Cbfa1 Isoforms Exert Functional Differences in Osteoblast Differentiation*

Hideyuki HaradaDagger , Shuzo TagashiraDagger , Masanori FujiwaraDagger , Shinji OgawaDagger , Takashi KatsumataDagger , Akira Yamaguchi§, Toshihisa Komori, and Masashi NakatsukaDagger parallel

From Dagger  Sumitomo Pharmaceuticals Research Center, Osaka 554-0022, the § Department of Oral Pathology, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8588, and the  Department of Medicine III, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan

    ABSTRACT
Top
Abstract
Introduction
References

Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation. We investigated functional differences among three isoforms of Cbfa1: Type I (originally reported as Pebp2alpha A by Ogawa et al. (Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6859-6863), Type II (originally reported as til-1 by Stewart et al. (Stewart, M., Terry, A., Hu, M., O'Hara, M., Blyth, K., Baxter, E., Cameron, E., Onions, D. E., and Neil, J. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8646-8651), and Type III (originally reported as Osf2/Cbfa1 by Ducy et al. (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, 747-754). A reverse transcriptase-polymerase chain reaction analysis demonstrated that these isoforms were expressed in adult mouse bones. The transient transfection of Type I or Type II Cbfa1 in a mouse fibroblastic cell line, C3H10T1/2, induced the expression of alkaline phosphatase (ALP) activity. This induction was synergistically enhanced by the co-introduction of Xenopus BMP-4 cDNA. In contrast, the transient transfection of Type III cDNA induced no ALP activity. In C3H10T1/2 cells stably transfected with each isoform of Cbfa1, the gene expression of ALP was also strongly induced in cells transfected with Type I and Type II Cbfa1 but not in cells with Type III Cbfa1. Osteocalcin, osteopontin,and type I collagen gene expressions were induced or up-regulated in all of the cells stably transfected with each isoform of Cbfa1, and Type II transfected cells exhibited the highest expression level of osteocalcin gene. A luciferase reporter gene assay using a 6XOSE2-SV40 promoter (6 tandem binding elements for Cbfa1 ligated in front of the SV40 promoter sequence), a mouse osteocalcin promoter, and a mouse osteopontin promoter revealed the differences in the transcriptional induction of target genes by each Cbfa1 isoform with or without its beta -subunit. These results suggest that all three of the Cbfa1 isoforms used in the present study are involved in the stimulatory action of osteoblast differentiation, but they exert different functions in the process of osteoblast differentiation.

    INTRODUCTION
Top
Abstract
Introduction
References

The gene targeting in mice of Cbfa1 (core-binding factor), originally identified as a T-cell differentiation regulator (1, 2), resulted in a complete lack of bone formation due to a maturational arrest of osteoblasts (3, 4). Cbfa1 is also the responsible gene for the human genetic disease of cleidocranial dysplasia (5, 6). The promoter region of the genes related to osteoblast differentiation such as osteopontin (OPN),1 osteocalcin (OSC), and bone sialoprotein contains binding sequences of Cbfa1 (7-9). The transfection of Cbfa1 gene into non-osteogenic cells such as C3H10T1/2 cells and primary skin fibroblasts directed the differentiation pathway of these cells toward the osteoblast lineage (10). These results indicated that Cbfa1 is one of the essential transcription factors that regulate osteoblast differentiation and bone formation (11). In addition, bone morphogenetic proteins (BMPs), one of the most potent stimulatory factors for osteoblast differentiation, induced or stimulated the expression of Cbfa1 mRNA (10, 12). This suggests that Cbfa1 is involved in the signaling pathway of BMP action.

