Biochemical and Biophysical Research Communications 394 (2010) 503–508

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

Sp7/Osterix up-regulates the mouse pro-a3(V) collagen gene (Col5a3) during
the osteoblast differentiation
Wu Yun-Feng, Noritaka Matsuo, Hideaki Sumiyoshi, Hidekatsu Yoshioka *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 26 February 2010
Available online 3 March 2010
Keywords:
Sp7/Osterix
Type V collagen
Bone
Osteoblast
Differentiation
Sp familya b s t r a c t
Type V collagen is a quantitatively minor collagen, but acts as critical regulator of ﬁbril formation in theextracellular matrix. The purpose of this study is to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Sp7/Osterix is a transcription factor speciﬁcally expressed by osteoblasts and is important for osteoblast differentiation. The overexpression of
Sp7/Osterix signiﬁcantly increased the promoter activity and the endogenous mRNA level of the Col5a3
gene in osteoblastic cells. Conversely, a reduction of Sp7/Osterix by siRNA treatment decreased thepromoter activity and the endogenous mRNA level of the Col5a3 gene. A CHIP assay conﬁrmed that
Sp7/Osterix interacted with the Col5a3 core promoter in vivo at the Sp1 binding site. The data from theexperiments using the osteoblast differentiation model and the co-overexpression of Sp7/Osterix with
Sp1 suggest that Sp7/Osterix promotes the expression of the collagen gene, Col5a3, and thereby playing
a role in bone formation.
Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction
Collagens, which are the major constituents of extracellularmatrices, are critical for the formation and function of the organs
in the body [1]. Type V collagen is a member of the ﬁbrillar collagen group, which is a minor subgroup based on the relative expression level and presence in tissues where type I collagen isexpressed. Type V collagen is incorporated into the ﬁbrils of themore abundant type I collagen, and acts as regulator of the size
and the shape of the ﬁbrils [2]. There are several type V isoforms
that differ in chain composition. The major isoform is[a1(V)]2a2(V). Defects in the human COL5A1 and COL5A2 genes
have been identiﬁed so far in half of the cases of classic Ehlers–
Danlos syndrome (EDS, type I/II) [3,4] . In addition, the [a1(V)]3
homotrimer and the a1(V)a2(V)a3(V) heterotrimer have been also
reported. The a1(V)a2(V)a3(V) isoform was isolated from humanplacenta as well as the uterus, skin, and synovial membranes[5–7]. The pro-a3(V) chain is also expressed in bone and the N-terminal globular domain inﬂuences bone formation by osteoblasts
through proteoglycans on the cell surface [8].
Sp7/Osterix is a zinc ﬁnger-containing protein which belongs to
the Sp/KLF (Kruppel like Factor) family of transcription factors [9].
The murine Sp7/Osterix protein contains 428 amino acids yielding
Abbreviation: CHIP assay, chromatin immunoprecipitation assay.
* Corresponding author. Fax: +81 97 586 5674.
E-mail address: hidey@med.oita-u.ac.jp (H. Yoshioka).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.02.171

a molecular weight of 44.7 kDa. It is distinctly expressed in all of
the developing bones and is important for osteoblast differentiation. It binds to GC box elements and activates mRNA synthesis
from the genes containing functional recognition sites. In null
mice, endochondral and intramembranous bone formation does
not occur.
Characterization of the core promoter of the human COL5A3
gene has been demonstrated that the CBF/NF-Y acts as a transcriptional activator of the COL5A3 gene in a human Schwannoma cellline [10]. In a previous study, a GC-rich domain was found in thecore promoter of the mouse Col5a3 gene [11]. The Sp1 bound to
the GC-rich domain and activated the Col5a3 gene in combination
with CBF/NF-Y in osteoblastic cells. In this study, the involvementof Sp7/Osterix, which binds to the Sp1 binding site, in the activation of the Col5a3 gene during osteoblast differentiation was
examined.
2. Materials and methods
2.1. Cell culture
The cell lines used in this study was mouse preosteoblast
MC3T3-E1 cells. Cells were cultured in Dulbecco’s modiﬁed Eagle’s
medium containing 10% heat-inactivated fetal bovine serum (Sanko Junyaku, Tokyo, Japan) at 37 °C in a 5% CO2/air environment. For
differentiation studies, MC3T3-E1 cells were fed at conﬂuence(time 0) with the above medium additionally containing 10 lM

