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First published online March 22, 2007
Stem Cells Vol. 25 No. 6 June 2007, pp. 1462 -1468
doi:10.1634/stemcells.2006-0391; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Cbfb Enhances the Osteogenic Differentiation of Both Human and Mouse Mesenchymal Stem Cells Induced by Cbfa-1 via Reducing Its Ubiquitination-Mediated Degradation

Chun-Yang Liena, Oscar K. Leea,b,c, Yeu Sua

aInstitute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan, China;
bDepartment of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, China;
cInstitute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, China

Key Words. Mesenchymal stem cells • Cbfa-1 • Cbfb • Osteogenic differentiation • Ubiquitination

Correspondence: Yeu Su, Ph.D., Institute of Biopharmaceutical Science, National Yang-Ming University, Shi-Pai, Taipei 11221, Taiwan, China. Telephone: 886-2-28267143; Fax: 886-2-28250883; e-mail: yeusu{at}ym.edu.tw; or Oscar K. Lee, M.D., Ph.D., Institute of Clinical Medicine, National Yang-Ming University, Taipei Veterans General Hospital, 201, Sec 2, Shi-Pai Road, Taipei 11221, Taiwan, China. Telephone: 886-2-28757557; Fax: 886-2-28757657; e-mail: kslee{at}vghtpe.gov.tw

