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Vol. 20, Issue 21, 4541-4551, November 1, 2009

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Sox9 Family Members Negatively Regulate Maturation and Calcification of Chondrocytes through Up-Regulation of Parathyroid Hormone–related Protein

Katsuhiko Amano^*,, Kenji Hata^*, Atsushi Sugita^*, Yoko Takigawa^*, Koichiro Ono^*, Makoto Wakabayashi^*, Mikihiko Kogo, Riko Nishimura^*, and Toshiyuki Yoneda^*

Departments of *Molecular and Cellular Biology and Oral and Maxillofacial Surgery, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan

Submitted March 20, 2009; Revised August 18, 2009; Accepted September 9, 2009
Monitoring Editor: Carl-Henrik Heldin

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sox9 is a transcription factor that plays an essential role in chondrogenesis and has been proposed to inhibit the late stages of endochondral ossification. However, the molecular mechanisms underlying the regulation of chondrocyte maturation and calcification by Sox9 remain unknown. In this study, we attempted to clarify roles of Sox9 in the late stages of chondrocyte differentiation. We found that overexpression of Sox9 alone or Sox9 together with Sox5 and Sox6 (Sox5/6/9) inhibited the maturation and calcification of murine primary chondrocytes and up-regulated parathyroid hormone–related protein (PTHrP) expression in primary chondrocytes and the mesenchymal cell line C3H10T1/2. Sox5/6/9 stimulated the early stages of chondrocyte proliferation and development. In contrast, Sox5/6/9 inhibited maturation and calcification of chondrocytes in organ culture. The inhibitory effects of Sox5/6/9 were rescued by treating with anti-PTHrP antibody. Moreover, Sox5/6/9 bound to the promoter region of the PTHrP gene and up-regulated PTHrP gene promoter activity. Interestingly, we also found that the Sox9 family members functionally collaborated with Ihh/Gli2 signaling to regulate PTHrP expression and chondrocyte differentiation. Our results provide novel evidence that Sox9 family members mediate endochondral ossification by up-regulating PTHrP expression in association with Ihh/Gli2 signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In vertebrae, most of skeleton is formed by endochondral ossification. Endochondral ossification is a unique biological event comprised of multiple processes including mesenchymal cell condensation, differentiation of mesenchymal cells into chondrocytes, maturation of chondrocytes, vascular invasion into cartilage, chondrocyte apoptosis, and the replacement of cartilage with bone tissue (de Crombrugghe et al., 2000, 2001; Kronenberg, 2006). These processes are sequentially and harmoniously regulated by several cytokines and hormones that activate downstream signaling pathways and transcription factors (de Crombrugghe et al., 2000, 2001; Kronenberg, 2006). Genetic evidence has demonstrated that the HMG-type transcription factor Sox9 plays an essential role in endochondral ossification, and in particular, the regulation of chondrocytic matrix expression (Akiyama et al., 2002, 2004, 2007). Biochemical studies have also demonstrated that Sox9 directly regulates the expression of type 2 collagen (Col21), aggrecan, type 11 collagen (Col112), Sox5, and Sox6 initiates the commitment of mesenchymal cells into chondrocytes, and promotes the early stages of chondrogenesis (Lefebvre et al., 1996, 1997, 1998; Bridgewater et al., 1998; Akiyama, 2008).

Sox5 and Sox6 have been shown to regulate the expression of Sox9 target genes and to function as transcriptional partners of Sox9 (Lefebvre et al., 1998; Akiyama et al., 2002). The Sox9 knockin mutant mouse demonstrates dwarfism phenotypes associated with delayed hypertrophy of chondrocytes, suggesting a possible role for Sox9 in the late stages of endochondral ossification (Akiyama et al., 2004). Ikeda and coworkers have also reported that in cooperation with Sox5 and Sox6 cofactors, Sox9 inhibits terminal differentiation of chondrocytic cell lines (Ikeda et al., 2004; Saito et al., 2007). However, the precise functional role of Sox9 in chondrogenesis during the late stages remains unknown to date. In particular, it remains unclear whether Sox9 functions as a negative regulator during the late stages of chondrogenesis. Furthermore, the molecular mechanisms underlying the control of late-stage endochondral ossification by Sox9 remain unclear.

Indian hedgehog (Ihh) is expressed in the prehypertrophic chondrocytes and stimulates the expression of parathyroid hormone–related protein (PTHrP; Lanske et al., 1996; Vortkamp et al., 1996; St-Jacques et al., 1999). PTHrP has been shown to stimulate the proliferation of chondrocytes but to inhibit their maturation and calcification (Karaplis et al., 1994; Kronenberg, 2006). These findings suggest that Ihh and PTHrP function in a negative feedback system during late-stage chondrocyte development. Recently, it has also been shown that Ihh stimulates chondrocyte maturation and calcification in a PTHrP-independent manner (Kobayashi et al., 2005; Amano et al., 2008; Mak et al., 2008). These results indicate that the precise role of the PTHrP/Ihh negative feedback loop is more complicated than initially proposed and that the regulation of PTHrP expression is complex.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture
C3H10T1/2, Cos7, and BOSC23 cells were purchased from the Riken cell bank (Tsukuba, Japan) and cultured in modified -minimum Eagle's medium (-MEM, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). ATDC5 cells were also purchased from the Riken cell bank and cultured in DMEM/F12 (Sigma) supplemented with 10% FBS.

