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Endocrinology Vol. 142, No. 9 4026-4039
Copyright © 2001 by The Endocrine Society

ARTICLES

Differential Regulation of the Two Principal Runx2/Cbfa1 N-Terminal Isoforms in Response to Bone Morphogenetic Protein-2 during Development of the Osteoblast Phenotype

Chaitali Banerjee, Amjad Javed, Je-Yong Choi, Jack Green, Vicki Rosen, André J. van Wijnen, Janet L. Stein, Jane B. Lian and Gary S. Stein

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655; and Genetics Institute (V.R.), Cambridge, Massachusetts 02140

Address all correspondence and requests for reprints to: Dr. Jane B. Lian, Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655. E-mail: jane.lian{at}umassmed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cbfa1/Runx2 is a transcription factor essential for bone formation and osteoblast differentiation. Two major N-terminal isoforms of Cbfa1, designated type I/p56 (PEBP2aA1, starting with the sequence MRIPV) and type II/p57 (til-1, starting with the sequence MASNS), each regulated by distinct promoters, are known. Here, we show that the type I transcript is constitutively expressed in nonosseous mesenchymal tissues and in osteoblast progenitor cells. Cbfa1 type I isoform expression does not change with the differentiation status of the cells. In contrast, the type II transcript is increased during differentiation of primary osteoblasts and is induced in osteoprogenitors and in premyoblast C2C12 cells in response to bone morphogenetic protein-2. The functional equivalence of the two isoforms in activation and repression of bone-specific genes indicates overlapping functional roles. The presence of the ubiquitous type I isoform in nonosseous cells and before bone morphogenetic protein-2 induced expression of the type II isoform suggests a regulatory role for Cbfa1 type I in early stages of mesenchymal cell development, whereas type II is necessary for osteogenesis and maintenance of the osteoblast phenotype. Our data indicate that Cbfa1 function is regulated by transcription, cellular protein levels, and DNA binding activity during osteoblast differentiation. Taken together, our studies suggest that developmental timing and cell type- specific expression of type I and type II Cbfa isoforms, and not necessarily molecular properties or sequences that reside in the N-terminus of Cbfa1, are the principal determinants of the osteogenic activity of Cbfa1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RUNX/CBFA/AML/PEBP{alpha}A family of transcription factors includes three distinct genes that encode proteins with crucial roles in the regulation of cell fate decision and transcriptional control of critical genes for cellular differentiation and development (reviewed in Refs. 1 and 2). Several key studies have established that Cbfa1 is required for in vivo bone formation (3, 4, 5) as well as maturation of hypertrophic chondrocytes (6, 7) and osteoblast differentiation (3, 8, 9). There is a complete lack of intramembranous and endochondral bone formation in Cbfa1 null mice (3), and haploinsufficiency of this gene results in cleidocranial dysplasia (CCD), a dominantly inherited developmental disorder of bone (4, 5). The loss of bone formation is attributed to maturational arrest of the osteoblast differentiation process (3, 8, 9). Cbfa1 is expressed in mesenchymal condensations of developing bones during embryogenesis (3, 5), and the mRNA has been shown to increase in osteogenic tissues (8, 9, 10, 11).

In the past few years several isoforms for each of the three Cbfa transcription factors have been identified. A shared property of the Cbfa genes is that expression is regulated by at least two distinct promoters that generate two N-terminal isoforms (11, 12, 13). Additional isoforms arise as a result of alternative splicing, exon skipping, as well as deletions and frameshift mutations in the N-terminal, C-terminal, and internal regions of the gene (11, 14). Cbfa1 isoform structure and expression have been studied in rat, mouse, and human (9, 11, 13, 14) and in the context of CCD phenotype (15, 16) where patient genotypes reveal perturbation of the Cbfa1 gene structure. The first Cbfa1 isoform identified, PEBP2aA1 (17) and recently described as the type I isoform (18), is a 513-amino acid protein (designated p56/type I) that initiates in exon 2 at the sequence MRIPV. It was initially shown to be expressed in T cells and Ha-ras-transformed fibroblasts (17, 19) and thymus (20) and was detected in other nonosseous (21), chondrogenic (7, 22), and osteoblast cell lines (8, 11, 18). The second major isoform, til-1 (14) (designated p57 or type II isoform), initiates in exon 1 at the sequence MASNS (11, 14, 23) and is only 15 amino acids longer than the p56/type I isoform. Forced expression of these isoforms modulates transcription of skeletal genes (18, 24, 25, 26), indicating that both proteins are functionally active in osteoblasts and hypertrophic chondrocytes. While attention has focused on the functional activities of these isoforms, their expression in relation to osteoblast maturation and differentiation under control of osteo-inductive factors remains to be addressed. Members of the family of bone morphogenetic proteins, BMP-2 and BMP-4/-7, mediate the commitment of undifferentiated mesenchymal progenitor cells to the skeletal lineage (27, 28, 29) and are potent inducers of Cbfa1 transcription (9, 18, 21, 30). Thus, the early events of osteogenesis, regulated by BMP-2, are closely linked to Cbfa1 induction of the genes involved in bone formation and osteoblast differentiation.

To increase understanding of the role of Cbfa1 in regulating osteogenesis, we examined the expression of Cbfa1 isoforms at different stages of osteoblast development. We also determined the BMP-2 responsiveness of the two major N-terminal isoforms in primary rat calvarial osteoblasts, mouse MC3T3 preosteoblasts, and cells that represent earlier stages of osteoprogenitors. Here we show that the type I transcript is constitutively expressed in nonosseous mesenchymal tissues and during osteoblast differentiation. However, expression of the type II transcript is regulated during osteoblast differentiation and is induced by BMP-2. Our studies provide novel insights into the regulation of Cbfa1 activity in relation to the development of skeletal lineage cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Normal diploid osteoblasts obtained from 21-d-old fetal rat calvariae were isolated and maintained as previously described (31). Primary cell cultures were established from postnatal mouse lung, liver, muscle, and skin tissues after 5- and 15-min sequential digestions with collagenase P (Roche Molecular Biochemicals, Mannheim, Germany), plating cells from the second digest at a density of 0.5 x 106/100-mm dish. MC3T3-E1 cells were maintained in {alpha}MEM supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA). MLB13MYC clone 14 and MLB13MYC clone 17 cell lines were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (32). Cells at passage 12 were changed to DMEM with 1% FBS when treated with 100 ng/ml BMP-2 (Genetics Institute, Cambridge, MA). C2C12 cells were maintained in DMEM (33) supplemented with 5% FBS and treated with 300 ng/ml BMP-2 when required. Charcoal-stripped serum was prepared for BMP-2-treated cells by the addition of 5% activated charcoal to FBS at 4 C overnight, followed by filter sterilization.