Received June 27, 2006; accepted for publication March 10, 2007.
First published online in STEM CELLS EXPRESS   March 22, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Core-binding factors are a small family of heterodimeric transcription factors that play critical roles in development. Whereas Cbfa-1, one of the three {alpha} subunits in the family, is essential for osteogenesis, Cbfb, the only β subunit, forms heterodimers with different Cbfas to increase their DNA binding affinity by inducing conformational changes. Although defective bone formation was found in both Cbfa-1 and Cbfb knockout animals, the precise role of the latter in osteogenesis remains unclear. To dissect the contribution of Cbfb in osteogenic differentiation of mesenchymal stem cells (MSCs), recombinant adenoviruses carrying Cbfb (AdHACbfb) and Cbfa-1 (AdCbfa-1) were generated and used to infect both the mouse C3H10T1/2 cells and human bone marrow-derived MSCs. Although Cbfb alone failed to trigger osteogenesis of MSCs, it markedly enhanced the gene expression and enzyme activity of alkaline phosphatase as well as osteocalcin activation in those cells overexpressing Cbfa-1. Enhancement of the osteogenic differentiation-inducing effect of Cbfa-1 by Cbfb resulted from an increase in stability of the former due to the suppression of ubiquitination-mediated proteasomal degradation by the latter. Taken together, in addition to defining the role of Cbfb in osteogenic differentiation of MSCs, our results also suggest that the Cbfa-1 and Cbfb coexpressing MSCs might be an appropriate strategy for bone repairing and regeneration therapies.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play crucial roles in development and in human diseases [1]. CBFs contain a DNA-binding CBF{alpha} subunit and a CBFβ subunit, which does not contact DNA directly [2, 3]. The {alpha} subunit, encoded by the mammalian homolog of the runt gene from Drosophila (RUNX), contains an evolutionarily conserved Runt domain required for both its DNA binding and heterodimerization with the β subunit. In mammals, three distinct {alpha} subunit-coding genes, RUNX1 (Cbfa-2/PEBP2{alpha}B), RUNX2 (Cbfa-1/PEBP2{alpha}A), and RUNX3 (Cbfa-3/PEBP2{alpha}C), were identified [4]. Among RUNX family members, Cbfa-1 was shown to be essential for the generation and maturation of osteoblasts because bone formation was not detected in Cbfa-1-deficient (Cbfa-1–/–) mice [5]. This transcription factor, in addition, appears to play a critical role in the commitment of the mesenchymal stem cells (MSCs) to osteogenic differentiation, since calvarial cells derived from the Cbfa-1–/– animals failed to differentiate into osteoblasts even under the influence of bone morphogenetic protein (BMP)-2, a strong osteoblastic differentiation inducer [6, 7]. Not surprisingly, regulation of RUNX expression was proposed to have the potential for designing novel therapies for bone formation deficit [8]. In this respect, adenovirus-mediated Cbfa-1 transfer has been shown to enhance the osteogenic activity of mouse bone marrow stromal cells both in vitro and in vivo [9]. Meanwhile, a potent induction of the early chondrocyte differentiation markers in mouse embryonic mesenchymal cells by Cbfa-2 overexpression has been reported, whereas its knockdown resulted in a downregulation of type II collagen, alkaline phosphatase, and Cbfa-1 [10]. Interestingly, efficient chondrogenic differentiation of mouse mesenchymal cells in micromass culture by retroviral transfer of BMP-2 has also been documented [11]. The β subunit of CBFs, denoted as Cbfb/PEBP2β, induces a conformational change of the Cbfas and increases their DNA-binding affinity by forming heterodimers with them [12]. Cbfb is required not only for the emergence of hematopoietic stem cells [13] and normal differentiation of lymphoid as well as myeloid lineage cells [14], but also for bone formation, since homozygous deficiency of this gene leads to ossification defects in mice [15, 16]. However, the precise role of Cbfb, other than increasing the stability of Cbfas [17, 18], in osteogenesis remains unclear. To examine whether Cbfb stimulates osteogenesis by itself or it simply facilitates the effects of Cbfa-1, both a mouse MSC-like cell line C3H10T1/2 [19–22] and human bone marrow-derived MSCs (hMSCs) [23–26] were used. After being infected separately or simultaneously with recombinant adenoviruses carrying Cbfa-1 or Cbfb, osteogenic differentiation of these cells was examined. As expected, upregulation of Cbfa-1 not only increased the alkaline phosphatase (ALP) activity in both C3H10T1/2 and hMSCs, but it also stimulated the expression of osteocalcin, another osteoblast marker, in murine MSCs. Furthermore, osteogenic differentiation of both types of MSCs induced by Cbfa-1 was dramatically enhanced by Cbfb coexpression, even though this factor failed to induce osteogenesis by itself. More interestingly, we found that Cbfb markedly prolonged the half-life of Cbfa-1 by reducing its ubiquitination. Hence, in addition to enhancing the target recognition of Cbfa-1, Cbfb may facilitate its osteogenic differentiation-inducing effect by increasing the stability of this transcription factor. Taken together, our results not only clarify the role of Cbfb in osteoblastic differentiation of MSCs, but they also demonstrate the potential of combining these factors in MSC-based therapy of bone diseases.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
Murine C3H10T1/2 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel, http://www.bioind.com), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 mg/ml amphotericin B (PSA; Biological Industries). Human MSCs isolated from bone marrow aspirates were grown in Iscove's modified Dulbecco's medium (Biological Industries) supplemented with 10% FBS, PSA, 10 µg/ml epidermal growth factor (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), and 25 µg/ml basic fibroblast growth factor (R&D Systems, Inc.), as previously described [23–26]. Ad293 human kidney epithelial cells and MG63 human osteosarcoma cells were also grown in DMEM supplemented with 10% FBS and PSA.

Construction of the Recombinant Adenoviral Genomes
The Cbfa-1 and Cbfb fragments obtained respectively from plasmids pEF-Bos-PEBP2{alpha}A (provided by Dr. Yoshiaki Ito, National University of Singapore) and pCMV-SPORT6-Cbfb (obtained from the Genome Research Center of National Yang Ming University) were inserted into the pShuttle-CMV (AdEasy XL Adenoviral Vector System; Stratagene, La Jolla, CA, http://www.stratagene.com) and pShuttle-CMV-HA, respectively, to obtain pShuttle-CMV-Cbfa-1 and pShuttle-CMV-HACbfb. Above plasmids, after being linearized by PmeI (New England BioLabs Inc., Ipswich, MA, http://www.neb.com), were transformed into the BJ5183 cells, and successful recombination was verified by PacI (New England BioLabs Inc.) digestion.

