Matrix Biology 29 (2010) 701–706

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Matrix Biology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t b i o

Sp7/Osterix is involved in the up-regulation of the mouse pro-α1(V) collagen gene(Col5a1) in osteoblastic cells
Yun-Feng Wu, Noritaka Matsuo, Hideaki Sumiyoshi, Hidekatsu Yoshioka ⁎Department of Matrix 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 July 2010
Received in revised form 21 September 2010
Accepted 23 September 2010
Keywords:
Type V collagen
Sp7/Osterix
Gene regulation
Osteoblastic cell
Bone formationa b s t r a c t
Sp7/Osterix, a transcription factor whose expression is restricted in osteoblasts, belongs to the Sp family oftranscription factor that bind to G/C-rich sequences. Previous studies have identiﬁed a Sp1binding site in theproximal promoter region of the mouse Col5a1 gene, but it did not activate or repress this gene in a mouseﬁbroblast cell line and a human rhabdomyosarcoma cell line. The purpose of the present study was to clarify
the involvement of Sp7/Osterix in the mouse Col5a1 gene. A functional analysis revealed that mutation of theSp1 binding site speciﬁcally decreased the promoter activity in osteoblastic cells. An overexpression of Sp7/
Osterix signiﬁcantly increased the promoter activity and the endogenous mRNA levels of the Col5a1 gene inosteoblastic cells. Conversely, siRNA-mediated knockdown of Sp7/Osterix decreased the promoter activity
and the endogenous mRNA levels of the Col5a1 gene. These effects on promoter activity were canceled whenthe mutant construct of Sp1 binding site was introduced. Consistent with these data, the experiments using anosteoblast differentiation model showed increased promoter activity and endogenous mRNA levels, alongwith increased Sp7/Osterix during differentiation. Therefore, type V collagen appears to be involved in boneformation.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
Bone formation is a tightly regulated process which occurs whenmesenchymal precursor cells differentiate to osteoblasts. Osteoblasts
are responsible for two types of bone formation: intramembranousand endochondral ossiﬁcation. Osteoblasts can directly differentiate
from mesenchymal condensations in intramembranous ossiﬁcation,
whereas cartilage formation is an intermediate step in endochondral
ossiﬁcation. In both cases, osteoblasts play a central role in the
production of the extracellular matrix and the mineralization of thebone matrix. Recent molecular genetic studies in mice and humans
have demonstrated a role for transcription factors that govern boneformation (Karsenty, 2003; Nakashima and de Cromrrugghe, 2003;
Marie, 2008). Osteoblast differentiation from mesenchymal precursors is controlled by a hierarchy of transcription factors. Runx2 andSp7/Osterix are transcription factors that are expressed in osteoblasts.
Runx2 is a member of the Runt family of transcription factors and isexpressed in mesenchymal cells at the beginning of skeletal
development, and is present in osteoblasts throughout differentiation(Ducy et al., 1997; Komori et al., 1997; Otto et al., 1997). Sp7/Osterix
acts downstream of Runx2. Sp7/Osterix, which is a zinc ﬁngercontaining transcription factor, belongs to the Sp family of transcrip-

Abbreviation: CHIP assay, chromatin immunoprecipitation assay.
⁎ Corresponding author. Tel.: + 81 97 586 5670; fax: +81 97 586 5674.
E-mail address: hidey@oita-u.ac.jp (H. Yoshioka).
0945-053X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.matbio.2010.09.002

