Biochemical and Biophysical Research Communications 452 (2014) 531–536

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Sp7/Osterix induces the mouse pro-a2(I) collagen gene (Col1a2)
expression via the proximal promoter in osteoblastic cells
Hiroyuki Yano a,⇑, Ryoji Hamanaka b, Miki Nakamura-Ota b, Sawako Adachi c, Juan Juan Zhang c,
Noritaka Matsuo c, Hidekatsu Yoshioka cabcDivision of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593, Japan
Department of Cell Biology, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593, Japan
Department of Matrix Medicine, Faculty of Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593, Japana r t i c l e

i n f oArticle history:
Received 12 August 2014
Available online 27 August 2014
Keywords:
Type I collagen
Sp7/Osterix
Osteoblastic cell
Bone formation
Gene expressiona b s t r a c t
Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The
extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amountsof other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process inwhich many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-richsequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed
to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 genecontaining multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse
Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site speciﬁcally decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its speciﬁc siRNA showed that this G/C-rich sequence is
responsible for the speciﬁc expression in osteoblastic cells. Consistent with these data, Sp7/Osterix boundto the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.
Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction
Bone is formed via intramembranous and endochondral ossiﬁcation. Osteoblasts, which are cells of mesenchymal origin, areresponsible for both of these processes. Bone is directly derived
from mesenchymal condensation at sites of intramembranous
ossiﬁcation while the intermediate step, cartilage formation, isinvolved in endochondral ossiﬁcation. The former process isobserved in most craniofacial bones and the lateral region of theclavicles, while the latter process occur in the long bones of thelimbs, vertebrae, basal portion of the skull, medial area of the clavicle and ribs. Recent advancements in molecular biology andmouse genetics have helped to identify various transcription factors that regulate bone formation [1–3]. Runx2 is the a subunitof a heterodimeric transcription factor, and a member of the Runt
family. In mice, the expression of Runx2 begins in the notochordon embryonic day 9.5 (E9.5), and later at sites of prechondrogenic
Abbreviation: CHIP assay, chromatin immunoprecipitation assay.

⇑ Corresponding author. Fax: +81 97 586 6279.
E-mail address: yanoh@oita-u.ac.jp (H. Yano).

http://dx.doi.org/10.1016/j.bbrc.2014.08.100
0006-291X/Ó 2014 Elsevier Inc. All rights reserved.

mesenchymal condensation and in chondrocytes [2]. In addition toits essential functions in osteoblast differentiation, Ranx2 plays arole in the differentiation of hypertrophic chondrocytes [1].
Sp7/Osterix is a zinc ﬁnger-containing transcription factor speciﬁc
to osteoblasts in vivo [3] that acts downstream of Runx2 andstrongly binds to GC-rich sequences, including Sp1. The expressionof Sp7/Osterix is more speciﬁc to osteoblasts than that of Runx2, andthe transcripts are not detected before E13 in mice [3].
The extracellular matrix of bone is mostly composed of collagen, primary type I collagen with other minor types of collagen.
Collagen molecules assemble into heterotypic aggregates that subsequently affect the biological and mechanical properties of bone[4]. Collagens are classiﬁed according to the ﬁbrillar or non-ﬁbrillar
structure [5]. Banded ﬁbrils exhibiting 67-nm periodicity containheterogeneous molecules of various collagen types. Fibrillar collagen is divided into two groups: major ﬁbrillar collagens (types I, II
and III) and minor ﬁbrillar collagens (types V and XI). In bone, bothﬁbrillar collagen (types I, III, V and XXIV) [6] and non-ﬁbrillar
collagen (types VI and XII) are observed.
Over 90% of case of osteogenesis imperfecta (OI) involve
autosomal dominant bone disorders caused by mutations in both

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H. Yano et al. / Biochemical and Biophysical Research Communications 452 (2014) 531–536

the collagen a1(I) and a2(I) chains [7]. Recently, it was reported
that 5–10% of recessive cases are caused by non-collagen genes
containing Osterix/Sp7 [8].
It has also been previously demonstrated that Sp7/Osterix upregulates the mouse Col5a1 and Col5a3 genes [9,10], which areco-expressed with type I collagen in osteoblasts. The proximal promoter of the human COL1A2 gene has several GC-rich sequences towhich Sp1 binds in order to activate the gene in ﬁbroblasts [11,12].
These GC-rich sequences may also be involved in processes of regulation in association with Sp7/Osterix in osteoblasts. In the present study, we examined whether Sp7/Osterix binds to and activatesthe mouse Col1a2 gene via the proximal promoter region in
osteoblasts.