Primary chondrocytes were isolated from the ribs of 4-wk-old DDY mice by repetitive digestion with 0.2% collagenase at 37°C. The isolated cells were filtered through a 40-µm nylon mesh (BD Bioscience, San Jose, CA), collected by centrifugation and with the exception of the cells isolated following the first digestion, and then cultured (Kamiya et al., 2002; Gartland et al., 2005). Primary chondrocyte cell cultures were maintained in DMEM (Sigma) supplemented with 10% FBS, 0.1 mg/ml ascorbic acid, and 5 mM β-glycerophosphate (Sigma) at 37°C in a humidified 5% CO₂ incubator. Adenoviruses were infected at 40 multiplicity of infection (moi). Recombinant BMP2 was generated as described previously (Ichida et al., 2004). BMP2 (100 ng/ml), PTHrP (5 x 10^–8 M, Sigma), and cyclopamine (5 µM, Biomol International, Tokyo, Japan) were used in the experiments outlined below. All animal experiments were preapproved by the Osaka University Animal Ethics Committee.

Organ Culture
Three central metatarsal rudiments were isolated from each hind limb of 15.5-d-old (E15.5) DDY mouse embryos, placed into 24-well plates, and cultured in organ culture medium as described previously (Haaijman et al., 1997; Yasoda et al., 1998). The metatarsals were cultured at 37°C in a humidified 5% CO₂ incubator for 12 d. Infection of adenoviruses (80 moi) or the administration of anti-PTHrP neutralizing antibody (193 µg/ml, Chugai Pharmaceutical, Tokyo, Japan) was performed 1 d after dissection (Nifuji et al., 2004). For morphometric analysis, metatarsal explants were photographed under a dissecting microscope (microscope: Stemi 2000-C; camera: AxioCam MRc; acquisition software: AxioVision AC Rel. 4.5, Carl Zeiss, Thornwood, NY). The longitudinal length and dark calcified zone in the diaphysis and epiphysis were measured as described previously (Yasoda et al., 1998; Amano et al., 2008). For histological analysis, paraffin sections of metatarsals were prepared and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis was performed using the following primary polyclonal rabbit antibodies: -hemagglutinin (HA) antibody (MBL, Nagoya, Japan) at 1:500 dilution, -Col2 antibody (LSL Biolafitte, St. Germain en Laye, France) at 1:500 dilution, -Col10 antibody (LSL Biolafitte) at 1:500 dilution and anti-PTHrP antibody (Yanaihara Institute, Shizuoka, Japan) at 1:1000 dilution. Antigen retrieval was performed using 2.5% hyaluronidase for -Col2 and -Col10 and with microwave treatment for 5 min in 0.05 M citrate buffer for anti-PTHrP. Immunoreactivity was visualized with a biotinylated anti-rabbit IgG secondary antibody using the ABC Vectastain kit (Vector Laboratories, Burlingame, CA) and the peroxidase substrate DAB kit (Vector) according to the manufacturer's protocol. For the analysis of alkaline phosphatase activity, metatarsals were fixed in 4% buffered paraformaldehyde, decalcified in 10% EDTA, and immersed in 30% sucrose, and 10-µm frozen sections were collected using a cryostat. The sections were then washed with phosphate-buffered saline (PBS) and stained with a mixture of 330 µg/ml nitro blue tetrazolium (Sigma), 165 µg/ml bromochoroindoyl phosphatase (Sigma), 100 mM NaCl, 5 mM MgCl₂, and 100 mM Tris, pH 9.5, for 10 min at 37°C. For bromodeoxyuridine (BrdU) staining, BrdU-labeling reagent (Roche) was added to the organ culture medium at 1:1000 dilution. After 24-h incubation, the samples were embedded in paraffin and sectioned. Paraffin sections were then incubated with anti-BrdU monoclonal mouse antibody (1:1000, Roche, Indianapolis, IN). Antigen retrieval was performed with trypsin, and the sections were restained using Vectashield (Vector) with DAPI for normalization. Histological sections were photographed using a microscope attached to a digital camera (microscope: Axioskop 2; camera: AxioCam HRc; acquisition software: AxioVision Rel. 4.4, Carl Zeiss). The distal growth plates of the metatarsals were observed histologically because they allowed the clear analysis of hierarchical chondrocyte differentiation.

