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Abstract

Matrix metalloproteinase-13 (MMP-13), is a key protein of bone matrix degradation, and is highly expressed by osteoblasts. We used the osteoblast-like MC3T3-E1 cell line and compared the stimulatory effects of the bone resorptive agents 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) 3,3′,5-triido-l-thyronine (T3) on the expression of MMP-13 mRNA. We showed that the stimulatory effects were time and dose dependent, and were also transduced to the protein level, with 1,25-(OH)2D3being more potent.

MMP-13 expression in different mouse cells and its localization within developing bone from the onset of osteogenesis were also investigated. 1,25-(OH)2D3- and T3-regulated osteocalcin (Osc) expression in mouse osteoblasts was compared to hormonal effects on MMP-13 expression and activity. Here we show divergent and common roles of 1,25-(OH)2D3and T3 action on the expression of these marker proteins, depending on the stage of cell differentiation. In addition, we propose a role for MMP-13 in the bone collar of developing long bones. The results could help to more precisely characterize hormonal regulation in the developmental sequence of osteoblasts.

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¹Both authors contributed equally to this work.

^*Corresponding author. Tel.: +43-1-91021-86921; fax: +43-1-91021-86929

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1 Introduction

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and thyroid 3,3′,5-triido-l-thyronine (T3) are calciotropic hormones and are potent stimulators of osteoclastic bone resorption (Boehm et al., 1999; Klaushofer et al., 1989; Klein-Nulend et al., 1991). It has recently been shown that, like other bone resorptive factors, these hormones also trigger extracellular matrix degradation, a process driven by osteoblasts (Uchida et al., 2000; Thompsonet al., 1989) or chondrocytes (Ishikawa et al., 1998). Mouse osteoblasts degrade native collagen-I in response to 1,25-(OH)2D3treatment (Thompson et al., 1989), and T3 decreases the amount of collagen fibrils in the extracellular matrix of cultured osteoblasts (Fratzl-Zelman et al., 1997) and chondrocytes (Ishikawa et al., 1998). MMP-13 is the most abundant collagenase in bone; it is a key regulator linking osteoblastic matrix degradation and osteoclastic activation (Holliday et al., 1997). Recently, both hormones have been shown to enhance MMP-13 expression in preosteoblastic MC3T3-E1 cells (Pereira et al., 1999; Uchida et al., 2001).

Expression of MMP-13 is regulated in part by Runx2 (Jimenez et al., 1999), a bone-specific runt-domain transcription factor that plays a pivotal role in the induction of osteoblastic differentiation (Hoshi et al., 1999;Komori et al., 1997). Runx2-depleted mice mutants lack MMP-13 expression and do not develop mature osteoblasts (Jimenez et al., 1999). Transgenic mice over-expressing Runx2 express enhanced MMP-13 and surprisingly develop osteopenia, perhaps due to osteoclastogenesis being stimulated by increased osteoblastic receptor activation of NF-kappaB-ligand (RANKL) (Geoffroy et al., 2002). Interestingly, 1,25-(OH)2D3and T3 promote the formation of osteoclasts through expression of RANKL (Miura et al., 2002; Otsuka et al., 2000; Thomas et al., 2001). In co-cultures of mouse osteoblasts/osteogenic cells, 1,25-(OH)2D3is a critical factor for osteoclast differentiation, and T3 can positively modulate but not substitute for 1,25-(OH)2D3(Gruber et al., 1999; Miura et al., 2002; Schiller et al., 1998). One reason for this observation may be that osteoblastic cells at different stages of maturation respond differently to each hormone (Ohishi et al., 1994; Suda et al., 1992). Both 1,25-(OH)2D3and T3 increase alkaline phosphatase (ALP) activity in early cultures (Kurihara et al., 1986; Varga et al., 1997), and affect osteoblastic differentiation markers and degradation of extracellular matrix in long term cultures (Fratzl-Zelman et al., 1997; Kurihara et al., 1986; Luegmayret al., 1996).

