B

Af

TDF

R

lrmmomdifii

(rtrPhwpqaTigrgrt

C

f

Biochemical and Biophysical Research Communications 276, 813–816 (2000)

doi:10.1006/bbrc.2000.3460, available online at http://www.idealibrary.com on

REAKTHROUGHS AND VIEWS

Fundamental Transcription Factoror Bone and Cartilage

oshihisa Komoriepartment of Molecular Medicine, Osaka University Medical School, Suita, Osaka 565-0871, Japan; and “Form andunction,” Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Japan

eceived August 2, 2000

blasts, chondrocytes, and osteoclasts, were affected(dmCdatccc

iiofotcimtcipRstcCepmsp(cf

Cbfa1-deficient mice were found to show a completeack of bone formation owing to the maturational ar-est of osteoblasts. Cbfa1 plays key roles in the deter-ination of osteoblastic lineage from multipotentialesenchymal cells, their differentiation into mature

steoblasts, and transcriptional regulation of boneatrix-related genes. Cbfa1 positively regulates chon-

rocyte maturation and osteoclast differentiation ands required for vascular invasion into cartilage. There-ore, complete elucidation of the function of Cbfa1 andts signaling would be of great benefit in understand-ng skeletogenesis. © 2000 Academic Press

Cbfa1 (core binding factor a1), also called Pebp2aApolyoma enhancer binding protein) or Runx2 (runt-elated gene 2), is a transcription factor that belongs tohe runt-domain gene family (1). There are threeunt-domain genes, Cbfa1/Pebp2aA/Runx2, Cbfa2/ebp2aB/Runx1, and Cbfa3/Pebp2aC/Runx3, whichave a DNA-binding domain, runt, that is homologousith the Drosophila pair-rule gene runt (2–5). Theseroteins form heterodimers with Cbfb/Pebp2b and ac-uire enhanced DNA binding activity in vitro (6, 7),nd they specifically recognize a consensus sequence,GT/CGGT (8–11). The DNA-binding site of Cbf was

dentified in the promoter region of the osteocalcinene (12). Cbfa2 reacted to the osteocalcin promoteregion and transcriptionally activated the osteocalcinene (13, 14). Finally, targeted disruption of Cbfa1evealed that Cbfa1 is an osteoblast-specific transcrip-ion factor (15, 16).

bfa1-DEFICIENT MICE

Cbfa1-deficient mice showed multiple defects in boneormation, because three major component cells, osteo-

813

15–19). The mutant mice completely lacked both en-ochondral and intramembranous ossification, andature osteoblasts were absent throughout the body.hondrocyte maturation was also disturbed in Cbfa1-eficient mice, and it was blocked prior to the appear-nce of prehypertrophic chondrocytes in most parts ofhe skeleton (18, 19). Therefore, the failure of endo-hondral ossification in Cbfa1-deficient mice wasaused by the combined defects of osteoblasts andhondrocytes.Cbfa1-deficient mice showed no vascular invasion

nto cartilage (15–19). The regulation of many factors,ncluding VEGF, MMP13, MMP9, chondromodulin I,steopontin, and bone sialoprotein, seem to be requiredor vascular invasion into cartilage (18, 20, 21). Indeed,steoclasts, which produce MMP9, also contribute tohis vascular invasion. In Cbfa1-deficint mice, calcifiedartilage was observed in limited parts of the skeletonncluding tibia, fibula, radius, and ulna. Only a few

ononuclear TRAP (tartrate-resistant acid phospha-ase)-positive cells appeared adjacent to the calcifiedartilage at the perichondrium (15, 17). Calvarial cellssolated from Cbfa1-deficient embryos could not sup-ort osteoclast formation efficiently in vitro, andANKL (receptor activator of NF-kB ligand) expres-ion was absent in Cbfa1-deficient embryos, suggestinghat the lack of RANKL expression in osteoprogenitorells is one reason for the absence of osteoclasts inbfa1-deficient mice (22). Although chondromodulin Ixpression was normally regulated and VEGF was ex-ressed in hypertrophic chondrocytes of the mutantice, no expression of MMP13, osteopontin, or bone

ialoprotein was observed in their terminal hypertro-hic chondrocytes (18, T. Komori, unpublished data)Fig. 1). Thus, the failure of vascular invasion intoartilage seems to be caused by the lack of multipleactors.