Vector Construction
HA-tagged Sox9, Sox5, Sox6, Myc-tagged Gli1, Flag-tagged Gli2, and Flag-tagged Gli3 expression vectors were used as described previously (Shimoyama et al., 2007; Hata et al., 2008). Myc-tagged dominant-negative Sox9 (aa2-234) and Myc-tagged dominant-negative Gli2 (aa2-968) that lacked the corresponding transcriptional activation domains were also used as described previously (Shimoyama et al., 2007; Hata et al., 2008). The human PTHrP gene promoter including the 1120-base pair 5'-flanking region (Gallwitz et al., 2002) and the deletion mutants were ligated into the pGL4.10 vector (Promega, Madison, WI). Four deletion mutants were constructed with or without the P2 midregion GC-rich promoter (between –1120 and –611) or the P3 TATA promoter (between –513 and –505) as described previously (Vasavada et al., 1993). His-tagged Sox9 and His-tagged Venus were generated by subcloning the corresponding PCR products into pCold vector (Takara, Tokyo, Japan).

Generation of Adenoviruses
Recombinant adenoviruses carrying Sox5, Sox6, HA-tagged Sox9, a dominant-negative Myc-tagged Sox9, Ihh, Myc-tagged Gli2, or a dominant-negative Gli2 were constructed by homologous recombination between the expression cosmid cassette (pAxCAwt) and the parental virus genome in 293T cells (Riken Cell Bank) using the adenovirus construction kit (Takara) as described previously (Shimoyama et al., 2007; Hata et al., 2008). Mock adenovirus was used as a control adenovirus. The viruses showed no proliferative activity because of the lack of E1A-E1B. Virus titers were determined using a modified point assay (Shimoyama et al., 2007; Hata et al., 2008).

Western Blotting
The cells were rinsed twice with PBS and solubilized in lysis buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl₂, 10% glycerol, 1% Triton X-100, 10 µg/ml aporotinin, 10 µg/ml leupeptin, 1 mM AEBSF, and 0.2 mM sodium orthovanadate. The lysates were then centrifuged for 10 min at 15,000 x g at 4°C and boiled in SDS sample buffer containing 0.5 M β-mercaptoethanol for 5 min. The supernatants were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies, and immunoreactivity was visualized with horseradish peroxidase–coupled anti-mouse, -rabbit or -goat IgG antibody using the ECL detection kit. Anti-Sox5, -Sox6, -HA, and -Myc antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Real-Time PCR
Total RNA was isolated using the total RNA isolation kit according to the manufacturer's protocol (Macherey-Nagel, Bethlehem, PA). After denaturation at 70°C for 10 min, cDNA was synthesized using oligo dT primer and reverse transcriptase (Takara). Real-time PCR was performed using the Taqman PCR protocol and an ABI 7300 real-time PCR system (Applied Biosystems, Tokyo, Japan). Taqman primers and probes used for the amplification were as follows: mouse Col21 (sense primer, 5'-CCTCCGTCTACTGTCCACTGA-3'; anti-sense primer, 5'-ATTGGAGCCCTGGATGAGCA-3'; probe, 5'-CTTGAGGTTGCCAGCCGCTTCGTCC-3'), mouse Ihh (sense primer, 5'-GACTCATTGCCTCCCAGAACTG-3'; anti-sense primer, 5'-CCAGGTAGTAGGGTCACATTGC-3';probe, 5'-CCACAGCCAGCCTGGACATCCCGA-3'), mouse alkaline phosphatase (sense primer, 5'-ATCTTTGGTCTGGCTCCCATG-3'; anti-sense primer, 5'-TTTCCCGTTCACCGTCCAC-3'; probe, 5'-TGAGCGACACGGACAAGAAGCCCTT-3'), mouse β-action (sense primer, 5'-TTAATTTCTGAATGGCCCAGGTCT-3'; anti-sense primer, 5'-ATTGGTCTCAAGTCAGTGTACAGG-3'; probe, 5'-CCTGGCTGCCTCAACACCTCAACCC-3'), mouse PTHrP (sense primer, 5'-GAACATCAGCTACTGCATGACAAG-3'; anti-sense primer, 5'-TCTGATTTCGGCTGTGTGGATC-3'; probe, 5'-CCATCCAAGACTTGCGCCGCCGTT-3'), mouse aggrecan (sense primer, 5'-TCACTGTTACCGCCACTTTCC-3'; anti-sense primer, 5'-TGCTGCTCAGATGTGACTGC-3'; probe, 5'-ACCGTCTCTCCGCATCCACCCAGG-3'), mouse Sox5 (sense primer, 5'-AGGCAGGAAATGCGACAGTAC-3'; anti-sense primer, 5'- CTCGGAGGGCAGGTGAGG-3'; probe, 5'-ACGTTGGGCAACAAGCACAGATCCCC-3'), mouse Sox6 (sense primer, 5'-TACCCACAGCTCCCCTGAAG-3'; anti-sense primer, 5'-CTCACCTTCAGTGGCAAGAGC-3'; probe, 5'-TCAGCAGCAGCGTTCACGAGCAGC-3'), mouse Patched (sense primer, 5'-CTCCAAAAGAAGAAGGCGCTAATG-3', anti-sense primer, 5'-GCACAAATGTTCCAACTTCCATTG-3', probe, 5'-ACCACAGAGGCTCTCCTGCAACACCT-3'). mRNA expression levels were normalized to that of β-actin.