Plasmids and analysis of promoter activity
Several promoter-reporter constructs were used for transient transfection assays as previously described (25). Rat osteosarcoma ROS 17/2.8 cells and nonosseous HeLa cells plated on six-well plates were transiently transfected with 2.5 µg/well of either the 0.6-kb chick bone sialoprotein promoter-chloramphenicol acetyltransferase (CAT; a gift from Dr. L. Gerstenfeld, Musculoskeletal Research Laboratory, Boston University Medical Center, Boston, MA) (34), the 1.6-kb TGFß type I receptor (TGF-ßRI) promoter-Luc (gift from Dr. M. Centrella, Department of Surgery, Yale University School of Medicine, New Haven, CT) (35), or the 1.1-kb rat osteocalcin (OC) (36) promoter-CAT. The reporter constructs were cotransfected with 750 ng/well expression plasmid containing cDNAs of either PEBP2{alpha}A1 (type I; gift from Dr. Yoshiaki Ito, Department of Viral Oncology, Kyoto University, Kyoto, Japan) (17) or til-1(type II; gift from Dr. James Neil, Department of Veterinary Pathology, University of Glasgow, Glasgow, UK) (14) using Superfect transfection reagent (Life Technologies, Inc.). CAT activity was measured 24–36 h after transfection, and transfection efficiency was normalized by the luciferase activity of the internal control plasmid Rous sarcoma virus-luciferase (100 ng/well). Representative results of three independent studies are shown. Data shown are the mean ± SD (n = 9).

Protein-DNA interaction analysis
Nuclear extracts were prepared from proliferating (d 2 or 3), differentiated (d 14) and mineralized (d 20 or 23) primary rat osteoblasts as previously described (37) using 0.45 M KCl for extraction. For mouse MC3T3-E1 nuclear extracts, cells were collected on d 7, 10, 16, and 22. MLB13MYC clone 14 and MLB13MYC clone 17 cells [control and 48-h BMP-2-treated (100 ng/ml)] were collected on d 7, C2C12 cells [control and 48 h BMP-2-treated (300 ng/ml)] were collected on d 5. EMSAs were performed using conditions previously described (8). Four micrograms of nuclear extracts were incubated with 1 µg of the nonspecific competitor poly(dI-dC)(dI-dC) (Pharmacia Biotech, Piscataway, NJ) and 10 fmol 32P end-labeled, double stranded Cbfa binding consensus oligonucleotide (5'-CGAGTATTGTGGTTAATACG-3'). Protein-DNA complexes were resolved in 4% nondenaturing polyacrylamide gels using Tris-glycine-EDTA buffer. Antibody supershift experiments contained polyclonal antiserum raised against a C-terminal peptide of Cbfa1 (38) or preimmune serum (control). Gels were dried and exposed to Kodak films (Eastman Kodak Co., New Haven, CT) at -70 C for 6–12 h.

Western blot analysis
Nuclear extracts (30 µg/lane) were resolved in 10% SDS-PAGE and electroblotted (using a semidry electroblotter; Owl Scientific Plastics, Cambridge, MA) onto nitrocellulose membranes (0.2 µm, Protran, Schleicher & Schuell, Inc., Keene, NH) according to the manufacturer’s specifications. Western blot analyses were performed as previously described (8). Based on our data and those of others (35, 39, 40, 41), it is not possible to electrophoretically separate Runx2/Cbfa1 isoforms, type I (p56) and type II (p57), nor has it been possible to generate antibodies that can discriminate between these two proteins. Membranes were incubated at a 1:100 or 1:150 dilution of antibody in Tris-buffered saline containing 1% BSA. IgG-fractionated rabbit polyclonal antibody specific for Cbfa1 (38) or a mouse monoclonal antibody (16) was used in these studies. Membranes were incubated with secondary antibody for 45 min, followed by chemiluminescent detection using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s specifications. Membranes were exposed for 10 sec to 5 min to Amersham Pharmacia Biotech Hyperfilm for detection of signals. Lamin B antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Northern blot analysis
Total cellular RNA was isolated using TRIzol (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s specifications. Polyadenylated [poly(A)+] RNA was isolated using an mRNA isolation kit (Roche, Indianapolis, IN). Ten micrograms of total RNA or 2 µg poly(A)+ RNA/lane were separated in a 1% agarose-formaldehyde gel, transferred onto Zetaprobe membrane (Bio-Rad Laboratories, Inc., Hercules, CA), and hybridized to probes specific for Cbfa1 exon 1 (which detects the N-terminal region of type II isoform; GenBank accession no. AF155361), a full-length cDNA, or a 266-bp BamHI-NcoI fragment of PEBP2aA1 (GenBank accession no. D14636) that is common to all isoforms. Hybridization was performed as previously described (42) in the presence of buffer containing 50% formamide at 42 C, and the blots were washed extensively in buffer containing 1 x SSC (standard saline citrate) and 0.1% SDS at 55 C. Data were analyzed after overnight exposure using a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Ethidium bromide staining of the gels was used to assess equal loading of samples.

