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Molecular and Cellular Biology, March 2005, p. 1971-1979, Vol. 25, No. 5
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.5.1971-1979.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Departments of Biochemistry,1 Removable Prothodontics, Graduate School of Dentistry, Osaka University, Osaka,2 Department of Oral Pathology, Dental School, Graduate School of Medicine, Nagasaki University, Nagasaki, Japan3
Received 16 July 2004/ Returned for modification 9 September 2004/ Accepted 1 December 2004
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ABSTRACT |
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INTRODUCTION |
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) (37). A recent study showed that PPAR insufficiency enhances osteogenesis by stimulating osteoblast differentiation of bone marrow progenitors (2). Consistently, the introduction of PPAR2 or activation of PPAR by treatment with its ligand inhibits Runx2 expression and osteoblast differentiation (25). Moreover, impairment of Runx2 expression increases adipocyte differentiation (16, 23). These studies suggest that the relative expression level of Runx2 and PPAR is involved in the regulation of balance between osteoblastogenesis and adipogenesis.
C/EBPß, which belongs to the leucine zipper family of transcription factors, forms a homodimer or heterodimer complex with other C/EBP family members (11). Overexpression of C/EBPß in mesenchymal cells induces PPAR expression and adipocyte differentiation in cooperation with C/EBP
(43). C/EBPß-deficient mice manifest the reduction in adipogenesis (41). These biochemical and genetic studies indicate that C/EBPß plays contributive roles in the early stages of adipogenesis by regulating transcription of the genes necessary for adipocytic differentiation, such as the PPAR gene (11, 41). Interestingly, it has been described that C/EBPß is expressed in the osteoblastic cell lineages and up-regulated during osteoblast differentiation (3, 17, 32, 35). Gutierrez et al. also showed that C/EBPß and C/EBP regulate the osteocalcin gene promoter through physical interaction with Runx2 (17). Collectively, these studies suggest the importance of C/EBPß during osteoblast differentiation. In contrast, it has been reported that overexpression of C/EBPß stimulates the proliferation of a mouse osteoblast cell line, MC3T3-E1, but suppresses its osteoblast differentiation (21). Therefore, it remains unclear whether C/EBPß is implicated in osteoblast differentiation of mesenchymal cells.
An isoform of C/EBPß, liver-enriched inhibitory protein (LIP), which is translated from the same mRNA of C/EBPß (12), has been shown to play a unique role in several tissues (4, 6, 15, 39). Since LIP lacks a transcriptional activation domain, it usually functions as a transcriptional repressor (12). Likewise, LIP markedly inhibits the transcriptional function of C/EBPß, thereby suppressing adipocyte differentiation (8). Consistently, LIP expression is negatively regulated during the adipocyte differentiation of mesenchymal cells (42). Therefore, it is most likely that the ration of C/EBPß-LIP is a critical factor for biological phenomena that are controlled by C/EBPß.
To understand the molecular basis that regulates the balance between osteoblastogenesis and adipogenesis, we investigated the role of C/EBPß and LIP in osteoblast differentiation and adipocyte differentiation. In the present study, we found that C/EBPß promotes osteoblast differentiation of mesenchymal cells in Runx2-dependent and -independent mechanisms. Moreover, our results indicate that LIP functions as a coactivator for Runx2 and stimulates osteoblastic differentiation of mesenchymal cells. Thus, these findings provide support for the notion that LIP is an important key regulator that determines the commitment of osteoblasts and adipocytes.
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MATERIALS AND METHODS |
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Isolation of primary osteoblasts and mesenchymal cells. The calvaria were isolated from 2- or 3-day-old neonatal mice and digested with 0.1% collagenase and 0.2% dispase for 7 min at 37°C, and then the cells collected by centrifugation were used as the primary mesenchymal cells. These cells contained few alkaline phosphatase (ALP)-positive cells. The digested calvaria were sequentially digested four times with 0.1% collagenase and 0.2% dispase for 7 min at 37°C, and the last three fractionated cells were collected and used as the primary osteoblasts.
Constructs and transfection.