Production and Infection of the Recombinant Adenoviruses
The recombinant adenoviral genomes carrying either Cbfa-1 or HACbfb linearized by PacI were transfected into Ad293 cells by Lipofectamine (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Forty-eight hours later, cells were harvested and disrupted by repeated freezing-thawing, and the viruses in cytosol were collected and further amplified in Ad293 cells. To prepare a large amount of pure viruses, cesium chloride (0.55 g/ml) was added into the cytosol pooled from 10 150-mm dishes of infected cells, and this solution was then subjected to ultracentrifugation at 32,000g for 18 hours. Virus particles were collected and stored at –70°C until use. Virus's titer was determined by infecting Ad293 cells with serial logarithmic dilutions of viruses, and the endpoint titer (multiplicity of infection [MOI] = 1) was defined as the highest dilution that caused an apparent cytopathic effect 2 days after infection. The optimal titers of virus for transducing C3H10T1/2 cells and human MSCs, respectively, were determined by measuring the numbers of cells stained positively by 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal; Sigma-Aldrich) 48 hours after being infected with different amounts of viruses carrying a lacZ gene (AdLacZ).

Alkaline Phosphatase Activity Assay
C3H10T1/2 cells (1 x 105) and hMSCs (5 x 104) were plated respectively onto 100-mm dishes 1 day before infection. Cells were harvested 4, 7, and 14 days postinfection, and total lysate was prepared using a RIPA lysis buffer supplemented with a protease inhibitor cocktail (Calbiochem, San Diego, http://www.emdbiosciences.com). Protein concentration of the lysates was determined by a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, http://www.bio-rad.com), and alkaline phosphatase activity was then analyzed by incubating the lysates (25 and 15 µg for C3H10T1/2 cells and hMSCs, respectively) with p-nitrophenylphosphate (4.5 mg/ml) at 37°C for 2 hours before the OD405 was measured by a spectrophotometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real-Time RT-PCR
C3H10T1/2 cells were seeded at a density of 5 x 105 cells per 100-mm dish 1 day before infection. Total RNAs were extracted from C3H10T1/2 cells 4, 7, and 14 days postinfection, and cDNA was synthesized using a reverse transcriptase (Fermentas Life Sciences, Hanover, MD, http://www.fermentas.com). PCR amplification was subsequently performed with the following primer sets: for Cbfa-1, sense: 5'-ATGCGTATTCCTGTAGATC-3' and antisense: 5'-AGATATGGAGTGCTGCT G-3'; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense: 5'-GACCAC AGTCCATGCCATCAC-3' and antisense: 5'-TCCACCACCCTGTTG CTGTAG-3'. The real-time PCR amplification was conducted with the following primer sets: for alkaline phosphatase, sense: 5'-AGTTACTGGCGACAGCAAGC-3' and antisense: 5'-GAGTGGTGTTGCATCGCG-3'; for osteocalcin, sense: 5'-GAACAGACAAGTC CCACACAG-3' and antisense: 5'-GAGCTGCTGTGACATCCATAC-3'; for GAPDH, sense: 5'-GACCACAGTCCATGCCATCAC-3' and antisense: 5'-TCCAC CACCCTGTTGCTGTAG-3'. All PCR was carried out with an initial denaturation at 95°C for 5 minutes followed by 25 cycles at 95°C for 1 minute, 55°C for 40 seconds, 72°C for 1 minute, and a final extension at 72°C for 10 minutes.