RT and PCR
RT was performed on total RNA using Moloney murine leukemia virus reverse transcriptase (Roche Molecular Biochemicals) as specified by the manufacturer. Total RNA (1–2 µg) was incubated at 37 C for 60 min in the presence of deoxy-NTPs, ribonuclease inhibitor (Promega Corp., Madison, WI), oligo(deoxythymidine)15 primers (Promega Corp.), and reverse transcriptase. For PCR, Taq polymerase (Promega Corp.) was used in reactions containing cDNA from the RT reactions, deoxy-NTPs, 1 µM each of forward and reverse primers, and 1 mM MgCl2. The forward primer for the type I isoform (p56/PEBP2{alpha}A1) corresponds to the N-terminal end of the published cDNA sequence of this isoform (GenBank accession no. D14636). The forward primer for the type II (p57/til-1) isoform corresponds to sequences upstream of the translational start site of til-1. The reverse primer is common to both isoforms. PCR amplification primers for Cbfa1 and all other genes are listed in Table 1Go.


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  		Table 1. Primers used for RT-PCR

 
PCR was performed using a thermal controller, PTC-100 (MJ Research, Inc., Watertown, MA) under the following conditions: 95 C for 5 min, 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec, 30 cycles for Cbfa1 (which remains in the linear range) and 15 cycles for GAPDH, followed by 72 C for 10 min. Samples were separated on 1% agarose gels. PCR products were transferred onto Hybond N+ nylon membranes (Amersham Pharmacia Biotech) and hybridized at 65 C to a probe corresponding to the 266-bp BamHI-NcoI-digested fragment from the N-terminal region of Cbfa1 (PEBP2{alpha}A1) cDNA. Hybridized blots were washed at 65 C with 1 x SSC and 0.1% SDS. Data were analyzed using a PhosphorImager after exposure for 2–24 h.

Immunofluorescence
Osteoblasts were grown on glass coverslips (Fisher Scientific, Springfield, NJ) at 37 C in growth medium to 40% confluence for 24 h. Cells were fixed in 4% paraformaldehyde in PBS or subjected to in situ extraction of cytoskeletal and soluble chromatin proteins to reveal the nuclear matrix-intermediate filament scaffold (see below). Coverslips were processed using protocols previously described by our laboratory (25).

Antibody staining was performed using an affinity-purified Cbfa1 primary antibody (38) at a dilution of 1:200 and was incubated for 1–1.5 h at 37 C. Coverslips were then incubated with a fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (1:500; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and incubated for 1 h at 37 C to detect Cbfa1. In situ nuclear matrexes were prepared as previously described (43). Briefly, cells on coverslips were washed in PBS and extracted twice in cytoskeletal (CSK) buffer for 15 min each. CSK buffer contains 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 1.2 mM phenylmethylsulfonylfluoride, and 1% vanadyl ribonucleoside complex. Deoxyribonuclease I digestion was performed twice in digestion buffer (CSK buffer with 50 mM NaCl) containing 100 µg/ml deoxyribonuclease I for 30 min, followed by an extraction in digestion buffer containing 0.25 M (NH4)2SO4 for 10 min. DNA content was evaluated by staining with 4',6-diamidino-2-phenylindole (DAPI; 5 µg/ml in PBS containing BSA and 0.05% Triton X-100). Cells were mounted in Vectashield H-1000 (Vector Laboratories, Inc., Burlingame, CA). Images were obtained using a CCD camera interfaced with a digital microscope system (Carl Zeiss, Thornwood, NY). Images were analyzed by Metamorph software (Universal Imaging Corp., West Chester, PA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Cbfa1 isoforms during osteoblast growth and differentiation
We previously reported an increase in Cbfa1 protein and DNA binding activity by EMSA of nuclear extracts from the proliferating to differentiated stages of primary rat calvarial osteoblast culture (8). To determine whether the increased DNA binding (Fig. 1AGo) is regulated by modulation of protein levels, we performed Western blot analysis (Fig. 1BGo) with nuclear extracts isolated during growth and differentiation of primary rat osteoblasts. The Western analysis showed low Cbfa1 protein levels on d 2 and significant levels in postproliferative d 14 cells. Cbfa1 protein level was thereafter moderately increased on d 20 (Fig. 1BGo). These results were confirmed using different antibodies [described in Ref. 16 and obtained from Oncogene Research Products (Boston, MA)] and with different time courses. It is noteworthy that an additional Cbfa1 minor band is present in very heavily mineralized osteoblast cultures, perhaps indicating partial degradation (data not shown). At late stages of differentiation, secreted osteocalcin levels are still in the peak range (Fig. 1CGo), consistent with increased Cbfa DNA binding activity (Fig. 1AGo). Thus, the greatest increases in Cbfa1 cellular protein occur between proliferating (d 2) and differentiated cells (d 14). However, the changes in Cbfa1 protein between d 14 and d 20 or d 23 are less dramatic than the increase observed in DNA binding activity. This discordance may reflect posttranslational modifications in the osteoblast-specific complex (OBSC) that enhance Cbfa1 DNA binding activity, which can be observed in native EMSA gels, in contrast to the denaturing conditions of the Western analysis.



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  		Figure 1. Representation of Cbfa1 during the growth and differentiation of fetal rat calvarial osteoblasts. A, DNA binding activity of Cbfa1 during osteoblast differentiation. EMSAs were performed using rat osteoblast nuclear extracts (lanes 1 and 2, d 3; lanes 3 and 4, d 14; lanes 5 and 6, d 23), a probe containing the consensus Cbfa-binding site and Cbfa1 antiserum (38 ). Lanes 1, 3, and 5, Nuclear extracts incubated with preimmune serum; lanes 2, 4, and 6, nuclear extracts incubated with 1 µl Cbfa1 antiserum. Dried gels were exposed for 6 h. The Cbfa1-containing complex (OBSC) and the supershifted bands (supershift) are indicated. The graph to the right is an average of the densitometric analysis of the supershifted complex. B, Cbfa1-immunoreactive proteins during osteoblast differentiation. Western blots were performed using rat osteoblast nuclear extracts obtained from different stages of growth and differentiation (proliferating d 2, differentiating d 14, and mineralizing d 20) as indicated. The membrane was incubated with antibody as previously described (16 ). Similar results were obtained using antibody from Meyers et al. (38 ) and Oncogene Research Products. Relative migration of Cbfa1 and lamin B to the markers is indicated. The graph to the right of B presents densitometric quantitation of the Cbfa1-immunoreactive band. C, The extent of osteoblast differentiation as reflected by secreted osteocalcin measured by RIA for the experiments presented in A and B.