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b-glycerophosphate and 50 lg/mL of ascorbic acid. The medium
for differentiation was changed every 2 days.
2.2. RT-PCR and real-time PCR
Total RNA was isolated from cultured cells using Isogen (Nippon
Gene, Tokyo, Japan) according to the manufacturer’s instructions.
RNA was quantiﬁed by optical density (A260) and stored at
80 °C until used. Three micrograms of total RNA was reverse transcribed by random hexamer priming using SuperScript II reverse
transcriptase (Invitrogen, CA, USA). The single-stranded cDNA
was ampliﬁed by PCR using speciﬁc primer pairs (Table S1). PCR
was carried out for 25–30 cycles using a step cycle of 94 °C for
30 s, 55 °C for 30 s, 72 °C for 30 s, followed by 72 °C for 7 min.
The PCR products were analyzed by electrophoresis on a 1.5% ethidium bromide-stained agarose gel. The ampliﬁed fragment was
eluted from the gel and sequenced.
Real-time PCR was performed using a LightCycler TaqMan Master. 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 GAPDH
gene from the same RNA preparations, using a comparative threshold cycle method. The primer sets are listed in Table S1.
2.3. Construction of chimeric plasmids

Lipofectamine 2000 (Invitrogen) to achieve a ﬁnal siRNA concentration of 50 nM. The speciﬁcity of Sp1 or Sp7/Osterix knockdown
was conﬁrmed by a RT-PCR reaction. For the promoter analysis,transfections, including luciferase reporter construct, were allowed
to proceed for 48 h before the analysis. For the quantitative analysis of the expression levels of the Col5a3 mRNA, a real-time PCR
reaction was performed.
2.7. DNA sequencing
Nucleotide sequences were determined by automated DNA
sequencing (ABI PRISM 310 Genetic Analyzer, Applied Biosystems,
Foster City, CA, USA) using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems).
3. Results
3.1. Up-regulation of the promoter activity of the Col5a3 gene by Sp7/
Osterix
To characterize the effects of the Sp7/Osterix transcription
factor on the mouse Col5a3 gene, the Sp7/Osterix overexpression
vector pMAM-BSD-Sp7 was generated. It was cotransfected into
the MC3T3-E1 cells with the 337/+92-Luc promoter luciferase
reporter gene, which contains the basic promoter region of the

The luciferase constructs, pGL4.10 337/+92 and mut 1-Luc,and the pCMV-Sp1 expression plasmid were previously described[9]. The Sp7/Osterix expression vector, pMAM-BSD-Sp7, was generated by RT-PCR using RNA extracted from MC3T3-E1 cells. The
set of primers are listed in Table S1. The PCR product was cloned
into the pGEM-T Easy vector and sequenced. The correct plasmid
was digested with KpnI/NotI, and then subcloned into the mammalian expression vector, pMAM-BSD.
2.4. Transient transfection and luciferase Assays
The cells were plated at a density of 2  105 per 35-mm dish
18 h before transfection. For transient transfection, 0.5 lg of plasmid DNA was transfected into these cells by using the LipofectAMINE Plus reagent system. Plasmid pRL-TK vector (Promega)
was always cotransfected as an internal control for transfection efﬁciency. After an additional cultivation for 48 h, the transfected cells
were harvested, lysed, centrifuged to pellet the debris, and subjected to luciferase assay. The luciferase activities were measured
as chemiluminescence in a luminometer (Lumat LB 9507, PerkinElmer Life Sciences) using the Dual Luciferase Reporter Assay System(Promega) according to the manufacturer’s protocol. The cotransfection experiments were performed using 1.5 lg of Sp1 or Sp7/
Osterix expression plasmids. Mithramycin A was incubated with aconcentration of 100 nM for 24 h before the luciferase assays.
2.5. Chromatin immunoprecipitation (CHIP) assay
The CHIP assays were performed using a chromatin immunoprecipitation assay kit (Upstate Biotechnology, Lake Placid, NY)
according to the manufacturer’s protocol. The anti-Sp1 and antiSp7/Osterix antibodies were purchased (Cosmo Bio, Tokyo, Japan).
The primer sets are listed in Table S1. The procedure was described
previously [11].
2.6. siRNA transfection
The siRNA cocktails targeting mouse Sp1 or mouse Sp7/Osterix
were purchased (Santa Cruz Biotechnology Inc, Sant Cruz, CA, USA).
The mouse MC3T3-E1 osteoblastic cells were transfected using

