The Retinoblastoma Protein Is an Essential Mediator of Osteogenesis That Links the p204 Protein to the Cbfa1 Transcription Factor Thereby Increasing Its Activity*

Bone formation requires the coordinated activity of numerous proteins including the transcription factor core-binding factor α1 (Cbfa1). Deregulation of Cbfa1 results in metabolic bone diseases including osteoporosis and osteopetrosis. The retinoblastoma protein (pRb) that is required for osteogenesis binds Cbfa1. We reported earlier that the p200 family protein p204, which is known to be involved in the differentiation of skeletal muscle myotubes, cardiac myocytes, and macrophages, also serves as a cofactor of Cbfa1 and promotes osteogenesis. In this study we established that suppression of p204 expression by an adenovirus construct encoding p204 antisense RNA inhibited osteoblast-specific gene activation by Cbfa1 in an osteogenesis assay involving the pluripotent C2C12 mesenchymal cell line. Using protein-protein interaction assays we established that Cbfa1, pRb, and p204 form a ternary complex in which pRb serves as a linker connecting p204 and Cbfa1. Chromatin immunoprecipitation assays revealed the binding of such a p204-pRb-Cbfa1 transcription factor complex to the promoter of the osteocalcin gene. The pRb requirement of the stimulation of Cbfa1 activity by p204 was established in experiments involving p204 mutants lacking one or two pRb binding (LXCXE) motifs. Such mutants failed to enhance the Cbfa1-dependent transactivation of gene expression as well as osteogenesis. Furthermore, as revealed in reporter gene and in vitro osteogenesis assays p204 synergized with pRb in the stimulation of Cbfa1-dependent gene activation and osteoblast differentiation.

Bone formation requires the coordinated activity of numerous proteins including the transcription factor core-binding factor ␣1 (Cbfa1). Deregulation of Cbfa1 results in metabolic bone diseases including osteoporosis and osteopetrosis. The retinoblastoma protein (pRb) that is required for osteogenesis binds Cbfa1. We reported earlier that the p200 family protein p204, which is known to be involved in the differentiation of skeletal muscle myotubes, cardiac myocytes, and macrophages, also serves as a cofactor of Cbfa1 and promotes osteogenesis. In this study we established that suppression of p204 expression by an adenovirus construct encoding p204 antisense RNA inhibited osteoblast-specific gene activation by Cbfa1 in an osteogenesis assay involving the pluripotent C2C12 mesenchymal cell line. Using protein-protein interaction assays we established that Cbfa1, pRb, and p204 form a ternary complex in which pRb serves as a linker connecting p204 and Cbfa1. Chromatin immunoprecipitation assays revealed the binding of such a p204-pRb-Cbfa1 transcription factor complex to the promoter of the osteocalcin gene. The pRb requirement of the stimulation of Cbfa1 activity by p204 was established in experiments involving p204 mutants lacking one or two pRb binding (LXCXE) motifs. Such mutants failed to enhance the Cbfa1-dependent transactivation of gene expression as well as osteogenesis. Furthermore, as revealed in reporter gene and in vitro osteogenesis assays p204 synergized with pRb in the stimulation of Cbfa1-dependent gene activation and osteoblast differentiation.
The differentiation of uncommitted mesenchymal cells to osteoblasts is a fundamental process in embryonic development and bone repair. The bone morphogenetic proteins (BMPs) 2 are important regulators of this process (1,2). They function by binding cell surface receptors, signaling by Smad proteins, and activating bone-specific genes including core binding factor ␣1 (Cbfa1) (3)(4)(5). This process is positively or negatively regulated by a variety of coactivators and corepressors (3,5,6). Cbfa1, also known as Runx2, PeBP2␣A, Osf2, or AML3, is a member of the runt family of transcription factors. It is an essential transcription factor of osteoblast and bone formation (4). During skeletal development Cbfa1 was observed first in early mesenchymal condensations and is then principally expressed in osteoblasts (7). Cbfa1 Ϫ/Ϫ mice exhibit a complete lack of ossification and die immediately after birth (8). Cbfa1 maintains osteoblastic function by regulating the expression of several bone-specific genes such as osteopontin and osteocalcin, and by controlling bone extracellular matrix deposition (4). Mutations in Cbfa1 are found in 65-80% of individuals with cleidocranial dysplasia (9 -11). Some mutations of Cbfa1 abolish its DNA binding activity (9,11,12) and others disturb the association of Cbfa1 to its binding partners including Smads (13). It was found that several Cbfa1-binding proteins such as retinoblastoma protein (pRb) (14), p204 (15), and Core binding factor ␤ (Cbfb) (16,17) may also play critical roles in bone development.