 
To assess the cellular localization of endogenous Cbfa1 protein in proliferating osteoblasts, we performed in situ immunofluorescence analysis. Endogenous Cbfa1 is detected in whole cell and nuclear matrix preparations of proliferating rat osteoblasts as intense punctate staining of the protein(s) localized specifically in the nucleus (Fig. 2Go, A and B). Together, these results establish that all the Cbfa1 protein detected is present in the nuclei. In proliferating osteoblasts, Cbfa1 is completely localized in subnuclear domains; thus, the low protein levels and DNA binding activity are not related to partitioning between the nucleus and the cytoplasm.



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  		Figure 2. Immunofluorescence detection of endogenous Cbfa1 in rat calvarial osteoblasts. Whole cell and nuclear matrix preparations of primary osteoblasts (d 2) were fixed in 4% paraformaldehyde, washed, and incubated with anti-Cbfa1 primary antibody followed by FITC-conjugated goat antirabbit secondary antibody as described in Materials and Methods. Representation of Cbfa1 in whole cells (A) and in situ nuclear matrix (NMIF) localization (B) at 40x (top rows) and 100x (bottom rows) are shown. Left panels, FITC-stained Cbfa1; middle panels, DAPI-stained nuclei; right panels, differential interference contrast (DIC) image of the cells. The absence of DNA in DAPI in B indicates the complete removal of chromatin from nuclear matrix intermediate filament (NMIF) preparation.

 
To determine whether the increases in the DNA-binding complex and protein levels by Western analyses correlated with increased cellular synthesis of Cbfa1, Northern analysis was performed on RNA derived from time courses of primary rat osteoblasts (ROB) and mouse MC3T3-E1 preosteoblasts (Fig. 3Go). We hybridized blots of ROB total RNA (from d 2, 7, 11, 18, and 22; Fig. 3Go, A–C) with a full-length cDNA probe with the potential to recognize all the Cbfa1 mRNAs (Fig. 3AGo), as well as with an exon 1 probe (Fig. 3BGo), which should specifically detect mRNAs encoding type II isoforms (illustrated in Fig. 4AGo). Our results show that multiple mRNA transcripts ranging in size from 0.6–6 kb are detected with the full-length probe (Fig. 3AGo), and there is a developmental increase in the major transcript from growth to differentiation stages of rat osteoblasts. Using the exon 1-specific probe, the increase in the major transcript above the 28S marker becomes more evident (Fig. 3BGo). Northern analysis of poly A+ RNA using full-length Cbfa1 cDNA probe (Fig. 3DGo) confirmed the presence of multiple transcripts and their increased expression during osteoblast differentiation.



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  		Figure 3. Northern analysis of Cbfa1 transcripts during osteoblast differentiation. Total cellular RNA was isolated during different stages of growth and differentiation from primary ROB (d 2, 7, 11, 18, and 22; A–C) and mouse MC3T3-E1: d 4, 7, 10, 16, and 22 (E and F). RNA (10 µg) was electrophoretically resolved in a 1% agarose/formaldehyde gel and blotted onto a nylon membrane. Hybridizations were performed with a full-length Cbfa1 cDNA probe (A) or a 460-bp probe corresponding to the EcoRV-BamHI fragment containing the exon 1 sequence of Cbfa1 cDNA (B and E). The blot was analyzed in a PhosphorImager after 18-h exposure. Cbfa1 transcripts indicated by arrows; 28S and 18S rRNA markers are indicated. Ethidium bromide staining of the same gels is shown in C and F to demonstrate the amount of RNA loading in each sample. D (ROB cells) and G (MC3T3E1 cells), Northern blot analyses of poly(A)+ RNA from a different time course (the indicated days are shown) to verify the multiple transcripts and developmental changes during osteoblast differentiation observed with total cellular RNA. The full-length Cbfa1 probe was used for hybridization. H (ROB) and I (MC3T3E1) summarize the combined expression of the major Cbfa1 transcripts during osteoblast differentiation [uppermost two arrows in B (rat) and D (mouse) in three different time courses]. The values from independent time courses (n = 4, ROB; n = 3, MC3T3) are plotted as the percent maximal expression in each time course.

 


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  		Figure 4. Expression of Cbfa1 transcripts during growth and differentiation of rat osteoblasts. A, Runx2/Cbfa1 gene organization, indicating the derivation of the principal N-terminal isoforms. Schematic illustration of exons 1–8 that comprise the Cbfa1 gene and the exon origin of the Cbfa1 isoforms p57 (MASNS), type II and p56 (MRIPV), or type I. Exon 1 shows the ATG1 start site for the type II isoform (til-1) (14 ), starting with the sequence MASNS, and exon 2 contains the start site of type I isoform (PEBP2aA1) (17 ), starting with the sequence MRIPV. The solid black exons denote the runt homology DNA binding domain (RHD); NMTS designates the location of the 31-amino acid nuclear matrix-targeting signal (43 ). The conserved VWRPY motif at the extreme C-terminal region of Cbfa1 gene is indicated. B, Total RNA from primary rat osteoblasts (proliferating d 2, postproliferative d 14, and differentiated in a mineralizing matrix d 23) were reverse transcribed followed by PCR amplification using Cbfa1 type II and type I isoform-specific primers and separated in 1% agarose gels as described in Materials and Methods. Rat ALP and rat OC expression are shown for reference. The rat GAPDH transcript indicates equal loading of samples. Cbfa1 type II and type I RT-PCR products were further confirmed by Southern hybridization: shown in panels on the right (see Materials and Methods). C, Graphic representation of the developmental expression of the type II Cbfa1 isoform in ROB cells. Data from two different time courses are plotted as the percent maximal expression from quantitative densitometric analysis of Southern blots of the RT-PCR products. Values are the mean ± SD. D, RT-PCR performed on MC3T3 cell-derived total RNA from proliferating through differentiated stages (d 7, 10, 16, and 22) as indicated. PCR amplification was performed using type II and type I isoform-specific primers and separated on a 1% agarose gel as described in Materials and Methods. Mouse GAPDH serves as a control for equal loading, and OC was used as reference for differentiation. E, Graphic representation of the developmental expression of the type II Cbfa1 isoform in differentiating MC3T3-E1 cells. Calculations are the same as those described in C.