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

LM

astmBttcctcltmci

OO

ae(

diaboepdol(oCO2i3cMTtdittift

da

Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INEAGE DETERMINATION FROM MULTIPOTENTIALESENCHYMAL CELLS

Cbfa1-deficient calvarial cells completely lost theirbility to differentiate into osteoblasts because theyhowed a low level of alkaline phosphatase (ALP) ac-ivity, undetectable level of osteocalcin mRNA, and noineralization during long-term culture; further,MP-2 could not induce their differentiation into os-

eoblasts in vitro or in vivo (23). However, they spon-aneously differentiated into adipocytes, with in-reased expression of PPARg2, LPL, and aP2, and intohondrocytes in the presence of BMP-2. This indicateshat they are enriched with immature mesenchymalells, which can differentiate into mesenchymal cellineages other than osteoblasts. Thus, it is suggestedhat Cbfa1 plays an essential role in the lineage deter-ination of multipotential mesenchymal precursor

ells by inducing osteoblastic differentiation and inhib-ting adipocyte differentiation (Fig. 1).

STEOBLAST DIFFERENTIATION AND REGULATIONF BONE MATRIX-RELATED GENES

Cbfa1 is weakly expressed in osteoprogenitor cells,nd its expression increases during osteoblast differ-ntiation; however, it is downregulated in osteocytes15, 16, 18, 19, 24). The cells expressing Cbfa1 decrease

FIG. 1. Role of Cbfa1 in skeletogenesis. Cbfa1 determines osteobifferentiation, and controls the production of bone matrix proteinsnd controls the production of matrix proteins in hypertrophic chon

814

uring aging (T. Komori, unpublished data), but theyncrease in the process of fracture healing (25). Cbfa1 isble to induce both early and late markers for osteo-last differentiation, including ALP, type I collagen,steopontin, bone sialoprotein, and osteocalcin in sev-ral cell lines or fibroblasts (24, 26) (Fig. 1). The ex-ression of these markers is virtually absent in Cbfa1-eficient mice (15), and Cbfa1 also controls expressionf bone matrix proteins after birth (27). Cbfa1 has ateast two isoforms with different N-terminal sequences2, 28, 29). The first one, translated from exon 2, wasriginally named Pebp2aA (hereafter termed type Ibfa1) (2); the other, translated from exon 1, was calledSF2/til1 isoform (hereafter termed type II Cbfa1) (28,9). The Cbfa1 isoforms seem to have different capac-ties in their activation of target genes of Cbfa1 (26, 30,1). However, the data that have been reported are notonsistent, and further investigation will be needed.MP13 was induced by Cbfa1 in the presence ofGF-b (32), and parathyroid hormone induced activa-ion of MMP13 promoter by protein kinase A (PKA)-ependent phosphorylation of Cbfa1 (33). Cbfa1 alsoncreased the promoter activity of TGF-b type I recep-or (34). Runt-domain factors themselves have weakransactivation domains and regulate gene expressionn cooperation with other transcriptional regulatoryactors (35–41). For example, osteopontin is coopera-ively regulated by Cbfa1 and ets1 (42).

tic lineage and inhibits adipocyte differentiation, induces osteoblastature osteoblasts, is a positive inducer in chondrocyte maturation,

cytes.

lasin mdro

Cbfa1 AND CHONDROCYTE MATURATION

epttdsgeptsadcAtuocgccdsmi

F

p4edcypu4ltsdupifem

R

2. Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J.,

1

1

1

1

1

1

1

11

1

2

2

2

2

2

2

2

Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Both Cbfa1 isoforms (type I and type II) were weaklyxpressed in proliferating chondrocytes, and their ex-ression was upregulated during chondrocyte matura-ion, although type I Cbfa1 was upregulated earlierhan type II Cbfa1 (43). Terminal hypertrophic chon-rocytes as well as osteoblasts showed the most exten-ive expression of both isoforms of Cbfa1. In a chondro-enic cell line, ATDC5, type I Cbfa1 expression waslevated prior to differentiation to the hypertrophichenotype, and treatment with antisense oligonucleo-ides for type I Cbfa1 reduced type X collagen expres-ion in ATDC5 cells (43). However, the dominant neg-tive form of Cbfa1, which contains only the runtomain, inhibited the cellular condensation of ATDC5ells (44). Therefore, Cbfa1 has dual functions inTDC5 cells: the induction of cellular condensation at

he early stage and the induction of chondrocyte mat-ration at a later stage. Retrovirally forced expressionf either type I or type II Cbfa1 in chick immaturehondrocytes decreased cell proliferation but inducedlycosaminoglycan production, ALP activity, type Xollagen and MMP13 expression, and extensiveartilage-matrix mineralization (43). However, theyid not induce any osteocalcin expression. These re-ults, combined with the phenotype of Cbfa1-deficientice, indicate that Cbfa1 is a positive regulatory factor

n chondrocyte maturation (Fig. 1).