tion factors that bind to G/C-rich sequences and activate mRNA
synthesis from genes containing these functional recognition sites(Nakashima et al., 2002).
Collagens are the major constituents of extracellular matrices andare critical for the formation and function of the organs in the body(van der Rest and Garrone, 1991). Type V collagen is a quantitatively
minor ﬁbrillar collagen with a broad tissue expression. It is present intissues where type I collagen is expressed. Type V collagen isincorporated into the ﬁbrils of the more abundant type I collagen, andacts as a regulator of the size and the shape of the ﬁbrils (Birk, 2001).
There are several type V isoforms that differ in chain composition.
The major isoform is [α1(V)]2α2(V) which is present in many tissues. In
addition, the [α1(V)]3 homotrimer and the α1(V)α2(V)α3(V) heterotrimer have also been identiﬁed (Haralson et al., 1980; Rhodes andMiller, 1981). Defects in the human COL5A1 and COL5A2 genes havebeen identiﬁed in half of the cases of classic Ehlers–Danlos syndrome(EDS, type I/II) (Toriello et al., 1996; Michalickova et al., 1998).
Characterization of the core promoter of the mouse Col5a1 genehas revealed that the CBF/NF-Y factor acts as a transcriptional
activator of the Col5a1 gene in the mouse NIH3T3 cell and thehuman rhabdomyosarcoma A204 cell (Sakata-Takatani et al., 2004).
Two GC-rich domains were also found in the core promoter of themouse Col5a1 gene. Sp1 bound to the proximal GC-rich domain, but
did not activate or repress the Col5a1 gene in these cell lines. In thepresent study, Sp7/Osterix, which binds to GC-rich domains, in theactivation of Col5a1 gene during osteoblast differentiation, wasexamined.

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2. Results
2.1. Functional analysis of the Col5a1 gene using the MC3T3-E1 cell line
To examine the effect of the Sp1 binding site in the Col5a1 inosteoblastic cells, a functional analysis of Col5a1 gene was performed
using MC3T3-E1 cells. We used wild-type and mutant constructs thatwere previously generated (Fig. 1A) (Sakata-Takatani et al., 2004).
The m1-Luc, which has a mutated Sp1 binding site, caused an
approximately 50% decrease in promoter activity in MC3T3-E1 cells(Fig. 1B). However, it had no effect in A204 and NIH3T3 cells, as seen
in a previous study (Fig. 1C and D) (Sakata-Takatani et al., 2004). The
m2-Luc, which was mutated in the CBF/NF-Y site, had decreased
promoter activity, and the m3-Luc, which had a mutated downstream
Sp1-like binding site, had no effect on the promoter activity in any of
the three cell lines (Fig. 1B, C and D).

signiﬁcant differences were observed between the expressedamounts (Fig. 2A and B). The overexpression of both Sp1 and Sp7/
Osterix increased the endogenous expression of the Col5a1 gene in
MC3T3-E1 cells (Fig. 2C). The effect of Sp7/Osterix overexpression
was approximately two times greater than that of Sp1 overexpression.
Conversely, the selective inhibition of Sp1 and Sp7/Osterix byspeciﬁc siRNAs was performed. No differences were observed
between inhibition by siRNA knockdown of Sp1 and Sp7/Osterix(Fig. 3A and B). siRNA knockdown of Sp1 or Sp7/Osterix decreased theendogenous mRNA levels of the Col5a1 gene by 35 to 40% (p b 0.05)(Fig. 3C). In contrast to the overexpression experiments mentioned
above (Fig. 2C), the inhibition by siRNA-mediated knockdown of Sp1and Sp7/Osterix was similar. This may be due to the abundance ofsiRNA to Sp1 and Sp7/Osterix, comparing to the corresponding
mRNAs.
2.3. The effect of Sp7/Osterix on the promoter activity of the Col5a1 gene2.2. The effect of Sp7/Osterix on endogenous expression of the Col5a1
gene
To examine the effect of Sp1 and Sp7/Osterix, we overexpressed
Sp1 and Sp7/Osterix in MC3T3-1 cells. The amounts of overexpressed
Sp1 and Sp7/Osterix were examined by real-time PCR, and noFig. 1. Functional analysis of the proximal Col5a1 promoter. (A) A schematic illustrationof the luciferase reporter constructs. mut1, mut2 and mut3 are mutated in the Sp1, CBF/
NF-Y and Sp1-like binding site of the WT(−231/+39-Luc) construct (Sakata-Takatani
et al., 2004). Transient transfections with the wild-type and the mutant constructs werecarried out in MC3T3-E1 (B), NIH3T3 (C) and A204 (D) cells. The histograms indicatedthe percentage of activity normalized to the internal control and are shown relative tothe activity of the wild-type construct transfected into each cell line. Data are presented
by the mean ± S.D. of at least three independent experiments. *p b 0.05 compared withthe wild-type.