Western Blotting
C3H10T1/2 and MG63 cells were harvested 4 days postinfection, and total lysate was prepared using a RIPA buffer as described in previous section. Forty µg of proteins from each sample were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto nitrocellulose membranes and then probed with either a goat anti-human Cbfa-1 antibody (1:1,000, R&D Systems Inc.) or a rabbit anti-hemagglutinin (HA) antibody (1:3,000, for detecting the HA-tagged Cbfb; Viogene, Taipei, Taiwan, http://www.viogene.com) at 4°C overnight and followed by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat IgG (1:5,000; Sigma-Aldrich) and an HRP-conjugated anti-rabbit IgG (1:3,000; GE Healthcare U.K. Limited, Bucks, U.K., http://www.gehealthcare.com), respectively. Protein visualization was achieved by enhanced chemiluminescence (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) according to the manufacturer's protocol.

Protein Stability Assay
Four days after virus infection, C3H10T1/2 cells were treated with 50 µM cycloheximide (Sigma-Aldrich) for 2, 4, 6, 8, and 12 hours before being harvested for total lysate preparation. Western blotting was then performed as described above, and the half-life of Cbfa-1 was estimated by quantifying the amount of this protein (i.e., its signal intensity on the x-ray films) by a densitometer (Molecular Dynamics, Sunnyvale, CA, http://www.ump.com/mdynamic.html).

Immunoprecipitation-Western Blotting
C3H10T1/2 cells 4 days postinfection were harvested after being treated with 100 µg/ml MG132 for 4 hours. Two hundred µg of total lysates precleared by incubating with G/A beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) from each sample were incubated with 20 µg G/A beads conjugated previously by the anti-Cbfa-1 (3 µg; Santa Cruz) and anti-actin (3 µg; Santa Cruz) antibodies, respectively, at 4°C for 4 hours. Western blotting was then performed as above described using an anti-ubiquitin antibody (1:1,000; Sigma-Aldrich) as a probe to detect the ubiquitination of Cbfa-1.

Von Kossa Staining
Twenty-one days postinfection, human MSCs were washed twice with phosphate-buffered saline (PBS) and fixed in 4% formaldehyde at room temperature for 20 minutes. After 3 PBS washes, cells were treated with 0.1% Triton X-100 at room temperature for 10 minutes, followed by several PBS washes. UV irradiation was then applied on cells incubated in 1% silver nitrate solution for 45 minutes. After PBS washes, cells were incubated in 5% sodium thiosulfate for 5 minutes before being washed again by PBS. Finally, cells were treated with the Van Gieson solution (1% acid fuchsin in saturated picric acid) for 5 minutes before being washed with 100% ethanol at –20°C for 2 minutes. The calcium accumulation was indicated by dark color.

Mineralization Staining
Human MSCs were processed 14 days postinfection as follows. After being washed twice with PBS, cells were fixed in 10% neutral formaldehyde buffer (10% formaldehyde, 16 g/l Na2HPO4, 4 g/l Na2HPO4.H2O) at room temperature for 20 minutes. Cells were then washed with distilled water three times and stained with 2.5% AgNO3 at room temperature for 30 minutes followed by distilled water washing three times. Finally, cells were incubated with a sodium carbonate formaldehyde solution (25% formaldehyde, 50 mg/ml Na2CO3) for 1 minute before being washed with distilled water three times. The brownish cells represented the ones that underwent mineralization.