In the present study, we have further defined the effects of 1,25-(OH)2D3and T3 on extracellular matrix degradation, by investigating whether hormonally regulated MMP-13 collagenolytic activity depends on osteoblastic differentiation. To this end, we also studied a late marker of the osteoblastic phenotype expression of osteocalcin (Osc), which in the mouse is inhibited by 1,25-(OH)2D3(Lian et al., 1997; Zhang et al., 1997), but stimulated by T3 (Varga et al., 1997; Varga et al., in press). Expression studies were done using non-osteogenic and osteoblastic cells and bone tissue (calvaria, long bones) obtained from prenatal and postnatal mice. We showed that hormone-stimulated MMP-13 expression depends on the differentiation stage of the osteoblastic cell system. While 1,25-(OH)2D3potently induced MMP-13 in preosteoblastic cell populations, T3-stimulated expression was minor. Primary osteoblasts, as well as bone explants from the early stages of osteogenesis onwards, expressed MMP-13 constitutively. In contrast, Osc was constitutively expressed only in newborn calvaria, but was up-regulated in early fetal stages of osteogenesis by T3. Finally, immunohistochemistry on fetal long bones revealed that MMP-13 is not only expressed in hypertrophic chondrocytes and osteoblasts within and below the growth plate, but also in early osteogenic cells of the bone collar.

2 Materials and methods

2.1 Cell and tissue culture

Fibroblastic, non-osteogenic cells C3H10T1/2 (American Type Culture Collection, Rockville, MD, USA; CCL-226) and NIH3T3 (ATCC; CRL1658), plus the ST-2 cell line derived from bone marrow (Riken Cell Bank, Japan), were all kindly provided by R. Gruber (Institute of Pathophysiology, University of Vienna, Austria). The osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria were kindly donated by M. Kumegawa, (Department of Oral Anatomy, Meikai University School of Dentistry, Saitama, Japan). 1,25-(OH)2D3was a generous gift from Hoffmann–La Roche (Vienna, Austria) and T3 was purchased from Sigma and used at the concentrations indicated.

Primary osteoblasts from mouse calvaria were prepared by sequential digestion of calvaria of 2-day-old mice and were passaged once after 48 h by 0.01% Pronase/EDTA digestion, replated (10,000 cells/cm²) and grown to confluence (Zhang et al., 1997). All cells were kept in a humidified atmosphere under 5% CO2at 37 °C. Culture medium was αMEM supplemented with 5% FBS, 4.5 mg/ml glucose and 30 μg/ml gentamicin. All cells were subcultured twice a week before reaching confluence. The medium was then renewed and hormonal treatment was carried out for up to 48 h.

Calvarial explants were aseptically dissected from fetal (day F 16.5 and F 17.5) and newborn (2-day-old) mice. Long bones (radii and ulnae) were dissected from fetal mice (day F 17.5 and F 18.5). All mice were from strain HIM, Institute of Experimental Animal Research, University of Vienna, Himberg, Austria. Neonatal calvaria were cultured for 48 h in roller tubes (Klaushofer et al., 1989). Fetal calvaria and long bones were also cultured for up to 48 h in 6- or 24-well culture plates. Culture medium was always as described above and a medium change was performed after 24 h in culture.

2.2 Northern analysis

Total RNA from cell cultures was isolated as described by Wilkinson (1988). RNA from mouse tissues was isolated with TRIZOL Reagent (Invitrogen). 5–20μg of total RNA was applied to each lane of a 1% agarose gel containing 2.2 M formaldehyde. After electrophoresis, the RNA was transferred to a nylon filter (GeneScreen, New England Nuclear Corp., Boston, MA, USA). A mouse collagenase cDNA was used as a hybridization probe (Henriet et al., 1992) (kindly provided by Yves Eeckhout, Université Catholique de Louvain, Faculté de Médecine, Unité de Biologie Cellulaire, Bruxelles, Belgium).