To examine the effect of Sp7/Osterix on the promoter activity of
the Col5a1 gene, Sp7/Osterix was cotransfected with the wild-type
and three mutant luciferase constructs into MC3T3-E1 cells. Sp7/
Osterix signiﬁcantly activated the wild-type promoter of the luciferase reporter gene (Fig. 4A). However, transfection had no effect on thecells that were cotransfected with m1-Luc, in which the Sp1 bindingsite of the promoter of the luciferase reporter gene was mutated(Fig. 4B). However, Sp7/Osterix activated m2-Luc and m3-Luc
transcription, in which the CBF/NF-Y and Sp1-like binding sites,
respectively, was mutated (Fig. 4C and D). The activity using the m3Luc construct was similar to that using the wild-type construct, but
the luciferase activity using the m2-Luc construct was lower.
Conversely, the selective inhibition of siRNA Sp7/Osterix after Sp7/
Osterix overexpression was performed. The Col5a1 promoter activity
using the wild-type construct was decreased by nearly 50% (Fig. 5A).
Similarly, the levels using m2-Luc and m3-Luc also decreased

Fig. 2. (A, B) The real-time RT-PCR analysis was performed with total mRNA from
MC3T3-E1 cells which were transfected by overexpressing Sp1 (lane 2 in A) and Sp7/
Osterix (lane 2 in B). The empty vector was transfected as a control (lane 1 in A and B).
(C) Effects of the Sp1 and Sp7/Osterix expression constructs on the endogenous mRNAlevels of Col5a1. Data are presented by the mean ± S.D. of at least three independent
experiments. *p b 0.05 compared with the control.

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Fig. 3. (A, B) The real-time RT-PCR analysis was performed with total mRNA from theMC3T3-E1 cells, which were transfected with siRNA targeted against Sp1 (lane 2 in A)
and Sp7/Osterix (lane 2 in B). (C) The effect of selective inhibition of Sp1 and Sp7/
Osterix on the endogenous mRNA levels of Col5a1. Data are presented the mean ± S.D.
of at least three independent experiments. *p b 0.05 compared with the (−) control.

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Fig. 5. Effect of inhibition with siRNA against Sp7/Osterix on luciferase activity. The
siRNAs were cotransfected with the luciferase reporter constructs, WT (−231/+39-Luc)(A), m1-Luc (B), m2-Luc (C) and m3-Luc (D). The relative luciferase activities werenormalized to the non-treated (−) siRNA samples. Note that the inhibition is lesseffective in the construct that is mutated (C) than is intact (D) in the CBF/NF-Y bindingsite. Data are presented by the mean ± S.D. of at least three independent experiments.
*p b 0.05 compared with the (−) control.

approximately 20% and 40%, respectively (Fig. 5C and D). However, nochanges were seen in the activity of m1-Luc (Fig. 5B).
2.4. Activation of the Col5a1 gene during the process of MC3T3-E1 celldifferentiation by Sp7/Osterix
To further examine the activation of Col5a1 gene by Sp7/Osterix inosteoblast cells, the cell culture model of osteoblast differentiation
was used. MC3T3-E1 was differentiated with β-glycerophosphate forthis purpose. The Sp7/Osterix expression was markedly increased,
while the expression of Sp1 was consistent with a small level (Fig. 6A).
The expression of Col5a1 gene also increased during osteoblast
differentiation (Fig. 6B). The activity of the Col5a1 promoter withthe wild-type construct increased by approximately 3.5-fold on Day
8 after differentiation, but did not change with mut1-Luc (Fig. 6C).
Consistent with these data, a CHIP assay revealed that Sp7/Osterix
was clearly bound to the Col5a1 core promoter sequence in vivo on
8 day after differentiation (Fig. 6D and E).
3. Discussion

Fig. 4. Transactivation of the Col5a1 gene by the overexpression of Sp7/Osterix in
MC3T3-E1 cells. The Sp7/Osterix expression (Sp7) and empty construct (cont) were
cotransfected with the luciferase reporter constructs, WT (−231/+39)-Luc (A), m1-Luc(B), m2-Luc (C) and m3-Luc (D). The relative luciferase activities were normalizedagainst those of the samples transfected with an empty expression vector. Note that theactivity is lower in the mutated construct (C) than in the intact (D) in the CBF/NF-Y
binding site. Data are presented by the mean ± S.D. of at least of three independent
experiments. *p b 0.05 compared with the control.