C/EBPß and C/EBP (41) cDNA were kindly given by Shizuo Akira. To generate the LIP construct, a PCR product which contains the C-terminal domain of C/EBPß (amino acids 152 through 296) was synthesized and then subcloned into pcDNA3 (Invitrogen) tagged with a Flag or Myc epitope in the N terminus. Deletion mutants of LIP, LIP(
73-145), and LIP(1-70) were generated by subcloning the corresponding PCR products into pcDNA3 tagged with a Myc epitope in the N terminus. The sequences of the constructs were confirmed by DNA sequence analysis. Runx2/Cbfa1 (22) and Cbfß (5) cDNA were kindly given by Yoshiaki Ito and Paul Liu, respectively. Dominant-negative mutant Runx2 was constructed as previously described (29). The Flag-tagged Runx2 mutant constructs Runx2(242-513) and Runx2(1-207) were generated by subcloning the corresponding PCR products into pcDNA3 tagged with a Flag epitope in the N terminus. The sequences of the constructs were confirmed by DNA sequence analysis. Transfection of C3H10T1/2 cells was carried out using FuGENE6 (Roche) according to the manufacturer's protocol.
Generation of adenovirus. The recombinant adenovirus carrying a wild-type or mutant form of C/EBPß or Runx2 was constructed by homologous recombination between the expression cosmid cassette (pAxCAwt) and the parental virus genome in 293 cells (RIKEN cell bank) by using an adenovirus construction kit (Takara) as previously described (19). The viruses showed no proliferative activity due to a lack of E1A-E1B (26). Titers of the viruses were determined by a modified point assay (26). Infection of C3H10T1/2, C2C12, or ST2 cells, primary osteoblasts, or mesenchymal cells with recombinant adenoviruses was performed by incubation with adenoviruses at a multiplicity of infection (MOI) of 50 except where specifically indicated.
Immunoprecipitation and Western blotting. The cells were washed three times with ice-cold phosphate-buffered saline (PBS) and solubilized in lysis buffer (20 mM HEPES [pH 7.4], 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.2 mM sodium orthovanadate) (19). The lysates were centrifuged for 15 min at 4°C at 16,000 x g and incubated with antibodies for 4 h at 4°C, followed by immunoprecipitation with protein A-Sepharose (Zymed) or protein G-agarose (Roche). Immunoprecipitates were washed five times with lysis buffer and boiled in sodium dodecyl sulfate (SDS) sample buffer, and supernatants were recovered as immunoprecipitate samples. These samples were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, immunoblotted with corresponding antibodies, and visualized with horseradish peroxidase coupled to protein A (KPL) or horseradish peroxidase coupled to anti-mouse immunoglobulin G (IgG) antibodies (Cappel) with an enhancement by use of ECL detection kits (Amersham).
Immunocytochemistry. Cultured cells were washed three times with ice-cold PBS and fixed with 3.7% paraformaldehyde-PBS for 20 min. After a 20-min incubation with 0.1% Triton X-100-PBS, the cells were blocked with PBS containing 1% bovine serum albumin for 2 h, incubated with anti-C/EBPß polyclonal antibodies in 1% bovine serum albumin-PBS, washed six times with PBS, and incubated with fluorescein isothiocyanate-conjugated affinity-purified anti-rabbit IgG antibodies (Jackson Laboratory). The cells were washed extensively with PBS and visualized with a fluorescence microscope (Zeiss).
Luciferase assay. A total of 1.3 kb of the mouse osteocalcin gene promoter, which contains the OSE2 site (positions –137 to –131), was used as described previously (13). A mouse osteocalcin gene promoter construct which lacks a putative C/EBP binding element (positions –454 to –446) was generated by subcloning the corresponding PCR products. The osteocalcin gene promoter fused to firefly luciferase was cotransfected with the Runx2 and Cbfß expression vector and a thymidine kinase (TK)-renilla luciferase construct (Promega) into C3H10T1/2 cells. Two days after transfection, the cells were lysed and luciferase activity was determined by using specific substrates in a luminometer (Promega) according to the manufacturer's protocol. Transfection efficiency was normalized by determining the activity of renilla luciferase.