 
We also examined the developmental profile of Cbfa1 RNA expression during the differentiation of mouse osteoblasts on d 4, 7, 10, 16, and 22 (Fig. 3Go, E–G). The MC3T3-E1 cell line has been well characterized with respect to the stages of osteoblast maturation (44). Using the same exon 1-specific probe, we observed that type II Cbfa1 RNA levels increased from proliferating (d 4–7) to postproliferative (d 10) and mineralizing (d 16–22) MC3T3-E1 cultures. However, consistent with other time courses, we observed a modest decline after d 22 when the matrix was heavily mineralized in the mouse cell cultures. Other mRNAs (e.g. osteocalcin) also declined. Northern blot analysis of poly(A)+ RNA with a full-length cDNA probe (Fig. 3GGo) confirmed our findings that multiple Cbfa1 transcripts are expressed, similar to total cellular RNA in mouse osteoblasts (Fig. 3EGo). Thus, the cellular levels of Cbfa1 transcripts that are derived from exon 1 increase in expression and are dramatically up-regulated during the onset of differentiation in both calvarial rat and mouse osteoblasts (Fig. 3Go, H and I, summary of data from several time courses).

To further confirm the specific development patterns of expression of the Cbfa type I and type II transcripts during osteoblast differentiation, we performed RT-PCR analysis with primers (shown in Table 1Go) specific for exon 1 or exon 2 (Fig. 4Go). Semiquantitative RT-PCR of mRNA from primary rat osteoblasts (Fig. 4BGo) and mouse MC3T3 cells (Fig. 4CGo) indicated that transcripts of both Cbfa1 isoforms were detected throughout the courses of osteoblast differentiation. Although the mRNA encoding type I was constitutively expressed from growth to mineralization stages, the type II encoded transcript increased postproliferatively from d 2 to 14/d 23 in rat osteoblasts (Fig. 4Go, B and C) and from d 7 to d 10/d 16 in MC3T3E1 cells (Fig. 4DGo). Notably, we observed a decline in representation of the type II isoform in heavily mineralized mouse osteoblasts (d 22; Fig. 4Go, D and E), consistent with Northern blot analyses (Fig. 3Go, E and G). We confirmed the specificity of the rat osteoblast RT-PCR products (visualized in agarose gels) by Southern hybridization with a Cbfa1-specific cDNA probe (described in Materials and Methods; Fig. 4BGo, right panel). For comparison, osteoblast phenotypic markers alkaline phosphatase (ALP) and OC are shown to reflect osteoblast differentiation, and as expected, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is constitutive. Taken together, these results show constitutive expression of type I and developmental and preferential expression of type II in final stages of osteoblast differentiation Thus, it appears that the two isoforms together account for Cbfa1 protein levels by EMSA, Western, and immunofluorescence analyses.

To ascertain the tissue specificity of Cbfa1 type I and II isoforms, we examined their expression in nonosseous organs and primary cultures of cells from soft tissues of the newborn mouse. We examined total cellular RNA from four organs, liver, lung, muscle, and skin, and the primary cells cultured to confluence (Fig. 5Go). The type I isoform, but not the type II isoform, was present in primary cell cultures as well as in RNA from the tissues. The RT-PCR product for the type I isoform was consistently detected at very low levels in liver tissue, but at significant levels in the mesenchymal tissues, particularly skin. Thus, the Cbfa1 type I isoform, which was shown to be present in thymus and spleen (1, 20), appears to be more ubiquitously expressed than previously appreciated.



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  		Figure 5. Cbfa1 type I isoform, but not type II isoform, is present in mesenchymal tissues. RT-PCR for Cbfa1 isoforms in primary cultures of cells from the tissues indicated (A) and mouse tissues (B). The primers used are shown in Table 1Go, and the conditions for RT-PCR are described in Materials and Methods. GAPDH is shown for control levels.

 
Functional analyses of type I and type II isoforms
The type I and type II isoforms each have distinct N-termini (Fig. 4Go) regulated by distinct promoters and the N-terminus of the type II isoform is 15 amino acids longer. However, the functional consequences of these molecular differences have not been unequivocally established. To assess the transcriptional activities of the type II and type I isoforms, we performed transient cotransfection studies with these two isoforms and a panel of bone-related Cbfa-responsive target genes (e.g. OC, TGFß-RI, and bone sialoprotein). Transfections were performed with osteoblastic ROS 17/2.8 cells (Fig. 6BGo) and HeLa cells that do not contain Cbfa1 (Fig. 6AGo). In both cell types we observed that type I and type II isoforms activated the OC and TGF-ßRI promoters by 4- to 8-fold and repressed bone sialoprotein promoter activity by 4- to 8-fold. No statistically significant differences were observed in the activation or repression potential of the two isoforms, and both transfected proteins were expressed at similar levels (Fig. 6CGo). The similarities in transcriptional results for both isoforms suggest that the 15-amino acid extension of the type II isoform does not contribute to activation or repression of transcription. Our results demonstrate that the type I and type II isoforms have nearly equivalent transcriptional activities.