The extracellular matrix of bone is primarily composed of type I
collagen, with lesser amounts of other components such as proteoglycans. Type V collagen is cross-linked to type I collagen in the bone
(Niyibizi and Eyre, 1989). During the ossiﬁcation process in themouse, high proα1(V) expression was observed in the osteoblasts oflong bones, vertebrae and calvaria, which indicated that type V
collagen was involved in the bone formation of both endochondraland intramembranous ossiﬁcation (Roulet et al., 2007). Our previous
study identiﬁed the presence of Sp1 and Sp1-like binding sites in theproximal promoter of the Col5a1 gene (Sakata-Takatani et al., 2004).
However, Sp1 did not activate the gene in the NIH 3 T3 cells or in theA204 rhabdomyosarcoma cells. In the present study, we examined704

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Fig. 6. Expression of Sp1 and Sp7/Osterix during MC3T3-E1 cell differentiation. An RT-PCR analysis for Sp1and Sp7/Osterix (A) and Col5a1 (B) gene on 0, 2, 4 and 8 days ofdifferentiation. β-actin was included as a positive control for the PCR. PCR was carried out for 25 cycles. (C) The relative luciferase activity of Col5a1 promoter using WT (−231/+39Luc) and m1-Luc. Data are presented by the mean ± S.D. of three independent experiments. *p b 0.05 compared with the WT-Luc on 0 day. (D) Positions of primers used for the CHIP
assay. Primer sets, p1 and p2, ampliﬁed between the −226 and +70 in the proximal promoter region of the Col5a1 gene and another set of control primers, p3 and p4, approximately
4 kb upstream. (E) The CHIP assay was performed to examine the binding of Sp7/Osterix to the Col5a1 promoter at 0 and 8 days. IgG was used as a control.

whether Sp7/Osterix, which belongs to the Sp family, bound to theregion and activated the genes in the osteoblastic cells. The results
showed that Sp7/Osterix bound to the proximal promoter Col5a1 gene
and activated the gene in osteoblastic cells. However, it did not
activate in the NIH 3 T3 cells or in the A204 rhabdomyosarcoma cell as
previously shown. Conversely, selective treatment by targeted siRNAknockdown of Sp7/Osterix inhibited the expression of the Col5a1 genein MC3T3-E1 cells (Fig. 3C). This experiment also revealed that theactivation of the Col5a1 gene was dependent on increasing amounts of
Sp7/Osterix during the differentiation of MC3T3-E1 cells (Fig. 6).
These data therefore showed that Sp7/Osterix promotes the expression of the Col5a1 gene in osteoblastic cells.
Sp7/Osterix is a zinc ﬁnger transcription factor and forms acomplex with the nuclear factor of activated T cells (NFAT), which
activates the Wnt/β-catenin signaling pathway and regulates bonemass in osteoblasts (Winslow et al., 2006). β-catenin is essential forthe differentiation of mature osteoblasts and, consequently, for boneformation (Caetano-Lopes et al., 2007). A complex of Sp7/Osterix and
NFAT bound to and activated the humanCOL1A1 gene (Koga et al.,2005). We and other investigators have recently demonstrated that
Sp7/Osterix binds to an Sp1 binding site in the proximal promoters ofthe mouse Col5a3 and the human COL11A2 gene and up-regulate

those genes, which are expressed in osteoblasts (Wu et al., 2010a;
Goto et al., 2006). Many collagen genes are expressed in osteoblastic
cells. The complex of Sp7/Osterix and NFAT might be bind to Sp1
binding sites and activate those collagen genes.
Gene transcription is controlled by a series of complex interactions ofmany transcription factors, which are expressed with ubiquitous andcell speciﬁc manners. Previous studies identiﬁed that the CBF/NF-Y
binding factor had basal activity in the Col5a1 gene in NIH 3 T3 and A204
cells (Sakata-Takatani et al., 2004). A functional analysis using the
mutant of CBF/NF-Y site showed that the promoter activity decreased inosteoblast as well as A204 and NIH 3 T3 cells (m2 in Fig. 1B, C and D). In
an experiment of the overexpression with Sp7 and the inhibition withsiRNA, the effects were lower in the mutant construct than in the intact
construct of CBF/NF-Y site even if Sp1 binding site was normal (Figs. 4
and 5). These data showed the involvement of CBF/NF-Y in the promoter
activity of the Col5a1 gene (Fig. 7). The CBF/NF-Y is ubiquitously
expressed, but might be required to coordinate the expression of types I
and V collagen in osteoblastic cells as well as in non-osteoblastic cells(Nagato et al., 2004; Wu et al., 2010a). Runx2, which is also identiﬁed asa master transcription factor for osteoblast differentiation, acts
upstream of Sp7/Osterix and is required for early steps of osteoblast
differentiation. Runx2 is a positive regulator of the osteoblast-speciﬁc

