Cbfa1 Isoforms Exert Functional Differences in
Osteoblast Differentiation*
Hideyuki
Harada
,
Shuzo
Tagashira
,
Masanori
Fujiwara
,
Shinji
Ogawa
,
Takashi
Katsumata
,
Akira
Yamaguchi§,
Toshihisa
Komori¶, and
Masashi
Nakatsuka
From
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 |
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 Pebp2
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
-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 |
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
-subunit of Cbf (Cbfa1, Cbfa2, and Cbfa3) and
one subtype of the
-subunit (Cbf
) have been reported (13, 14).
The
-subunits of Cbf family transcription factors acquire enhanced
DNA binding activity when they heterodimerize with the
-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 Pebp2
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 (Pebp2
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
-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 Cbf
/Pebp2
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
-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
-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 ( X174/HaeIII).
Numbers indicate the size of PCR products. Primers: Type I,
Cbfa1 (Pebp2 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 -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, -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.
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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
-subunit induced no
synergistic increase in the reporter gene activity, even in the
presence of different amounts of their
-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
-subunit expression plasmid (Fig. 4b). The
dose-response analysis of Cbfa1 revealed that Type I
Cbfa1 induced the highest luciferase activity with no
exogenous
-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
-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
-subunit was expressed (Figs. 4c and
5c). With the
-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
-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 -subunit ( ) (0.1 µg) and 0.3 µg of the reference
plasmid pCH110. After 48 h of transfection, both the luciferase
and -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 -subunit ( ) (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.
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 |
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 TCR
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
-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
-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
-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 Cbf
(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 Cbf
expression.
Different activities of transcription factors often occur among
isoforms generated by alternative splicing. For example,
AML1c (a human homolog of mouse Pebp2
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
-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.
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).
 |
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