Interferons are cytokines with antimicrobial, immunomodulatory, and cell growth and differentiation regulatory activities (18,19). The activities of interferons are mediated by numerous interferon-inducible proteins. p204 is a member of the interferon-inducible p200 family proteins that are encoded by genes from the gene 200 cluster (20,21). p204 consists of 640 amino acid residues. In the N-terminal domain (amino acids 1-216), there is a basic amino acid-rich nuclear localization signal and a canonical export signal required for the translocation of p204 from the nucleus to the cytoplasm during skeletal and cardiac muscle differentiation (22,23). The C-terminal domain of p204 consists of two homologous, partially conserved 200-amino acid segments (a and b) in which pRb binding motifs (e.g. LXCXE) are located. Overexpression of p204 is growth inhibitory due in part to its inhibition of ribosomal RNA transcription by binding of p204 to the ribosomal DNA-specific upstream binding factor transcription factor and inhibiting its sequencespecific binding to DNA (24,25). The antiproliferative activity of p204 has been attributed to the binding of p204 to pRb by its pRb binding LXCXE motifs (24,26,27). However, the antiproliferative activity of p204 does not always depend on pRb (28). 3 Overexpression of p204 was found to delay the progression of cells from the G 0 /G 1 phase to the S phase of the cell cycle (29).
p204 was found to be an important regulator of both skeletal and cardiac muscle differentiation (22,23,30,31). In the course of these processes p204 is induced, phosphorylated, and translocated from the nucleus to the cytoplasm (23,30). Overexpression of p204 accelerates muscle formation (23). This is due at least in part to the binding of p204 to the inhibitor of differentiation (Id) proteins including Id1, Id2, and Id3, and overcoming the inhibition by the Id proteins of MyoD activity in the case of skeletal muscle differentiation, and Gata4 and Nkx2.5 activity in the case of cardiac myocyte formation (22,31). A recent publication revealed the involvement of p204 also in macrophage differentiation (32). The p204 gene was isolated as an Macrophage Colony-Stimulating Factor (M-CSF)-responsive gene using a gene trap approach in the interleukin 3-dependent myeloid FD-Fms cell line. Moreover forced expression of p204 strongly repressed the interleukin-3 and M-CSF-dependent proliferation, whereas it promoted the M-CSF-induced macrophage differentiation of FD-Fms cells (32). p204 may also play an important role in lymphocytic differentiation based on the observation that 204 mRNA levels are higher in less mature double-positive (CD4 ϩ CD8) thymocytes than in single-positive (CD4ϩ or Cd8ϩ) thymocytes (33). Notch1, an essential factor for promoting the maturation of T thymocytes can transcriptionally up-regulate p204 gene expression during the maturation to both CD4ϩ and CD8ϩ single positive lineages of CD4 ϩ CD8ϩ double positive thymocytes (33).
We previously reported that p204 is involved in osteoblast differentiation, it acts as a cofactor of Cbfa1 and enhances Cbfa1-dependent gene activation and osteogenesis (15). In this study we extend these findings by elucidating the molecular basis underlying the p204-pRb-Cbfa1 complex-regulated osteogenesis. The results we present here indicate that p204 and pRb synergistically enhance Cbfa1-dependent osteogenesis by forming a p204-pRb-Cbfa1 complex in which pRb bridges p204 and Cbfa1 and that acts in concert in promoting transcription and osteogenesis.

EXPERIMENTAL PROCEDURES
Preparation of Three pCMV204mt-type Expression Constructs in Which One or Two pRb Binding Motifs Are Mutated-Each of the a and b domains of p204 contains an LXCXE pRbbinding motif. One or both of these motifs were substituted by an LXGXK sequence in the pCMV204mt-type constructs using a site-directed mutagenesis kit (Stratagene). The three constructs were: 1) pCMV204mt-a (substitution in the a domain), 2) pCMV204mt-b (substitution in the b domain), and 3) pCMV204mt-a/b (substitutions in both the a and b domains). The constructs were verified by nucleotide sequencing. Subsequent analysis was performed using Curatools (Curagen, New Haven, CT) and BLAST software (available at www.ncbi.nlm.nih.gov/BLAST).
Reporter Gene Assays-To examine whether pRb and p204 synergistically transactivate Cbfa1-dependent reporter gene activity, C2C12 cells grown to 50% confluence in 60-mm tissue culture dishes were transfected with 1 g of Cbfa1-specific reporter construct (p-147luc or p6OSEluc) generously provided by P. Ducy (7,34), together with 1 g of pSVGal (internal control plasmid), and various amounts of pEF1 (vector, negative control), pEFCbfa1, pCMVRb, and/or pCMV204 as indicated. 48 h after transfection the cultures were harvested and lysed. Luciferase assays were performed using 20 l of cell lysate and 100 l of luciferin substrate (Promega). ␤-Galactosidase assays were performed using a kit (Tropix, Inc., Foster City, CA) following the manufacturer's protocol. ␤-Galactosidase and luciferase activities were measured using a Mini-Lum luminometer (Bioscan, Washington, D. C.).
To examine whether Rb is required for the p204-mediated enhancement of Cbfa1 activity, first Rb-deficient SAOS-2 cells were transfected with 1 g of a Cbfa1-specific reporter construct (either p6OSE2-luc or p-147luc), 1 g of pSVGal plasmid, 1 g of pEF-Cbfa1 expression plasmid, and 1 g of p204 expression plasmid, as indicated. Next C2C12 cells were transfected with 1 g of a Cbfa1-specific reporter construct (either p6OSE2-luc or p-147luc), 1 g of pSVGal plasmid, 1 g of pEF-Cbfa1 expression plasmid, and 1 g of pCMV-p204mt-a/b encoding the p204 mutant in which two Rb-binding sites located in the a and b segments of p204 were mutated. To determine whether both Rb-binding sites in p204 are needed for the p204 action in stimulating Cbfa1 activity, pCMV-p204mt-a (Rb-binding site in the a segment is mutated), pCMV-p204mt-b (Rb-binding site in the b segment is mutated), and pCMV-p204mt-a/b and pCMV-204 were used with the Cbfa1 expression construct in the reporter gene assay. The sample was processed as described above.
