Induction of Osteoclast Differentiation by Runx2 through Receptor Activator of Nuclear Factor-κB Ligand (RANKL) and Osteoprotegerin Regulation and Partial Rescue of Osteoclastogenesis in Runx2–/– Mice by RANKL Transgene

Abstract

Receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2–/–) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2–/– calvaria-derived cell line (CA120–4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120–4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2–/– livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2–/– mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development. Receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2–/–) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2–/– calvaria-derived cell line (CA120–4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120–4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2–/– livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2–/– mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development. In the process of endochondral ossification, chondrocytes mature to hypertrophic chondrocytes, matrix around terminally differentiated chondrocytes (terminal hypertrophic chondrocytes) is mineralized, blood vessels invade into the calcified cartilage, and cartilage is replaced by bone (1Gilbert S.F. Developmental Biology. 5th Ed. Sinauer Associates Inc., City, MA1997Google Scholar). Osteoclasts accelerate these processes by resorption of the calcified matrix leading to bone marrow formation. Osteoclasts differentiate from hematopoietic precursor cells through direct contact with osteoblastic/stromal cells (2Suda T. Takahashi N. Martin T.J. Endocr. Rev. 1992; 13: 66-80Google Scholar). Recently, osteoprotegerin (OPG) 1The abbreviations used are: OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-κB; RANKL, RANK ligand; sRANKL, soluble RANKL; EGFP, enhanced green fluorescence protein; TRAP, tartrate-resistant acid phosphatase; M-CSF, macrophage-colony stimulating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tg, transgenic; E, embryonic day. 1The abbreviations used are: OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-κB; RANKL, RANK ligand; sRANKL, soluble RANKL; EGFP, enhanced green fluorescence protein; TRAP, tartrate-resistant acid phosphatase; M-CSF, macrophage-colony stimulating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tg, transgenic; E, embryonic day./osteoclastogenesis inhibitory factor, which is an inhibitor of osteoclast differentiation (3Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. 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Dev. 1998; 8: 494-499Google Scholar) and functions by forming a heterodimer with Cbfb (core binding factor β) (20Yoshida C.A. Furuichi T. Fujita T. Fukuyama R. Kanatani N. Kobayashi S. Satake M. Takada K. Komori T. Nat. Genet. 2002; 32: 633-638Google Scholar, 21Kundu M. Javed A. Jeon J.P. Horner A. Shum L. Eckhaus M. Muenke M. Lian J.B. Yang Y. Nuckolls G.H. Stein G.S. Liu P.P. Nat. Genet. 2002; 32: 639-644Google Scholar, 22Miller J. Horner A. Stacy T. Lowrey C. Lian J.B. Stein G. Nuckolls G.H. Speck N.A. Nat. Genet. 2002; 32: 645-649Google Scholar). Runx2–/– mice completely lack bone formation because of the maturational arrest of osteoblasts, indicating that Runx2 is an essential factor for osteoblast differentiation (23Komori 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. Kishimoto T. Cell. 1997; 89: 755-764Google Scholar, 24Otto 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. Owen M.J. Cell. 1997; 89: 765-771Google Scholar). In addition, chondrocyte maturation is also disturbed in Runx2–/– mice (25Inada M. Yasui T. Nomura S. Miyake S. Deguchi K. Himeno M. Sato M. Yamagiwa H. Kimura T. Yasui N. Ochi T. Endo N. Kitamura Y. Kishimoto T. Komori T. Dev. Dyn. 1999; 214: 279-290Google Scholar, 26Kim I.S. Otto F. Zabel B. Mundlos S. Mech. Dev. 1999; 80: 159-170Google Scholar), and Runx2 has been shown to be an important factor for chondrocyte maturation (27Enomoto H. Enomoto-Iwamoto M. Iwamoto M. Nomura S. Himeno M. Kitamura Y. Kishimoto T. Komori T. J. Biol. Chem. 2000; 275: 8695-86702Google Scholar, 28Ueta C. Iwamoto M. Kanatani N. Yoshida C. Liu Y. Enomoto-Iwamoto M. Ohmori T. Enomoto H. Nakata K. Takada K. Kurisu K. Komori T. J. Cell Biol. 2001; 153: 87-100Google Scholar, 29Takeda S. Bonnamy J.P. Owen M.J. Ducy P. Karsenty G. Genes Dev. 2001; 15: 467-481Google Scholar). Although chondrocytes had matured, and the matrix was mineralized in restricted parts of the skeleton of Runx2–/– mice, osteoclasts were completely absent, and no vascular invasion into the calcified cartilage occurs (23Komori 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. Kishimoto T. Cell. 1997; 89: 755-764Google Scholar). Therefore, Runx2 plays important roles in multiple processes of endochondral ossification, including chondrocyte maturation, vascular invasion into the cartilage, osteoclast differentiation, and osteoblast differentiation (30Komori T. J. Cell. Biochem. 