In Vitro Cell.Dev.Biol.—Animal (2016) 52:235–242
DOI 10.1007/s11626-015-9959-y

Sp1 upregulates the proximal promoter activity of the mousecollagen α1(XI) gene (Col11a1) in chondrocytes
Keijirou Watanabe 1,2 & Mariko Hida 1 & Takako Sasaki 1 & Hiroyuki Yano 3 &Kenji Kawano 2 & Hidekatsu Yoshioka 1 & Noritaka Matsuo 1

Received: 13 August 2015 / Accepted: 10 September 2015 / Published online: 20 October 2015 / Editor: Tetsuji Okamoto# The Society for In Vitro Biology 2015

Abstract Type XI collagen is a cartilage-specific extracellular matrix, and is important for collagen fibril formation andskeletal morphogenesis. We have previously reported that NFY regulated the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes (Hida et. al.
In Vitro Cell. Dev. Biol. Anim. 2014). However, the mechanism of the Col11a1 gene regulation in chondrocytes has notbeen fully elucidated. In this study, we further characterized
the proximal promoter activity of the mouse Col11a1 gene inchondrocytes. Cell transfection experiments with deletion andmutation constructs indicated that the downstream region of
the NF-Y binding site (-116 to +1) is also necessary to regulate
the proximal promoter activity of the mouse Col11a1 gene.
This minimal promoter region has no TATA box and GC-richsequence; we therefore examined whether the GC-rich sequence (-96 to -67) is necessary for the transcription
regulation of the Col11a1 gene. Luciferase assays using aseries of mutation constructs exhibited that the GC-rich sequence is a critical element of Col11a1 promoter activity in* Noritaka Matsuo
matsuon@oita-u.ac.jp
1

Department of Matrix medicine, Faculty of medicine, Oita
University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593,
Japan

2

Department of Oral and Maxillo-Facial Surgery, Faculty of medicine,
Oita University, 1-1, Idaigaoka, Hasama-machi, Yufu,
Oita 879-5593, Japan

3

Division of Radioisotope Research, Department of Research
Support, Research Promotion Project, Faculty of medicine, Oita
University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593,
Japan

chondrocytes. Moreover, in silico analysis of this region suggested that one of the most effective candidates was transcription factor Sp1. Consistent with the prediction, overexpressionof Sp1 significantly increased the promoter activity. Furthermore, knockdown of Sp1 expression by siRNA transfection
suppressed the proximal promoter activity and the expressionof endogenous transcript of the mouse Col11a1 gene. Taken
together, these results indicate that the transcription factor Sp1
upregulates the proximal promoter activity of the mouse
Col11a1 gene in chondrocytes.
Keywords Type XI collagen . Chondrocyte . Sp1 .
Transcription . Promoter

Introduction
The collagen superfamily plays an important role, not only instabilizing the tissues as structural components but also in
regulating a variety of biological functions, such as development, differentiation, proliferation, and morphogenesis. There
are more than 46 distinct α-chains of collagen genes, which
form at least 28 types of collagen proteins (Ricard-Blum2012). The fibrillar collagens (types I, II, III, V, XI, XXIV,
and XXVII) are divided into three subgroups, major (I, II, andIII), minor (V and XI), and new (XXIV and XXVII) fibrillar
collagens on the basis of their contents in tissues and phylogenetic analyses. Vertebrate fibrillar collagens are either restricted to cartilage (II, XI, and XXVII) or widely distributed
in non-cartilage tissues (I, III, V, and XXIV) (Exposito et al.
2010; Fang et al. 2012). Cartilage is a highly specialized tissue
and serves as a template for the growth and development ofbones. It contains an extensive extracellular matrix, which
included types II, IX, and XI collagen, and provides

236

WATANABE ET AL.