Three subtypes of the alpha -subunit of Cbf (Cbfa1, Cbfa2, and Cbfa3) and one subtype of the beta -subunit (Cbfbeta ) have been reported (13, 14). The alpha -subunits of Cbf family transcription factors acquire enhanced DNA binding activity when they heterodimerize with the beta -subunit (1, 15). In addition, several isoforms of Cbfa1 have been identified by differential promoter usage or differential splicing (16, 17). One isoform, originally cloned from ras-transformed NIH3T3 cells, was named Pebp2alpha A (referred to as Type I Cbfa1 hereafter). Recently, three groups of investigators independently identified two other isoforms of Cbfa1, from osteoblasts and lymphoblasts (5, 10, 16); in these isoforms, two translational start sites are suggested: the shorter isoform (referred to as Type II isoform hereafter) and the longer isoform (referred to hereafter as Type III isoform). Although Ducy et al. (10) demonstrated that the transfection of Type III Cbfa1 into non-osteogenic cells induced gene expression related to osteoblast differentiation, functional differences among the isoforms of Cbfa1 have not been clarified.

We investigated the functional differences among three isoforms of Cbfa1 (Type I, II, and III) by the generation of stably transfected cells with each Cbfa1 isoform and a transient transcriptional assay using Cbfa1 target gene promoter-driven luciferase reporter genes. We demonstrate here that these three isoforms of Cbfa1 have different functions in osteoblast differentiation.

    MATERIALS AND METHODS

Cell Culture-- The mouse embryonic fibroblast cell line, C3H10T1/2, was purchased from Riken Cellbank (Saitama, Japan). This cell line was maintained in BME medium (Life Technologies, Inc.) containing 10% fetal calf serum (Life Technologies, Inc.) and antibiotics.

Detection of Alkaline Phosphatase Activity-- Alkaline phosphatase (ALP) activity was detected histochemically using an Alkaline Phosphatase Substrate Kit IV (Vector Laboratories, Burlingame, CA). The ALP activity of the cell lysates was determined using p-nitrophenyl phosphate as a substrate as described previously (29-32).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)-- The poly(A+) RNA purification, first-strand cDNA synthesis, and PCR were performed as described (18). The PCR conditions were as follows. After 1 min of preincubation at 94 °C, amplification was performed for 35 cycles consisting of 20 s of denaturing at 94 °C, 1 min of annealing, and extension at 66 °C. The primers used for each isoform were as follows (small letters; restriction enzyme site and Kozak sequence).

Mouse Type I Cbfa1 (Pebp2alpha A (1)): sense, 5'-ggatc caccA TGCGT ATTCC TGTAG ATCCG AG-3' (nucleotides +1016/1038); antisense, 5'-CATCA TTCCC GGCCA TGACG GTAAC-3' (nucleotides +1475/1451). Mouse Type II/III Cbfa1 (Osf2/Cbfa1 (10)): sense, 5'-ggatc caccA TGCTT CATTC GCCTC ACAAA CAACC-3' (nucleotides +1/26); antisense, 5'-TGGTG CGGTT GTCGT GCGGC-3' (nucleotides +529/510).

For the detection of ALP mRNA by RT-PCR in the transient transfection assay, 1 µg of total RNA purified from transfected cells (6 days after transfection) was used. The PCR conditions were as follows. After 1 min of preincubation at 94 °C, amplification was performed for 30 cycles consisting of 20 s of denaturing at 94 °C, 30 s of annealing at 56 °C, and 30 s of extension at 72 °C. The primers used were as follows. Sense, 5'-GCAGG ATTGA CCACG GACAC TATG-3' (+1183/1206); antisense, 5'-TTCTG CTCAT GGACG CCGTG AAGC (+1615/1592) (19). As an internal control, a PCR analysis was also performed with beta -actin specific primers for 25 cycles (sense, 5'-CATCA CTATT GGCAA CGAGC-3' (+821/840); antisense, 5'-ACTCA TCGTA CTCCT GCTTG-3' (+1154/1173)) (20).