Reverse transcriptase-PCR (RT-PCR). After denaturation of total RNA at 70°C for 10 min, cDNA was synthesized with the oligo-dT primer and reverse transcriptase (Gibco). PCR amplification was performed by using the specific primers for mouse osteocalcin (sense primer, 5'-GACAAAGCCTTCATGTCCAAGC-3'; antisense primer, 5'-AAAGCCGAGCTGCCAGAGTTTG-3'), ALP (sense primer, 5'-GCTGATCATTCCCACGTTTT-3'; antisense primer, 5'-CTGGGCCTGGTAGTTGTTGT-3'), type IA collagen (sense primer, 5'-CCTGGTAAAGATGGTGCC-3'; antisense primer, 5'-CACCAGGTTCACCTTTCGCACC-3'), or Osterix (sense primer, 5'-GAAAGGAGGCACAAAGAAG-3'; antisense primer, 5'-CACCAAGGAGTAGGTGTGTT-3'). PCR products were loaded onto an agarose gel and stained with ethidium bromide. After the PCR products were subcloned into a TA cloning vector, DNA sequences of the PCR products were determined.
Oligonucleotide precipitation assay. Cells were lysed in lysis buffer (10 mM HEPES [pH 7.9], 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.5% NP-40, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml, 1 mM PMSF, 0.2 mM sodium orthovanadate). The lysates preincubated with streptavidin-agarose beads (Vector) were incubated with 1 mg of a biotinylated double-stranded oligonucleotide probe that contains three repeats of C/EBP binding element present in the osteocalcin gene promoter (sense primer, 5'-GATCGGACATTACTGAACACTACGGGACATTACTGAACACTCCCGGGACATTACTGAACACT-3'; antisense primer, 5'-GATCAGTGTTCAGTAATGTCCCGGGAGTGTTCAGTAATGTCCCGTAGTGTTCAGTAATGTCC-3') and 10 mg of poly(dI-dC) · poly(dI-dC) for 16 h. DNA-bound proteins were collected with streptavidin-agarose beads for 1 h, washed with the lysis buffer, separated on an SDS-polyacrylamide gel, and identified by Western blotting.
Chromatin immunoprecipitation analysis. C3H10T1/2 cells were cross-linked with 1% formaldehyde for 15 min at room temperature and washed three times with ice-cold PBS containing 1 mM PMSF and 1 µg of aprotinin/ml. The cells were collected by scraping and centrifugation and lysed in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8], 1 mM PMSF, 1 µg of aprotinin/ml). The lysates were vortexed and sonicated with a Bioruptor (Cosmo Bio). The average length of DNA fragments ranged between 300 and 800 bp. The lysates were then clarified by centrifugation and diluted fivefold in chromatin immunoprecipitation buffer (15 mM Tris [pH 8], 1% Triton X-100, 0.01% SDS, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, 1 µg of aprotinin/ml, 1 µg of leupeptin/ml). The samples were precleared using protein G-Sepharose beads for 1 h at 4°C. Next, 0.1, 0.5, and 1% of each sample were used for input control. The samples were then immunoprecipitated with anti-Flag antibody or control IgG, and the immune complexes were washed with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8], 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8], 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8]), and TE buffer (10 mM Tris-Hcl [pH 8], 1 mM EDTA) and eluted with elution buffer (50 mM Tris-HCl [pH 8], 10 mM EDTA, 1% SDS). Immunoprecipitated DNA was reverse cross-linked at 65°C for 12 h and purified using a PCR purification kit (QIAGEN). A total of 5 µl of the purified DNA was subjected to PCR amplification using the specific primer for the C/EBP binding element present in the osteocalcin gene (sense primer, 5'-TGCCCTACAACCGGATCTTA-3'; antisense primer, 5'-AAACTGGGCTCCAACTCTCA-3').
Determination of ALP activity. ALP activity was determined as described previously (31). C3H10T1/2, C2C12, or ST2 cells were washed with PBS and lysed with 0.05% Triton X-100 solution. The ALP activities of the lysates were determined using p-nitrophenol-phosphate as a substrate. Protein contents of the lysates were measured by use of the Bradford protein assay reagent (Bio-Rad). For cytochemical analysis, cells were washed with PBS, fixed with 3.7% formaldehyde, and stained with a mixture of 330 µg of Nitro Blue Tetrazolium/ml, 165 µg of bromochloroindolyl phosphate/ml, 100 mM NaCl, 5 mM MgCl2, and 100 mM Tris (pH 9.5).