Fig. 1. Transactivation of the Col5a3 by overexpression of Sp7/Osterix in MC3T3-E1.
(A) An RT-PCR analysis of Sp7/Osterix transcripts from MC3T3-E1 cells transfectedwith the Sp7/Osterix expression construct (lane 2) or with the control empty
plasmid (lane 1). (B) A schematic illustration of the luciferase reporter constructs.
The mut1-Luc is mutated in the Sp1 binding site of pGL4.10 337/+92-Luc
construct. (C) The effect of Sp7/Osterix expression construct and mithramycin A(100 nM) on the activity of Col5a3 promoter constructs. The relative luciferase
activities were normalized against the activity of the pGL4.10 337/+92-Luc
construct without any additional treatment. Data are means ± S.D. of three
independent experiments. The asterisks indicate statistically signiﬁcant results(p < 0.05).

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Col5a3 gene [11]. The overexpression of Sp7/Osterix was veriﬁed
by a PCR reaction (Fig. 1A). Sp7/Osterix greatly activated the337/+92 promoter luciferase reporter gene (p < 0.05) (Fig. 1B
and C). This increase was cancelled by the addition of mithramycin
A, which recognizes GC-rich promoter regions and interferes withthe binding of the Sp family [12]. However, it had no effect on thecells that were cotransfected with mut1-Luc, in which the Sp1binding site of the promoter of the luciferase reporter gene wasmutated (Fig. 1B and C).
3.2. The effect of Sp7/Osterix on the promoter activity and endogenous
expression of the Col5a3 gene
We previously demonstrated that Sp1 binds to the Col5a3 promoter and that Sp1 increased the activity [11]. To compare the505

effect of Sp1 and Sp7/Osterix, we overexpressed Sp1 and Sp7/
Osterix. The expression was detected by a RT-PCR reaction withprimers that are speciﬁc for either Sp1 or Sp7/Osterix (Fig. 2A).
The amount of overexpressed Sp1 and Sp7/Osterix gene was
examined by real-time PCR and no difference was observed between both expressed amounts (data not shown). The overexpression of both Sp1 and Sp7/Osterix increased the promoter
activities (Fig. 2B) and endogenous expression (Fig. 2C) of the
Col5a3 gene in MC3T3-E1 cells. The effect of Sp7/Osterix overexpression was more substantial than that of Sp1 overexpression.
The overexpression of Sp7/osterix transactivated the Col5a3 promoter by approximately 4-fold, while overexpression of Sp1transactivate the Col5a3 by approximately 2-fold. Similarly, theoverexpression of Sp7/Osterix signiﬁcantly increased the endogenous mRNA levels of Col5a3 by approximately 3-fold, while

Fig. 2. (A) RT-PCR was performed with total mRNA from MC3T3-E1 cells which were transfected with overexpression vector of Sp1 (lane 2) and Sp7/Osterix (lane 3). The
empty vector was transfected as a control. (lane 1). Comparison of effect of the Sp1 and Sp7/Osterix expression constructs on (B) the activity of Col5a3 promoter using pGL4.10
337/92-Luc construct and on (C) the endogenous mRNA levels of Col5a3. (D) RT-PCR was performed with total mRNA from the MC3T3-E1 cells, which were transfected withsiRNA against Sp1 (lane 2), Sp7/Osterix (lane 3), and both (lane 4). A comparison of the effect of selective inhibition of Sp1 and Sp7/Osterix on (E) the activity of the Col5a3
promoter using the pGL4.10 337/92-Luc construct and (F) on the endogenous mRNA levels of Col5a3. Real-time PCR was performed to examine the amount of endogenous
mRNAs. GAPDH were used to normalize Col5a3 expression levels. Data are means ± S.D. of at least three independent experiments. *p < 0.05 compared with the control.