Plasmids-- A reporter plasmid containing 6 repeats of the consensus Cbfa1 binding site (6XOSE2) was constructed to insert a blunt-ended PCR fragment containing AACCACA-based direct repeats (21) into the SmaI site of the pGL3 promoter vector (Promega, Madison, WI). A reporter plasmid containing a mouse OSC promoter (-147/+13) (21) was constructed to insert a PCR fragment with the NheI-HindIII sites into the cognate site of the pGL3 basic vector (Promega). A reporter plasmid containing a mouse OPN promoter (-253/+28) (7) was constructed to insert a PCR fragment with the BamHI-HindIII sites into the BglII-HindIII sites of the pGL3 basic vector. A reporter plasmid for a mouse ALP promoter (-1838/+81) (22) was constructed to insert a PCR fragment with the HindIII sites into the cognate site of the pGL3 basic vector. An expression plasmid of each Cbfa1 isoform was generated to insert the entire coding sequence with the Kozak sequence into the BamHI (Type I) or BglII (Type II and III) site of the mammalian expression vector pSG5 (Stratagene, La Jolla, CA), respectively. Since our expression plasmids for Type II and III Cbfa1 were constructed using Type I as the template, they have one glutamine deletion in the Q-stretch region compared with the originally reported Osf2/Cbfa1 (1, 10), but we verified the sequence of the Q-stretch region in our genomic clone (3). An expression plasmid of mouse Cbfbeta /Pebp2beta cDNA (14) was generated to insert the entire coding sequence with the Kozak sequence into the EcoRI site of the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA). Xenopus BMP-4 (xBMP-4) cDNA was a kind gift from Dr. N. Ueno (National Institute for Basic Biology, Okazaki, Japan), and the expression plasmid of xBMP-4 cDNA was generated to insert the entire coding sequence with the Kozak sequence into the EcoRI site of pSG5.

Generation of Stable Transformants of Cbfa1-- C3H10T1/2 cells grown to 40-60% confluence in a 9-cm Petri dish were transfected with a total of 25 µg of DNA by calcium phosphate co-precipitation (23). Each Cbfa1 expression plasmid or mock pSG5 (24 µg/dish) was co-transfected with 1 µg of pSV2neo (Life Technologies, Inc.), and the cells were treated with 450-500 µg/ml G418 (Life Technologies, Inc.) from 2 days after the transfection.

Transient Transfection and Luciferase Assay-- C3H10T1/2 cells grown to 40-60% confluence in a 12-well multiplate were transfected with a total of 1 µg of DNA, using the transfection reagent LT-1 (Panvera Corp., Madison, WI). The reporter plasmid (0.2 µg/well) was co-transfected with the indicated amount of each expression vector for each type of Cbfa1, with or without its beta -subunit (0.1 or 0.2 µg), and 0.3 µg of the reference plasmid pCH110 (Amersham Pharmacia Biotech, Uppsala, Sweden). Bluescribe M13+ (Stratagene) was used as the carrier to adjust the DNA amount to 1 µg. After 48 h, the luciferase activity was measured using a luminometer (ML-3000, Dynatec Laboratories Inc., Chantilly, VA). Relative luciferase activity was calculated after normalizing the transfection efficiency by beta -galactosidase activity expressed by pCH110 (23).

RNA Isolation and Northern Blots-- RNA isolation and Northern hybridization were performed as described (18). After final washing, the membrane was exposed to a BAS imaging plate (Fuji Film, Tokyo, Japan), and the relative signal intensity was calculated. The partial-length cDNAs of rat ALP (24) and rat type I collagen (ColI) (25) were cloned by PCR. Mouse OPN and OSC cDNA were kind gifts from Dr. S. Nomura (Osaka University Medical School, Osaka, Japan) (26, 27).