Statistical Analysis
Arithmetic means and standard deviations were calculated for our triplicate data, and statistical significance was defined as p < .05 using Student's t test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Osteogenic Differentiation of C3H10T1/2 Cells Under the Influence of Cbfb Alone
The optimal viral titers for transducing C3H10T1/2 murine mesenchymal stem cells and human bone marrow-derived MSCs were first determined by infecting them with different amounts of the viruses carrying a lacZ gene (AdLacZ), and X-Gal-positive (blue) cells were counted under a microscope 48 hours postinfection. As can be seen, more than 80% of C3H10T1/2 cells (Fig. 1A, left) and human MSCs (Fig. 1A, right) were bluish after being infected with AdLacZ at MOI of 800 and 300, respectively. To examine whether Cbfb and Cbfa-1 were expressed in cells infected with the recombinant adenoviruses carrying their corresponding genes, C3H10T1/2 cells were transduced with increasing titers of each virus, and the levels of Cbfb and Cbfa-1 were analyzed by Western blotting. As shown in Figure 1B, the expression levels of Cbfb and Cbfa-1 were indeed proportional to the amount of viruses infected. Alkaline phosphatase activity was then measured to assess the osteogenic differentiation of C3H10T1/2 cells induced by each factor. In agreement with several earlier reports [2–7], ALP expression in these cells was induced by Cbfa-1 in a dose- and time-dependent manner. By contrast, no stimulation of ALP activity was found in C3H10T1/2 cells transduced by Cbfb (Fig. 1C).


Figure 1
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  		Figure 1. Osteogenic differentiation of C3H10T1/2 cells under the influence of Cbfb alone. (A): C3H10T1/2 cells (left) and human MSCs (right) were infected with different titers of AdLacZ. Two days later, cells were fixed with 4% paraformaldehyde, and the β-galactosidase activities were determined by 5-bromo-4-chloro-3-indolyl-β-D-galactoside (1 mg/ml) staining for 4 hours. (B): Forty µg of total lysates prepared from C3H10T1/2 cells 4 days postinfection were subjected to Western blot analysis using rabbit anti-HA and goat anti-human Cbfa-1 antibodies as probes, respectively. (C): ALP activity was determined by incubating a mixture containing 25 µg of total lysates prepared 4, 7, and 14 days postinfection and p-nitrophenylphosphate (10 mM) at 37°C for 2 hours before OD405 was measured. *, p < .01 in comparison with that of the AdLacZ-infected cells by Student's t test. Abbreviations: ALP, alkaline phosphatase; hMSC, human bone marrow-derived MSC; MOI, multiplicity of infection.

 
Osteogenic Differentiation of C3H10T1/2 Cells Induced by Cbfa-1 Plus Cbfb
To examine whether Cbfb coexpression could facilitate the osteogenic differentiation of C3H10T1/2 cells induced by Cbfa-1, ALP activity was determined after they were coinfected with AdCbfa-1 and AdHA-Cbfb for 14 days. As can be seen in Figure 2A, upon being coinfected with increasing amounts of AdHA-Cbfb, dramatic increases of ALP activity in these cells were observed, which was associated with a dose-dependent increase in its mRNA (Fig. 2B). Moreover, a Cbfb dose-dependent upregulation of the gene encoding osteocalcin (OCN), another osteoblast marker, in C3H10T1/2 cells 14 days postinfection was also detected (Fig. 2C).


Figure 2
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  		Figure 2. Osteogenic differentiation of C3H10T1/2 cells induced by Cbfa-1 plus Cbfb. (A): Cells were infected with different combinations of AdLacZ, AdCbfa-1, and AdHACbfb, simultaneously. ALP activity in cells 14 days postinfection was determined. *, p < .01 when compared with that of the cells infected with both AdCbfa-1 and AdLacZ by Student's t test. Total RNAs were isolated from cells 14 days postinfection, and the expression of ALP (B) and osteocalcin (C) was analyzed by real-time reverse transcription-polymerase chain reaction. Abbreviations: ALP, alkaline phosphatase; MOI, multiplicity of infection; ND, nondetectable.