2002; 87: 1-8Google Scholar). We have shown that Runx2–/– calvaria-derived cells have less ability to support osteoclast differentiation from normal spleen cells, and RANKL expression is severely diminished in Runx2–/– mice, suggesting that Runx2 is involved in osteoclastogenesis through the regulation of RANKL expression in osteoblastic/stromal cells (31Gao Y.H. Shinki T. Yuasa T. Kataoka-Enomoto H. Komori T. Suda T. Yamaguchi A. Biochem. Biophys. Res. Commun. 1998; 252: 697-702Google Scholar). However, Runx2 binding elements are also present in the promoter region of OPG, and Runx2 increased the activity of the OPG promoter, suggesting that Runx2 inhibits osteoclast differentiation and activation through OPG induction (32Thirunavukkarasu K. Halladay D.L. Miles R.R. Yang X. Galvin R.J. Chandrasekhar S. Martin T.J. Onyia J.E. J. Biol. Chem. 2000; 275: 25163-25172Google Scholar). Further, Runx2 failed to stimulate the transcriptional activity of the 0.7-kb 5′-flanking region of the RANKL gene (33O'Brien C.A. Kern B. Gubrij I. Karsenty G. Manolagas S.C. Bone. 2002; 30: 453-462Google Scholar). Therefore, the role of Runx2 in osteoclast differentiation remains to be clarified. In the present study, we investigated the involvement of Runx2 in RANKL and OPG expression and osteoclast differentiation in vitro and in vivo. Runx2 induced RANKL expression and suppressed OPG expression in vitro, leading to the promotion of osteoclast differentiation. Further, overexpression of soluble RANKL (sRANKL) partially rescued the blockage of osteoclast differentiation in Runx2–/– mice, indicating the involvement of Runx2 in osteoclastogenesis by regulating RANK-RANKL signaling. Establishment of Calvaria-derived Runx2–/–Cell Lines—Runx2+/– mice (23Komori 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. Kishimoto T. Cell. 1997; 89: 755-764Google Scholar) were mated with p53–/– mice (34Gondo Y. Nakamura K. Nakao K. Sasaoka T. Ito K. Kimura M. Katsuki M. Biochem. Biophys. Res. Commun. 1994; 202: 830-837Google Scholar) to generate Runx2+/–p53–/– mice. Runx2–/–p53–/– mice were generated by mating Runx2+/–p53–/– mice. Calvarial cells derived from E18.5 Runx2–/–p53–/– embryos were prepared and cultured as described previously (35Kobayashi H. Gao Y.H. Ueta C. Yamaguchi A. Komori T. Biochem. Biophys. Res. Commun. 2000; 273: 630-636Google Scholar). Colonies of the calvarial cells were isolated by digestion with trypsin/EDTA for 5 min at 37 °C within stainless steel cloning rings. Isolated cells were then expanded and recloned by limiting dilution. Prior to the study, all experiments were reviewed and approved by Osaka University Medical School Animal Care and Use Committee. RNA Extraction and Northern Blot Analysis—Total RNA was extracted from cellular samples using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Aliquots of 20 μg of total RNA were separated by electrophoresis and transferred onto nylon membrane filters. A 1.5-kb fragment of mouse RANKL cDNA (8Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Google Scholar), a 1.5-kb fragment of OPG cDNA (4Yasuda H. Shima N. Nakagawa N. Mochizuki S. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; 139: 1329-1337Google Scholar), and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (36Himeno M. Enomoto H. Liu W. Ishizeki K. Nomura S. Kitamura Y. Komori T. J. Bone Miner. Res. 2002; 17: 1297-1305Google Scholar) were labeled with [α-32P]dCTP using a Megaprime DNA labeling kit (Amersham Biosciences), and hybridization was performed as described previously (27Enomoto H. Enomoto-Iwamoto M. Iwamoto M. Nomura S. Himeno M. Kitamura Y. Kishimoto T. Komori T. J. Biol. Chem. 2000; 275: 8695-86702Google Scholar). The intensities of RANKL, OPG, and GAPDH bands were quantitated by densitometry using FMBIO analysis software (Hitachi Software Engineering Co. Ltd., Tokyo, Japan). Construction of Adenovirus—A mouse cDNA containing the entire open reading frame of Runx2 (37Liu W. Toyosawa S. Furuichi T. Kanatani N. Yoshida C. Liu Y. Himeno M. Narai S. Yamaguchi A. Komori T. J. Cell Biol. 2001; 155: 157-166Google Scholar) was inserted into the BamHI site of pIRES2-EGFP (Clontech), and a DNA fragment containing Runx2, internal ribosome entry site, and enhanced green fluorescence protein (EGFP) was inserted into the BamHI-XbaI sites of pACCMV.pLpA shuttle vector (38Herz J. Gerard R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2812-2816Google Scholar). The constructed vector was co-transfected with the adenovirus cloned plasmid pJM17 (39McGrory W.J. Bautista D.S. Graham F.L. Virology. 