Generation of Recombinant Viruses and Infection of C2C12 Cells-The AdEasy adenoviral vector system (Invitrogen) was used to construct, according to the protocol of the manufacturer, adenoviruses expressing various proteins. The viral constructs include Ad-pRb, Ad-p204, Ad-p204mt-a Ad-p204mt-b, Ad-p204mt-a/b, and Ad-204AS (encoding 204 antisense RNA). All constructs were verified by DNA sequencing. The expression of all recombinant viruses in infected C2C12 cells was tested by Western blotting with appropriate antibodies. C2C12 cells were infected with recombinant Ad-Cbfa1 (kindly provided by Dr. R. Franceschi) and as specified with Ad-pRb, Ad-p204, Ad-p204mt-a, Ad-p204mt-b and Ad-p204mt-a/b. The viruses (1 ϫ 10 7 plaque-forming units) were added to 5 ϫ 10 5 cells in 2% fetal bovine serum, and incubated at 37°C for 1 h. The dishes were rotated every 5 min for the first 15 min to ensure that all of the cells were exposed to the virus. After 1 h the medium was aspirated and the cultures were rinsed twice with serum-free medium and then supplemented with fresh medium containing 10% fetal bovine serum. 4 days after the infection the C2C12 cells were lysed for alkaline phosphatase (ALP) assay and the media were collected for osteocalcin (OCL) assay.
Alkaline Phosphatase and Osteocalcin Assays-Cells were sonicated in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM MgCl 2 , 1% Triton X-100) supplemented with protease inhibitor mixture (Sigma) and centrifuged at 10,000 ϫ g for 15 min. The supernatant fractions were collected and protein concentrations were determined using the Bio-Rad assay kit. ALP activities were assayed by a standard method with p-nitrophenyl phosphate as a substrate (Sigma). Enzyme activity was calculated using a p-nitrophenyl standard curve and expressed as micromoles of p-nitrophenyl phosphate/g of protein. OCL in the culture medium was assayed by an enzyme-linked immunosorbent assay using a mouse osteocalcin assay kit (Biomedical Technologies, Staughton, MA) according to the manufacturer's protocol.
Co-immunoprecipitation and Western Blotting-Total cell extract proteins (300 g) prepared from C2C12 cells transfected with pCMV204, pCMVRb, and pEF-Cbfa1 expression plasmids or infected with Ad-BMP2, from osteoblast MC-3T3-E1 or from osteocyte-like MLO-Y4 cells, were incubated with either rabbit polyclonal antibodies to p204 (25 g/ml), rabbit antibodies to pRb (25 g/ml), goat antibodies to Cbfa1 (25 g/ml), or control rabbit IgG (25 g/ml), respectively, at room temperature for 2 h. Immune complexes were collected with protein A-Sepharose (Amersham Biosciences), washed five times with lysis buffer, released by boiling in 40 l of 2ϫ SDS loading buffer for 10 min and examined by Western blotting with antibodies to p204, pRb, or Cbfa1. The signals were detected using the ECL chemiluminescent system.
To assay whether pRb is required for the binding of p204 to Cbfa1 in vivo, pRb-deficient Saos-2 cells were transfected with pCMV204 and pEF-Cbfa1 together with or without pCMVRb. Immunoprecipitation with antibodies to p204 or Cbfa1 was performed as described above. The immunoprecipitated proteins were subjected to 10% SDS-PAGE and detected by Western blot analysis using antibodies to p204, pRb, or Cbfa1.
GST Pull-down Assay in Vitro-To assay whether pRb is required for the binding of p204 to Cbfa1 in vitro, glutathione-Sepharose beads (50 l) preincubated with purified recombinant GST-204 (500 ng) or GST (500 ng) (serving as negative control) were incubated with 500 g of cell extract from Saos-2 cells, if so indicated, transfected with pCMVRb or C2C12 cells that had been transfected with a pEF-Cbfa1 expression plasmid (positive control). The beads were washed 5 times with washing buffer (0.3% Triton X-100 in phosphate-buffered saline). The bound proteins were denatured in sample buffer, released, and analyzed by 10% SDS-PAGE, and the Cbfa1 protein was detected by Western blotting with antibodies to Cbfa1.
Knockdown of pRb by Specific siRNA Silencing-For silencing the expression of Rb, 19 nucleotides (AAGTT AAAGGTAC-CCGATC) of murine Rb were targeted with small interfering RNA (siRNA) using pSuper mammalian expression vector (OligoEngine, Seattle, WA) according to the manufacturer's instructions. To generate siRNAs, equimolar amounts of complementary sense and antisense strands were mixed and annealed by slowly cooling to 10°C in 50 (or is it 100?) l of reaction buffer (100 mM NaCl and 50 mM HEPES, pH 7.4). The annealed oligos were inserted into the BglII/HindIII sites of pSuper vector. The resulting plasmids and the pSuper control vector were transfected into MC3T3-E1 cells using Lipofectamine 2000 reagent (Invitrogen) and the level of pRb was determined using Western blotting with anti-pRb antibody. MC3T3-E1 cells transfected with either pSuper-pRb or pSuper vector control (CTR) were cultured in minimal essential medium, a medium supplemented with 10% fetal bovine serum under standard cell culture conditions for 3-4 days and harvested for co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP) assays.
