrocyte maturation is completely blocked in

alized. In the tibia, ﬁbula, radius, and ulna in Runx2 embryos

−/− −/−

Runx2 Runx3 mice [11]. Therefore, Runx2 is a major transcrip-

at embryonic day (E) 18.5, chondrocytes terminally differentiate

tion factor for chondrocyte maturation and Runx3 has a redundant

and express Spp1, Ibsp, and Mmp13, and the matrix is mineralized

function with Runx2 in chondrocyte maturation (Fig. 1).

without vascular invasion. However, chondrocyte hypertrophy and

−/−

The lengths of limbs are shortened in Runx2 mice and fur-

Col10a1 expression are virtually absent in the rest of the skeleton.

−/− −/−

ther shortened in Runx2 Runx3 mice [11]. As chondrocyte

Furthermore, the expression of Ihh and Pth1r is severely reduced

−/−

proliferation is reduced in Ihh mice, Ihh positively regu-

[2]. These ﬁndings indicate that Runx2 is required for chondrocyte

lates chondrocyte proliferation [18]. Ihh expression is severely

−/−

reduced in Runx2 mice. There are several perfect Runx2-binding

sequences in the Ihh promoter, and Runx2 directly regulates Ihh

expression (Fig. 1). Although Ihh expression is undetectable in

−/− −/−

Runx2 Runx3 mice, Runx3 cannot regulate Ihh expression. The

−/− −/−

further reduction of limb length in Runx2 Runx3 mice com-

−/−

pared with that in Runx2 mice is due to the reduction in the size

of chondrocytes because chondrocyte maturation is completely

−/− −/−

blocked in Runx2 Runx3 mice [11].

3. Runx2 and osteoblast differentiation

Intramembranous bones and bone collar formation are com-

−/−

pletely absent in Runx2 mice. Osteoblast differentiation is

blocked at an early stage, and weak alkaline phosphatase activ-

ity is detected only in the periphery of the calciﬁed cartilage in the

−/−

tibia, ﬁbula, radius, and ulna [4]. Furthermore, Runx2 calvarial

cells spontaneously differentiate into adipocytes and differentiate

into chondrocytes in the presence of bone morphogenetic pro-

tein (BMP)-2 in vitro, but they do not differentiate into osteoblasts

even in the presence of BMP-2 in vitro or in vivo [19]. Therefore,

−/−

Runx2 mesenchymal cells have the potential to differentiate into

adipocytes and chondrocytes, but they completely lack the poten-

tial to differentiate into osteoblasts (Fig. 2).

−/−

Fig. 1. Regulation of chondrocyte maturation and proliferation by Runx2 and Osteoblast differentiation is also completely blocked in Sp7

Runx3. Runx2 enhances chondrocyte maturation and Runx3 is partly involved it. mice and mice with the deletion of Ctnnb1 (␤-catenin) in mes-

Runx2 induces the expression of Ihh, which enhances chondrocyte proliferation.

enchymal cells (Fig. 2). In both types of mice, perichondral

Ihh induces the expression of Pthlh, which inhibits Runx2 expression. Further, Ihh

mesenchymal cells, which express Runx2, condense and differen-

is required for the expression of Runx2 in mesenchymal cells in the perichondrium,

−/−

in which bone collar formation occurs. tiate into chondrocytes, indicating that Sp7 mesenchymal cells

T. Komori / Oral Science International 12 (2015) 1–4 3

osteoblasts. Furthermore, the histone variant H2A.Z is enriched

in the enhancer. These are typical chromatin modiﬁcations in

enhancers. The enhancer is sufﬁcient to direct Runx2 expression

speciﬁcally to osteoblasts [29].

5. Cbfb and Runx family transcription factors

Inversion of chromosome 16 involves breakage of CBFB, and it

is associated with acute myeloid leukemia in humans. Cbfb forms

a heterodimer with Runx1, and both Runx1 and Cbfb are essential

−/−

for hematopoiesis in the fetal liver [30]. Cbfb mice die at midges-

tation due to the absence of fetal liver hematopoiesis. Rescue of

Fig. 3. The functions of Runx family transcription factors and Cbfb in skeletal devel-

−/−

opment. Runx1 and Runx2 have a redundant function in chondrocyte differentiation, hematopoiesis in Cbfb mice by mating with Cbfb transgenic

at least in some parts of the skeleton. Runx2 is a major transcription factor for mice under the control of Gata1 promoter, which directs reporter

chondrocyte maturation, and Runx3 is partly involved in it. Runx2 is an essential

gene expression to primitive and deﬁnitive erythroid cells, revealed

transcription factor for osteoblast differentiation, and the 343-bp enhancer regulates

the requirement for Cbfb in skeletal development [31]. Rescue of

Runx2 expression in osteoblasts. The contribution of Runx1 and Runx3 in osteoblast

−/−

hematopoiesis in Cbfb mice using Tek promoter Cbfb transgenic

differentiation remains to be clariﬁed. Cbfb forms heterodimers with Runx family

transcription factors, enhances their DNA binding, and inhibits their degradation. mice also showed similar results [32]. In another mouse model in

which GFP was inserted into exon 5 of Cbfb to generate a Cbfb–GFP

fusion protein, the skeletal development was also impaired [33].