    RESULTS

Both Type I and Type II/III Isoforms of Cbfa1 Are Expressed in Adult Mouse Bone-- We first examined whether Cbfa1 isoforms (Type I and Type II/III) are expressed in bone by RT-PCR analysis using specific primers for each isoform (Fig. 1a) because both Type II and Type III Cbfa1 isoforms are suggested to be translated from the same mRNA (28). As shown in Fig. 1b, both types of transcripts were expressed in adult mouse bone. Note that the duplicate signals were detected by Type II/III-specific primers because of alternative splicing in this region, as reported by Xiao et al. (29) (insertion of 33 bp compared with the reported Osf2/Cbfa1 sequence, data not shown).


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Fig. 1.   Expression of Cbfa1 isoforms in adult mouse bone. RT-PCR analysis: 0.1 µg of poly(A+) RNA was reverse-transcribed, and a PCR was performed for 35 cycles with primers specific to these isoforms of Cbfa1 as described under "Materials and Methods." PCR products were resolved in 6.5% polyacrylamide gels and visualized with ethidium bromide. a, scheme of the structural difference of Type I, II, and III of Cbfa1. Coding regions are shown as white boxes. M stands for the methionine residue. Primers for PCR are shown by arrows. b, polyacrylamide gel electrophoresis. M stands for the molecular marker (phi X174/HaeIII). Numbers indicate the size of PCR products. Primers: Type I, Cbfa1 (Pebp2alpha A); Type II/III, Osf2/Cbfa1. Template: N, no reverse transcription; G, genomic DNA; C, cDNA.

Transient Transfection with Type I and Type II Cbfa1, but Not with Type III Cbfa1, Induced ALP Activity in C3H10T1/2 Cells-- Since ALP is one of the early differentiation markers for osteoblasts (30-33), we investigated ALP activity in C3H10T1/2 cells transiently transfected with each isoform of Cbfa1 and/or xBMP-4. No ALP-positive cells were found in C3H10T1/2 cells without transfection. Six days after transfection with Cbfa1 isoforms, many ALP-positive cells appeared in the cells transfected with Type I or Type II Cbfa1 (Fig. 2, a-1). Transfection with xBMP-4 also induced ALP activity in C3H10T1/2 cells. The co-introduction of Type I Cbfa1 and xBMP-4 synergistically increased the number of ALP-positive cells and activity (Fig. 2, b-1 and b-2), suggesting some functional linkage between Cbfa1 and BMP-4. No ALP-positive cells were induced by transfection with Type III Cbfa1 or by transfection with mock pSG5 (Fig. 2, a-1 and b-1). The effect of the transient transfection of Cbfa1 and/or xBMP4 on ALP mRNA expression was also verified by RT-PCR (Fig. 2, a-2 and b-2).


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Fig. 2.   Effects of Cbfa1 and/or BMP4 overexpression on the induction of ALP activity and ALP mRNA in C3H10T1/2 cells. Six days after the transfection of the expression plasmid(s), cells were fixed and stained as described under "Materials and Methods." From one culture plate, cell lysates were prepared and ALP activity was measured using p-nitrophenyl phosphate as a substrate. From another culture plate, total RNA was purified from the transfected cells. 1 µg of total RNA was reverse-transcribed, and a PCR analysis was performed with specific primers for mouse ALP (ALP) and beta -actin (ACT). The representative results of at least four independent experiments are shown. a-1, ALP staining of the transfected cells of each Cbfa1 isoform. Type-I, Type I Cbfa1; Type-II, Type II Cbfa1; Type-III, Type III Cbfa1; MO, mock pSG5; NT, no transfection; ATRA, treatment with 1 µM all-trans retinoic acid. a-2, induction of ALP mRNA and activity by the transient transfection of each Cbfa1 isoform. The number indicates the ALP activity (nmol/min/mg of protein) of the cell lysate. MO, mock pSG5; I, Type I Cbfa1; II, Type II Cbfa1; III, Type III Cbfa1. b-1, ALP staining of transfected cells with Cbfa1 and/or BMP4. MO, mock pSG5; BMP, Xenopus(x) BMP4; Cbfa1, Type I Cbfa1; Cbfa1/BMP4, Type I Cbfa1 and xBMP4; NT, no transfection; ATRA, treatment with 1 µM all-trans retinoic acid. b-2, induction of ALP mRNA and activity by the transient transfection of Cbfa1 and/or BMP4. The number indicates the ALP activity (nmol/min/mg of protein) of the cell lysate. MO, mock pSG5; C, Type I Cbfa1; B, xBMP4; C/B, Type I Cbfa1 and xBMP4.