The cotransfection experiments were performed using 1.5 lg of
Sp1 or Sp7/Osterix expression vectors [10].

2.5. siRNA transfection
The siRNAs targeting mouse Sp1 or Sp7/Osterix were purchased(Santa Cruz Biotechnology, CA, USA). The mouse MC3T3-E1 cells
were transfected using Lipofectamine2000 (Invitrogen) with a ﬁnal
siRNA concentration of 50 nM. The procedure was described for
RT-PCR and luciferase assay using siRNAs previously [10].

2. Materials and methods
2.1. Cell culture
Mouse MC3T3-E1 and mouse NIH-3T3 cells were cultured in
Dulbecco’s modiﬁed Eagle’s medium containing 10% fetal bovine
serum (Sanko Junyaku, Tokyo, Japan) at 37 °C in humidiﬁed 5%
CO2 and 95% air. After the MC3T3-E1 cells were cultured at conﬂuent, 10 lM b-glycerophosphate and 50 lg/mL of ascorbic acid wereadded in the medium for cell differentiation experiment. The medium was changed every 2 days.
2.2. Real time PCR
Total RNA was extracted from cultured cells using Isogen
extraction kit (Nippon Gene, Tokyo, Japan). RNA was stored at
80 °C until used. One micrograms of total RNA was reverse transcribed using ReverTra Ace (Toyobo, Tokyo, Japan) with random
primers. For a quantiﬁcation of mRNA, real-time PCR was performed using a LightCycler TaqMan Master (Roche, IN, USA). The
thermal cycling conditions included 1 cycle at 95 °C for 10 min,40 cycles at 95 °C for 15 s, 60 °C for 1 min. The relative mRNA
expression levels were normalized against that of that of GAPDH
gene using a comparative threshold cycle method [13]. The primer
sets are listed in Table S1.
2.3. Construction of chimeric plasmids
To generate the luciferase constructs, mouse genomic DNA
were derived from pBACe3.6RP23 clone (BACPAC Resources Children’s Hospital Oakland, CA, USA). Wild type construct was
obtained by using Sac site-linked 50 and Xho site-linked 30 speciﬁc
for the mouse Col1a2 sequence. Ampliﬁed product was inserted
into the pGEM-T Easy vector (Promega, WI, USA), digested with
Sac and Xho, and subcloned into pGL4.1 basic luciferase vector(Promega, WI, USA). The Xba site, TCTAGA, was introduced into
wild type fragment to generate various mutant constructs. The primer sets are listed in Table S1.
2.4. Transient cell transfection and luciferase Assay
Approximately 2  105 cells were plated in 35-mm dish for 18 h
before transfection. Five lg of plasmid DNA was transfected into
cells using calcium phosphate precipitation methods. PlasmidpRL-TK vector (Promega, WI, USA) was cotransfected as an internal
control for transfection efﬁciency. After 48 h, the transfected cells
were harvested, lysed, centrifuged to remove the debris, and subjected to luciferase assay. The luciferase activities were measured
with a luminometer (Lumat LB 9507, Perkin-Elmer Life Sciences,
Waltham, MA, USA) using the Dual Luciferase Reporter Assay System (Promega, WI, USA) according to the manufacturer’s protocol.

Fig. 1. Luciferase assay of the proximal promoter of the mouse Col1a2 gene. (A) The
luciferase reporter constructs. Transient transfection with the wild-type and six
mutant constructs was carried out in the MC3T3-E1 (B) and NIH-3T3 (C) cells. Eachvalue was normalized to the internal control and is shown relative to the activity of
the wild-type construct in each cell line. The data represent the mean ± S.D. of atleast three independent experiments. ⁄p < 0.05 compared with the wild-type.

H. Yano et al. / Biochemical and Biophysical Research Communications 452 (2014) 531–536

2.6. Chromatin immunoprecipitation (CHIP) assay

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The Sp1 binding sites, G/C rich sequences, in the proximal promoter region of the human COL1A2 gene have been previously