−/−

and Ctnnb1 mesenchymal cells maintain the potential to dif-

Chondrocyte maturation is delayed, chondrocyte proliferation

ferentiate into chondrocytes [20–23]. However, the mesenchymal

is reduced, and osteoblast differentiation is inhibited in Cbfb condi-

condensation and the differentiation of the mesenchymal cells into

tional knockout mice using Dermo1-Cre knock-in mice, in which Cre

−/−

chondrocytes are not observed in Runx2 mice [4]. As Sox9 is

recombinase is expressed in mesenchymal cells giving rise to both

essential for mesenchymal condensation and chondrocyte differ-

chondrocyte and osteoblast lineages [34]. The proteins of Runx1,

entiation during embryogenesis [24], these ﬁndings suggest that

Runx2, and Runx3 are reduced in the cartilaginous limb skeletons of

Runx2 is required for mesenchymal condensation, which is a pre-

Cbfb conditional knockout embryos at E15.5. The proteins of Runx1,

requisite for chondrocyte differentiation, in the cells that do not

Runx2, and Runx3 are also reduced in calvarial tissues of Cbfb con-

express enough Sox9.

ditional knockout embryos, although the reduction of the Runx2

Runx2 directly regulates Sp7 expression [25,26]. It also inhibits

protein in calvarial tissues is much milder than that in cartilagi-

adipocyte differentiation and directs the mesenchymal cells to

nous limb skeletons [34] (Fig. 3). Therefore, the Runx2 protein is

osteoblasts. Runx2 together with Sp7 and canonical Wnt signal-

more stable in calvariae than cartilaginous limb skeletons in the

ing completes the lineage determination of mesenchymal cells into

absence of Cbfb. This suggests that some unknown proteins com-

osteoblasts, completely blocking the ability of mesenchymal cells

pensate for the lack of Cbfb in the calvariae to protect against the

to differentiate into chondrocytes [27] (Fig. 2).

degradation of the Runx2 protein. Although the development of

+/−

endochondral bones is virtually normal in Runx2 mice, the cal-

+/−

4. Transcriptional regulation of Runx2 gene for osteoblast variae and clavicles are more severely affected in Runx2 mice

differentiation than in Cbfb conditional knockout mice [34]. The relative stability

of the Runx2 protein in the calvariae of Cbfb conditional knock-

Runx2 messenger RNA (mRNA) is transcribed from two pro- out mice can explain why Cbfb conditional knockout mice show a

moters, distal (P1) and proximal (P2) promoters, and speciﬁcally severe delay in endochondral ossiﬁcation but milder deformities

expressed in osteoblasts and chondrocytes [28]. Runx2 expression in the calvariae and the lateral parts of clavicles, which are formed

in osteoblasts is mainly transcribed from the P1 promoter. How- through intramembranous ossiﬁcation. All Runx family proteins are

ever, neither promoter drives reporter genes to osteoblasts in mice, involved in endochondral ossiﬁcation, and Cbfb is likely to be nec-

suggesting the presence of an enhancer for the osteoblast-speciﬁc essary for all of the Runx family proteins in endochondral bone

expression. Green ﬂuorescent protein (GFP) reporter mice using a formation (Fig. 3). In particular, Runx2 and Runx3 have redundant

bacterial artiﬁcial chromosome (BAC) clone of the Runx2 gene locus function in chondrocyte maturation, and Runx3 partly compen-

successfully recapitulated the endogenous expression of Runx2. sates for the lack of Runx2. Runx1 is involved in the development

An enhancer for osteoblast-speciﬁc expression has been identi- of sternum, occipital bone, and palate by regulating chondrocyte

ﬁed by deletion of the BAC clone. GFP is speciﬁcally expressed in differentiation [15,16]. However, the impaired development of the

+/−

osteoblasts in GFP reporter mice driven by the 343-bp enhancer calvariae and clavicles in Runx2 mice indicates that Runx1 and

and a minimal promoter (Fig. 3). The sequence of this enhancer is Runx3 cannot compensate for the haplodeﬁciency of Runx2 in their

highly conserved among mouse, human, dog, horse, opossum, and development. It explains why CCD patients show typical pheno-

chicken. Dlx5/6 and Mef2 directly bind to the enhancer. Dlx5/6 and types in the calvariae and clavicles [34].

Mef2 form an enhanceosome with Tcf7, Ctnnb1, Sox5/6, Smad1, and Recently, other groups also reported various Cbfb conditional

Sp7, and they activate the enhancer (Fig. 2). Although both Msx2 knockout mice. Wei’s group generated Cbfb conditional knockout

and Dlx5 bind to the homeobox motif in the enhancer, Msx2 inhibits mice using Prrx1-Cre mice, Sp7-Cre mice, Col1a1-Cre mice, Dermo1-

the enhancer activity. The binding of Msx2 to the enhancer is Cre mice, and Col2a1-Cre mice [35–38]. They also revealed that Cbfb

detected in uncommitted mesenchymal cells, C3H10T1/2, whereas is required for chondrocyte maturation and osteoblast differentia-

the binding of Dlx5 to the enhancer is detected in osteoblastic tion and for both intramembranous and endochondral ossiﬁcation.

MC3T3-E1 cells. Therefore, the switching of the binding from Msx2 However, they did not observe the reduction of Runx family pro-

ﬂ/ﬂ

to Dlx5 is important for activation of the enhancer [29]. teins in the cartilaginous skeletons of Cbfb Col2a1-Cre mice and

fl/fl fl/fl

The enhancer is highly enriched for histone H3 mono- and in the calvaria of Cbfb Sp7-Cre mice and Cbfb Col1a1-Cre mice

dimethylated at Lys4 and acetylated at Lys27 and Lys18, but [35,38]. Lim et al. also reported Cbfb conditional knockout mice

it is depleted for histone H3 trimethylated at Lys4 in primary using Col1a1-Cre mice [39]. They showed that cortical bone is

4 T. Komori / Oral Science International 12 (2015) 1–4

ﬂ/ﬂ

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