mechanical strength to resist compression in joint (Mendler
et al. 1989).
Type XI collagen is a fibrillar collagen mainly foundin the cartilage, and consists of three polypeptide
chains, α1(XI), α2(XI), and α3(XI). The α1(XI) andα2(XI) chains are genetically distinct products, whereas
the α3(XI) chain is an overglycosylated variant of theα1(II) chain, which is encoded by the type II collagengene (Ricard-Blum 2012). Type XI collagen copolymerizes with type II collagen within collagen fibrils
and is important for regulation of the fibril diameter andfunction in connective tissues (Mendler et al. 1989).
Therefore, the absence and/or mutations of the α1(XI)
chain result in abnormally thickened cartilage collagen
fibrils. In human disease, mutations in the COL11A1
gene have been identified in Stickler and Marshall syndromes, which are clinically characterized by alteredfacial appearance, eye abnormalities, joint alterations,and hearing loss (Annunen et al. 1999; Myllyharju
and Kivirikko 2001). Furthermore, fibrochondrogenesis,
which is short-limbed skeletal dysplasia, is also caused
by mutations in the COL11A1 gene (Tompson et al.
2010). These observations suggest that the expressionof Col11a1 is critical for maintaining normal structureand function of cartilage.
Among cartilage collagens, type II collagen is well characterized as a model gene of chondrogenic marker. The
COL2A1 gene expression is regulated by the transcriptional activator and/or repressor such as Sox9 (Bell et al. 1997;
Zhou et al. 1998), Sp1/3 (Ghayor et al. 2001; Renard et al.
2012), Egr-1 (Tan et al. 2003), c-Krox (Ghayor et al.
2000), and Ap2 (Niebler and Bosserhoff 2013) in boththe proximal promoter and the first intron. It has been reported that two chondrocyte-specific enhancer elements
were identified in the proximal promoter and the first intron of the Col11a2 gene and were regulated by Sox9
(Bridgewater et al. 1998; Liu et al. 2000). In addition, we
have first reported that NF-Y regulates the proximal promoter activity of the Col11a1 gene in chondrocytes (Hidaet al. 2014). However, the molecular mechanism of transcriptional regulation of the Col11a1 gene in chondrocytes
is not completely characterized yet.
In this study, we have additionally investigated the transcriptional regulation of the mouse Col11a1 gene inchondrocytes. Transient transfection assays, using deletionand mutation constructs, revealed that the downstream regionof the NF-Y binding site, especially in the GC-rich sequence,is necessary for the activation of basal transcription of themouse Col11a1 gene. In silico analysis of the GC-rich sequence suggested that a potential candidate was transcription
factor Sp1, and it was confirmed by cell transfection experiments with overexpression and/or knockdown of Sp1. Taken
together, these results demonstrated that Sp1 upregulates theproximal promoter activity of the mouse Col11a1 gene inchondrocytes.

Materials and Methods
Cell cultures. Rat chondrosarcoma (RCS) was used in thisstudy. Cells were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% heat-inactivated fetal bovine
serum at 37°C in humidified 5% CO2 and 95% air as described previously (Hida et al. 2014).
Construction of chimeric plasmids. All mouse Col11a1
promoter-luciferase constructs were derived from thepBACe3.6 RP24-512D13 clone as previously reported (Hidaet al. 2014). Nucleotide substitutions were generated by sitedirected mutagenesis as described previously (Matsuo et al.
2003).
For overexpression experiments, mouse full-length Sp1
cDNA was generated by RT-PCR using mouse embryoniccDNA library as a template. A sense primer was designedwith KpnI site sequence, and an antisense primer was
designed with NotI site sequence, respectively. The PCR
product was cloned into the pGEM-T Easy vector(Promega, Fitchburg, WI), followed by digestion with
KpnI and NotI, and subcloned into the KpnI/NotI site of
pcDNA3 mammalian expression vector (Invitrogen,
Carlsbad, CA).
For knockdown experiments, synthetic oligonucleotides
were inserted between the mouse U6 promoter and theterminator sequence of the pBAsi-mU6 vector (TAKARA,Tokyo, Japan) to generate a stem-loop type of smallinterfering RNA (siRNA) in the transfected cells. siDirect2.0
siRNA design software (http://sidirect2.rnai.jp/) was used to
select highly effective siRNA sequences of the targeting gene.
The sense target sequence for rat Sp1 (5’-GAGTAATGCCTAATATTCAGT-3’), followed by the hairpin loop sequence(5’-CTGTGAAGCCACAGATGGG-3’), and the antisense
target sequence were synthesized and cloned into the
BamHI/HindIII site of pBAsi-mU6 vector. The sense scramble sequence for negative control (5’-AGATCTCAAG
TTCCTCACACC-3’) was described previously (Huang
et al. 2008). All constructs were sequenced with an ABIPRISM 3100 Genetic Analyzer (Applied Biosystems,
Waltham, MA).
Cell Transfection and luciferase assay. The cells were plated
at a density of 1.5×105 cells per 35-mm dish 18 h before
transfection. For transient transfection, each of the plasmid
DNA was transfected into the cells by using the calcium
phosphate-mediated precipitation methods with somemodification (Chen and Okayama 1987).