The Effects of the Stable Transfection of Cbfa1 on the Expression of Osteoblast-related Genes Vary among Isoforms-- To further investigate functional differences in the effects of Cbfa1 isoforms on osteoblast differentiation, we examined gene expressions related to osteoblast differentiation using stably transfected C3H10T1/2 cells with the three isoforms (Type I, II, and III) of Cbfa1. The expression of each exogenous Cbfa1 isoform was ensured by Northern hybridization (Fig. 3a). C3H10T1/2 cells transfected with Type I or Type II Cbfa1 exhibited the expression of ALP mRNA, but no ALP mRNA was detected in the cells transfected with Type III Cbfa1 (Fig. 3a). These results were consistent with those observed in the transient transfection experiments. OSC, OPN, and ColI gene expressions were induced or up-regulated in all cell types transfected with respective isoforms of Cbfa1. The highest induction of ALP gene expression was observed in Type I Cbfa1-transfected cells, and the highest induction of OSC gene expression was observed in the Type II Cbfa1-transfected cells, when each expression level was normalized by that of the corresponding transfected isoform of Cbfa1 (Fig. 3b). There were no apparent changes in the expression levels of OPN and ColI among isoforms of Cbfa1 (Fig. 3, a and b).


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Fig. 3.   Effects of the overexpression of each Cbfa1 isoform on the osteoblast differentiation marker gene expressions. a, Northern blot analysis. Stable transformants of each Cbfa1 isoform were generated as described under "Materials and Methods," and the results of two independent clones of each isoform are shown. 20 µg of total RNA was separated on a formaldehyde/agarose gel, and a Northern blot analysis was performed by using partial cDNA fragments of various osteoblast differentiation markers. Arrows indicate the signal of the mRNA from transfected Cbfa1 expression vector. MO, mock pSG5; NT, no transfection; I, Type I Cbfa1; II, Type II Cbfa1; III, Type III Cbfa1; ColI, Type I collagen; OSC, osteocalcin; ALP, alkaline phosphatase; OPN, osteopontin; ACT, beta -actin. b, relative signal intensity of various osteoblast differentiation markers are shown after the normalizing of each Cbfa1 isoform expression level. The signal intensity of each kind of mRNA of the first Type I Cbfa1 transfected clone was regarded as 1 fold. MO, mock pSG5; Type I, Type I Cbfa1; Type II, Type II Cbfa1; Type III, Type III Cbfa1.