ChIP-To assay whether a p204-pRb-pCbfa1 complex is associated with the osteocalcin promoter in vivo we performed ChIP assays. Using a ChIP assay kit (Upstate Biotechnology, Inc., Lake Placid, NY) and murine C3H 10T1/2 progenitor cells transiently transfected with expression plasmids encoding Cbfa1, pRb, and p204. Murine C3H 10T1/2 progenitor cells were cultured in 10-mm dishes and treated with formaldehyde (1% final concentration) to cross-link the p204-pRb-Cbfa1 complex to the DNA. The cells were washed with cold, phosphate-buffered saline and lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1). The lysates were sonicated to shear the DNA to a length between 200 and 1000 bp. The sonicated supernatant fraction was diluted 10-fold with ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 2 mM Tris-HCl, pH 8.1), and 150 mM NaCl) and incubated with antibodies to Cbfa1 (Stratagene, La Jolla, CA), pRb (Santa Cruz Biotechnology, Santa Cruz, CA), p204, or preimmune serum with rotation at 4°C overnight. To collect the DNA-protein complexes a salmon sperm DNA-protein A-agarose slurry was added to the mixture and incubated with rotation at 4°C for 1 h, and the DNA-protein-agarose was pelleted by centrifugation. After extensive washing of the pellet with a series of buffers (Low Salt Wash Buffer, High Salt Wash Buffer, LiCl Immune Complex Wash Buffer, and 1ϫ TE Buffer (10 mM Tris, pH 8.1, 1 mM EDTA)) the pellet was dissolved in 250 l of elution buffer (1% SDS, 0.1 M NaHCO 3 , 0.01 mg/ml herring sperm DNA), and centrifuged to remove the agarose. The supernatant fraction was treated with 20 l of 5 M NaCl and heated to 65°C for 4 h to reverse the p204-pRb-Cbfa1-DNA cross-linking. After treatment with EDTA and proteinase K, the supernatant fraction was extracted with phenol-chloroform and precipitated with ethanol to recover the DNA. For amplification of the osteocalcin promoter region, 10% of the chromatin-immunopre-cipitated DNA was PCR amplified using as forward primer 5Ј-CTGCAATCACCAACCACAGC, and as reverse primer 5Ј-CTGCACCCTCCAGCATCCAG. 40 cycles of PCR (at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s) were performed. The PCR products were analyzed by electrophoresis in a 2% agarose gel. The validity of the ChIP assay was strengthened by interrogating the input and ChIP DNAs with two pairs of negative control primers located roughly 2 kb upstream and downstream from the Cbfa-1-binding site in the osteocalcin promoter. The sequences of the upstream primer pair were: forward primer, 5Ј-CCCTCCAAACAAA-ATCATGG, and reverse primer, 5Ј-AGGAGGAAGTATG-CCACTGT. The sequences of the downstream primer pair were: forward primer, 5Ј-ATGCCTGGGACCCCTGATAA, and reverse primer, 5Ј-GTTTTGGGGCTGGAGAAATG.

Lowering the Level of Endogenous p204 Inhibits the Cbfa1
Transcription Factor-mediated Osteogenesis-The pluripotent mesenchymal C2C12 cells are a well established model for studying osteogenesis in vitro (35)(36)(37). Our earlier data (15) demonstrated that overexpression of p204 enhances the BMP2-induced osteogenesis of C2C12 cells (38). We now tested whether endogenous p204 is required for the Cbfa1-mediated expression of osteocalcin, a marker gene for osteoblasts. For this purpose we generated a recombinant adenovirus encoding p204 antisense RNA (Ad-204As). As shown in Fig. 1A infection of C2C12 cells with Ad-204As, but not control Ad-GFP, decreased the expression of endogenous p204 in a dosedependent manner. As revealed in Fig. 1, B and C, infection of C2C12 cells with the recombinant adenovirus encoding Cbfa1 (Ad-Cbfa1) led to enhanced expression of ALP and a robust production of osteocalcin. The expression of ALP and production of osteocalcin were strongly inhibited by coinfection with Ad-p204As, but not control Ad-GFP, in a dose-dependent manner. These results indicated the requirement for p204 of Cbfa1-triggered osteogenesis in C2C12 cells.
p204 Synergizes with pRb in C2C12 Cells in the Stimulation of Cbfa1-dependent Gene Activation and Osteoblast Differentiation-Both p204 and pRb bind Cbfa1 and act as its cofactors (15). We tested whether these two cofactors synergistically enhance the activity of Cbfa1 in assays of the expression in C2C12 cells of two Cbfa1-specific reporter genes: p6OSE2LUC and p-147luc. As shown in Fig. 2 transfection of plasmids encoding p204 or pRb resulted in an approximately C2C12 cells were infected with either Ad-GFP (serving as control) or Ad-204AS at different multiplicity of infections. The p204 levels were assayed in the cells by Western blotting with anti-p204. B, a decrease in the p204 level resulted in a decrease in the activity of ALP in C2C12 cells. C2C12 cells were infected with Ad-GFP or Ad-204AS as in A, and the activities of endogenous ALP protein in the C2C12 cells were determined. C, a decrease in the p204 level resulted in a decrease in the production of OCL in C2C12 cells. C2C12 cells were infected with Ad-GFP or Ad-204AS as in A, and the levels of endogenous OCL protein in the culture medium were measured. FIGURE 2. p204 synergized with pRb in the enhancement of the Cbfa1dependent expression of Cbfa1-specific reporter constructs. C2C12 cells were transfected with the reporter constructs p6OSE2luc or p-147luc, as well as plasmids encoding beta galactosidase (internal control), Cbfa1, and if so indicated pCMV vector or plasmids pCMV204 and/or pCMVRb. Cell extracts were prepared 48 h after transfection and luciferase and ␤-galactosidase assays were performed. Luciferase activities were normalized to ␤-galactosidase activities. The leftmost bar corresponds to a relative luciferase activity of 1.