237

TRANSCRIPTIONAL REGULATION OF THE MOUSE COL11A1 GENE IN CHONDROCYTESFor overexpression and/or knockdown experiments, thecells (1×106 cells) were mixed with the expression or knockdown plasmids with luciferase constructs. The cell suspension
with plasmid DNAs was added to 2-mm gap cuvettes and waselectroporated at 175 V for 5 ms using a NEPA electroporator
according to the manufacturer’s protocol (Nepa Gene, Chiba,
Japan).
Luciferase activity was assayed 24 h later by the DualluciferaseTM reporter assay system (Promega, WI) using aluminometer (Lumat, 9507, Berthold Technologies, Bad
Wildbad, Germany). The pRL-TK Renilla reniformis luciferase expression vector was used as an internal control for transfection efficiency. The relative luciferase activity of each construct was compared with the proximal promoter construct,
pGL3+1+68 for Figs. 1b and 3b or pGL3-366+68(wt) for
Figs. 2b and 4, respectively. Results were expressed asmeans±SE of five independent experiments.
RNA analysis. Total RNA was isolated from RCS using Isogen(Nippon Gene, Tokyo, Japan) according to the manufacturer’s
instruction. Reverse transcriptions were carried out using
Rever Tra Ace reverse transcriptase (TOYOBO, Tokyo,
Japan) with Random and Oligo(dT) primers, and the resulting

single-stranded cDNA molecules were PCR amplified using
gene-specific primers. For a quantification of mRNA, realtime PCR was performed using a LightCycler TaqMan Master(Roche, Indianapolis, IN). 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 using acomparative threshold cycle method (Page and Stromberg
2011).
The primers for Col11a1, Sp1, and GAPDH were as follows: Col11a1 sense, 5’-CCCGACTGTGACTTAACATCC3’, antisense, 5’-GTCCTCTTCTTCTTTGTGTAATTGG-3’;
Sp1 sense, 5’-GCTATAGCAAACACCCCAGGT-3’, antisense, 5’-CAGGGCTGTTCTCTCCTTCTT-3’; and GAPDH
sense, 5’-TGGGAAGCTGGTCATCAAC-3’, antisense, 5’GCATCACCCCATTTGATGTT-3’.

a-150
aggcctgtgA TTGGgtatga ccttcagtcg gcttgtcagt ttctcgggca< CCAAT(ATTGG) box >-100
cacaGGAGGC GGGAGGAGTG GGCGGAGGAG GGGGctgcgc gagcctcttg< GC rich sequence; -96 > -67 >-96-87-366+68(m1)
AAAAAT TTTT
-86
-77
-366+68(m2)
AAAAAT TTTT
-76
-67
-366+68(m3)
AAAAAT TTTTatgATTGGgt-366+68 (wt)aaatacggg-366+68 (mut)-50-256+68+1
tccagcccac ccacagcatg aagcctttag gggtgctcag tgctttcggc-116+68

b

+1+68

b

-366+68 (wt)-366+68 (wt)-366+68 (m1)-366+68 (mut)-366+68 (m2)-256+68-366+68 (m3)-116+68

0
+1+68

0.2

0.4

0.6

0.8

1

Relative Luciferase activity
0

2

4

6

8

10

Relative Luciferase activity

Figure 1. Determination of the minimal promoter region of the mouse
Col11a1 gene. (a): Schematic representation of the 5’-deletion andmutation constructs of the mouse Col11a1 promoter. pGL-366+68(mut)was mutated to the NF-Y binding site. (b): Deletion and mutation analysisof the mouse Col11a1 proximal promoter. Relative luciferase activities ofeach construct were assessed in transiently transfected RCS cells. A seriesof mouse Col11a1 promoter-luciferase constructs were transfected into
the cells with the pRL-TK vector as an internal control for transfection
efficiency. Relative luciferase activities were normalized against theactivity of the pGL+1+68 vector, and results are expressed as mean±
SE of five independent experiments.