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  		Figure 6. Activity of Cbfa1 isoforms on promoters of genes expressed in osteoblasts. A, Transfections were performed in HeLa cells that do not endogenously express Cbfa factors. The cells were plated in six-well plates and transiently cotransfected with expression vector for type I or type II isoforms (750 ng/well) together with either 2.5 µg/well 1.1 kb OC promoter (OC), 1.6 kb TGF-ßRI, or 0.6 kb bone sialoprotein promoter (BSP) fused to the luciferase reporter gene. The fold activation for OC and TGF-ßRI and the fold repression for BSP are shown for n = 6. B, Rat osteosarcoma ROS 17/2.8 cells were transiently transfected as described for HeLa cells. Fold activation or repression for six values are shown. C, Western analyses shows Cbfa1 protein expression in HeLa cell lysates (30 µg protein) harvested 24 h after transfection with either the type II or type I Cbfa1 isoforms and compared with tubulin as a control.

 
Cbfa1 type I and type II isoforms are both expressed in skeletal progenitor cells and are differentially regulated by BMP-2
The presence of significant levels of Cbfa1 protein in proliferating osteoblasts, which are precommitted cells derived from calvarial bone, led us to assess whether the Cbfa1 gene is expressed at earlier stages of osteoblast development. We examined cellular Cbfa1 protein levels in the skeletal progenitor cells MLB13 MYC clone 14 and MLB13MYC clone 17 (32) derived from mouse limb bud. MLB13MYC clone 14 represents an undifferentiated early skeletal progenitor that differentiates into chondroblasts and then into osteoblasts in response to BMP-2. MLB13MYC clone 17 represents a prechondroblast cell line that directly differentiates into osteoblasts after BMP-2 treatment. Cbfa1 proteins were initially analyzed by whole cell in situ immunofluorescence microscopy. In both cell lines, endogenous Cbfa1 proteins were detected throughout the cell layer and exhibited intense punctate FITC staining in the nucleus (Fig. 7Go, A and B). Thus, Cbfa1 proteins are uniformly and highly expressed in these endochondral skeletal cells, which are not yet committed to the osteoblast lineage.



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  		Figure 7. Cellular levels of Cbfa1 proteins in mouse marrow-derived progenitor cell lines. Cells were fixed with 4% paraformaldehyde, washed, and incubated with Cbfa1 primary antibody followed by FITC-conjugated goat antirabbit secondary antibody as described in Materials and Methods. Endogenous expression of Cbfa1 in whole cells of MLB13MYC clone 14 (A) and MLB13MYC clone 17 (B) at 40x and 63x magnifications are shown as indicated. Cbfa1 expression is shown in the left panels; DAPI staining of nucleus (middle panels) and differential interference contrast (DIC) (right panels) images for both fields are shown.

 
The presence of Cbfa1 proteins in early mesenchymal progenitor cells suggests that it may have an important role in early stages of lineage commitment, but our data do not discriminate whether one or both Cbfa1 isoforms are expressed. Therefore, we examined representation of Cbfa1 type I and type II transcripts in these two cell lines after BMP-2-induced osteoblast differentiation. Semiquantitative RT-PCR analyses (Fig. 8AGo) showed that both type I and type II transcripts were detected at low levels in the clone 14 and clone 17 cells before BMP-2 treatment, when cell phenotype-specific markers are not expressed (e.g. mALP and mOC). BMP-2 treatment for 48 h significantly enhanced the expression of the type II transcript, but not the type I transcript, in both cell lines. In parallel, expression of bone phenotype transcripts mOC and mALP increased with BMP-2 treatment, indicating differentiation of the skeletal progenitor cells, whereas expression of the housekeeping gene mouse GAPDH remained constant. Hence, our data clearly show selective up-regulation of the type II isoform along with onset of expression of bone phenotypic markers.



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  		Figure 8. Detection and BMP-2 regulation of Cbfa1 isoforms in early mouse progenitor cells. A, RT-PCR analysis of Cbfa1 transcripts. MLB13MYC clone 14 and MLB13MYC clone 17 cells with or without 48-h BMP-2 treatment (100 ng/ml) were harvested on d 7. Total RNA isolated from untreated (Control) and treated (BMP-2) cells were reverse transcribed, followed by PCR amplification using type I and type II Cbfa1isoform-specific primers (Table 1Go) as described in Materials and Methods. For type I and type II Cbfa1, PCR products were hybridized with Cbfa1 cDNA (lower panels) to indicate the specificity of the reaction products. Mouse OC (mOC), mALP, and mouse collagen I (mCollI) are used as reference for osteoblast differentiation and equal sample loading represented by mGAPDH. B, BMP-2 increases the DNA binding activity of Cbfa1 in skeletal progenitor cells. EMSA nuclear extracts obtained from skeletal progenitor cell lines MLB13MYC clone 14 and MLB13MYC clone 17 were incubated with anti-Cbfa1 antiserum in binding reactions using a probe containing the consensus Cbfa binding site. Lanes 1 and 2, Nuclear extracts from untreated control cells; lanes 3 and 4, nuclear extracts from cells treated 48 h with BMP-2 (100 ng/ml). Reactions in lanes 2 and 4 were incubated with Cbfa1 antiserum (38 ). Dried gels were exposed for 12–16 h. The OBSC and the Cbfa1 supershifted bands (supershift) are indicated.

 
To evaluate whether up-regulation of type II expression after BMP-2 treatment increased Cbfa1 DNA binding activity, we analyzed nuclear extracts from these two skeletal progenitor cells for formation of DNA binding complexes with the Cbfa1 consensus binding site as the probe. EMSAs coupled with Cbfa1 antibody supershift assays demonstrate that both of these cell lines form Cbfa1-containing DNA binding complexes (Fig. 8BGo). Treatment of cells with BMP-2 substantially increased the Cbfa1 protein-DNA complex. This result parallels the observed increase in the type II transcript in differentiated rat and mouse osteoblasts detected by RT-PCR (Fig. 4Go). Thus, these biochemical studies reveal that Cbfa1 proteins are expressed in very early stage mesenchymal progenitors, before their differentiation into the osteoblast phenotype. The enhanced expression of the type II transcript in response to BMP-2 correlates with increased DNA binding activity that parallels induction of osteoblast differentiation by BMP-2.