 
Stability and Ubiquitination of Cbfa-1 in the Absence and Presence of Cbfb
Since osteogenic differentiation of C3H10T1/2 cells induced by Cbfa-1 was notably enhanced by Cbfb, the underlying mechanism was next elucidated. We first examined whether the amount of Cbfa-1 protein was increased by Cbfb using Western blot analysis. Indeed, a Cbfb dose-dependent increase in Cbfa-1 was detected (Fig. 3A). However, no parallel increase in its RNA could be found (Fig. 3B). To examine whether Cbfb upregulates the expression of an endogenous Cbfa-1, Western blotting was also performed using total lysates prepared from MG63 human osteosarcoma cells infected with AdHACbfb. To no surprise, Cbfa-1 levels in these cells were also increased by Cbfb in a dose-dependent manner (Fig. 3C). Since Cbfb has been reported to increase the stability of all three Cbfas [17], we measured the half-life of Cbfa-1 in the presence and absence of Cbfb. As can be seen in Figure 4, Cbfb markedly prolonged the half-life of Cbfa-1. To examine whether Cbfa-1 is degraded by a proteasomal pathway, we incubated the AdCbfa-1-transduced C3H10T1/2 cells with MG132, a proteasome inhibitor, for 6 hours before the levels of this factor were determined. Indeed, MG132 treatment increased Cbfa-1 levels (Fig. 5A). To examine whether Cbfb increases the stability of Cbfa-1 by preventing it from ubiquitin-mediated proteasomal degradation, immunoprecipitation-Western blot analysis was performed as follows. C3H10T1/2 cells were coinfected with or without AdHACbfb for 4 days, and MG132 was added 2 hours before cell harvesting. Total lysates prepared from these cells were immunoprecipitated by an anti-Cbfa-1 and an anti-actin (as a negative control) antibody, respectively. The precipitates were then subjected to Western blot analysis using an anti-ubiquitin antibody as a probe. As can be seen in Figure 5B, ubiquitination of Cbfa-1 in mouse MSCs was drastically diminished when Cbfb was coexpressed.


Figure 3
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  		Figure 3. Examination of the protein and mRNA levels of Cbfa-1 under the influence of Cbfb. C3H10T1/2 cells were infected with different combinations of AdLacZ, AdCbfa-1, and AdHA-Cbfb, simultaneously. (A): Forty µg of total lysates prepared 4 days postinfection were subjected to Western blot analysis using a goat anti-human Cbfa-1 antibody as a probe. (B): RNA levels of Cbfa-1 in cells 4 days postinfection were analyzed by reverse transcription-polymerase chain reaction. (C): Cbfa-1 protein levels in MG63 human osteosarcoma cells infected by different amounts of AdHACbfab were analyzed by Western blotting, and the values shown below were measured by densitometry. Abbreviations: Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOI, multiplicity of infection.

 


Figure 4
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  		Figure 4. The stability of Cbfa-1 under the influence of Cbfb. C3H10T1/2 cells were infected either with AdCbfa-1 (multiplicity of infection [MOI] 800) plus AdLacZ (MOI 800) or AdCbfa-1 (MOI 800) plus AdHACbfb (MOI 800) for 4 days. Cells were then treated with 100 µg/ml cycloheximide for 0, 2, 4, 6, 8, and 12 hours before being harvested for total cell lysate preparation. (A): Fifty µg of total lysates were subjected to Western blot analysis using a goat anti-human Cbfa-1 antibody as a probe. (B): Protein levels of Cbfa-1 were quantified by densitometry (mean ± SD from three independent experiments). Abbreviations: CHX, cycloheximide; hr, hours; L, infected only by AdLacZ.

 


Figure 5
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  		Figure 5. The ubiquitination of Cbfa-1 in the absence and presence of Cbfb. (A): C3H10T1/2 cells were infected either with AdCbfa-1 (MOI 800) plus AdLacZ (MOI 800) or AdCbfa-1 (MOI 800) plus AdHACbfb (MOI 800) for 4 days. One sample infected by AdCbfa-1 plus AdLacZ was treated with 100 µg/ml MG132 for 6 hours before being harvested for total lysate preparation. Forty µg of proteins were then subjected to Western blot analysis using a goat anti-human Cbfa-1 antibody as a probe. (B): Total lysates (200 µg) prepared from C3H10T1/2 cells treated as described above were immunoprecipitated respectively with anti-Cbfa-1 (right) and anti-actin (left) antibodies, and the precipitated proteins were subjected to Western blot analysis using a mouse anti-ubiquitin antibody as a probe. Abbreviations: IP, immunoprecipitate; MOI, multiplicity of infection.