1988; 163: 614-617Google Scholar) into human kidney 293 cells by the SuperFect transfection reagent (Qiagen) according to the manufacturer's instructions. A recombinant adenovirus was generated by homologous recombination events between the two plasmids. Amplified crude viral stocks were purified by CsCl gradient ultracentrifugation and used for the infection. The recombinant adenovirus carrying only internal ribosome entry site-EGFP was also generated and used for the control infection. Adenoviral Introduction of Runx2 into CA120–4 Cells—CA120–4 cells were plated at a density of 1 × 106 cells/dish in collagen-coated 60-mm plates (Iwaki Glass, Chiba, Japan). On the following day, cells were infected with either EGFP-expressing (control) or Runx2- and EGFP-expressing virus. After the viral infection, cells were cultured with or without 1α,25(OH)2D3 and harvested for RNA extraction. Osteoclast Differentiation in Vitro—CA120–4 cells were inoculated in 24-well plates at a density of 2 × 104 cells/well. Twenty-four h after plating, cells were infected by either EGFP-expressing (control) or Runx2- and EGFP-expressing virus for 2 h. Thereafter, cells were rinsed twice with phosphate-buffered saline to eliminate adenovirus and subsequently cultured with bone marrow cells (2 × 105 cells/well) in α-minimum Eagle's medium (0.5 ml/well) containing 10% fetal calf serum and 10–8m 1α,25(OH)2D3. Cultures were fed every 3 days by replacing 0.4 ml of old medium with fresh medium (40Suda T. Jimi E. Nakamura I. Takahashi N. Metods Enzymol. 1997; 282: 223-235Google Scholar). Recombinant human OPG and sRANKL proteins (R&D Systems, Minneapolis, MN) were administrated at a final concentration of 100 and 30 ng/ml, respectively. On the seventh day of the co-culture, adherent cells were fixed with 10% formaldehyde in phosphate-buffered saline, treated with ethanol-acetone (50:50), and stained for tartrate-resistant acid phosphatase (TRAP) using the acid phosphatase, leukocyte kit (Sigma-Aldrich) according to the manufacturer's protocols. Western Blot Analysis—Equal amounts of proteins (20 μg) were separated on a 10% gel by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking the membrane with 5% skimmed milk, the membrane was incubated with a mouse monoclonal antibody against Runx2 (41Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y Ryoo H.M. Bae S.C. Mol. Cell. Biol. 2000; 20: 8783-8792Google Scholar) at a 1:300 dilution and then with peroxidase-conjugated goat anti-mouse IgG antibody. Bound antibody was visualized by an ECL detection system (Amersham Biosciences). Generation of Runx2–/–tg Mice—sRANKL transgenic mice were generated using the hSAP (human serum amyloid P component) gene promoter (42Yoshimoto T. Wang C.R. Yoneto T. Waki S. Sunaga S. Komagata Y. Mitsuyama M. Miyazaki J. Nariuchi H. J. Immunol. 1998; 160: 588-594Google Scholar) and a sRANKL DNA fragment containing immunoglobulin κ-chain leader sequence and the extracellular domain sequence of RANKL RANKL A. Kanno T. M. Yano K. Fujise N. Kinosaki M. Yamaguchi K. Tsuda E. Murakami A. Yasuda H. Higashio K. J. Bone Miner. 2002; 20: Scholar). The sRANKL transgenic mice were mated with mice to generate sRANKL transgenic mice. Runx2–/– sRANKL transgenic mice were generated by mating transgenic mice with mice. were by analysis for Runx2 and analysis for sRANKL, as described previously (23Komori 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. Kishimoto T. Cell. 1997; 89: 755-764Google Scholar, A. Kanno T. M. Yano K. Fujise N. Kinosaki M. Yamaguchi K. Tsuda E. Murakami A. Yasuda H. Higashio K. J. Bone Miner. 2002; 20: Scholar). sRANKL was by using a antibody against mouse RANKL K. Shima N. Mochizuki S. Yamaguchi K. Kinosaki M. Yano K. Udagawa N. Yasuda H. Suda T. Higashio K. Biochem. Biophys. Res. Commun. 