reported [11,12]. Sp7/Osterix binds to G/C rich sequence in theproximal promoter region of the Col5a1 and Col5a3 genes in osteoblastic cells [9,10]. Therefore, we performed an experiment inorder to examine whether this transcription factor also binds to
any Sp1 binding sites in the promoter region of the Col1a2 genein osteoblastic cells. Consequently a wild-type and ﬁve mutant
constructs that link to the luciferase gene were generated to assess
the promoter activity in MC3T3-E1 cells (Fig. 1A). m2-Luc, which
was mutated in the second G/C-rich sequence from the transcription start site, caused an approximately 50% decrease in the promoter activity of the MC3T3-E1 cells (Fig. 1B), but not the NIH3T3 cells (Fig. 1C). In addition, mCAT-Luc, which was mutated in
the CBF/NF-Y binding site, decreased the promoter activity in both
the MC3T3-E1 cells and NIH-3T3 cells (Fig. 1B and C). In contrast,
m1-Luc, m3-Luc, m4-Luc and m5-Luc had no effect on the promoter activity in the osteoblastic cells (Fig 1B). On the other hand,
m1-Luc, m3-Luc and m5-Luc exhibited a tendency to decrease theFig. 2. Overexpression of Sp1 and Sp7/Osterix in the MC3T3-E1 cells. The
expression of Sp1 and Sp7/Osterix following the overexpression of Sp1 and Sp7/
Osterix expression vector, respectively (A and B). The expression of Col1a2 following
the overexpression of Sp1 and Sp7/Osterix (C). The luciferase activity obtainedusing WT-Luc (D) and m2-Luc (E). An empty expression construct was transfected
as a control in each experiment. The data represent the mean ± S.D. of at least three
independent experiments. ⁄p < 0.05 compared with the wild-type.

Fig. 3. Inhibition with siRNA against Sp1 and Sp7/Osterix. The expression of Sp1and Sp7/Osterix after inhibition by speciﬁc siRNA of Sp1 and Sp7/Osterix,
respectively (A and B). The expression of Col1a2 following the inhibition of Sp1and Sp7/Osterix (C). The luciferase activity obtained using WT-Luc (D) and m2-Luc(E). Scramble siRNA was transfected as a control in each experiment. The data
represent the mean ± S.D. of at least three independent experiments. ⁄p < 0.05
compared with the wild-type.

The CHIP assays were performed using a chromatin immunoprecipitation assay kit (Upstate Biotechnology, NY, USA) according
to the manufacturer’s protocol. The anti-Sp1 and anti-Sp7/Osterix
antibodies were purchased (Cosmo Bio, Tokyo, Japan). The procedure was described previously [14]. The primer sets are listed inTable S1.

3. Results
3.1. Functional analysis of the proximal promoter region of the Col1a2
gene

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H. Yano et al. / Biochemical and Biophysical Research Communications 452 (2014) 531–536

Fig. 4. Gene expression during MC3T3-E1 cell differentiation. The RT-PCR analysis of the Col1a2 (A), Osteocalcin (B), Sp1 (C) and Sp7/Osterix (D) genes. Each value wasnormalized to the level of GAPDH. The value observed on day 0 in each gene is set at 1.0. The data represent the mean ± S.D. of at least three independent experiments.
⁄
p < 0.05 compared with the value of each gene on day 0. The luciferase assay of the promoter of the Col1a2 gene using WT-Luc and m2-Luc constructs during differentiation(E). The value of wild-type on day 0 is set at 1.0. The data represent the mean ± S.D. of at least three independent experiments. ⁄p < 0.05 compared with the WT-Luc on 0 day.
Position of primers used for the CHIP assay (F). A CHIP assay was performed to examine the binding of Sp1 and Sp7/Osterix to the Col1a2 proximal promoter on days 0 and 8.
IgG was used as a control. A schematic illustration of the bindings of Sp7/Osterix and CBF to the proximal promoter of the mouse Col1a2 gene in the osteoblastic cells (H).