Figure 2. The GC-rich sequence was necessary to regulate the proximal
promoter of the mouse Col11a1 gene. (a): Nucleotide sequence of theproximal promoter region of the mouse Col11a1 gene. The GC-richsequence (total 30 bp) was divided into three regions (10 bp): m1(-96
to -87), m2(-86 to -77), and m3(-76 to -67), respectively, and theseregions were replaced with the AAAAATTTTT sequence. The CCAAT(ATTGG) box and GC-rich sequences are shown by a capital letter. (b):Mutation analysis of the mouse Col11a1 proximal promoter. A series ofmutation luciferase constructs (m1, 2, 3) were transfected into RCS cellswith the pRL-TK vector as an internal control for transfection efficiency.
Relative luciferase activities were normalized against the Wt (pGL-366+
68) vector, and results are expressed as mean±SE of five independent
experiments.

238
Figure 3. Transcription factor
Sp1 upregulated the proximal
promoter activity of the mouse
Col11a1 gene. (a) Nucleotide
sequence of the putative Sp1binding sites in the proximal
promoter region. The JASPAR
database indicated three bindingsites: -96 to -86, -83 to -73, and 77 to -67, and the Genomax
database indicated one binding
site: -86 to -70, respectively. The
putative Sp1 binding sites areshown by a bold letter. (b) Effectof overexpression of Sp1 on themouse Col11a1 promoter activity.
A series of 5’-deletion luciferase
constructs were co-transfectedwith the Sp1 expression vector(pcDNA-Sp1) or pcDNA3 empty
vector into RCS cells. Relative
luciferase activities werenormalized against pGL+1+68
treated with the pcDNA3 empty
vector, and results are expressed
as mean±SE of five independent
experiments. White: cotransfection with the pcDNA-Sp1
expression vector, Black: cotransfection with the pcDNA3
empty vector.

WATANABE ET AL.

a

-100
-61
cacaGGAGGCGGGAGGAGTGGGCGGAGGAGGGGGctgcgc( GC rich sequence; -96 > -67 )-96-87-366+68(m1) AAAAATTTTT-86-77-366+68(m2)
AAAAATTTTT-76-67-366+68(m3)
AAAAATTTTT<JASPAR database>-96-86
Sp1 site
ggaggcgggag-83-73
Sp1 site
gtgggcggagg-77-67
Sp1 site
ggaggaggggg<Genomax database>Sp1 site-86-70
ggagtgggcggaggaggb-366+68-256+68-116+68
+1+68
0

10

20

Relative Luciferase activity
Results
Downstream region of the NF-Y binding site is necessary to
upregulate the proximal promoter activity of the mouse Col11a1 gene. We previously reported that NF-Y regulates theproximal promoter activity of the mouse Col11a1 gene inchondrocytes (Hida et al. 2014). As shown in Fig. 1b, however, pGL-116+68 had slightly increased the luciferase activity compared with pGL+1+68, which has no promoter region. Furthermore, pGL-366+68(mut), in which the NF-Y
binding site was mutated (Fig. 1a), failed to inhibit the proximal promoter activity completely, and the degree of luciferase
activity was almost the same as pGL-116+68 (Fig. 1b). These
results indicated that the additional region between +1 and -116 was also necessary to the proximal transcriptional activityof the mouse Col11a1 gene.
GC-rich sequence is necessary to upregulate the proximal
promoter activity of the mouse Col11a1 gene. In eukaryotes,
the TATA box is a highly conserved element, and locatedapproximately -35 to -25 bp upstream of the transcriptional
start site. However, the Col11a1 gene has no TATA sequencesin the proximal promoter region as shown in Fig. 2a. In thisstudy, we focused on the 30-bp GC-rich sequence (-96 to -67)
in the downstream region of the NF-Y binding site, because
the GC-rich sequences are also important regulatory elementsin the promoter region of many genes, especially the TATAless genes. A series of mutation constructs (m1, -96 to -87;

Relative mRNA expression Relative Luciferase activity Relative mRNA expression

TRANSCRIPTIONAL REGULATION OF THE MOUSE COL11A1 GENE IN CHONDROCYTES

Sp1

1

0

Luc

1

0

Col11a1

1239

both databases indicated that one of the most promising candidate was transcription factor Sp1, which is probably helping
recruit the TATA-binding protein in the TATA-less gene.
To confirm whether Sp1 regulated the proximal promoter
activity of the mouse Col11a1 gene in chondrocytes, a seriesof 5’-deletion luciferase constructs were co-transfected with
Sp1 overexpression plasmid into RCS cells. As shown in
Fig. 3b, all luciferase constructs with the GC-rich sequence
had significant increase in the luciferase activity, whereas
pGL+1+68 lacking the GC-rich sequence had no effect on
the luciferase activity by overexpression of Sp1. The ratio of
the relative luciferase activity was also summarized in Table 1.
The maximum ratio was exhibited by pGL-116+68, and theratio was not increased in both pGL-256+68 and pGL-366+
68, suggesting that the upstream region of the NF-Y bindingsite was not affected by overexpression of Sp1.
Consistent with these observations, knockdown of Sp1 expression by siRNA transfection also significantly reduced theproximal promoter activity of the mouse Col11a1 gene andthe expression of the endogenous mouse Col11a1 transcript(Fig. 4). Taken together, our data indicate that the transcription
factor Sp1 upregulates the proximal promoter activity of themouse Col11a1 gene in chondrocytes.