Alizarin Red Staining
Cultured murine primary chondrocytes were rinsed twice with PBS, fixed in 4% buffered paraformaldehyde and then 95% ethanol, and stained with 1% alizarin red solution (Wako, Osaka, Japan) for 10 min. Stained samples were scanned using an Epson GT-9500 (, Long Beach, CA), and the alizarin red–positive area of the cells measured using Image Proplus (Media Cybernetics, Bethesda, MD).

MTT Assay
Cell proliferation was examined using reagent WST-1 according to the manufacturer's protocol (Roche). The WST-1 reagent was added to cultured cells at a final dilution of 1:10. The samples were then incubated at 37°C for 2 h, and the absorbance of the samples was measured against a background control at 450 nm using a microplate reader (model 550, Bio-Rad).

Luciferase Assay
The luciferase reporter construct for the human PTHrP gene promoter and the TK-renilla luciferase construct (Promega) were cotransfected with expression vectors into Cos7 or ATDC5 cells using Fugene6 reagent (Roche). After 48-h incubation, cells were lysed, and the luciferase and renilla activity was measured in the substrates on a luminometer (Promega) according to the manufacturer's instructions. Renilla was used to normalize the transfection efficiency.

Oligonucleotide Pulldown Assay
Protein lysates were prepared from BOSC23 transfected with HA-Sox9 or C3H10T1/2 cells infected with Sox5, Sox6, and Sox9 adenoviruses. Lysates were then incubated with the biotinylated double-stranded DNA oligonucleotide 5'-GCTCGCCCCGCGCGCGTTCCTAGGGCGCCA-3' for 3 h, and incubated with streptavidin beads (Vector) for 1 h. After five washes in lysis buffer, the precipitated samples and the beads were boiled in 20 µl sample buffer and subjected to Western blotting. Competition was performed in the presence of a 40-fold amount of nonbiotinylated oligonucleotide.

In Vitro Binding Assay
His-tagged Sox9 and His-tagged Venus proteins were generated in the BL21 Escherichia coli strain using the pCold system (Takara). Cell lysates were then incubated with His-tagged Sox9 or Venus protein for 4 h, and the His-tagged Sox9 or Venus protein was precipitated with TALON beads (Clontech, Palo Alto, CA). The proteins associated with His-tagged Sox9 or Venus were determined using immunoblotting.

Statistical Analysis
Data were analyzed using the Student's t test, multiple comparisons (Tukey procedure) of one- or two-way ANOVA. Data are presented as the mean ± SD.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sox9 Family Induces PTHrP Expression in Chondrocytes
To understand the role of the Sox9 family members in chondrocyte differentiation, we first examined the effects of Sox9 family protein overexpression on the commitment of undifferentiated mesenchymal cells to chondrocytes in the C3H10T1/2 mesenchymal cell line. Overexpression of Sox5, Sox6, and Sox9 (Figure 1A) markedly induced Col2a1 and aggrecan expression in the C3H10T1/2 cells (Figure 1, B and C). Interestingly, Sox9 family proteins also induced the expression of PTHrP in the cells (Figure 1D). In contrast, overexpression of a dominant-negative Sox9 inhibited PTHrP expression (Figure 1, E and F). These results suggested that Sox9 family members are involved in not only the promotion of chondrocyte differentiation, but also the regulation of PTHrP expression. To confirm this finding, we next examined the role of Sox9 on PTHrP expression in primary chondrocytes isolated from mouse ribs. We found that overexpression of Sox9 increased PTHrP expression in the primary chondrocytes along with up-regulation of Col2a1, Sox5, and Sox6 expression (Figure 2, A–D). In addition, overexpression of Sox5 or Sox6 alone also significantly increased PTHrP expression in the cells (Figure 2D). Furthermore, overexpression of a dominant-negative Sox9 suppressed PTHrP expression in primary murine chondrocytes (Figure 2E). These results suggested that Sox9 controls chondrocyte differentiation via PTHrP. PTHrP is known to play an important role in the regulation of proliferation, maturation, and apoptosis processes in chondrocytes (Weir et al., 1996; Amling et al., 1997; Zerega et al., 1999; Beier et al., 2001; Yamanaka et al., 2003). Furthermore, previous genetic studies have suggested an inhibitory role for Sox9 in the maturation of chondrocytes (Akiyama et al., 2004). However, it remains unknown as to whether Sox9 directly regulates chondrocyte maturation and calcification. Thus, we next examined the effects of Sox9 protein on the calcification of chondrocytes. As shown in Figure 3, A and B, overexpression of Sox9 proteins abolished BMP2-induced calcification of primary chondrocytes. In addition, overexpression of Sox9 markedly suppressed the expression of ALP (alkaline phosphatase) in primary chondrocytes (Figure 3C). In contrast, Sox9 overexpression was found to significantly stimulate proliferation of primary chondrocytes (Figure 3D). As expected, treatment with PTHrP also inhibited the BMP2-induced calcification of primary chondrocytes (Figure 3, E and F). In combination, these results suggested that the Sox9 family members may negatively control chondrocyte calcification via the up-regulation of PTHrP.