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overexpression of Sp1 increased the levels by approximately 1.5fold.
3.3. Selective inhibition of Sp7/osterix by siRNA
To examine the relative contribution of Sp1 and Sp7/Osterix
to Col5a3 transcription in MC3T3-E1 cells, selective inhibitionby speciﬁc siRNA was performed. Before inhibition, the expression of Sp1 was approximately ﬁve times higher than that of
Sp7/Osterix in the cells (data not shown). The siRNA treatmentof Sp1 or Sp7/Osterix decreased the promoter activity and theendogenous mRNA levels of the Col5a3 gene (p < 0.05)(Fig. 2D–F). The inhibition of Sp1 or Sp7/Osterix, singly and combination, decreased the promoter activity as well as theFig. 3. (A) Expression of Sp1 and Sp7/Osterix during MC3T3-E1 osteoblast differentiation. An RT-PCR analysis for Osteocalcin (Oc), Sp1, Sp7/Osterix and Col5a3 gene on
0, 2, 4 and 8 days of differentiation. b-Actin was included as a positive control for the PCR. PCR was carried out for 25 cycles. (B) The relative luciferase activity ofCol5a3 promoter using pGL4.10 –337/92-Luc and mut1-Luc. Data are means ± S.D. of three independent experiments. Asterisks indicate statistically signiﬁcant results(p < 0.05). (C) A CHIP assay was performed to examine the binding of Sp1 and Sp7/Osterix to the Col5a3 promoter at 0 and 8 days. All of the immunoprecipitated
DNA fragments were analyzed by a PCR reaction with primer sets, p1 and p2, for the 293 to 68 in the proximal promoter region of the Col5a3 gene and anotherset of control primers, p3 and p4, which amplify the segments that were located approximately 4 kb upstream. IgG was used as a control. (D) The co-overexpressionof Sp1and Sp7/Osterix in MC3T3-E1. RT-PCR analysis after the transfection with different ratios of Sp7(Osterix)/Sp1. (E) The relative luciferase activity of Col5a3 core
promoter using pGL4.10 337/92-Luc construct. (F) The endogenous mRNA levels of Col5a3. Real-time PCR was performed to examine the amount of endogenous
mRNAs. GAPDH were used to normalize Col5a3 expression levels. Data are means ± S.D. of at least three independent experiments. *p < 0.05 compared with the ratios
1:5 of Sp7(Osterix)/Sp1.