To control RNA loading, we hybridized the same Northern blots using rat glyceraldehyde–phosphate dehydrogenase (GAPDH) cDNA (kindly provided by Meinrad Busslinger, Institute of Molecular Pathology, Vienna, Austria) or a human 18S rDNA probe. Probe labeling was performed by random primed labeling of the excised insert, using a commercially available kit (Roche Diagnostics) with [32P] dCTP (3000 Ci/mmol) (NEN, Boston, MA, USA). Hybridization was performed overnight in a solution consisting of 0.33 M sodium phosphate buffer (pH 7.2) and 6.66% SDS at 65°C. After thorough washes with 2×SSC, 1% SDS at 65°C for 1 h and 0.2×SSC, 0.1% SDS at 60°C for 45min, the membranes were quantified in an Instantimager (Packard Instrument Co., Meriden, CT, USA). Northern blot analyses were performed using at least two independent cultures, and a representative autoradiogram from one experiment that yielded typical results is shown.

2.3 MMP-13 activity assay

For measurements of MMP-13 activity in the culture supernatants, MC3T3-E1 cells were seeded at a density of 20,000/cm²in 24-well plates and cultured for 6 days. Thereafter, the culture medium was changed to serum-free OPTIMEM-1 (250 μl/well; Invitrogen) and hormonal treatments were carried out in quadruplicate for40h. The culture supernatants were assayed with an MMP-13 activity assay kit (Chemicon Inc., Temecula, CA, USA). Thirty μl of each supernatant was activated for 1 h at 37 °C with a mercury compound. A fluorogenic peptide substrate was then added and incubation was continued for a further 30 min at room temperature. Fluorescence was measured at Ex 360 nm/Em 465 nm in a Spectrafluor Plus (Tecan GmbH, Groedig, Austria). Statistical significance was analyzed with StatView 4.5 (Abacus Concepts Inc., Berkeley, CA, USA) using ANOVA (post hoc test; Scheffel).

2.4 Immunohistochemistry

Long bones from fetal (day F 18.5) mice were fixed in 4% phosphate-buffered formaldehyde at 4 °C for 24 h and then decalcified with EDTA for a further 24 h. The specimens were then dehydrated with increasing concentrations of ethanol and embedded in paraffin. The sections were treated with 0.3% hydrogen peroxide in PBS for 15 min at room temperature and then rinsed in PBS. Further immunohistochemistry was performed with the Vector “MOM Immunodetection Kit” (Vector Laboratories, Burlingame, CA, USA) using slight modifications to the manufacturer's instructions. Briefly, slides were incubated in a working solution of MOM Mouse IgG Blocking Reagent for 1 h and rinsed in PBS. The sections were equilibrated with the diluted protein solution (MOM Diluent). A monoclonal antibody against MMP-13 was purchased from Oncogene Research Products (Cambridge, MA, USA) and diluted 1:50 in MOM Diluent. The sections were incubated overnight with this antibody solution at 4 °C, rinsed and incubated for 10 min with the secondary antibody (MOM biotinylated Anti-Mouse IgG Reagent) and rinsed in PBS. For detection, the ABC peroxidase detection system (Vectastain Elite ABC Reagent, Vector Laboratories) was used. After washing with PBS, the sections were immersed in diaminobenzidine solution (DAB substrate kit for peroxidase, Vector Laboratories, Burlingame, CA, USA), counterstained with haematoxylin (Innogenex), dehydrated with increasing concentrations of ethanol and xylene, mounted in permanent mounting medium (Vecta Mount, Vector Laboratories) and viewed with an Axiophot Zeiss Microscope (Oberkochen, Germany); pictures were taken with an Axiocam Zeiss video camera and printed without further processing.

3 Results

3.1 1,25-(OH)2D3and T3 stimulated MMP-13 expression in MC3T3-E1 cells

When grown in the absence of 1,25-(OH)2D3or T3, osteoblast-like MC3T3-E1 cells express minimal amounts of MMP-13. Addition of 1,25-(OH)2D3or T3 to the culture medium resulted in a profound increase in MMP-13 mRNA expression. After 48 h in culture, induction of MMP-13 mRNA with 1,25-(OH)2D3was detectable at doses as low as 10⁻¹⁰M (2-fold), and was maximal at 10⁻⁸M (about 40-fold). Treatment with T3 also resulted in dose-dependent stimulation of MMP-13 mRNA, although only the higher concentrations of T3 (2×10⁻⁸M to 2×10⁻⁷M) were effective (Fig. 1). The maximal stimulatory effect of 1,25-(OH)2D3at 10⁻⁸M was more pronounced than that of T3 at the top dose (2×10⁻⁷M). Time course experiments revealed a 4-fold increase in MMP-13 mRNA expression after 6 h of 1,25-(OH)2D3treatment. A plateau was reached (about 30- to 40-fold of the initial level) at 24 h that was sustained out to 48 h. Indicating a different mechanism of action, T3 (2×10⁻⁷M) increased MMP-13 mRNA levels 3-fold only after a lag period of at least 24 h, and 5- to 10-fold after 48 h of treatment. However, the effects of both hormones were reversible: 24 h after withdrawal of the hormones from the cultures, MMP-13 mRNA levels returned to baseline (Fig. 2).