BMP-2 induces selective expression of the type II Cbfa1 isoform during osteogenic differentiation of myogenic progenitor C2C12 cells
The presence of Cbfa1 type II isoform in endochondral progenitor cells suggests that expression of the type II isoform precedes cellular commitment to osteoblasts and/or is a marker for cells destined to differentiate into bone cells. To address this question, we examined the C2C12 cell model, a premyoblastic cell line of mesenchymal origin, because muscle cells lack expression of Cbfa1 type II isoform (Fig. 5Go), and the C2C12 line is capable of trans-differentiating into the osteoblast lineage in response to BMP-2 (33). We used RT-PCR to assess expression of the Cbfa1 type I and type II isoforms in the myoblast stage and during BMP-2-induced osteoblast differentiation (Fig. 9Go). These studies were carried out in 5% serum, which supports myogenesis, and 10% serum, which favors osteogenic differentiation. In parallel, cells were also analyzed in the presence of charcoal treated FBS (stripped serum). Figure 9Go demonstrates that expression of the type II isoform was not detected in control C2C12 cells cultured in either 5% or 10% serum (regular or stripped). Interestingly, after 48 h of treatment with BMP-2, we observed a significant increase in expression of the type II transcript in cultures grown in either 5% or 10% serum, although the increase was greater in 10% serum. Notably, the type I transcript was expressed in the C2C12 cell line before treatment with BMP-2 (see control lanes). Expression of type I did not significantly differ between control and BMP-2-treated cells in either 10% or 5% serum in repeated studies; however, in stripped serum, BMP-2 stimulated the type I RT-PCR product. This finding indicates that factors present in regular serum may influence the expression of this isoform. As expected, the expression of myogenic MyoD was suppressed by BMP-2 in cultures supplemented with either 5% or 10% serum. This down-regulation of MyoD with BMP-2 treatment occurred concomitant with induction of the osteoblast marker OC, whereas Smad 1, a downstream target of the BMP-2 signaling pathway, showed no appreciable change in expression, as expected (45). Together, these results demonstrate that the expression of Cbfa1 type II isoform is induced with onset of the osteoblast phenotype (that is, expressing OC), whereas the type I isoform is expressed in mesenchymal progenitors and is not restricted to skeletal tissues.



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  		Figure 9. Detection of Cbfa1 transcripts and protein in mesenchymal C2C12 cells. C2C12 cells were grown for 3 d in 5% or 10% serum (regular or stripped serum) and then supplemented with 300 ng/ml BMP-2 for 48 h. Total RNA from these cultures were used to perform semiquantitative RT-PCR using type I and type II Cbfa1 primers (Table 1Go) as described in Materials and Methods. Blots were hybridized to Cbfa1-specific cDNA probe to indicate the specificity of the reaction products. MyoD, Smad 1, mouse OC (phenotypic markers), as well as mouse GAPDH (for quantitation purposes) were reverse transcribed, followed by PCR using the primers listed in Table 1Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although genetic studies have shown that Cbfa1 is essential for bone formation (3, 4, 5), these approaches did not distinguish the biological roles of the two different N-terminal isoforms of Cbfa1 in transcriptional control of osteoblast differentiation. In this study we have characterized the expression pattern of each of the major N-terminal isoforms in models of osteogenic differentiation. Our findings can be summarized as follows. First, the type I isoform is expressed before the type II; second, type I transcript expression is ubiquitous and constitutive in both nonosseous and mesenchymal cells and during all stages of osteoblast differentiation; and third, a developmental increase in type II transcript expression occurs during differentiation of primary osteoblasts and after BMP-2 mediated differentiation of progenitor cells. We propose that the Cbfa1 type I isoform represents a marker of early stage stromal mesenchymal cells, and that type II isoform defines a cell that has the potential for commitment to the osteoblast lineage. Our data also suggest that Cbfa1 contributes to gene regulation for osteoblast differentiation through multiple levels of control.

The intricate regulatory pattern of Cbfa1 isoform expression is generated at least in part by utilization of alternative ATG start codons located in different exons and transcribed from two distinct promoters (11, 14, 19). We detected at least two major long transcripts and several minor shorter transcripts by Northern analysis using the full-length Cbfa1 cDNA as probe. Different mRNA variants, which represent utilization of the two promoters as well as alternative splicing and exon skipping (11, 14, 19), have been documented for other Cbfa factors (12, 46). Cbfa2, a key determinant for hemopoiesis (47), is also encoded by multiple transcripts produced through alternate promoter usage and exon skipping (46). Interestingly, alternatively spliced smaller transcripts of the Cbfa1 type II form are predominant in the testis (48). Differential regulation by two alternative promoters has also been observed for other genes. The human c-src gene (49) and the rat bone/liver/kidney/placenta ALP gene (50) each contain two alternative promoters and associated exons that splice to a common downstream exon, resulting in identical coding regions with different 5'-ends. Also, tissue-specific expression of the c-myc gene in Xenopus is controlled by two differentially regulated promoters (51). The expression of Cbfa1 variants from distinct promoters represents a versatile mechanism for temporal and cell-type specific control of Cbfa1 activity during development and for responsiveness to osteogenic factors during bone formation.

The results from our studies as well as those of others have indicated that there is considerable complexity in the expression, molecular characteristics, and biological roles of Cbfa proteins (1, 2, 11, 18, 24). Our studies show that Cbfa-dependent gene regulation involves transcriptional control of Cbfa1, cellular levels of the protein, and posttranslational formation of multiple Cbfa1-containing protein-DNA complexes. Northern blot and RT-PCR analyses of total cellular RNA from primary rat osteoblasts and mouse MC3T3E1 cells revealed increased type II RNA during progressive development of the osteoblast phenotype. The developmental increase in type II mRNA is paralleled by a similar increase in Cbfa1 protein and DNA binding activity of the osteoblast-specific Cbfa1-containing complex during early stages of mature osteoblast differentiation. However, there is a decline in type II expression in heavily mineralized stages of mouse MC3T3 osteoblasts. This decrease may be due to autoregulation of Cbfa1 (13) or may result from apoptosis of cells in vitro. A decline in Cbfa1 mRNA levels has been observed during cellular aging of human trabecular osteoblasts (52).