Discussion0pBA-scrpBA-rSp1Figure 4. Knockdown of Sp1 expression downregulated the Col11a1
promoter activity and the expression of endogenous Col11a1 transcript
in chondrocytes. pGL-366+68(wt) construct was co-transfected with theSp1 siRNA expression vector (pBA-Sp1) or the scramble siRNA
expression vector (pBA-scr) into RCS cells. Relative luciferase
activities and the expression level of the endogenous Col11a1 transcript
were normalized against pGL-366+68 co-transfected with pBA-scr, andresults are expressed as mean±SE of five independent experiments.

m2, -86 to -77; and m3, -76 to -67) were generated as shown in
Fig. 2a, and then luciferase assays were carried out. Figure 2b
showed that all mutation constructs have significant reductions in the luciferase activity, and especially, m2 (-86 to 77, mutated in the center of the GC-rich sequence) has a dramatic reduction (70–80%) compared with pGL-366+68(wt).
These results suggested that the GC-rich sequence was also
important for the promoter activity of the Col11a1 gene.
Sp1 upregulates the proximal promoter activity of the mouse
Col11a1 gene. Next, we performed in silico analysis using
JASPAR database (http://jaspardev.genereg.net/) andGenomatix (http://www.genomatix.com/) to identify putativetranscriptional factors and their binding sites involved in the
GC-rich sequence. As shown in Fig. 3a, in silico analysis using

Chondrogenesis is an important biological event in skeletal
development and tissue formation. It starts from multipotent
mesenchymal cells, and then these cells proliferate, followedby differentiating into chondrocytes. During these processes,
the cells secrete chondrogenic matrices such as types II, IX,
XI, and XXVII collagens that surround themselves. Therefore, to understand chondrocyte differentiation and cartilage
formation, it is necessary to study the regulation of cartilage
collagens in chondrocytes (de Crombrugghe et al. 2000).
Among them, type XI collagen, particularly in Col11a1, playa critical role in chondrocyte differentiation and skeletal organization. That is because the α1(XI) chain-deficient mice (chomice), although α1(II) chain is produced normally, showed
Table 1.
Sp1

Ratio of the relative luciferase activity by overexpression of

Col11a1 construct

Ratio of relative luciferase activity

pGL+1+68
pGL-116+68

1.02±0.12
1.89±0.06

pGL-256+68

1.60±0.29

pGL-366+68

1.63±0.18

Data represented the ratio of pcDNA-Sp1 to pcDNA3 as referred to in
Fig. 3b. Results were expressed as means±SE of five independent
experiments

240

WATANABE ET AL.