pRb Links p204 to Cbfa1 in Osteogenesis 2-3-fold increase in the Cbfa1-dependent activity that is in accord with an earlier report (15). Cotransfection of plasmids encoding p204 and pRb, however, led to an approximately 9 -10-fold increase in reporter gene activity. These data revealed the synergy between p204 and pRb in the stimulation of Cbfa1dependent transcription.
We next explored whether p204 cooperates with pRb in the Cbfa1dependent osteogenesis of C2C12 cells. We generated adenovirus constructs encoding pRb (Ad-pRb) and p204 (Ad-p204) and infected with these C2C12 cells (shown in the Western blots in Fig.  3A). The effects of the infection of C2C12 cells with Ad-p204, Ad-pRb, or both were tested on the Cbfa1-dependent expression of ALP and OCL, i.e. proteins that are expressed in osteoblasts. In C2C12 cells infected with an adenovirus construct encoding Cbfa1 (Ad-Cbfa1) infection with Ad-p204 or Ad-pRb increased the production of ALP slightly (between 1.5-2-fold), whereas coinfection with Ad-p204 and Ad-pRb resulted in a stronger increase (approximately 4-fold) (Fig. 3B). OCL expression in Ad-Cbfa1-infected C2C12 cells was increased upon infection with Ad-p204 or Ad-pRb approximately 2and 7-fold, respectively, whereas coinfection with both Ad-p204 and Ad-pRb resulted in an approximate 20-fold increase (Fig. 3C). These results indicate that pRb synergizes with p204 also in the promotion of Cbfa1-dependent osteoblastic differentiation.
Association of p204 with pRb Is Required for the p204-mediated Stimulation of Cbfa1-dependent Transactivation-As noted, p204 contains an LXCXE pRb binding motif in both the a and b domains (Fig. 4A, top scheme). We generated p204 derivatives with mutations in the pRb binding motifs in the a domain (Ad-p204mt-a), the b domain (Ad-p204mt-b), or both domains (Ad-p204mt-a/b) (Fig. 4A, three lower schemes). In C2C12 cells infected with Ad-Cbfa1 and trans- . pRb and p204 synergistically stimulated the Cbfa1-mediated osteoblast differentiation as revealed by increased ALP activity and OCL production. A, adenovirus encoding pRb (Ad-pRb) and adenovirus encoding p204 (Ad-p204) expressed pRb and p204 proteins, respectively, in C2C12 cells. C2C12 cells were infected with Ad-GFP (serving as a control), Ad-pRb, or Ad-p204, and the levels of pRb and p204 protein were determined by Western blotting. The pRb and p204 bands are indicated with arrows. B, overexpression of p204 and/or pRb enhanced the Cbfa1-mediated ALP activity. C2C12 cells were infected with Ad-Cbfa1 without or together with Ad-pRb, Ad-p204, or both as indicated, and ALP activity was assayed in the cell lysates 4 days after infection. C, overexpression of p204 and/or pRb enhanced the Cbfa-mediated OCL production. Cultures were treated as described in B and the medium was collected for assaying the level of OCL. In the p204mt-a mutant the LXCXE in the a domain was substituted by LXGXK, in the p204mt-b mutant the LXCXE in the b domain was substituted by LXGXK, and in the p204mt-a/b double mutant the LXCXE motifs in both a and b domains were replaced by LXGXK. B, p204 mutants failed to enhance the Cbfa1-dependent expression of the Cbfa1specific reporter construct p-147luc. C2C12 cells were transfected with a plasmid encoding Cbfa1 together with pCMV vector, or the pCMV204, pCMV204mt-a, pCMV204mt-b, or pCMV204mt-a/b plasmids. 48 h after transfection the cultures were harvested and ␤-galactosidase and luciferase activities were determined. Luciferase activities were normalized to ␤-galactosidase activities. The leftmost bar indicates a relative luciferase activity of 1. C, p204 did not stimulate the Cbfa-dependent transcription in pRb-deficient Saos-2 cells. Saos2 cells were transfected with the indicated reporter genes (p6OSE2luc or p-147luc) and a plasmid encoding Cbfa1 together with pCMV or pCMV204 plasmid and the cultures were processed as described in B.
pRb Links p204 to Cbfa1 in Osteogenesis fected with a p-147luc reporter construct infection with Ad-p204 stimulated luciferase activity approximately 3.2-fold (over Cbfa1 alone), whereas p204 in which either of the two pRb binding motifs was mutated failed to stimulate luciferase activity (Fig. 4B). These results indicate that both LXCXE motifs are needed for stimulating Cbfa1-dependent transcription.
Unexpectedly mutant p204 in which both LXCXE sequences were altered strongly inhibited Cbfa1-dependent gene activation (Fig. 4B). It is conceivable that this double mutant might inhibit the association of endogenous p204 with Cbfa1.