1998; Scholar). was performed as described previously K. Komori T. Ozawa H. Bone. 1999; Scholar). In E18.5 embryos were fixed in in for and a of and in for of were used for and or bone were at the of the plates to the using a system Japan). and used are by the of the for Bone and H. P.J. R.R. J. Bone Miner. Res. Scholar). following with in the were in and into These were a Tokyo, Japan) after with and Establishment of Runx2–/–Cell showed previously (35Kobayashi H. Gao Y.H. Ueta C. Yamaguchi A. Komori T. Biochem. Biophys. Res. Commun. 2000; 273: 630-636Google Scholar) that Runx2–/– calvarial cells are to differentiate into and into chondrocytes with We isolated from the calvarial cells of Runx2–/–p53–/– mice. cell line had its with to to differentiate into either chondrocytes or In all of the Runx2–/– cell RANKL expression was but OPG expression was by Northern and of the cell was used for the following experiments cell line the ability to differentiate into chondrocytes or but phosphatase activity was induced by with the expression of RANKL and OPG in CA120–4 cells, osteoclast differentiation was severely in the of CA120–4 cells and normal bone marrow cells Runx2 RANKL and Osteoclast the role of Runx2 in the regulation of RANKL expression and we the Runx2 gene into CA120–4 cells by gene We that the infected cells Runx2 protein by As shown in a of in the Runx2 cells to that in Runx2 in cells, which were used as a Adenoviral introduction of the Runx2 gene induced RANKL expression in the presence of and the induction was at h after and for at whereas the induction was in the control in the presence of 1α,25(OH)2D3 and In the of however, Runx2 failed to RANKL 1α,25(OH)2D3 suppressed OPG expression as described previously (31Gao Y.H. Shinki T. Yuasa T. Kataoka-Enomoto H. Komori T. Suda T. Yamaguchi A. Biochem. Biophys. Res. Commun. 1998; 252: 697-702Google Scholar), but the introduction of Runx2 gene suppressed OPG expression at h after without 1α,25(OH)2D3 and The of OPG by Runx2 was at 3 days after infection, but Runx2 and 1α,25(OH)2D3 OPG expression for at Although the presence of CA120–4 cells completely suppressed osteoclast differentiation of normal bone marrow cells the introduction of Runx2 into CA120–4 cells restored the formation of osteoclast-like cells from the bone marrow cells and However, the addition of OPG abolished the induction of osteoclast-like cells by the introduction of Runx2 and These indicate that Runx2 induces osteoclast differentiation through RANK-RANKL signaling. the involvement of RANKL in we sRANKL and the on the formation of osteoclast-like cells and In the presence of CA120–4 cells, sRANKL also restored the formation of osteoclast-like cells from normal bone marrow cells indicating that RANKL the inhibitory of CA120–4 cells on osteoclast differentiation. these findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL expression and inhibiting OPG Generation of with sRANKL in vitro that RANKL was a gene of Runx2 and RANKL osteoclast differentiation in the of Runx2. we RANKL osteoclast differentiation in Runx2–/– mice. of sRANKL the control of the promoter and early which direct the expression from an early in at the A. Kanno T. M. Yano K. Fujise N. Kinosaki M. Yamaguchi K. Tsuda E. Murakami A. Yasuda H. Higashio K. J. Bone Miner. 2002; 20: Scholar). Therefore, we generated transgenic which sRANKL the control of the hSAP promoter (42Yoshimoto T. Wang C.R. Yoneto T. Waki S. Sunaga S. Komagata Y. Mitsuyama M. Miyazaki J. Nariuchi H. J. Immunol. 1998; 160: 588-594Google Scholar). The the control of promoter was expressed in the and its expression increased at the X. K. Miyazaki J. K. J. Biochem. 1992; Scholar), in an in A. Kanno T. M. Yano K. Fujise N. Kinosaki M. Yamaguchi K. Tsuda E. Murakami A. Yasuda H. Higashio K. J. Bone Miner. 2002; 20: Scholar). The sRANKL transgenic mice were mated with mice and then Runx2–/–tg mice were the activity of the hSAP promoter at the embryonic was than in we into mice with embryos at embryonic day to and the promoter the embryonic The enhanced the expression of sRANKL in the livers of transgenic embryos but of embryos and the serum sRANKL in the transgenic embryos were to the we used for in vitro The of sRANKL transgenic mice were and the number of cells was increased with cells analysis using E18.5 showed that the induction of sRANKL in fetal in of bone and than of osteoclast surface and number These findings indicate that sRANKL expression in the embryos was for the promotion of osteoclast differentiation and concentration of concentration in a Forced of sRANKL Osteoclast Differentiation but to Osteoclasts in a