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endogenous mRNA levels by approximately 20% and 50%,
respectively.
3.4. Activation of Col5a3 gene during the process of MC3T3-E1 celldifferentiation by Sp7/Osterix
To further examine the activation of Col5a3 gene by Sp7/Osterix
in osteoblast cells, the cell culture model of osteoblast differentiation was used. Preosteoblast cells, MC3T3-E1, were differentiatedfor this purpose. As shown in Fig. 3A, osteocalcin, an osteoblast differentiation marker, was observed to markedly increase in the process of the differentiation of MC3T3-E1 cell. The Sp7/Osterix
expression was markedly increased, while that of Sp1 constantly
demonstrated a small amount. The expression of Col5a3 gene also
increased during osteoblast differentiation. The activity of Col5a3
promoter with 337/+92-Luc construct increased approximately
3.5-fold, but it did not change with mut1-Luc (Fig. 3B). Consistent
with these data, CHIP assay revealed that Sp7/Osterix increasinglybind to the Col5a3 core promoter sequence in vivo during differentiation (Fig. 3C).
Finally, to conﬁrm that Sp7/Osterix is responsible for activationof the Col5a3 gene rather than Sp1, the co-overexpression of theSp7Osterix and Sp1 with the different ratios was performed(Fig. 3D–F). The activity of Col5a3 core promoter as well as theendogenous mRNA levels increased along with the change in theratio of Sp7(Osterix)/Sp1from 1:5 to 5:1 (Fig. 3E and F).
4. Discussion
Sp7/Osterix is a recently identiﬁed zinc ﬁnger-containing transcription factor that belongs to Sp family [9]. It is expressed in all of
the developing bones, and it is essential for osteoblast differentiation and bone formation. Therefore, Sp7/Osterix was thought to be
involved in regulating the Col5a3 gene in osteoblastic cells. In thisstudy, we compared the contribution of both Sp1 and Sp7/Osterix
to the regulation of the Col5a3 gene. For this purpose, the osteoblast differentiation model was used. The results showed the activation of the Col5a3 gene was dependent on increasing amounts ofamount of Sp7/Osterix but not Sp1 during osteoblast differentiation (Fig. 3A–C). This result was conﬁrmed with experiments inwhich the cells overexpressed different ratios of Sp7(Osterix)/Sp1(Fig. 3D–F). Conversely, selective inhibition by speciﬁc siRNA of
Sp1 and Sp7/Osterix showed similar degree of inhibition although
the expression of Sp1 was dominant in MC3T3-E1 cells (Fig. 2D–F).
These data therefore showed that Sp7/Osterix promotes theexpression of the collagen gene, Col5a3, in comparison to Sp1 in
osteoblastic cells.
Sp7/Osterix activated Col1a1 promoter activity by forming acomplex with NFAT (nuclear factor of activated T cells) [13]. Goto
et al. showed that the transcription factors, Sp1, Sp3, and Sp7/
Osterix, regulate COL11A2 gene expression through its proximal
promoter in osteoblastic cells [14]. Other a chains of type V collagen, a1(V) and a2(V), are expressed in osteoblastic cells [15]. The
Sp1 site was identiﬁed in the mouse Col5a1 gene [16]. Therefore,
Sp7/Osterix might bind to the Sp1 site of the Col5a1 gene andthereby regulate it in osteoblastic cells. Sp7/Osterix should be involved in the regulation of various collagen genes which are expressed in bone.
Gene transcription is controlled by a series of complex interactions of many transcription factors. The inhibition with siRNA(Fig. 2D–F) and deletions and/or mutations that removed the binding sites of Sp1 and CBF/NF-Y [11] did not completely eliminate thepromoter activity of the Col5a3 gene. This suggested that other factors or mechanisms are involved in the activation of the Col5a3
gene in bone tissues. Runx2/Cbfa1 is also identiﬁed as a mastertranscription factor for osteoblast differentiation. It acts upstream

Fig. 4. A schematic illustration of the activation of the Col5a3 gene by the Sp family
during osteoblast differentiation.

of Sp7/Osterix and is needed for an early step of osteoblast differentiation [17]. Runx2/Cbfa1 contributes to the osteoblast-speciﬁc
expression of both type I collagen genes. It binds to the regions
at around 1347 bp in the mouse a1(I) and +12 bp in the mousea2(I) promoters [18]. ATF4 and b-catenin also act downstream of
Runx2/Cbfa1 [17]. ATF4 is a ubiquitous protein that is expressedin many cells, but it also interacts with Runx2/cbfa1 to regulate
the transcriptional activity of osteocalcin. One Wnt protein,
Wnt10b, was found to induce the expression of Runx2/cbfa1 andSp7/Osterix. Runx2/Cbfa1 could be responsible for the activationof the Col5a3 promoter in the early stages of osteoblast differentiation. The factors, such as ATF4 and b-catenin, that are ubiquitously expressed but which also interact with speciﬁc factors inosteoblastic cells may also be involved in the regulation of the
Col5a3 gene. In conclusion, Sp7/Osterix was observed to participate
in the activation of the Col5a3 promoter during osteoblast differentiation (Fig. 4). This ﬁnding should provide some clues regarding
the mechanism of bone formation.
Acknowledgments
We thank Ms. S. Adachi for her valuable technical support, andthe staff members of Research Promotion Project, Oita University.
This work was supported by Grants-In-Aid for Scientiﬁc Research(No. 20390402 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 doi:10.1016/j.bbrc.2010.02.171.
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