irregular collagen fibrils in their cartilage, resulting in achondrodystrophic phenotype (Li et al. 1995).
We previously reported that the transcription factor NF-Y
regulates the proximal promoter activity of the Col11a1 genein chondrocytes. NF-Y specifically binds to the CCAAT (orATTGG) sequences, and regulates a wide range of genes, both
ubiquitous and/or tissue-specific genes. In most cases, NF-Y
cooperatively works together with other transcription factors
in the promoters, resulting in the transcriptional activation
and/or repression (Dolfini et al. 2012). In this study, we provided additional evidence that the transcription factor Sp1bound to the GC-rich sequence in the proximal promoter,and upregulated the transcription of the Col11a1 gene inchondrocytes. It has been assumed that the transcription initiation of the eukaryotic gene could be directed by the TATAbox or by the CG/GT box in the TATA-less gene (Dikstein2011). In this context, it is one possibility that Sp1 may recruit
TATA-binding proteins and locates them in the correct transcription start site, since the Col11a1 gene has no TATA box
as shown in Fig. 2. In addition to the machinery of recruiting
TATA-binding proteins, the Sp1-like family is able to regulate
many eukaryotic promoters as the transcriptional activators or
repressors. Their regulatory mechanisms of Sp1 transcription
factor is as follow: as activators, Sp1 binds to the GC elementsin the promoter and/or enhancer regions with other transcription mediators, and as repressors, Sp1-like repressors such as
Sp3 or other transcription mediators compete the binding to
GC elements with Sp1 (Zhao and Meng 2005). Many transcription factors were shown to regulate the transcription with
the Sp1-like family, and the combination between NF-Y andSp1 has been also reported to activate and/or repress the transcription synergistically (Yamada et al. 2000; Ge et al. 2002).
For other fibrillar collagen genes, the previous reports demonstrated that Sp1 regulates the proximal promoter activity oftypes I (García-Ruiz et al. 2002; Kypriotou et al. 2007), II(Dharmavaram et al. 1997; Ghayor et al. 2001), V (Wu et al.
2010a, b), and XI (Goto et al. 2006) collagen genes. The
proximal promoter of Col2a1 is regulated by the combinationof Sp1 and Sp3, which bind to the GC-rich sequence. Sp1 is apotent activator of Col2a1 transcription by binding to the GCrich element in the promoter and/or enhancer, whereas Sp3
inhibits the promoter activity by competing with Sp1 as described above (Ghayor et al. 2001). Sp1, Sp3, and Sp7 upregulate the promoter activity of the Col11a2 gene in osteoblasts,
but not in chondrocytes, and the degree of their transcription
activities are almost identical. In this case, Sp3 does not inhibit
the promoter activity of the Col11a2 gene by competing withSp1 (Goto et al. 2006). On the other hand, in non-cartilage
collagens, the transcription of type I collagen genes are also
regulated by Sp1 and Sp3 in fibroblasts and hepatic stellate
cells (García-Ruiz et al. 2002; Kypriotou et al. 2007). In addition, Sp7/Osterix, one of the Sp1-like transcription factor,
upregulates the transcriptional activity of types I (Ortuño et al.

2013; Yano et al. 2014) and V (Wu et al. 2010a, b) genes inosteoblasts. These studies suggested that the Sp1-like family
regulates the expression of collagen genes in a gene-specificand/or a tissue-specific manner. Furthermore, the Sp1-likefamily may regulate, at least in part, in chondrocyte differentiation, cartilage formation, and skeletal development. Theinteraction between NF-Y and Sp1-like transcription factorsof the Col11a1 gene is not clarified in this study; however,further studies will be required to address these questions.
Another transcriptional regulation is modulated by tissuespecific cis-acting element, such as enhancers and/or silencers.
In cartilage, Sox9, one of the HMG-box containing a transcription factor, is essential for chondrocyte differentiation and skeletal development (Akiyama 2008). As described previously,
cartilage collagens are composed of types II, IX, and XI collagens. In the Col2a1 gene, it has been reported that thechondrocyte-specific enhancer element is identified, and Sox9
regulates the Col2a1 gene expression (Bell et al. 1997; Zhou
et al. 1998). The Col11a2 gene has also two chondrocytespecific enhancer elements in the proximal promoter and thefirst intron and is regulated by Sox9 similar to those of the
Col2a1 gene (Bridgewater et al. 1998; Liu et al. 2000). The
Col9a1 gene also has Sox9 binding elements in the proximal
promoter, resulting in activation of the proximal promoter by
Sox9 (Zhang et al. 2003; Genzer and Bridgewater 2007). On
the other hand, there is no direct evidence that Sox9 regulates
the expression of the Col11a1 gene. However, Lincoln et al.
have shown that Sox9 is required for heart valve development
as well as the Col11a1 and Col5a1 genes, suggesting that it ispossible that Sox9 may regulate the expression of the Col11a1
and Col5a1 genes (Lincoln et al. 2006, 2007). In addition, it
has been reported that lymphocyte enhancer-binding factor 1(Lef1) indirectly activates the proximal promoter of the
Col11a1 gene in osteoblasts, and suppresses terminal osteoblast
differentiation (Kahler et al. 2008). However, the chondrocytespecific cis-acting elements have not been identified in the
Col11a1 gene yet. Thus, further study on the identification oftissue-specific transcription factor would clarity the mechanismof transcriptional regulation of the Col11a1 gene, chondrocyte
differentiation, cartilage formation, and skeletal development.
Acknowledgments We thank Ms. H Satoh for the technical support, andthe staff members of the Research Promotion Institute, Oita University.
This work was supported by Grants-In-Aid for Scientific Research (No.
21591952 to N.M.) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.

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