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promoter activity of the NIH-3T3 cells (Fig. 1C). Therefore Sp1
likely binds to these sites [11,12].
3.2. Endogenous expression of the Col1a2 gene overexpressed by
Sp7/Osterix
The overexpression of Sp1 and Sp7/Osterix was induced inorder to examine the effects of Sp1 and Sp7/Osterix in the
MC3T3-E1 cells (Fig. 2A and B). Consequently, the mRNA levelsof the Col1a2 gene signiﬁcantly increased following the overexpression of both factors signiﬁcantly (p < 0.05) (Fig. 2C). The
wild-type and m2-Luc luciferase constructs were subsequently
cotransfected with Sp1 or Sp7/Osterix to order to examine theeffects of these transcription factors on the promoter activity of
the Col1a2 gene in the MC3T3-E1 cells. Both Sp7/Osterix and Sp1
signiﬁcantly activated the wild-type promoter of the luciferase
reporter gene (Fig. 2D). However, neither activity was canceledby the cotransfection of the m2-Luc construct (Fig. 2E).
Conversely, the inhibition of Sp1 and Sp7/Osterix with speciﬁc
siRNAs was also performed (Fig. 3A and B). As a result the knockdown of Sp1 or Sp7/Osterix decreased the endogenous mRNA levels of the Col1a2 gene by 50% to 40%, respectively (p < 0.05)(Fig. 3C).The promoter activity obtained using the wild-type construct was also decreased by both speciﬁc siRNAs (Fig. 3D). Similar
to the results of overexpression mentioned above, the knockdown
experiments (p < 0.05) showed almost no effects on the promoter
activity when using the m2-Luc construct (Fig. 3E).
3.3. Activation of the Col1a2 gene during osteoblast cell differentiationinduced by Sp7/Osterix
MC3T3-E1 was differentiated with b-glycerophosphate to order
to examine the level of activation of the Col1a2 gene induced by
Sp7/Osterix in the osteoblasts up to day 8. The expression levelsof Col1a2, osteocalcin, Sp1 and Sp7/Osterix were examined using
real-time RT-PCR, which showed that the expression of Col1a2
gradually increased (Fig. 4A), while that of Osteocalcin remarkably
increased during differentiation (Fig. 4B). Concerning the two transcription factors, the expression of Sp1 was slightly up-regulated(Fig 4C), while that of Sp7/Osterix increased by approximately
3.5-fold (Fig. 4D) on day 8 after differentiation. Furthermore theactivity of the Col1a2 promoter associated with the wild-type construct increased by approximately 4.5-fold on day 8 after differentiation, although it did not change with m2-Luc, the Sp7/Osterix
binding site of which was mutated (Fig. 4E). In order to examine
the binding of the transcription factors in vivo, a CHIP assay was
performed (Fig. 4F). The results subsequently showed that
Sp7/Osterix clearly bound to the Col1a2 proximal promoter region
on day 8 after differentiation (Fig. 4G).

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Sp7/Osterix forms a complex with NFATc1, resulting in upregulation of the human COL1A1 gene [16]. Collagen ﬁbrils consist of type
I and V collagen molecules, which are cross-linked together in bone
[7,17]. Therefore, type I and V collagens are expressed in a coordinate fashion during osteogenesis. This study clearly demonstrated
that Sp7/Osterix is responsible for the speciﬁc expression of both
molecules during ﬁbril formation in bone. Many other types of collagen including type XII, which belongs to the FACIT family localized on the surface of ﬁbrils, is expressed in a small amount inbone. Since mice without type XII collagen have fragile bones witha disorganized collagen ﬁbers [18], Sp7/Osterix may be involved in
the expression of the mouse Col12a1 gene.
Other transcription factors including Runx2 and ATF4 are
known to play important roles in osteogenesis. Runx2 is
expressed in Sp7/Osterix – deﬁcient mice, indicating that Runx2
works upstream of Sp7/Osterix [3]. Runx2 has also been shownto induce the expression of the mouse Col1a1 gene by bindingat 1347 and 372 bp upstream from the transcription site[19]. In addition, it has been reported that Runx2 binds to the element at the ﬁrst exon of the Col1a2 gene and that multimer ofthis element confer an osteoblast-speciﬁc activity. However, thisspeciﬁc activity was not observed in the current study (data notshown). This discrepancy may be due to differences in the constructs, which contain multimer or monomer of the element.
ATF4 regulates osteoblast terminal differentiation and is phosphorylated by the kinase RSK2, the inactivation of which resultin Cofﬁn-Lowry syndrome, an X-linked disorder associated withmental retardation and skeletal abnormalities [20]. The ATF4 geneis ubiquitously expressed, although its protein is degraded via
ubiquitination in many cells except osteoblasts [21]. Type I collagen synthesis is decreased in the osteoblasts of ATF4-deﬁcent
mice, which indicates that the activity of ATF4 is regulated posttranslationally [20]. In conclusion, osteogenesis is a multi-stepprocess in which mesenchymal cells become osteocytes. Further
research is needed to clarify the complex mechanisms underlying
the regulation of bone formation.
Acknowledgments
We thank the staff members of Research Promotion Project,
Oita University. This work was supported by Grants-In-Aid for
Scientiﬁc Research (No. 25462375 to H.Y.) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bbrc.2014.08.100.
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4. Discussion
Sp7/Osterix is a zinc ﬁnger transcription factor that strongly
binds to GC-rich sequence. The DNA-binding domain of Sp7/Osterix is similar to those of Sp1 and Sp3, which are ubiquitously
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