Oil red O staining. C3H10T1/2 cells were washed with PBS and fixed with 10% formalin for 20 min. After the cells were washed twice with PBS and once with 60% isopropyl alcohol, they were stained with Oil red O solution (Sigma). The area of the cells stained with Oil red O was measured with an Image Pro Plus analyzer (Palmerton, Inc.).
Determination of osteocalcin production. Osteocalcin production in the culture media was determined by using a mouse osteocalcin enzyme immunoassay kit (Biomedical Technologies, Inc.) according to the manufacturer's protocol.
Statistical analysis. All data were analyzed by analysis of variance, followed by a paired t test. Values are given as means ± standard deviations.
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RESULTS |
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(17). We therefore examined whether C/EBPß functionally collaborates with Runx2 in the regulation of osteoblast differentiation. As previously reported (14, 29), overexpression of Runx2 induces ALP activity (Fig. 3A) and osteocalcin, type IA collagen, and ALP expression in C3H10T1/2 cells (Fig. 2D). When C/EBPß and Runx2 were cooverexpressed in C3H10T1/2 cells, ALP activity was significantly increased (Fig. 3A). These data show that C/EBPß and Runx2 functionally collaborate in promotion of osteoblast differentiation of mesenchymal cells. To verify the cooperative role of C/EBPß and Runx2, we next performed a reporter assay using the osteocalcin gene promoter, which is directly regulated by Runx2 though the specific DNA binding element OSE2 (14). As described previously (17), overexpression of C/EBPß significantly increases osteocalcin gene promoter activity in the absence of Runx2 (Fig. 3B) and further enhances its activity in the presence of Runx2 (Fig. 3B). We have confirmed that C/EBPß overexpression does not induce Runx2 expression in C3H10T1/2 cells (Fig. 3C). Since there is a consensus sequence of C/EBP binding element in the osteocalcin gene promoter, we determined whether C/EBPß binds to the element using a biotinylated oligonucleotide probe. As shown in Fig. 3D, C/EBPß binds to the element. The deletion of the element suppressed C/EBPß-induced osteocalcin gene promoter activity, whereas the deletion did not affect Runx2-induced promoter activity (Fig. 3E). These data indicate that C/EBPß regulates the osteocalcin gene promoter through association with its DNA binding element. Furthermore, a coimmunoprecipitation experiment confirmed that C/EBPß physically associates with Runx2 (Fig. 3F), as shown previously (17). Together with the cooperative effects of C/EBPß and Runx2 on ALP activity, these results indicate that C/EBPß controls osteoblast differentiation of mesenchymal cells through a physical association with Runx2.
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Regulation of osteoblast and adipocyte commitment by LIP.
It has been shown that a naturally occurring isoform of C/EBPß, LIP, exhibits a dominant-negative effect on adipogenesis due to a lack of the transcriptional activation domain (18) (Fig. 4A). We observed that overexpression of LIP inhibits C/EBPß-induced adipocytic differentiation in C3H10T1/2 cells (Fig. 4B and C). Moreover, LIP overexpression also inhibits the PPAR gene promoter activity transactivated by C/EBPß and C/EBP (Fig. 4D). These data support the notion that LIP inhibits adipogenesis in a dominant-negative fashion. Notably, we found that LIP was expressed in primary osteoblasts (Fig. 1A) and induced in C3H10T1/2 cells in response to BMP2 treatment (Fig. 4E), suggesting that LIP plays a role in osteoblast differentiation. To define whether LIP affects osteoblast differentiation as well as adipocytic differentiation of mesenchymal cells, we examined the effects of LIP overexpression on osteoblast differentiation of C3H10T1/2 cells in the presence of BMP2 that concomitantly induced osteoblastic and adipocytic differentiation of C3H10T1/2 cells (1, 19). As expected, overexpression of LIP markedly inhibited adipogenesis induced by BMP2 (Fig. 4F). However, surprisingly, LIP overexpression enhanced the osteogenic action of BMP2 on osteoblast differentiation (Fig. 4G and H) without affecting Runx2 expression (Fig. 4I). Consistently, LIP increased BMP2-induced ALP activity in C2C12 cells (Fig. 4J). We found that overexpression of LIP did not induce osteocalcin, Runx2, and Osterix expression (Fig. 5A), indicating that LIP alone is unable to promote osteoblast differentiation. Since C/EBPß physically and functionally associates with Runx2 (Fig. 3F) (17), we next examined whether LIP has physical and functional interactions with Runx2. Although the effect of overexpression of LIP alone had no effects on