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Fig. 1

Dose dependence of 1,25-(OH)2D3(10⁻¹¹M to 10⁻⁷M) and T3 (2×10⁻¹¹M to 2×10⁻⁷M) stimulated MMP-13 expression in MC3T3-E1 cells after 48 h of induction (Northern analysis). Ten μg of total RNA was applied to each lane and hybridized with MMP-13 cDNA probes. Hybridization using a GAPDH cDNA probe was included as a control for RNA loading.

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Fig. 2

Time-course studies of 1,25-(OH)2D3(10⁻⁸M) and T3 (2×10⁻⁷M) stimulated MMP-13 expression in MC3T3-E1 cells (Northern analysis). Cells were treated with the hormones for up to 48 h, thereafter the cultures were continued in control media for an additional 2 h (+2) or 24 h (+24). Total RNA was isolated at the various time points of stimulation. Ten μg were applied to each lane and hybridized with the MMP-13 cDNA probe. Hybridization using a GAPDH cDNA probe was included as a control for RNA loading.

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To investigate whether the increment in MMP-13 mRNA expression also results in increased MMP-13 secretion by MC3T3-E1 cells, we used an MMP-13 activity assay. In line with the mRNA results, treatment for 40 h with 1,25-(OH)2D3(10⁻⁸M and 10⁻⁷M) or T3 (2×10⁻⁶M and 2×10⁻⁷M) resulted in a significant increase in the amount of MMP-13 activity in the culture supernatants. Again, the effect of 1,25-(OH)2D3(10⁻⁸M) was more pronounced than that of T3 (2×10⁻⁷M and 2×10⁻⁶M) (Fig. 3).

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Fig. 3

MMP-13 activity was measured in the culture supernatant of MC3T3-E1 cells. After treatment with or without 1,25-(OH)2D3(10⁻⁷M to 10⁻⁹M) or T3 (2×10⁻⁶M to 2×10⁻⁸M) for 40h, supernatants were activated and measured as described in Materials and Methods. Bars represent mean S.E. (n=4) and are given as relative fluorescence units (FU). ***P<0.001 (treated vs. control).

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3.2 Differential expression of MMP-13 in non-osteoblastic and osteoblast-like cell-lines and primary osteoblasts; comparison to Osc

To further investigate the effects of 1,25-(OH)2D3and T3 on MMP-13 expression, we added the hormones to the culture media of various mouse non-osteoblastic and osteoblastic cell lines, as well as to cultures of primary osteoblasts derived from calvaria of newborn mice. As non-osteoblastic cells we used the fibroblastic NIH3T3 and C3H10T1/2 cells. ST-2 and MC3T3-E1 and primary cells were chosen as osteogenic cell systems. ST-2 cells, a bone marrow derived cell line, represent a preosteoblastic phenotype, as differentiation of these cells into osteoblast-like cells is initiated by the presence of ascorbic acid (Otsuka et al., 1999). Mouse primary osteoblasts, isolated from calvaria of 2-day-old mice, were used as a finite system comparable in their differentiation pattern to the immortalized MC3T3-E1 cell line (Zhang et al., 1997).

In agreement with Runx2-dependent expression of MMP-13, we did not detect MMP-13 mRNA in NIH3T3 or C3H10T1/2 cells, either under control conditions or after treatments (Fig. 4). Neither MC3T3-E1 nor ST-2 cells expressed MMP-13 in detectable amounts under control conditions, but both cell lines expressed MMP-13 mRNA after 48 h treatment with 1,25-(OH)2D3(10⁻⁸M). T3 also increased expression of MMP-13 in MC3T3-E1 cells, but not in ST-2 cells. These results indicate that induction of MMP-13 mRNA expression by hormonal treatments depends on the developmental state of the osteoblastic cells.