UTURE DIRECTIONS

Cbfa1 was induced by BMP-2, -4/7, and -7, and sup-ressed by 1a,25(OH)2D3 and glucocorticoid (24, 30,5). However, other factors will also influence Cbfa1xpression, because the level of Cbfa1 expression isifferent depending on the maturational stage of bothhondrocytes and osteoblastic cells. Cbfa1 is phosphor-lated by the MAPK pathway or PKA, and is sup-ressed by proteolytic degradation involving anbiquitin/proteasome-dependent mechanism (33, 46,7). The pathways for Cbfa1 induction and posttrans-ational modification are important aspects that needo be further investigated. Clarification of the down-tream genes for osteoblast differentiation and chon-rocyte differentiation, the most important task for thenderstanding of skeletogenesis, remains to be accom-lished. Finally, the findings on Cbfa1-deficient calvar-al cells raise the question as to whether transcriptionactors that are responsible for the determination ofach lineage regulate one another in the lineage deter-ination of mesenchymal cells.

EFERENCES

1. Komori, T., and Kishimoto, T. (1998) Curr. Opin. Genet. Dev. 8,494–499.

815

Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad.Sci. USA 90, 6859–6863.

3. Bae, S. C., Yamaguchi-Iwai, Y., Ogawa, E., Maruyama, M., Inu-zuka, M., Kagoshima, H., Shigesada, K., Satake, M., and Ito, Y.(1993) Oncogene 8, 809–814.

4. Bae, S. C., Takahashi, E., Zhang, Y. W., Ogawa, E., Shigesada,K., Namba, Y., Satake, M., and Ito, Y. (1995) Gene 159, 245–248.

5. Kania, M. A., Bonner, A. S., Duffy, J. B., and Gergen, J. P. (1990)Genes Dev. 4, 1701–1713.

6. Ogawa, E., Inuzaka, M., Maruyama, M., Satake, M., Naito-Fujimoto, M., Ito, Y., and Shigesada, K. (1993) Virology 194,314–331.

7. Wang, S., Wang, Q., Crute, B. E., Melnikova, I. N., Keller, S. R.,and Speck, N. A. (1993) Mol. Cell Biol. 13, 3324–3339.

8. Kamachi, Y., Ogawa, E., Asano, M., Ishida, S., Murakami, Y.,Satake, M., Ito, Y., and Shigesada, K. J. (1990) Virology 64,4808–4819.

9. Wang, S., and Speck, N. A. (1992) Mol. Cell Biol. 12, 89–102.0. Melnikova, I. N., Crute, B. E., Wang, S., and Speck, N. A. (1993)

J. Virol. 67, 2408–2411.1. Meyers, S., Lenny, N., Sun, W., and Hiebert, S. W. (1996) Onco-

gene 13, 303–312.2. Merriman, H. L., van Wijnen, A. J., Hiebert, S., Bidwell, J. P.,

Fey, E., Lian, J., Stein, J., Stein, G. S. (1995) Biochemistry 34,13125–13132.

3. Geoffroy, V., Ducy, P., and Karsenty, G. (1995) J. Biol. Chem.270, 30973–30979.

4. Banerjee, C., Hiebert, S. W., Stein, J. L., Lian, J. B., Stein, G. S.(1996) Proc. Natl. Acad. Sci. USA 93, 4968–4973.

5. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K.,Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M.,Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S., and Kishimoto,T. (1997) Cell 89, 755–764.

6. Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour,K. C., Rosewell, I. R., Stamp, G. W. H., Beddington, R. S. P.,Mundlos, S,. Olsen, B. R., Selby, P. B., and Owen, M. J. (1997)Cell 89, 765–771.

7. Hoshi, K., Komori, T., and Ozawa, H. (1999) Bone 25, 639–651.8. Inada, M., Yasui, T., Nomura, S., Miyake, S., Deguchi, K., Him-

eno, M., Sato, M., Yamagiwa, H., Kimura, T., Yasui, N., Ochi, T.,Endo, N., Kitamura, Y., Kishimoto, T., and Komori, T. (1999)Dev. Dyn. 214, 279–290.

9. Kim, I. S., Otto, F., Zabel, B., and Mundlos, S. (1999) Mech. Dev.80, 159–170.

0. Gerber, H. P., Vu, T. H., Ryan, A. M., Kowalski, J., Werb, Z., andFerrara, N. (1999) Nature Med. 5, 623–628.

1. Vu, T. H., Shipley, J. M., Bergers, G., Berger, J. E., Helms, J. A.,Hanahan, D., Shapiro, S. D., Senior, R. M., and 1erb, Z. (1998)Cell 93, 411–422.

2. Gao, Y. H., Shinki, T., Yuasa, T., Kataoka-Enomoto, H., Komori,T., Suda, T., and Yamaguchi, A. (1998) Biochem. Biophys. Res.Commun. 252, 687–702.

3. Kobayashi, H., Gao, Y. H., Ueta, C., Yamaguchi, A., and Komori,T. (2000) Biochem. Biophys. Res. Commun. 273, 630–636.

4. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G.(1997) Cell 89, 747–754.