ALP activity (Fig. 5B), ALP activity was dramatically increased when LIP was cooverexpressed with Runx2 (Fig. 5B). Furthermore, overexpression of LIP together with Runx2 also transactivated the osteocalcin gene promoter activity more than Runx2 alone (Fig. 5C). In addition, LIP also stimulated Runx2-dependent ALP activity in C2C12 cells (Fig. 5D). Coimmunoprecipitation experiments showed the physical interaction between LIP and Runx2 (Fig. 5E). To gain further insight into the molecular interaction between LIP and Runx2, we examined the association by using the deletion mutants of LIP or Runx2. As shown in Fig. 5F, the leucine zipper domain of LIP is responsible for the binding to Runx2. Figure 5G indicates that Runx2 associates with LIP through the C-terminal portion containing the transcriptional activation domain. To address whether LIP can directly bind to the osteocalcin gene, we performed an oligonucleotide precipitation assay and a chromatin immunoprecipitation experiment. As shown in Fig. 5H and I, LIP has the capacity to bind to the osteocalcin gene promoter. These data suggest that LIP functions as a coactivator for Runx2 during osteoblastogenesis. To explore the importance of LIP as a coactivator for Runx2, we examined the effects of a mutant of LIP, namely, LIP(1-70), which lacks both transcriptional activation and DNA binding domains (Fig. 4A). Because the mutant retains the ability to associate with Runx2 (Fig. 5F) and C3H10T1/2 cells slightly express C/EBPß (Fig. 1C), overexpression of the mutant profoundly inhibited Runx2-induced ALP activity (Fig. 5J) by inhibiting the interaction of C/EBPß with Runx2. These data suggest that LIP functions as a coactivator for Runx2 during osteoblast differentiation. To further understand the relationship between LIP and Runx2 during osteoblast differentiation, we next evaluated the effects of LIP on osteoblast differentiation by using Runx2-deficient mesenchymal cells. Consistent with the results shown in Fig. 5B and C, overexpression of LIP failed to induce osteoblast differentiation of Runx2-deficient mesenchymal cells (Fig. 5K), whereas overexpression of Runx2 or C/EBPß was able to promote osteoblast differentiation of Runx2-deficient mesenchymal cells (Fig. 3H). In contrast, when Runx2 was introduced into Runx2-deficient mesenchymal cells, LIP overexpression stimulated osteoblast differentiation (Fig. 5K). These data indicate that LIP, which lacks transcriptional activity, requires Runx2 to induce osteoblast differentiation of mesenchymal cells.
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DISCUSSION |
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during osteoblast and adipocyte differentiation (2, 16, 23). Recently, Cheng et al. (9) and Ichida et al. (20) indicated that a homeobox gene, the Msx2 gene, stimulates the commitment of mesenchymal cells into an osteoblast lineage in association with inhibition of adipogenesis. Because transgenic mice overexpressing the FosB transcription factor showed increases in bone formation and decreases in adipogenesis (36), it is likely that AP-1 transcription factors are also involved in the commitment of bone marrow mesenchymal cells. However, to date, investigation of the transcriptional events involved in the commitment of mesenchymal cells has not been accomplished. In the present study, we have shown that LIP preferentially stimulates osteoblast differentiation of undifferentiated mesenchymal cells but inhibits adipocyte differentiation. To support this, we found that expression of LIP is up-regulated during osteoblast differentiation. In contrast, Tang et al. demonstrated that LIP expression is down-regulated during adipocyte differentiation of 3T3-L1 cells (42). Previous studies (18, 45) and our results also indicate that LIP blocks the adipogenic function of C/EBPß in a dominant-negative mechanism. Thus, LIP is one of several key transcription factors that define the balance between osteoblastogenesis and adipogenesis in bone marrow. Since LIP is an isoform of C/EBPß that lacks a transcriptional activation domain, it how LIP stimulates osteoblastogenesis is an interesting question. We have demonstrated that LIP physically associates with Runx2 and binds to the DNA binding element present in the osteocalcin gene promoter which is one of the targets for Runx2. These biochemical experiments indicate that LIP functions as a coactivator for Runx2. Indeed, LIP enhances the transcriptional activity of Runx2 and its osteogenic action. Consistently, unlike C/EBPß, LIP was not able to promote osteoblast differentiation in Runx2-deficient cells. Collectively, LIP has dual functional roles in the regulation of osteoblastogenesis and adipogenesis.