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Fig. 4

Cell type specificity of stimulated MMP-13 and Osc mRNA expression (Northern blot) in the mouse. Comparison of NIH3T3, 10T1/2, ST-2, and MC3T3-E1 cell lines and primary cells derived from neonatal (2-day-old) calvaria. Total RNA from the cells was isolated as indicated, and cultured with or without T3 (2×10⁻⁷M) or 1,25-(OH)2D3(10⁻⁸M) for 48 h. 5–15 μg total RNA was applied per lane and hybridized with MMP-13 and Osc specific cDNA probes. Hybridization using a GAPDH cDNA probe was included as a control for RNA loading.

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A different pattern of MMP-13 expression was found in cultures of primary osteoblasts isolated from calvaria of newborn mice. As expected, high levels of MMP-13 mRNA were detected in untreated cultures, reflecting a more mature osteoblast population. Subsequent stimulation of MMP-13 mRNA expression by the addition of 1,25-(OH)2D3or T3 was insignificant (Fig. 4, upper lane).

We then examined the expression of Osc, a late marker of the osteoblastic phenotype. As expected, Osc mRNA was not detected in non-osteoblastic cells (NIH3T3 and C3H10T1/2). Neither the osteogenic ST2 cells nor the osteoblast-like MC3T3-E1 cells or primary osteoblasts expressed significant amounts of Osc under control conditions. However, in MC3T3-E1, as well as primary osteoblastic cells, but not in the preosteoblastic ST-2 cells, 48 h treatment with T3 (2×10⁻⁷M) induced Osc mRNA expression. Consistent with earlier reports (Lian et al., 1997; Zhang et al., 1997), 1,25-(OH)2D3did not stimulate Osc expression in mice (Fig. 4, second lane). Taken together, these results indicate that transcriptional mechanisms leading to hormonally stimulated MMP-13 and Osc expression may depend on cofactors provided by the osteoblast through its developmental sequence.

3.3 Expression of MMP-13 in fetal and neonatal calvaria; comparison to Osc expression

To determine whether the high basal expression of MMP-13 found in primary osteoblasts cultured from newborn mice was related to the developmental stage of the osteoblasts in situ, we cultured calvaria from newborn mice and from fetuses at the beginning of osteogenesis (F 16.5 and F 17.5).

MMP-13 was highly expressed under control conditions from the onset of skeletal formation, throughout mouse development to the postnatal stages (2-day-old) (Fig. 5), whereas basal Osc expression was increased in calvaria from newborn mice. Treatment of calvarial tissue cultures with 1,25-(OH)2D3and T3 did not result in significant changes in the amount of MMP-13 transcripts. In contrast, Osc mRNA levels were slightly lower after 1,25-(OH)2D3treatment, but augmented by T3 treatment in bone tissue.

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Fig. 5

Expression of MMP-13 and Osc in tissue cultures from fetal (day F16.5, F17.5) and new born (2-day-old) mouse calvaria, and of MMP-13 in tissue cultures from fetal (day F 18.5) mouse metatarsals. In control cultures, Osc was only found in neonatal calvaria, while MMP-13 was expressed at much earlier timepoints in osteogenesis. Total RNA (10 μg) prepared from calvaria or metatarsals cultured with or without T3 (2×10⁻⁷M) and 1,25-(OH)2D3(10⁻⁸M) for 48 h was applied to each lane and hybridized with MMP-13 and Osc specific cDNA probes. Hybridization using an 18S rDNA probe was included as a control for RNA loading.

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3.4 MMP-13 expression in fetal long bones

We further extended our investigation to the study of MMP-13 expression in long bones (metatarsal, tibia and ulna), where, in contrast to the direct or membranous bone formation process in calvaria, endochondral ossification prevails. Fetal mouse metatarsals were dissected at day F 18.5 and cultured for 2 days in the presence or absence of hormones. Again, MMP-13 mRNA was already expressed at relatively high levels in fetal long-bones (Fig. 6) and the stimulatory effect by 1,25-(OH)2D3or T3 was only marginal.