Cbfa1 Isoforms Induce Different Transcriptional Activity of the Target Genes-- Cbfa1 has the ability to enhance the expression of target genes by binding to its target sequence in the promoter and/or enhancer region (7-9, 34-37). Thus, we next examined whether the difference of the ability to enhance target gene expression (Figs. 2 and 3) is caused at the transcriptional level, using a luciferase reporter gene assay system. When p6XOSE2-luc was used as a reporter plasmid (10, 21), the transfection of expression vector for each Cbfa1 isoform efficiently induced reporter gene activity (Fig. 4a). The dose-response analysis of Cbfa1 plasmid revealed that Type II Cbfa1 induced the highest luciferase activity among the Cbfa1 isoforms (Fig. 5a). The co-introduction of each Cbfa1 isoform and its beta -subunit induced no synergistic increase in the reporter gene activity, even in the presence of different amounts of their beta -subunits (Figs. 4a and 5a). When the reporter plasmid used was pOSC(-147/+13)-luc (10, 21), which includes the mouse OSC promoter region with one functional Cbfa1 binding site, each isoform of Cbfa1 induced luciferase activity when co-transfected with its beta -subunit expression plasmid (Fig. 4b). The dose-response analysis of Cbfa1 revealed that Type I Cbfa1 induced the highest luciferase activity with no exogenous beta -subunit, and that each isoform induced luciferase activity similarly except for that at the highest amount of Type III Cbfa1 expression plasmid with the co-introduction of its beta -subunit (Fig. 5b). When pOPN(-253/+28)-luc (which includes the mouse OPN promoter region having one functional Cbfa1 binding site near the Ets-1 binding site (7, 38)) was used as a reporter plasmid, each isoform induced luciferase activity similarly, when no exogenous beta -subunit was expressed (Figs. 4c and 5c). With the beta -subunit expression plasmid, Type III Cbfa1 effectively induced luciferase activity especially with the highest amount of Cbfa1 expression plasmid, as was observed with pOSC(-147/+13)-luc (Fig. 5c). With the use of pALP(-1838/+81)-luc, which includes the bone/liver/kidney (B/L/K)-type ALP gene promoter region (22, 39) and one putative Cbfa1 binding site, no apparent enhancement of luciferase activity was observed by the transfection of each Cbfa1 expression plasmid alone or in combination with its beta -subunit (Figs. 4d and 5d). A dose-response analysis of Cbfa1 also revealed no obvious enhancement of luciferase activity (Fig. 5d).


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Fig. 4.   Effect of Cbfa1 isoforms on the expression of target genes at the transcriptional level. 0.2 µg of various Cbfa1 target gene reporter plasmids were co-transfected with the indicated amount of the expression vectors for each Cbfa1 isoform (I, II, III) and/or its beta -subunit (beta ) (0.1 µg) and 0.3 µg of the reference plasmid pCH110. After 48 h of transfection, both the luciferase and beta -galactosidase activity were measured as described under "Materials and Methods," to express normalized luciferase activity. Values are the means of duplicate wells, and representative results of at least four independent experiments are shown. a, p6XOSE2-luc; b, pOSC(-147/+13)-luc; c, pOPN(-253/+28)-luc; d, pALP(-1838/+81)-luc.


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Fig. 5.   Dose-response analysis of each Cbfa1 isoform on the transcription of various Cbfa1 target genes. 0.2 µg of various Cbfa1 target gene reporter plasmids were co-transfected with the indicated amount of the expression vectors for each Cbfa1 isoform in the absence or presence of its beta -subunit (beta ) (0.2 µg) and 0.3 µg of the reference plasmid pCH110. Relative luciferase activity was calculated as described in Fig. 4. Values are the means of duplicate wells, and representative results of at least four independent experiments are shown. a, p6XOSE2-luc; b, pOSC(-147/+13)-luc; c, pOPN(-253/+28)-luc; d, pALP(-1838/+81)-luc.


    DISCUSSION

As noted earlier, there are several isoforms of Cbfa1 (1, 10, 16), but the function of each isoform has not been clarified. We first examined the expression pattern of isoforms of Cbfa1 in mouse bones by RT-PCR using specific primers for the Type I and Type II/III isoforms, and we found that these isoforms were expressed in adult bones (Fig. 1). This result suggested important roles of each isoform in bones. Ducy et al. (10) and Xiao et al. (29) reported that PEBP2a/Cbfa1 (Type I Cbfa1) was not expressed in osteoblasts. In contrast to these previous studies, we detected both Type I and Type II/III transcripts, because we used cDNA-transcribed mRNA from whole bone in our RT-PCR analysis.