The C/EBPß gene has only one exon, and both C/EBPß and LIP are transcribed from the common mRNA (12). Calkhoven et al. showed the molecular regulatory mechanisms of expression of C/EBPß isoforms in an adipogenic cell line, 3T3-L1, during adipogenesis (8). However, the regulation of LIP expression during osteoblast differentiation is presently unknown. Because we found that BMP2, one of most powerful osteogenic cytokines (44), induces LIP expression, investigation of the role of BMP2 signaling in LIP expression may solve this issue.
Large bodies of evidence indicate that Runx2 is an essential transcription factor for bone formation (13, 44). However, it has been suggested that other transcriptional regulators are cooperatively involved in the osteogenic action of Runx2 because the transcriptional activity of Runx2 itself is relatively weak (22). As described previously (17), we have also observed that C/EBPß physically interacts with Runx2. In addition, we showed that C/EBPß induces ALP activity and osteogenic markers as type IA collagen and osteocalcin. Furthermore, a mutant of C/EBPß, which lacks a DNA binding domain but retains the activity of binding to Runx2, markedly inhibited the osteogenic action of Runx2 presumably by competing with endogenous C/EBPß or its related family. Together with the results from a previous study (17), we show that C/EBPß is an important transcriptional partner for Runx2 during osteogenesis. In conflict with our data, an earlier study reported that osteoblast differentiation was inhibited in MC3T3-E1 cells stably transfected with C/EBPß (21). Although we cannot completely exclude the possibility that this discrepancy is due to a difference in the experimental model or culture condition, we believe that C/EBPß positively regulates osteoblastogenesis based on the following reasons: (i) overexpression of C/EBPß by using an adenovirus system, which is one of the most powerful, efficient, and reproducible technologies to introduce the exogenous genes into the variety type of cells without making a clonal difference, promoted osteoblast differentiation of ST2, C2C12, and primary mesenchymal cells as well as C3H10T1/2 cells; (ii) these effects of C/EBPß adenovirus are dose dependent (data not shown); (iii) C/EBPß physically interacts with Runx2 and enhances its transcriptional activity and osteogenic action; and (iv) in our hands, we did not observe the stimulative effects of C/EBPß on the proliferation of osteoblasts. Generation and analyses of appropriate transgenic mice or knockout mice as described below solve this issue.
Interestingly, our results, using a dominant-negative Runx2 mutant or Runx2-deficient mesenchymal cells, indicate that C/EBPß itself can induce osteoblast differentiation. Thus, C/EBPß regulates osteoblast development in Runx2-dependent and -independent mechanisms. However, bone abnormality in C/EBPß-deficient mice has not yet been reported (41). We have observed that C/EBP
and C/EBP are able to promote osteoblast differentiation of mesenchymal cells (R. Nishimura, K. Hata, and M. Ueda, unpublished data). Gutierrez et al. reported that C/EBP as well as C/EBPß activates the rat osteocalcin gene promoter (17). It is therefore likely that other C/EBP family members would compensate for C/EBPß deficiency during osteoblast differentiation. Since we have presently been generating transgenic mice in which the pan type of dominant-negative C/EBP mutant is specifically expressed in an osteoblast lineage, the analyses of the mouse model may allow further dissection of the roles of C/EBPß in bone development in vivo.
In conclusion, the identification of dual functional roles of LIP in osteoblast and adipocyte differentiation provides a novel insight into understanding the molecular mechanism of the commitment of mesenchymal stem cells in bone marrow and may allow us to develop new therapeutic agents for bone disease such as osteoporosis.
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ACKNOWLEDGMENTS |
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cDNA. This work was supported in part by Ministry of Education, Science, Sports and Culture Grants-in-Aid for Scientific Research A 11307041(T.Y.), B 15390560 (R.N.), and C 10671739 (R.N.) Support was also received from a Grant-in-Aid for Scientific Research on Priority Areas B 12137205 (T.Y) and from the Senri Life Science Foundation (R.N.) and The 21st Century COE Program (T.Y. and R.N.).
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FOOTNOTES |
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