View larger version (29K): [in this window] [in a new window]   Figure 1. Induction of PTHrP by Sox9 family members in C3H10T1/2. (A) C3H10T1/2 was infected with adenoviruses carrying Sox5, Sox6, and HA-tagged Sox9 (HA-Sox9) at 40 moi and cultured for 2 d. Expression of each Sox protein after infection of the adenoviruses was examined by immunoblotting with anti-Sox5 (top), anti-Sox6 (middle), or anti-HA antibody (bottom). (B–D) C3H10T1/2 cells infected with control (Cont) or Sox5/6/9 adenoviruses were cultured for 3 d. Total RNA isolated from the cells was determined by real-time PCR analyses for Col21 (B), Aggrecan (C), and PTHrP (D). (E) C3H10T1/2 cells infected with adenovirus carrying Myc-tagged dominant-negative Sox9 (DN-Sox9) at 40 moi were cultured for 2 d. Expression of DN-Sox9 was examined by immunoblotting with anti-Myc antibody. (F) C3H10T1/2 cells infected with control (Cont) or dominant-negative Sox9 (DN-Sox9) adenovirus were cultured for 3 d. Total RNA of the cells was subjected to real-time PCR analysis for PTHrP. Data represent mean ± SD (n = 3). *p < 0.01 vs. control; Student's t test.

View larger version (17K): [in this window] [in a new window]   Figure 2. Induction of PTHrP by Sox9 in murine primary chondrocytes. (A–C) Primary chondrocytes were infected with control (Cont) or HA-Sox9 adenoviruses at 40 moi and cultured for 3 d. Total RNA isolated from the cells was determined by real-time PCR analyses for Col21 (A), Sox5 (B), and Sox6 (C). (D) Primary chondrocytes infected with the adenoviruses (40 moi) as indicated were cultured for 3 d. Total RNA isolated from the cells was determined by real-time PCR analyses for PTHrP. (E) Primary chondrocytes infected with control (Cont) or DN-Sox9 adenovirus at 40 moi were cultured for 3 d. Total RNA of the cells was determined by real-time PCR analysis for PTHrP. Data represent mean ± SD (n = 3). *p < 0.01, **p = 0.01, ***p < 0.05 vs. control; Student's t test.

View larger version (29K): [in this window] [in a new window]   Figure 3. Inhibition of calcification by Sox9 family members in primary chondrocytes. (A and B) Primary chondrocytes cultured with or without BMP2 (100 ng/ml) were infected with control (Cont) or Sox5/6/9 adenoviruses, cultured for 7 d, and stained with alizarin red (A). Alizarin red–positive area of the cells was measured by Image Proplus (B). Data represent mean ± SD (n = 3). *p < 0.01 vs. BMP2; one-way ANOVA. (C) Primary chondrocytes infected with control (Cont) or HA-Sox9 adenovirus at 40 moi were cultured for 3 d. Total RNA of the cells was determined by real-time PCR analyses for ALP. Data represent mean ± SD (n = 3). *p < 0.01 vs. control; Student's t test. (D) Primary chondrocytes were placed at a density of 10 x 104/well of 24-multiwell plates. The cells were infected with control (Cont) or HA-Sox9 adenovirus and cultured for 3 d. MTT assay was performed to evaluate proliferation of the cells. Data represent mean ± SD (n = 4). *p < 0.05 vs. control; Student's t test. (E and F) Primary chondrocytes cultured with or without BMP2 (100 ng/ml) were treated with or without PTHrP (5 x 10–8 M) for 7 d and then stained by alizarin red (E). The alizarin red–positive area of the cells was measured with Image Proplus (F). Data represent mean ± SD (n = 3). *p < 0.01 vs. BMP2; one-way ANOVA.

View larger version (42K): [in this window] [in a new window]   Figure 4. Suppression of calcification of chondrocytes by Sox9 family members in organ culture. (A) Mouse metatarsals isolated from 15.5-d-old embryo (E15.5), 1 d after birth (P1), and 14-d-old mice (P14) were observed under a dissecting microscope (top). Metatarsals were histologically analyzed by H&E staining (bottom, magnification, x100). Bars (top) show calcified region. R, resting zone; P, proliferating zone; H, hypertrophic zone; C, calcified zone; S, secondary ossified area. (B) Mouse metatarsal explants isolated from E15.5 embryo were cultured for 6 and 12 d and then macroscopically (top) and histologically (bottom; magnification, x100) examined. Bars (top) show calcified region. R, resting zone; P, proliferating zone; H, hypertrophic zone; C, calcified zone; S, secondary ossified area. (C) Mouse metatarsal explants isolated from E15.5 embryo were infected with control (Cont) or HA-Sox9, Sox5, and Sox6 adenoviruses at 80 moi and cultured for 12 d. Organ cultured metatarsals were immunostained with anti-HA antibody. (D) Mouse metatarsal explants isolated from E15.5 embryo were infected with control (Cont) or Sox5/6/9 adenoviruses at 80 moi and cultured for 12 d. Left, red bars, calcified area; black arrows, secondary ossification. Right, longitudinal length and calcified zone were measured under a microscope. Data represent mean ± SD (n = 12). *p < 0.01 vs. control; Student's t test.