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Fig. 6

Expression of MMP-13 in cultured metatarsals of fetal mice. Long bones from fetal mice (day F18.5) were cultured with or without T3 (2×10⁻⁷M) or 1,25-(OH)2D3(10⁻⁸M) for 48 h. Five μg of total RNA was probed with MMP-13 cDNA and 18S rDNA probes.

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To verify the localization of MMP-13 protein within developing long bone, immunohistochemistry on decalcified paraffin sections was performed. Metatarsal bone from fetal mice (day F 18.5) cultured for 48 h in the absence (Fig. 7A) or presence of T3 (Fig. 7B) or 1,25-(OH)2D3(Fig. 7C) was used. At this stage of osteogenesis, the bone rudiments consist of a primitive mineralized core in the short diaphysis. Cartilage displayed all stages of endochondral differentiation (proliferating, maturating, pre-hypertrophic and hypertrophic chondrocytes). The developing long bones appeared to be enveloped by perichondrium and periosteum, and a thin layer of osteoid was visible toward the diaphyses. Metatarsals cultured in the presence of 1,25-(OH)2D3and T3 showed a slightly enlarged calcified area in the center of the diaphyses compared to the control bones, but otherwise appeared very similar.

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Fig. 7

Immunolocalization of MMP-13 in long bones of fetal (day F18.5) mice on paraffin sections. Bars=200 μm. (A, B and C) Metatarsals cultured without (A) or with T3 (B) (2×10⁻⁷M) and (C) 1,25-(OH)2D3(10⁻⁸M) for 48 h. (C and D) Tibia and ulna (non-cultured). Immunolocalization of MMP-13 is seen as brown staining in the perichondrium, hypertrophic chondrocytes and primary spongiosa. Proliferating cartilage cells are not stained. Note the enlarged calcified cartilage zone in the central part of the bone rudiment cultured in the presence of 1,25-(OH)2D3and T3, compared to the control bone.

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In all cultured bones, MMP-13 staining (brownish precipitate) appeared in some pre-hypertrophic cells, but was mainly seen in the area of hypertrophic cartilage. Interestingly, MMP-13 expression was also observed in the cells of the periosteum and perichondrium. In addition, the mineralized cores, indicative of osteoblastic activity in the center of the diaphyses, were immuno-positive for MMP-13. Tibiae (Fig. 7D) and ulnae (Fig. 7E) of fetal mice at day F 18.5 were also immediately processed for immunohistochemistry. As expected, their MMP-13 expression pattern was similar to metatarsals. Hypertrophic chondrocytes, as well as cells of the bone collar and periosteum, were immuno-positive for MMP-13.

4 Discussion

The calciotropic hormones 1,25-(OH)2D3and T3 play an essential role in maintaining bone homeostasis and are important modulators of osteoblastic gene expression. In this study we assessed whether mouse osteogenic cells respond differently to these hormones depending on their maturation state.

From our results, it is evident that the studied cell systems had different ratios of MMP-13 and Osc expression, with the higher states of differentiation corresponding to high levels of MMP-13 and Osc. The apparent co-expression of MMP-13 and Osc in the culture systems used may reflect the heterogeneity of osteoblastic phenotypes in in vitro cultures and within tissues, rather than co-expression within a single cell (Candeliere et al., 2001; Fratzl-Zelman et al., 1997). Indeed, it has recently been shown that the expression of MMP-13 and Osc are mutually exclusive in osteoblasts (Tuckermann et al., 2000; Yamagiwa et al., 1999). Thus, the target cells for 1,25-(OH)2D3and T3 may reside in different osteoblastic developmental stages. Our results show that 1,25-(OH)2D3modulates the differentiation of pre-osteoblasts to express MMP-13, but inhibits the development of a fully mature osteoblastic phenotype (Osc expression).