The above results suggest that both pRb binding motifs in p204 are required to allow proper interaction between p204 and endogenous pRb that is required for the synergistic stimulation of Cbfa1-mediated transcription.
Functioning pRb Is Needed for the p204-dependent Enhancement of Cbfa1-dependent Transcription-We tested the need of functional pRb for the enhancement by p204 of the Cbfa1-dependent transcription in experiments with human Saos-2 osteosarcoma cells that are known to be deficient in pRb activity (39). As revealed in Fig. 4C, transfection of a p204 expression plasmid failed to stimulate the Cbfa1-mediated expression of either the p6OSE2luc or the p-147luc reporter construct in these cells lacking pRb.
pRb Is Required for p204-mediated Enhancement of Osteoblast Differentiation-To establish whether pRb is required for enhancement of Cbfa-1-mediated osteoblast differentiation by p204, we constructed Ad-p204mt-a/b encoding a p204 derivative lacking the LXCXE pRb binding motifs from both the a and b domains. In C2C12 cells infected with Ad-Cbfa1, Ad-p204 enhanced the production of ALP 2.5-fold more and OCL 7-fold more than in cells infected only with Ad-Cbfa1 (Fig. 5, A and B). Infection with Ad-p204mt-a/b, however, failed to increase the production of ALP and OCL (Fig. 5, B and C). These results indicated that proper binding of p204 to pRb is also needed for the stimulation of Cbfa1-mediated osteogenesis.
p204, pRb, and Cbfa1 Form a Ternary Complex in Vivo-Both pRb and p204 were shown earlier to associate with Cbfa1 and act as transcriptional cofactors of Cbfa1. To determine FIGURE 5. pRb was required for the enhancement of Cbfa1-induced osteoblast differentiation by p204. A, Western blotting assays of the expression of a p204 mutant encoded by adenovirus Ad-p204mt-a/b. C2C12 cells were infected with Ad-p204mt-a/b or Ad-p204 (serving as a control) and the expressions of p204mt-a/b and p204 were assayed by Western blotting with anti-p204 antibodies. B, mutant p204 (p204mt-a/b) failed to enhance the Cbfa1-mediated ALP activity in C2C12 cells. C2C12 cells were infected with Ad-Cbfa1, if so indicated together with Ad-p204mt-a/b or Ad-p204, incubated for 4 days and the cell lysates were assayed for ALP activity. C, mutant p204 (p204mt-a/b) failed to enhance the Cbfa1-mediated production of OCL. Cultures were treated as described in B and the medium was assayed for OCL production. whether these three proteins form a ternary complex, first we prepared cell lysates from C2C12 cells transfected simultaneously with mammalian constructs encoding p204, pRb, and Cbfa1. Immunoprecipitation of the cell lysates with antibodies to either one of the three proteins, but not control IgG, immunoprecipitated all three proteins (Fig. 6A). The relevance of these observations for osteoblast differentiation was revealed using lysates from C2C12 cells induced to undergo osteogenesis with BMP2 (Fig. 6B), and also lysates from osteoblast MC3T3-E1 cells and osteocyte-like MLO-Y4 cells (Fig. 6, C and  D). The results obtained clearly indicated that endogenous p204, pRb, and Cbfa1 also form a ternary complex in vivo.
pRb Is Required for the Association of p204 and Cbfa1 Both in Vivo and in Vitro-Functional studies revealed that pRb is required for the p204-mediated enhancement of Cbfa1dependent transcription and osteoblast differentiation. Furthermore, as noted above, p204, pRb, and Cbfa form a ternary complex in the course of osteogenesis. These findings prompted us to determine whether pRb is needed for the association of p204 and Cbfa1. For this purpose we first performed co-IP assays using lysates from BMP2-treated C2C12 cells (serving as a positive control) and pRb-deficient Saos-2 cells transfected with the p204 and Cbfa1 expression plasmids. As shown in Fig. 7A anti-p204 antibodies efficiently immunopre-cipitated Cbfa1 from a lysate of C2C12 cells but not from Saos-2 cells, although Cbfa1 is expressed in Saos-2 cells, as revealed by immunoprecipitation (not shown). These findings were verified with a co-IP assay (Fig. 7B). In this case, anti-Cbfa1 antibody, but not the control IgG, immunoprecipitated p204 from lysates of C2C12 cells but not from pRb-deficient Saos-2 cells. Intriguingly, the association between p204 and Cbfa1 was rescued in the Saos-2 cells transfected with a pCMV-pRb expression plasmid (Fig. 7, A and B, lanes 7-9). These results revealed that pRb is an essential mediator for the interaction between p204 and Cbfa1 in vivo.