View larger version (58K): [in this window] [in a new window]   Figure 5. Stimulation of PTHrP expression and suppression of chondrocyte maturation of by Sox9 family members in organ culture. (A) Organ-cultured metatarsals were histologically analyzed by H&E staining and immunostained with anti-Col2 and anti-Col10 antibodies. S, secondary ossified region; H+C, hypertrophic and calcified areas. Red bars, immunostained areas. Magnification, x100. (B) Metatarsals infected with green fluorescent protein (GFP) adenovirus or Sox5/6/9 adenoviruses were cultured for 12 d. The cultured metatarsals were frozen with dry ice for a moment and ground with lysis buffer to isolate mRNA. Total RNA of the cells was subjected to real-time PCR analysis for PTHrP (left), Col2a1 (middle), and Col10a1 (right) expression. p < 0.01 vs. GFP control; Student's t test. (C) Organ cultured metatarsals infected with control (Cont) or Sox5/6/9 adenoviruses were histologically analyzed by immunostaining with anti-PTHrP antibodies. No Ab, not incubated with PTHrP primary antibody. Magnification, x100. (D) Organ cultured metatarsals infected with control (Cont) or Sox5/6/9 adenoviruses were subjected to ALP staining. Magnification, x100. (E and F) Organ-cultured metatarsals were subjected to BrdU and DAPI staining (E), and BrdU-positive chondrocytes were counted and normalized by DAPI-stained chondrocytes present at the resting and proliferating zone (F). Magnification, x100. Bars, immunostained areas. Data represent mean ± SD (n = 3). *p < 0.05 vs. control; Student's t test.

View larger version (53K): [in this window] [in a new window]   Figure 6. Rescue of chondrocyte maturation and calcification by anti-PTHrP antibody. (A) Metatarsals infected with control (Cont) or Sox5/6/9 adenovirus were cultured for 12 d with or without anti-PTHrP-antibody (193 µg/ml). Organ-cultured metatarsals were photographed (left; blue bars, calcified area; black arrows, secondary ossification) and measured (right). Data represent mean ± SD (n = 5). N.S., no significant difference between Sox5/6/9 and Sox5/6/9+Anti-PTHrP. *p < 0.01 versus Sox5/6/9 **p < 0.05 versus control; one-way ANOVA. (B and C) The organ-cultured metatarsals shown in A were subjected to H&E staining (B) and immunostained with anti-Col10 antibody (C). S, secondary ossification; H+C, hypertrophic and calcified areas. Red bars, immunoreactive area. Magnification, x100.

View larger version (21K): [in this window] [in a new window]   Figure 7. Regulation of the PTHrP gene promoter by Sox9 family members. (A) Cos7 cells were transfected with a PTHrP gene promoter luciferase (1120 bp) and TK-renilla luciferase constructs together with empty vector (Cont) or Sox5, Sox6, or Sox9 expression vectors, as indicated. After 48 h, luciferase activity of the cell lysates was measured. Transfection efficiency was normalized by renilla luciferase activity. Data represent mean ± SD (n = 4). *p < 0.01 (vs. control), **p < 0.05 (vs. Sox5 or Sox6), ***p < 0.01 (vs. Sox6 or Sox9), ****p < 0.01 (vs. Sox5+Sox6, or Sox6+Sox9); one-way ANOVA. (B) Cos7 cells were transfected with PTHrP gene promoter luciferase (1120 bp) and TK-renilla luciferase constructs with empty vector (Cont) or a combination of Sox5/6/9 or dominant-negative (DN)-Sox9 expression vector as indicated. After 48 h, luciferase activity of the cell lysates was measured. Transfection efficiency was normalized by renilla luciferase activity. Data represent mean ± SD (n = 4). *p < 0.01 vs. Sox5/6/9; one-way ANOVA. (C) Schematic diagram of a series of human PTHrP gene promoter luciferase constructs. (D) Cos7 cells were transfected with a series of PTHrP gene promoter luciferase and TK-renilla luciferase constructs together with empty vector (Cont) or combination of Sox5/6/9 as indicated. After 48 h, luciferase activity of the cell lysates was measured. Transfection efficiency was normalized by renilla luciferase activity. Data represent mean ± SD (n = 4). *p < 0.01 vs. control, –440, –611 and –803; two-way ANOVA. (E) Lysates of BOSC23 transfected with empty or HA-Sox9 vector were incubated with biotinylated oligonucleotide (Bio-oligo) containing a putative Sox9 binding element of the PTHrP gene promoter. After precipitation with streptavidin beads, associated proteins with the oligonucleotide were determined by immunoblotting with anti-HA antibody. Comp, competition with 40-fold of nonbiotinylated oligonucleotide. (F) Lysates of C3H10T1/2 infected with control or Sox5/6/9 adenoviruses were incubated with biotinylated oligonucleotide (Bio-oligo) containing a putative Sox9-binding element of the PTHrP gene promoter. After precipitation with streptavidin beads, associated proteins with the oligonucleotide were determined by immunoblotting with anti-Sox5 antibody. Comp, competition with 40-fold nonbiotinylated oligonucleotide.