We next examined the effects of the transfection of each Cbfa1 isoform on ALP activity in C3H10T1/2 cells, because Cbfa1-deficient mice exhibited extremely low levels of ALP activity in skeletal tissues (3). With the present transient transfection of each isoform into C3H10T1/2 cells, two Cbfa1 isoforms, Type I and Type II, induced ALP activity, but Type III isoform did not (Fig. 2a). This result was confirmed by the RT-PCR analysis (Fig. 2b). The results were also confirmed at the mRNA expression level by experiments using stably transfected C3H10T1/2 cells with each isoform (Fig. 3). These findings suggest that the Type I and Type II isoforms of Cbfa1 are involved in the early differentiation process of osteoblasts, because ALP activity is one of the early markers during osteoblast differentiation. In addition, the induction of ALP activity by transfection with the Type I isoform was synergistically increased by the co-transfection of xBMP-4 (Fig. 2b). This suggests that Cbfa1 is involved in a signaling pathway of BMP-4 in the early differentiation process of osteoblasts.

We investigated whether Cbfa1 directly regulates the transcriptional activity of the ALP gene in a luciferase reporter gene assay (Fig. 4d). For this experiment, we cloned the ALP gene and used about 2 kb of the mouse ALP promoter region. Our sequence analysis and the previous report by Terao et al. (22) indicated that our reporter plasmid contains one consensus Cbfa1 binding site and three consensus-like Cbfa1 binding sites (data not shown, (8, 22)). Unexpectedly, none of the three Cbfa1 isoforms used in the present study induced any apparent transcriptional activity of the ALP gene (Figs. 4d and 5d). These results raise the possibility that Cbfa1 binding sites might not be well functioning because they exist far away from the putative transcriptional start site, even if Cbfa1 directly binds there. Kobayashi et al. (39) demonstrated that the deletion of these putative Cbfa1 binding sites from a B/L/K-ALP promoter construct did not cause a significant decrease of promoter activity. Banerjee et al. (40) reported that two distal putative Cbfa binding sites did not function well in the rat osteocalcin promoter. Cbf family transcriptional factors are known to function as both negative and positive transcriptional regulators, and it is also known that the context of the binding sequences of transcription factors is essentially important for the regulation of gene expressions (35, 41, 42). In addition, a recent analysis of the in vivo promoter activity of the B/L/K-type ALP gene revealed that the essential region for whole skeletal tissue expression existed in the upstream region (-4.3/-2.0 kb) of this ALP(-1838/+81) construct (43). Thus, it is likely that functional Cbfa1 binding sequences exist in the 5'-upstream region of ALP(-1838/+81), which we used in this study. Alternatively, other functional binding sites for Cbfa1 or Cbfa1-inducible factors may exist upstream or downstream of ALP(-1838/+81), as shown in the analysis concerning the TCRalpha enhancer region (35, 36).

Ducy et al. (10) reported that the transient transfection of Osf2/Cbfa1 (Type III) into non-osteogenic cells such as C3H10T1/2 cells induced or increased the expression levels of mRNAs related to osteoblast differentiation. We also investigated, using stable transformants obtained from C3H10T1/2 cells, whether three Cbfa1 isoforms (Type I, II, and III) have similar activity (Fig. 3). We confirmed that all of these isoforms induced the mRNA expression of OSC and OPN and increased the expression levels of ColI, although the potency regulating the expression of these mRNAs differed among the isoforms. The Type II isoform more effectively induced OSC expression compared with the Type I and Type III isoforms, but the stimulatory effects on OPN and ColI mRNAs were not so different among each isoform. These results may be closely related to those of the luciferase reporter gene assay (Fig. 4), i.e. a higher induction rate of p6XOSE2-luc by the Type II isoform compared with the Type I and Type III isoforms, and similar induction rates of pOPN(-253/+28)-luc among the three isoforms without an exogenous beta -subunit. In the transcriptional activation of the OSC gene, the discrepancy between the results of the promoter analysis and those of the stable transfection experiment may be due to the region of the construct we used in luciferase assay, i.e. endogenous OSC gene expression is regulated by many factors and many functional elements in the gene, such as CREB/CRE (44), MSX (45), and GR/GRE (46). In the transcriptional activation of the OPN gene, the results of the promoter analysis and those of the stable transfection experiment correlated very well; this reflected that the important regulatory element in osteoblastic lineage cells exists in the regions of our construct (38, 47). In addition, the highest transcriptional activation of the OPN gene was demonstrated with Type III Cbfa1 transfection when its beta -subunit was co-transfected. Taken together, our findings suggest that Cbfa1 isoforms have different functions in the regulation of osteoblast-related gene expression.