 
Osteogenic Differentiation of Human Mesenchymal Stem Cells Under the Influence of Cbfa-1 Plus Cbfb
To examine whether Cbfb could facilitate the osteogenesis of hMSCs, cells were infected by AdCbfa-1 plus AdLacZ or plus AdHACbfb, and their ALP activities were determined 7 days postinfection. In good agreement with murine MSCs, ALP activity in human cells cotransduced with the Cbfa-1 and Cbfb was twofold higher than that only expressing the former (Fig. 6A). Although a typical osteoblastic morphology was observed in human MSCs transduced by Cbfa-1 alone or together with Cbfb (Fig. 6B, top panels), higher levels of calcium accumulation (Fig. 6B, middle panels) as well as mineralization (Fig. 6B, bottom panels) were found in cells coexpressing these genes.


Figure 6
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  		Figure 6. Osteogenic differentiation of human mesenchymal stem cells under the influence of Cbfa-1 plus Cbfb. Human mesenchymal stem cells were infected with different combinations of AdLacZ, AdCbfa-1, and AdHA-Cbfb, simultaneously. (A): ALP activity in cells 7 days postinfection was assessed using 15 µg of total lysates. #, p < .01 when compared with that of the AdLacZ-infected cells, Student's t test. *, p < .01 when compared with that of the AdCbfa-1 plus AdLacZ-infected cells by a similar method. (B): Morphological changes (top), calcium accumulation determined by Von Kossa staining (middle), and mineralization determined by alizarin red S staining (bottom) of MSCs induced by AdCbfa-1 and AdCbfa-1 plus AdCbfb. Abbreviations: ALP, alkaline phosphatase; Ctrl, control; MOI, multiplicity of infection.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Both Cbfa-1 and Cbfb appear to be crucial for bone formation [5–7, 15–16], and the former, when overexpressed, could stimulate the in vitro osteogenic differentiation of skin fibroblasts [28] and mesenchymal stem cells [29, 30]. However, the precise role of the latter in osteogenesis of MSCs has as yet been delineated. In this study, we found that Cbfb, when introduced alone into both murine and human MSCs, failed to induce their osteogenic differentiation, but transduction of Cbfa-1 triggered a dramatic increase of alkaline phosphatase activity in these cells (Figs. 1, 6). Distinct functions of Cbfa-1 and Cbfb might be explained by the fact that the former is a bona fide transcription factor capable of activating its target genes by itself, whereas the latter, residing primarily in the cytoplasm, only facilitates the target recognition of Cbfa-1 by dimerizing with it [31]. Interestingly, we found a dose-dependent enhancement of Cbfa-1-induced ALP expression by Cbfb in mouse MSCs (Fig. 3A), suggesting that Cbfb is important for the optimal (or even maximal) osteogenic activity of Cbfa-1. Although the combination of Cbfa-1 and BMP-2 has been shown to induce a higher ALP expression in C3H10T1/2 cells [29], we attribute this to a suboptimal osteogenesis-inducing effect of each factor, because the osteoblastic differentiation of C2C12 mesenchymal precursor cells stimulated by BMP-2 was mediated mainly by Cbfa-1 [32].