Collaboration between Sox9 Family and Ihh/Gli2 Signaling
Given that Ihh regulates hypertrophic conversion by induction of PTHrP (Lanske et al., 1996; Vortkamp et al., 1996), we next examined whether Ihh signaling is involved in PTHrP induction by Sox9. Consistent with previous reports, Ihh was found to increase the expression of PTHrP in primary chondrocytes in a manner similar to that of Patched1, one of the direct target genes of Ihh (Figure 8A). These effects of Ihh were inhibited after treatment with a hedgehog inhibitor cyclopamine (Figure 8A). We also found that cyclopamine significantly inhibited PTHrP expression induced by Sox9 (Figure 8B). In contrast, Sox9 did not show any effects on the expression of Ihh and Patched1 in the primary chondrocytes (Figure 8, C and D). These results suggested that Sox9 proteins regulate PTHrP expression in association with Ihh signaling, but not through the up-regulation of Ihh and Patched1 expression.

View larger version (16K): [in this window] [in a new window]   Figure 8. No effects of Sox9 on Ihh and Patched 1 expression in primary chondrocytes. (A) Primary chondrocytes infected with control (Cont) or Ihh adenovirus were cultured in the presence or absence of cyclopamine (Cy; 5 µM) for 3 d. Total RNA of the cells was determined by real-time PCR analysis for PTHrP (left) and Patched1 (right). Data represent mean ± SD (n = 3). *p < 0.01 vs. control; one-way ANOVA, **p < 0.01 vs. Ihh by one-way ANOVA. (B) Primary chondrocytes infected with control (Cont) or HA-Sox9 adenovirus were cultured with or without cyclopamine (5 µM) for 3 d. Total RNA of the cells was determined by real-time PCR analysis for PTHrP. Data represent mean ± SD (n = 3). ***p < 0.05 vs. Sox9; one-way ANOVA. (C) Primary chondrocytes infected with control (Cont) or HA-Sox9 adenovirus were cultured for 3 d. Total RNA of the cells was determined by real-time PCR analysis for Ihh. N.S., no significant difference; Student's t test. (D) Primary chondrocytes infected with control (Cont) or Sox9 adenovirus were cultured in the presence or absence of cyclopamine (Cy; 5 µM) for 3 d. Total RNA of the cells was determined by real-time PCR analysis for Patched1. Data represent mean ± SD (n = 3). N.S., no significant difference as determined; one-way ANOVA.