5. Ferguson, C., Alpern, E., Miclau, T., and Helms. J. A. (1999)Mech. Dev. 87, 57–66.

6. Harada, H., Tagashira, S., Fujiwara, M., Ogawa, S., Katsumata,T., Yamaguchi, A., Komori, T., and Nakatsuka, M. (1999) J. Biol.Chem. 274, 6972–6978.

27. Ducy, P., Starbuck, M., Priemel, M. Shen, J., Pinero, G., Geof-

2

2

3

3

3

3

3

3

3

3

38. Zhang, D., Hetherington, C. J., Meyers, S., Rhoades, K. L., Lar-

34

4

4

4

4

4

4

4

Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

froy, V., Amling, M., and Karsenty, G. (1999) Genes Dev. 13,1025–1036.

8. Stewart, M., Terry, A., Hu, M., O’Hara, M., Blyth, K., Baxter, E.,Cameron, E., Onions, D. E., and Neil, J. C. (1997) Proc. Natl.Acad. Sci. USA 94, 8646–8651.

9. Thirunavukkarasu, K., Mahajan, M., Mclarren, K. W., Stifani,S., and Karsenty, G. (1998) Mol. Cell Biol. 18, 4197–4208.

0. Tsuji, K., Ito, Y., and Noda, M. (1998) Bone 22, 87–92.

1. Xiao, Z. S., Hinson, T. K., and Quarles, L. D. (1999) J. Cell.Biochem. 74, 596–605.

2. Jimenez, M. G., Balbin, M., Lopez, J. M., Alvarez, J., Komori, T.,and Lopez-Otin, C. (1999) Mol. Cell. Biol. 19, 4431–4442.

3. Selvamurugan, N., Pulimati, M. R., Tyson, D. R., and PartridgeN. C. (2000) J. Biol. Chem. 275, 5037–5042.

4. Ji, C., Casinghino, S., Chang, D. J., Chen, Y., Javed, A., Ito, Y.,Hiebert, S. W., Lian, J. B., Stein, G. S., McCarthy, T. L., andCentrella, M. (1998) J. Cell. Biochem. 69, 353–363.

5. Wotton, D., Ghysdael, J., Wang, S., Speck, N. A., and Owen,M. J. (1994) Mol. Cell Biol. 14, 840–850.

6. Giese, K., Kingsley, C., Kirshner, J. R., and Grosschedl, R. (1995)Genes Dev. 9, 995–1008.

7. Wargnier, A., Legros-Maida, S., Bosselut, R., Bourge, J. F.,Lafaurie, C., Ghysdael, J., Sasportes, M., and Paul, P. (1995)Proc. Natl. Acad. Sci. USA 92, 6930–6934.

816

son, C. J., Chen, H., Hiebert, S. W., and Tenen, D. G. (1996) Mol.Cell Biol. 16, 1231–1240.

9. Zaiman, A. L., and Lenz, J. (1996) J. Virol. 70, 5618–5629.0. Sasaki-Iwaoka, H., Maruyama, K., Endoh, H., Komori, T.,

Kato, S., and Kawashima, H. (1999) J. Bone Miner. Res. 14,248 –255.

1. Hanai, J., Chen, L. F., Kanno, T. Ohtani-Fujuta, N., Kim, W. Y.,Guo, W. H., Imamura, T., Ishidou, Y., Fukuchi, M., Shi, M. J.,Stavnezer, J., Kawabata, M., Miyazono, K., and Ito, Y. (1999)J. Biol. Chem. 274, 31577–31582.

2. Sato, M., Morii, E., Komori, T., Kawahara, H., Sugimoto, M.,Terai, K., Shimizu, H., Yasui, T., Ogihara, H., Yasui, N., Ochi, T.,Kitamura, Y., Ito, Y., and Nomura, S. (1998) Oncogene 17, 1517–1525.

3. Enomoto, H., Enomoto-Iwamoto, M., Iwamoto, M., Nomura, S.,Himeno, M., Kitamura, Y., Kishimoto, T., and Komori, T. (2000)J. Biol. Chem. 275, 8695–8702.

4. Akiyama, H., Kanno, T., Ito, H., Terry, A., Neil, J., Ito, Y., andNakamura, T. (1999) J. Cell. Physiol. 181, 169–178.

5. Chang, D. J., Ji, C., Kim, K. K., Casinghino, S., McCarthy, T. L.,and Centrella, M. (1998) J. Biol. Chem. 273, 4892–4896.

6. Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K.,Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275,4453–4459.

7. Tintut, Y., Parhami, F., Le, V., Karsenty, G., and Demer, L. L.(1999) J. Biol. Chem. 274, 28875–28879.