This observation is consistent with published data (Ecarot and Desbarats, 1999; Ishida et al., 1993; Owenet al., 1991), where continuous or transient exposure of proliferating osteoblasts to 1,25-(OH)2D3blocks the formation of the mature osteoblastic phenotype as well as matrix mineralization. In addition, 1,25-(OH)2D3has been found to be a negative regulator of mouse Osc expression, probably via inhibition of Runx2 activity on the mouse Osc promoter (Lian et al., 1997; Varga et al., in press; Zhang et al., 1997). In contrast, T3 is a potent stimulator of osteoblast differentiation and Osc expression (Varga et al., 1997; Varga et al., in press), but, like 1,25-(OH)2D3,inhibits extracellular matrix deposition (Fratzl-Zelman et al., 1997; Luegmayr et al., 1996; Luegmayr et al., 2000). Here we show that, in sharp contrast to the hormonal regulation of Osc, 1,25-(OH)2D3and T3 are both able to significantly increase MMP-13 mRNA expression and activity in mouse osteoblast-like cells, reflecting the inhibitory effect of both hormones on extracellular matrix maturation.

Comparison of MMP-13 and Osc expression patterns in osteoblast-like cells suggests that these marker genes may be inversely regulated, as high MMP-13 expression is associated with low Osc levels and vice-versa. In the context of osteoblastic differentiation this could indicate a functional dissociation between osteoblasts expressing MMP-13 and those expressing Osc regulated at the hormonal level. The mechanism underlying this regulation is unclear, but differential use of cofactors recruited to hormone-receptor complexes in a promoter-dependent fashion might explain the opposing effects of 1,25-(OH)2D3on MMP-13 and Osc expression in mouse osteoblasts with distinct differentiation states (Kraichely and MacDonald, 1998).

With regards to T3, our present data add support to previous findings showing that in vitro T3 enhances the expression of genes associated with differentiation (MMP-13) and maturation (Osc) of osteoblasts (Fratzl-Zelman et al., 1997; Glantschnig et al., 1996; Kasonoet al., 1988; Klaushofer et al., 1995; Luegmayr et al., 1996; Varga et al., 1997, 1999). We deduce from our findings, supported by other recent studies, that 1,25-(OH)2D3drives early stages and inhibits late stages of mouse osteoblast development (Ecarot and Desbarats, 1999), whereas T3 can favor both preosteoblastic and osteoblastic phenotypes (Ohishi et al., 1994; Varga et al., 1997).

As we have demonstrated profound MMP-13 expression in developing osteoblasts of the calvaria, we became interested in the localization of MMP-13 in limb bones where endochondrial ossification prevails. Consistent with recent reports (D'Angelo et al., 2000; Johanssonet al., 1997), immunohistochemical detection of MMP-13 is predominant in hypertrophic chondrocytes of the growth plate and further within the calcified cartilage. However, the first steps of osteogenesis in limb bones are directed by osteogenic cells outside the cartilage rudiment leading to a primitive bone collar, thus more closely resembling intramembranous bone formation (Haaijman et al., 1997; Pechak et al., 1986). These cells are known to express alkaline phosphatase, to secrete collagen I and to form an osteoid layer. Interestingly, we detected MMP-13 expression in cells surrounding the cartilage, i.e. the cells of the periosteum and perichondrium. It is conceivable that MMP-13 activity within the bone collar might act to degrade the non-mineralized matrix, which would play an important role in continuous bone remodeling during developmental bone growth. The concerted action of these collagenolytic activities in the bone collar may go hand in hand with MMP-13-mediated degradation of non-calcified ECM within the growth plate.

In summary, our results show that MMP-13 is differentially expressed, as well as regulated, by calciotropic hormones, depending on the developmental status of the osteoblastic cell culture systems. This in vitro pattern is probably a reflection of MMP-13 expression during skeletal formation at very early to late stages of osteogenesis, while Osc-expression and its hormonal regulation is restricted to mature osteoblasts. In addition to its role within the growth plate, MMP-13 expression, and presumably its hormonal sensitivity, could play a role in skeletal remodeling in the bone collar during bone growth.

Acknowledgments

We would like to thank Prof. Dr M. P. M. Erlee for helpful discussion and critical reading of this manuscript. We are grateful to Prof. Dr K. Klaushofer for helpful discussion, critical comments and for his continuous support.

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Received 28 May 2002/16 December 2002; accepted 12 February 2003

doi:10.1016/S1065-6995(03)00037-4

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