View larger version (18K): [in this window] [in a new window]   Figure 9. Functional cooperation of Sox5/6/9 and Ihh/Gli2 signaling in regulation of PTHrP expression. (A) Cos7 (left) or ATDC5 cells (right) were transfected with a PTHrP gene promoter luciferase (1120 bp) and TK-renilla luciferase constructs with empty vector (Cont), Gli1, Gli2, or Gli3 vectors. After 48 h, luciferase activity of the cell lysates was measured. Transfection efficiency was normalized by renilla luciferase activity. Data represent mean ± SD (n = 4). *p < 0.01 or **p < 0.05; one-way ANOVA (vs. control, Gli1, and Gli3). (B) Cos7 cells were transfected with a series of PTHrP gene promoter luciferase and TK-renilla luciferase constructs together with empty (Cont) or Gli2 vector. After 48 h, luciferase activity of the cell lysates was measured. Transfection efficiency was normalized by renilla luciferase activity. Data represent mean ± SD (n = 4). *p < 0.01; two-way ANOVA (vs. control, –440, –611 and –803). (C) Primary chondrocytes infected with control (Cont) or Gli2 adenovirus were cultured for 3 d. Total RNA of the cells was determined by real-time PCR analysis for PTHrP. Data represent mean ± SD (n = 3). *p < 0.01 vs. control; Student's t test. (D) Cos7 (left) or ATDC5 cells (right) were transfected with a PTHrP gene promoter luciferase (1120 bp) and TK-renilla luciferase constructs with empty vector (Cont), Gli2, or Sox5/6/9 as indicated. After 48 h, luciferase activity of the cell lysates was measured. Transfection efficiency was normalized by renilla luciferase activity. Data represent mean ± SD (n = 3). *p < 0.01; one-way ANOVA (vs. control, Sox5/6/9, or Gli2). (E) Primary chondrocytes infected with control (Cont), HA-Sox9, or dominant-negative (DN)-Gli2 adenoviruses as indicated were cultured for 3 d. Total RNA of the cells was determined by real-time PCR analysis for PTHrP. Data represent mean ± SD (n = 3). *p < 0.01 vs. Sox9; one-way ANOVA. (F) Lysates of 293FT cells transfected with or without Myc-Gli2 were precipitated by TALON beads with His-tagged Sox9 or Venus protein, and then the precipitates were determined by immunoblotting with anti-Myc antibody (top). Inputs of Myc-Gli2 (middle) and His-tagged Sox9 protein (bottom) were determined by immunoblotting with anti-Myc and Sox9 antibodies, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we identified PTHrP as a direct transcriptional target of Sox9. This result is supported by the reported finding that PTHrP is down-regulated in Sox9 conditional knockout mice driven off the Col2a1-Cre transgene (Akiyama et al., 2002). These mice exhibited severe chondrodysplasia, suggesting that Sox9 controls chondrogenesis, at least in part, through PTHrP. Given that PTHrP stimulates phosphorylation of Sox9 via protein kinase A (PKA), a reaction that subsequently results in the up-regulation of the DNA binding affinity and transcriptional activity of Sox9 (Huang et al., 2000; Huang et al., 2001), we hypothesized that Sox9 and PTHrP may generate a positive loop required for their function. In addition, we also suggest that Sox5 and Sox6 function as cofactors with Sox9 on the PTHrP gene promoter, as is the case for Col2a1 (Lefebvre et al., 1998). Overexpression of either Sox5 or Sox6 in primary chondrocytes also marginally increased PTHrP expression, although overexpression of Sox9, which could induce both Sox5 and Sox6 and subsequently form protein complexes on specific chondrogenic genes, dramatically increased PTHrP expression. Using the described luciferase assay, we found that the most efficient effects of Sox9 were observed in the presence of Sox5 and Sox6. These results are supported by the finding that Sox9 null mutants exhibit severe chondrodysplasia, whereas Sox5 and Sox6 single null mice are born with mild skeletal abnormalities only (Smits et al., 2001; Smits et al., 2004).

Our ex vivo experiments using isolated mouse metatarsals indicated that the Sox9 family inhibited secondary ossification. Therefore, it appears likely that the Sox9 family not only inhibits terminal differentiation of chondrocytes at the growth plate, but also prevents the hypertrophy of resting and periarticular chondrocytes during secondary ossification processes via the up-regulation of PTHrP. Consistent with this finding, LacZ transgenic mice driven off the PTHrP gene promoter demonstrated PTHrP expression in the resting or periarticular chondrocytes, as well as in the perichondrium (Chen et al., 2006). Indeed, secondary ossification appears to be initiated at the PTHrP-rich periarticular region (Chen et al., 2006). Moreover, PTHrP-positive chondrocytes not only participated in the proximal proliferation stage in the growth plate, but were also separated into distal and differentiated subarticular chondrocytes (Chen et al., 2006). As secondary ossification is thought to be a physiologically important process that results in the organization of articular cartilage and spongiosa in the epiphysis, we suggest that Sox9 family proteins may be involved in the development of secondary ossification by up-regulating PTHrP expression.

There appears to be a discrepancy in the literature regarding the expression pattern of PTHrP and Sox9 in cartilage tissues. As described by Chen et al. (2006), PTHrP is expressed in the perichondrium, an area where Sox9 does not appear to be expressed. In contrast, the proliferating columnar chondrocytes express Sox9, but not PTHrP (Akiyama et al., 2004). A potential explanation for this discrepancy is that Sox9 may not be essential for the induction of PTHrP. It is also possible that the Sox9 and Ihh dose gradient is involved in the regulation of PTHrP. Further investigations using appropriate in vivo systems may be required to further our understanding of the roles of Sox9 in PTHrP regulation.

We also observed that Gli2 may serve as an effective positive regulator of PTHrP gene promoter activity. Gli3 has also been demonstrated to be a negative regulator of Ihh-dependent PTHrP expression (Hilton et al., 2005). Distinct roles for Gli2 and Gli3 have also been described previously in other systems (Motoyama et al., 1998; Ruiz i Altaba, 1998; Shimoyama et al., 2007). In combination, these findings suggest that PTHrP expression may be regulated by Gli2 and Gli3 in a dose-dependent manner.

In summary, we demonstrate that the Sox9 gene family regulates terminal differentiation of chondrocytes in association with a Ihh/PTHrP loop. These findings advance our understanding of the molecular mechanisms underlying the spatial and temporal regulation of endochondral ossification by the Sox9 gene family.

	ACKNOWLEDGMENTS

 
We thank Chugai Pharmaceutical for providing the anti-PTHrP antibody. This work was supported in part by the Ministry of Education, Science, Sports, and Culture Grants-in-Aid for Scientific Research A (T.Y.) and B (R.N.), and the 21st Century COE Program (T.Y., R.N.).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-03-0227) on September 16, 2009.

Address correspondence to: Riko Nishimura (rikonisi{at}dent.osaka-u.ac.jp).

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