In addition to facilitating the DNA binding of different Cbfas by forming heterodimers with them [33, 34], Cbfb has been shown to reduce their turnover rates by protecting them from the ubiquitin-mediated proteasomal degradation [17]. Therefore, it is not too surprising to find a dose-dependent increase in the protein levels of Cbfa-1, regardless of whether it was encoded by a transduced (Fig. 3A) or an endogenous (Fig. 3C) gene, by Cbfb without a concomitant increase in their RNAs (Fig. 3B). Accordingly, the half-life of Cbfa-1 in mouse MSCs was also dramatically prolonged (from approximately 2.5 hours to 7 hours, Fig. 4B) by Cbfb, suggesting a marked increase in its stability. This speculation was further supported by our immunoprecipitation Western blot analysis, which showed a significant reduction in ubiquitinated Cbfa-1 when Cbfb was coexpressed in mouse MSCs (Fig. 5B). Not surprisingly, a Cbfb-mediated decrease of Cbfa-1 ubiquitination was also found in human MSCs (data not shown). Prevention of Cbfa-1 ubiquitination by Cbfb might be explained by the fact that the latter, by binding to the Runt domain of the former, interferes with ubiquitination of the five lysine residues (126, 133, 168, 187, and 210) it contains. Since the lysine residues in the Runt domain of Cbfa-2 have previously been reported to be critical for its turnover [17], by hiding the similar lysine residues in Cbfa-1 from Smuf1, the ubiquitin E3 ligase responsible for its modification [35–38], either through a physical blockade or an induced conformational change, Cbfb could prevent the proteasomal degradation of Cbfa-1. On the other hand, four lysine residues (225, 230, 350, and 351) outside the runt domain of Cbfa-1 have recently been shown to be crucial for its turnover mediated by Smuf1 [37]. Although a direct interaction between Cbfa-1 and Smurf1 is still under debate [37, 39], interfering with the complex formation between these proteins by Cbfb is by far the best explanation for its protection on Cbfa-1, which is supported by our unpublished observation. Finally, a positive effect of Cbfb on Cbfa-1-induced osteogenic differentiation was also found in human MSCs, suggesting that the cooperation between these factors is evolutionarily conserved.

Previous studies have suggested that bone marrow-derived MSCs may be good materials for bone regeneration therapy [40, 41] because of their high osteogenic differentiation potential, which can be stimulated further by various factors, especially the bone morphogenetic proteins. Accordingly, recombinant human BMP-2 and -7 have been approved by the U.S. Food and Drug Administration for certain clinical applications [42]. In the meantime, BMP-based cell therapies have been extensively studied in various animal models to evaluate their clinical utilities [43–48]. However, potential side effects of BMP-overproducing cells should be carefully considered, because they are secreted molecules capable of triggering paracrine and endocrine responses. For instance, BMPs may suppress both folliculogenesis and ovulation by inhibiting progesterone production and/or ovulation rate [49, 50]. BMP-4 and -6 may compromise certain antihypertensive therapies [51]. Another major concern for the clinical usage of BMPs and/or cells overexpressing these cytokines is their potential tumorigenic effects. Indeed, BMP-2 has been reported to induce not only tumor angiogenesis [52] but also transformation and in vivo growth of lung cancer cells [53, 54]. More recently, the BMP/SMAD pathway was found to be activated in breast and prostate cancers, which may play a role in their malignant progression [55, 56]. By contrast, transplantation of MSCs coexpressing Cbfa-1 and Cbfb, two nonsecreted osteogenic-promoting factors, may be much safer, even though upregulation of the former has been postulated to be responsible for the malignant progression of several types of cancer [57–59]. Although further studies are necessary to fully elucidate the protective effect(s) of Cbfb on Cbfa-1, our present results suggest that Cbfb plays a crucial role in stabilizing Cbfa-1 by reducing the ubiquitination, hence facilitating its osteogenic activity; these results also raise the possibility of transplanting the Cbfa-1 and Cbfb coexpressing MSCs to achieve an efficient bone repair (regeneration) therapy with little (or no) adverse effects.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by Grants C1-93-8 and CI-94-7 from the Yen Tjing Ling Medical Foundation (to YS), from Taipei Veterans General Hospital (Grant number 94-365-13, to OKL), and from the National Science Council (Grant numbers NSC95-2314-B-075-014-MY2 and 95-2475-B-002-MY3, to OKL).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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