The interaction between p204 and Cbfa1 in the presence or absence of functional pRb was also examined in GST pull-down assays in vitro (Fig. 7C). Briefly, affinity purified GST and purified GST-p204 that were immobilized on glutathione-Sepharose beads were incubated with cell extract, prepared from either BMP2-treated C2C12 cells or Saos-2 cells transfected with a Cbfa1 expression plasmid together with or without a pRb expression plasmid. After washing the glutathione-Sepharose beads the retained proteins were resolved by 10% SDS-PAGE, and detected by Western blotting. GST-p204 but not control GST pulled down Cbfa1 protein from a lysate from BMPtreated C2C12 cells and from Cbfa1-and pRb-double trans-  A, siRNA against pRb dramatically suppressed pRb expression, assayed by Western blot. Cell lysates were prepared from MC3T3-E1 cells transfected with pSuper plasmid or pSuper-pRb encoding a siRNA against pRb and the total protein was analyzed by Western blot with anti-pRb antibody. Tubulin was used as an internal control. B and C, p204 failed to associate with Cbfa1 in pRb-knockdown MC3T3-E1 cells, whereas the interaction was rescued when pRb was re-expressed (co-IP assay). Cell lysates prepared from MC3T3-E1 cells (lanes 1-3), MC3T3-E1 cells transfected with pSuper-pRb (lanes 4 -6), or MC3T3-E1 cells transfected with pSuper-pRb first and then with pCMV-pRb (lanes 7-9), were incubated with control IgG, or antibodies to p204 (B) or Cbfa1 (C), as indicated, and the immunoprecipitated protein complexes and cell lysates (positive control) were examined by Western blotting with antibodies to Cbfa1 (B) or p204 (C).
fected Saos-2 cells, but not that from Cbfa1-transfected Saos-2 cells. These results indicate that the interaction between p204 and Cbfa1 in vitro also requires the presence of functional pRb.
The requirement of pRb for the binding of p204 to Cbfa1 was further tested in experiments involving silencing by siRNA. We identified a 19-nucleotide gene-specific sequence for pRb and generated a pSuper-pRb construct encoding an siRNA targeting this sequence in the Rb gene. As revealed in Fig. 8A, this construct dramatically reduced the endogenous pRb level in MC3T3-E1 osteoblasts. Next we examined whether the suppression of pRb expression by this siRNA affects the interaction of p204 and Cbfa1 in co-IP assays using lysates from MC3T3-E1 cells (serving as a positive control), from MC3T3-E1 cells transfected with pSuper-pRb (MC3T3-E1/pSuper-pRb) and from MC3T3-E1/pSuper-pRb cells transfected with a pCMVRb construct. As shown in Fig. 8, B and C, anti-p204 or anti-Cbfa1 antibodies efficiently immunoprecipitated Cbfa1 or p204 from the cell lysate of MC3T3-E1 cells (lanes 1-3), but not from pRb-knockdown MC3T3-E1 cells (lanes 4 -6), clearly indicating that pRb is needed for the association of p204 with Cbfa1. Furthermore, interaction between p204 and Cbfa1 was restored when pRb was re-expressed in MC3T3-E1/pSuper-pRb cells by transfection of pCMVRb (Fig. 8, B and C, lanes 7-9). Collectively, this set of assays indicated that pRb is strictly required for the binding of p204 to Cbfa1.
The Cbfa1-pRb-p204 Transcription Factor Complex Is Bound to the Osteocalcin Promoter in Vivo-To test whether the p204-pRb-Cabf1 complex associates with the osteocalcin promoter in vivo we performed ChIP assays. These can identify the chromatin segments to which particular transcription factors are bound in vivo. ChIP was first carried out using 10T1/2 cells transfected with an OCL-specific reporter construct (p-147luc) and expression plasmids encoding Cbfa1, pRb, and p204. After cross-linking the proteins to the DNA they were associated with using formaldehyde, we lysed the cells, sheared the chromatin by sonication, and immunocoprecipitated protein-DNA complexes using control IgG (negative control) as well as anti-Cbfa1, anti-pRb, or anti-p204 antibodies. We examined the DNA samples purified from the various immunoprecipitates by PCR with primers that span the Cbfa1 binding site in the OCL promoter. The PCR products of the DNA samples purified from the immunoprecipitates with antibodies to Cbfa1, pRb, or p204 (but not that obtained from immunoprecipitation with the IgG control) were identical and of the expected size (Fig. 9A). Note that segments located ϳ2 kb both upstream or downstream from the Cbfa-1-binding site were not co-immunoprecipitated by the anti-Cbfa1 antibodies (Fig.  9B), indicating the sequence-specific binding of Cbfa1 to DNA. Similar ChIP assays were performed using C2C12 cells induced to osteogenic differentiation by infection with an adenovirus construct encoding BMP-2 (Ad-BMP2, generously provided by T.-C. He), MC3T3-E1 osteoblast cells as well as osteocyte-like MLO-Y4 cells. As revealed in Fig. 9, C-E, the products of the PCR analyses with the same primers as in Fig. 9A of the DNA samples in the immunoprecipitates with antibodies to Cbfa1, pRb, or p204 (but not with the IgG control) were of uniform and appropriate size. These results, together with the demonstration that Cbfa1, pRb, and p204 form a ternary complex in vivo and in vitro (Figs. 7 and 8), indicate that the endogenous p204-pRb-Cabf1 transcription factor complex is associated with the osteocalcin promoter.
The following results established that, as expected, endogenous pRb was required for the formation of the p204-pRb-Cbfa1 complex on the osteocalcin promoter in MC3T3-E1 osteoblasts. In a ChIP assay, using a lysate from MC3T3-E1 cells, in which pRb expression was inhibited by transfection with pSuper-pRb, the gene segment involving the Cbfa1 binding site was co-immunoprecipitated by anti-Cbfa1, but not by anti-p204 (Fig. 10A). However, in a ChIP assay, using a lysate from MC3T3 cells, in which pRb expression was inhibited by transfection with pSuper-pRb, but, in addition, pRb expression was restored by transfection with pCMVRb, anti-p204 did co- immunoprecipitate the Cbfa1 binding site as well as anti-Cbfa1 (Fig. 10B).