The results of the present reporter gene analysis suggested that at least two kinds of regulatory mechanisms are involved in the Cbfa1-induced transcription of target genes (Fig. 4). First, Cbfa1 itself may be able to activate target genes effectively in the case of the OPN gene and p6XOSE2 construct. Second, Cbfa1 may regulate the transcription of the target genes in collaboration with its beta -subunit in the case of the OSC and OPN genes. However, Ducy et al. (10) reported that a single transfection of Osf2/Cbfa1 (Type III Cbfa1) induced the transcription activity of the OSC gene, using the same region of the mouse OSC promoter as that used in the present study. The discrepant results between the Ducy study and ours might arise from the different experimental protocols or different cell usage (i.e. different interaction with cell-specific co-factors, as described below), judging from previous observations (15, 34-37). One of the candidate co-factors is Cbfbeta (also known as heterodimerizing partner of Cbfa), which is reported to be abundantly expressed in various kinds of cells (14), because we detected stronger activation by far of the pOSC(-147/+13)-luc and pOPN(-253/+28)-luc in the presence of exogenous Cbfbeta expression.

Different activities of transcription factors often occur among isoforms generated by alternative splicing. For example, AML1c (a human homolog of mouse Pebp2alpha B), one of the runt domain family gene transcripts, is 14 amino acids longer than AML1a in the N-terminal region like til-1 (Type II Cbfa1). No functional difference between AML1a and AML1c has as yet been uncovered, although many functional domains concerning DNA binding, heterodimerization, and transactivation in its C-terminal region have been identified (15). As for SREBP1 (sterol regulatory element binding protein), one of the essential transcription factors in fatty acid metabolism, its two isoforms with different N-terminal structures (SREBP1a and SREBP1c) were shown to have different abilities to induce the target gene expression in experimental animals and cultured cells (48), as is also the case for progesterone receptor (A and B), which is one of the ligand-dependent transcription factors and is essential for sex hormone function (49). Different activities of transcription factors among isoforms generated by alternative splicing might result from the different interaction with cofactors such as co-activators and co-repressors (50). Cbfa1 has been reported to interact directly or indirectly with cofactors including not only its beta -subunit (14, 15) but also C/EBP (34), Ets-1 (35, 38), Myb (37), and ALY (36) on the target gene enhancers and/or promoters. There have been no reports concerning cofactors interacting with Cbfa1 in its N-terminal region; the identification of the coupling protein in this region might clarify the reasons for the functional differences among the isoforms of Cbfa1.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Y. Koga and Dr. T. Nakatani for their encouragement.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence and reprint requests should be addressed: Sumitomo Pharmaceuticals Research Center, 3-1-98, Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan. Tel.: 81-6-466-5222; Fax: 81-6-466-5483; E-mail: mnakatsu{at}sumitomopharm.co.jp.

    ABBREVIATIONS

The abbreviations used are: OPN, osteopontin; Cbf, core binding factor; OSC, osteocalcin; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; Pebp, polyoma enhancer binding protein; Osf, osteoblast-specific factor; OSE, osteocalcin-specific element; RT, reverse transcriptase; PCR, polymerase chain reaction; CRE, cAMP-responsive element; CREB, CRE-binding protein; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; AML, acute myeloid leukemia factor; SREBP, sterol regulatory element binding protein; C/EBP, CCAAT/enhancer binding protein; ALY, ally of AML-1 and LEF-1; bp, base pair(s); kb, kilobase(s).

    REFERENCES
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Abstract
Introduction
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