Y.-F. Wu et al. / Matrix Biology 29 (2010) 701–706

Fig. 7. Schematic illustration of the core promoter of Col5a in osteoblastic cells. The
bindings of the Sp7(Osterix)/Sp1 and CBF/NF-Y. The indicated sequences are critical forbinding the factors.

expression of both type I collagen genes, and binds to the regions
approximately 1.3 kb upstream in the α1(I) collagen gene and in theﬁrst exon of α2(I) collagen gene (Kern et al., 2001). It should also
regulate the Col5a1 gene, although it could not be identiﬁed in thecurrent study. ATF4 is another important transcription factor controlling
osteoblasts (Ameri and Harris, 2008). The mRNA of ATF4 is expressed inmany cells, but the protein is unstable and degraded in the majority ofcell types through ubiquitination except in osteoblasts, where it
accumulates (Yang and Karsenty, 2004). It interacts with Runx2 toregulate the transcriptional activity of osteocalcin. It affected thesynthesis of type I collagen, which was reduced in the ATF4-deﬁcientmice. These factors may also be involved in the regulation of the Col5a1
gene. In conclusion, Sp7/Osterix was observed to participate in theactivation of the Col5a1 promoter during osteoblast differentiation.
Further study is, however, still required to clarify the complexity of gene
regulations regarding bone formation.
4. Experimental procedures

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DNA was transfected into these cells by using the LipofectAMINE Plusreagent system. Plasmid pRL-TK vector (Promega) was always
cotransfected as an internal control for transfection efﬁciency. Afteran 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 chemiluminescencein 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 μg of Sp1 or Sp7/Osterix expression plasmids(Wu et al., 2010b).
4.5. 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-Sp7/Osterix antibodies werepurchased (Cosmo Bio, Tokyo, Japan). The primer sets are listed in Table
S1. The procedure was described previously (Matsuo et al., 2003).
4.6. siRNA transfection
The siRNA cocktails targeting mouse Sp1 or mouse Sp7/Osterix
were purchased (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).
The mouse MC3T3-E1 osteoblastic cells were transfected using
Lipofectamine 2000 (Invitrogen) to achieve a ﬁnal siRNA concentration of 50 nM.
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.matbio.2010.09.002.

4.1. Cell culture
The cell lines used in this study were mouse preosteoblast MC3T3-E1,
human rhabdomyosarcoma A204 and mouse NIH3T3 cells. Cells were
cultured in Dulbecco's modiﬁed Eagle's medium containing 10% heatinactivated fetal bovine serum (Sanko Junyaku, Tokyo, Japan). For
differentiation studies, MC3T3-E1 cells were fed at conﬂuence (time 0)
with the above medium additionally containing 10 μM β-glycerophosphate and 50 μg/mL of ascorbic acid. The medium for differentiation
was changed every 2 days.
4.2. 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 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). 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, and 60 °C for 1 min. The relative mRNA expression
levels were normalized against that of the GAPDH gene from the same
RNA preparations, using a comparative threshold cycle method. The
primer sets are listed in Table S1.
4.3. Chimeric plasmids
The luciferase constructs, −231/+39-Luc, m1-Luc, m2-Luc andm3-Luc, and the Sp1 and Sp7/Osterix expression plasmid werepreviously described (Sakata-Takatani et al., 2004; Wu et al., 2010b).
4.4. Transient transfection and luciferase Assays
The cells were plated at a density of 2 × 105 per 35-mm dish for
18 h before transfection. For transient transfection, 0.5 μg of plasmidAcknowledgements
We thank Ms. S. Adachi for her valuable technical support, and thestaff 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, andTechnology of Japan.
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