DISCUSSION
p204 is a multifunctional protein involved in the control of cell proliferation and the differentiation of numerous types of cells and tissues including skeletal muscle myotubes, cardiac myocytes, macrophages, chondrocytes, and osteoblasts. We have reported earlier that p204 associates with the key osteogenic transcription factor Cbfa1 and serves as its coactivator (15). Overexpression of p204 stimulates osteogenesis triggered by BMP2.
Here we established that inhibition of the induction of p204 expression (by 204 antisense RNA) decreased the Cbfa1-dependent formation of the osteoblast protein osteocalcin (Fig. 1). We also demonstrated that p204 and pRb, known to bind each other, synergistically stimulated Cbfa1-dependent gene expression and thereby promoted osteogenesis (Figs. 2 and 3).
p204 controls cell proliferation and differentiation by associating with various proteins. By associating, e.g. with UBF1 and/or pRb, p204 acts as an inhibitor of cell proliferation (24,26,27). By associating with the Id proteins and accelerating their degradation, p204 promotes the differentiation of skeletal muscle myotubes and cardiac myocytes (22,30,31). p204 also mediates the differentiation of macrophages, although the proteins with which it interacts in the process remain to be identified (32).
As noted, Cbfa1 (also known as Runx2, Osf2, PeBP2␣, or AML3) a member of the Runt family of transcription factors, is a central regulator of osteoblast differentiation and bone formation. A variety of Cbfa1-binding proteins that positively or negatively regulate osteoblast gene expression have been identified (14, 17, 40 -47). It has been reported that Cbfa1 interacts with histone acetylase (44), as well as with histone deacetylase, including histone deacetylase 3, 4, and 6 (43,48). This suggests that chromatin remodeling is essential for regulating the osteoblast-specific genes. BMP2 and its downstream signaling proteins Smad1 and Smad5, as well as Cbfa1 stimulate osteoblast differentiation. Smads interact with Cbfa1 both in vitro and in vivo and enhance the transactivation of gene expression by Cbfa1. A Cbfa1 mutation in cleidocranial displasia impairs its association with Smads (45). Smurf1 (Smad ubiquitin regulatory factor), an E3 ubiquitin ligase, was reported to interact directly with both Smad1 and Cbfa1, and to mediate the degradation of Cbfa1 in a ubiquitin-and proteasome-dependent manner (46,47). Core binding factor ␤ (Cbfb, also called PEP2B), another protein of the Runt family, forms heterodimers with Runx1 and Cbfa1, and this is involved in skeletal development (13,49). TAZ (transcriptional coactivator with PDZ binding domain) was identi-  fied to bind Cbfa1 in a yeast two-hybrid assay. Cbfa1 affects the nuclear localization of TAZ and this affects osteoblast differentiation (40). Although there is no direct interaction between activating transcription factor 4 and Cbfa1 the two proteins can be associated in vivo. Activating transcription factor 4 and Runx2 activate the expression of osteocalcin via cooperative interactions. A groucho homolog, Grg5, was isolated as a Runx2-interacting protein in a genetic screen. Grg5 modulates Runx2 activity during postnatal development in mice (50,51). Stat1 is one of the mediators of type I and type II interferon responses. Unphosphorylated Stat1 in the cytoplasm binds Cbfa1, blocks its translocation to the cytoplasm, and thereby inhibits Cbfa1 activity and bone remodeling in postnatal mice (52). HES, a basic loop helix transcription factor interacts with Cbfa1, and thereby antagonizes the binding of Cbfa1 to mammalian corepressors of the groucho family including Grg5. This suggests that groucho and HES modulate the functioning of mammalian Runt-related proteins (53). As described earlier pRb, whose mutants have been found in osteosarcoma patients, binds Cbfa1 and acts as its direct transcriptional coactivator (14). p204, which binds pRb, is involved in the inhibition of cell proliferation and also in the differentiation of various cells and tissues (28, 54 -59). As reported earlier p204 also associates with Cbfa1, acts as its coactivator, and promotes osteoblast differentiation (15).
In this study we further established that p204, pRb, and Cbfa1 form a ternary complex in which pRb serves as the linker. This complex can be associated in vivo with the promoter of the osteocalcin gene (Figs. 6 -10). pRb is needed for the stimulation of the Cbfa1-mediated gene expression and osteogenesis by p204 (Figs. 4 and 5).
Based on the data in the literature (4), our earlier findings (15), and the results of this study we propose a model for the role of p204 in osteogenesis (Fig. 11). The binding of BMPs to their type I and II receptors triggers the association of the two types of receptors. This results in the phosphorylation and activation of the type I receptor. This in turn elicits the phosphorylation of the MH2 domains of the R-Smads (Smad1 and Smad5). These bind Co-Smad (Smad4) and the resulting complex enters the nucleus. In the nucleus the Smad complex activates the transcription of the Smad target genes. These include the genes encoding the osteoblast-specific Cbfa1 factor (60 -62) and p204 (15). Serving as a bridge pRb links p204 and Cbfa1 (this study). In this ternary complex both p204 and pRb are activating cofactors of Cbfa1 (14,15). The ternary complex associates with osteoblast-specific promoters and activates the transcription of Cbfa1 target genes including those encoding alkaline phosphatase and osteocalcin. Although not shown here, as noted in the previous paragraph, p204 might also have further functions in osteogenesis.