Although it is not possible to correlate specific transcripts with proteins identified by antibodies (by Western or gel shift analyses), our data indicate that transcription of Cbfa1 is rate limiting for cellular protein and DNA binding activity from the immature osteoblast stage (proliferating) to postproliferative differentiated cells. However, we demonstrate a discordance between expression and protein levels relative to DNA binding in mature osteoblasts only after d 14/d 16. We suggest that Cbfa1 cellular protein is rate limiting for formation of the DNA binding complex in early stage osteoblasts (d 2–14), whereas in late stage osteoblasts, protein-protein interactions or the known posttranslational modifications of Cbfa1 may contribute to enhanced DNA binding activity. Indeed, studies have shown that phosphorylation by MAPK pathways plays a role in regulation of Cbfa1 transcriptional activity (53). Alternatively, the more significant increase in DNA binding occurring in mature osteoblasts may involve Cbfa1 interactions with numerous coregulatory proteins (2, 25). Posttranscriptional regulation or protein-protein interactions with cell type-specific cofactors may also play a major role in tissue-specific expression and activity of the Cbfa1 isoforms. Thus, the significant increase in Cbfa1 DNA binding activity in mature osteoblasts indicates that Cbfa1-interacting proteins may contribute to the regulation of a broad spectrum of genes in osteoblasts and other cell types (25, 26).

Previous in vivo studies have documented that during embryonic development and fracture repair, Cbfa1 is expressed in mesenchymal cell condensations of the early developing mouse (embryonic day 10) skeleton (3, 4, 5, 6, 7, 9, 10, 54). Cbfa1 is present in abundant levels in thymus and T and B cells (17, 55) and is also detected in stromal populations derived from human and mouse bone marrow (56), representing both fully differentiated and intermediate preosteoblastic cells (57), as well as in nonosteogenic clonal colonies (58). The significance of our findings relative to those of previous studies is that we have clearly distinguished the expression patterns of Cbfa1 type I and type II transcripts during commitment and development of the osteoblast phenotype. We demonstrate the presence of the Cbfa1 type I isoform in nonosseous cells of mesenchymal origin and constitutive expression in uninduced skeletal progenitors and early stage proliferating osteoblasts that do not yet express genes reflecting mature osteoblasts (e.g. bone sialoprotein, ALP, and OC) (31, 32, 44). Thus, our studies show that the type I isoform is expressed ubiquitously among cells of mesenchymal origin. We suggest that the Cbfa1 type I isoform appears in early stages of fetal development to provide pluripotent stem cells with the option for commitment to the mesenchymal lineage.

We have provided novel evidence to support the concept that p57/type II isoform of Cbfa1 is specifically related to osteogenic commitment and differentiation, because the type II transcript is selectively up-regulated by BMP-2 and during development of the osteoblast phenotype. Interestingly, we have examined 3 kb of the Cbfa1 type II promoter (13) for BMP-2 responsiveness, and under the conditions of these experiments in which endogenous mRNA and protein are increased, BMP-2 did not mediate a change in the activity of this segment of the promoter (unpublished observations). Our finding that the Cbfa1 type II isoform is selectively induced during BMP-2-mediated osteogenesis in skeletal progenitor cells and nonosseous mesenchymal C2C12 cells clarifies the differences reported in other studies. Gori et al. (29) showed up-regulation of Cbfa1 in response to BMP-2 in immortalized marrow stromal osteoprogenitor cells that already expressed osteoblast phenotypic markers (59). Another study showed that Cbfa1 mRNA is unaffected by BMP-2 treatment in both skeletal and nonskeletal cells, whereas BMP-4/-7 treatment up-regulates Cbfa1 expression in the same cells (21). Thus, future studies must consider interpretation of results within the context of a particular BMP, the cell type, and the specific Cbfa1 isoform.

In summary, our studies demonstrate expression of the p56/type I isoform of Cbfa1 in a spectrum of nonosseous, pluripotent, and committed osteoprogenitor cells before expression of the p57 type II isoform. We observe the regulated expression of the p57/type II isoform in committed osteoprogenitor cells and in osteoblasts as well as after BMP-2-mediated osteoblastic induction of stromal cells or trans-differentiation of C2C12 cells. The data indicate that BMP2-mediated induction of the Cbfa1 type II isoform through utilization of promoter 1 is critical for differentiation to osteoblasts. Our comparative expression studies indicate that although the two N-terminal isoforms are differentially expressed, they have functionally equivalent transcriptional activity on promoters in both osseous and nonosseous cell lines. Recent studies in vivo support this concept (60). Thus, the timing of expression of each Cbfa1 isoform, perhaps in conjunction with responses to BMPs in different subpopulations of cells during specific stages of bone development, must be an important component of the mechanism by which Cbfa1 regulates osteogenesis.


   Acknowledgments
 


   Footnotes
 
This work was supported in part by NIH Grants AR-39588, DE-12528, and AR-45688. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The nomenclature committee of the Human Genome Organization has recently adopted the following designations for Runt-related transcription factors: RUNX1 (AML1/CBFA2/PEBP2{alpha}B), RUNX2 (AML3/CBFA1/PEBP2{alpha}A), and RUNX3 (AML2/CBFA3/PEBP2{alpha}C).

Abbreviations: ALP, Alkaline phosphatase; BMP, bone morphogenetic protein; CCD, cleidocranial dysplasia; CSK, cytoskeletal; DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mALP, mouse alkaline phosphatase; OBSC, osteoblast-specific complex; OC, osteocalcin; poly(A)+, polyadenylated; ROB, rat osteoblasts; TGFß-RI, TGFß type I receptor.

Received October 9, 2000.

Accepted for publication May 10, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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