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Keywords:

  • plasminogen activators;
  • mice;
  • osteoblast;
  • matrix degradation;
  • bone formation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Plasminogen activators tPA and uPA are involved in tissue remodeling, but their role in bone growth is undefined. Mice lacking tPA and uPA show increased bone formation and bone mass. The noncollagenous components of bone matrix are also increased, probably from defective degradation. This study underlines the importance of controlled bone matrix remodeling for normal endochondral ossification.

Introduction: Proteolytic pathways are suggested to play a role in endochondral ossification. To elucidate the involvement of the plasminogen activators tPA and uPA in this process, we characterized the long bone phenotype in mice deficient in both tPA and uPA (tPA−/−:uPA−/−).

Materials and Methods: Bones of 2- to 7-day-old tPA−/−:uPA−/− and wild-type (WT) mice were studied using bone histomorphometry, electron microscopy analysis, and biochemical assessment of bone matrix components. Cell-mediated degradation of metabolically labeled bone matrix, osteoblast proliferation, and osteoblast differentiation, both at the gene and protein level, were studied in vitro using cells derived from both genotypes.

Results: Deficiency of the plasminogen activators led to elongation of the bones and to increased bone mass (25% more trabecular bone in the proximal tibial metaphysis), without altering the morphology of the growth plate. In addition, the composition of bone matrix was modified in plasminogen activator deficient mice, because an increased amount of proteoglycans (2×), osteocalcin (+45%), and fibronectin (+36%) was detected. Matrix degradation assays showed that plasminogen activators, by generating plasmin, participate in osteoblast-mediated degradation of the noncollagenous components of bone matrix. In addition, proliferation of primary osteoblasts derived from plasminogen activator-deficient mice was increased by 35%. Finally, osteoblast differentiation and formation of a mineralized bone matrix were enhanced in osteoblast cultures derived from tPA−/−:uPA−/− mice.

Conclusions: The data presented indicate the importance of the plasminogen system in degradation of the noncollagenous components of bone matrix and suggest that the accumulation of these proteins in bone matrix—as occurs during plasminogen activator deficiency—may in turn stimulate osteoblast function, resulting in increased bone formation.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Endochondral bone formation is a fundamental process for longitudinal bone growth during vertebrate development. During this process, a cartilage anlage is replaced by bone through a tight coordination of matrix removal and bone deposition. There is evidence that certain proteolytic enzymes, such as matrix metalloproteases, are involved in endochondral bone formation.(1–3) Little is known, however, about the involvement in this process of neutral serine proteases. The major enzymatic pathway included in this group is the plasminogen activator/plasmin pathway, whereby plasminogen is activated into plasmin. This process is induced by tPA and uPA and is inhibited by their specific inhibitor PAI-1.(4) The participation of this pathway in endochondral bone formation is suggested by the presence of uPA and PAI-1 in osteoblasts and chondrocytes in human bone(5) and by the synthesis of all the components of the plasminogen system by osteoblasts(6–9) and osteoclasts(10) in vitro.

Plasmin may be involved in bone matrix removal because it can degrade several components of the extracellular matrix, such as fibronectin, laminin, and the protein core of proteoglycans.(11) Of particular importance to bone may be the ability of plasmin to cleave osteocalcin and release it from hydroxyapatite.(12) Besides these direct effects, uPA-generated plasmin is an activator of several zymogen matrix-degrading proteinases (MMPs)(13) and might participate indirectly in degradation of extracellular bone matrix. Supporting the proteolytic role of plasmin in bone, we and others have found that the plasminogen activator/plasmin pathway participates in degradation of non-mineralized bone-like matrix in vitro.(9,14,15) In line with these observations, Everts et al.(16) have recently shown that plasmin is required for the activity of bone lining cells to degrade the collagen fragments remaining in Howship's lacunae after osteoclastic resorption. This restricted role in matrix resorption may explain earlier observations showing that the global resorption process in explanted fetal metatarsal bones is not affected by plasmin or the plasminogen activators.(17,18)

The other crucial step during endochondral ossification is the formation and mineralization of new bone matrix composed of collagenous and noncollagenous extracellular matrix proteins synthesized by the osteoblasts. Previous studies have suggested a role for the aminoterminal fragment of uPA in inducing osteoblast proliferation.(19) In addition, growth factors play an important role during bone formation, and plasmin has been shown to activate latent transforming growth factor β (TGF-β)(20) and to dissociate insulin-like growth factors (IGFs) from their binding proteins,(21) making them able to exert biological activity. The release and activation by plasmin of growth factors stored in bone matrix could provide a mechanism by which the plasminogen activator/plasmin pathway is involved in bone formation and its coupling to bone resorption.

Because plasminogen activators can affect processes related to bone formation as well as bone resorption, we wanted to determine their role in endochondral bone formation by analyzing the long bone phenotype of neonatal mice deficient in both plasminogen activators tPA and uPA (tPA−/−:uPA−/−).(22) By using a combination of histological and biochemical analyses, electron microscopy evaluation, and in vitro models of bone resorption and formation, we show that bone formation is enhanced and that bone matrix composition is altered in plasminogen activator-deficient mice. The degradation of the noncollagenous proteins of bone matrix is decreased when plasminogen activators are lacking, and together with alterations in osteoblast function, this may contribute to the bone phenotype of the knockout mice.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animals

Mice with combined deficiency of tPA and uPA were generated as previously described.(22) Deletion of the genomic sequences was demonstrated by Southern blot analysis of genomic tail-tip DNA.

The animals were bred in our animal housing facilities (Proefdierencentrum Leuven, Belgium) and kept under conventional housing conditions. They were fed ordinary chow and water ad libitum.

Bone histology

Bones from 5-day-old mice were removed and processed for bone histomorphometry as previously described.(23) Briefly, the bones were embedded undecalcified in methylmetacrylate, and 4-μm-thick longitudinal sections were cut with a rotary microtome (RM 2155; Leica, Heidelberg, Germany) equipped with a tungsten carbide 50° knife. Sections were stained with hematoxylin and eosin (H&E) for general morphological analysis or according to von Kossa to assess mineralized bone.

For immunohistochemistry, bones were fixed in 2% paraformaldehyde in PBS, decalcified in EDTA, and embedded in paraffin. Bone sections were stained with Safranin O-fast green for identification of proteoglycans(24,25) or immunostained for CD-34 as described.(26)

The measurements were performed in a standardized area comprising most of the proximal tibial metaphysis, using a Kontron Image Analyzing System (KS 400 V 3.00; Kontron Electronic, Eching bei Munchen, Germany). All parameters comply with the recommendations of the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research.(27)

Alcian blue/Alizarin red staining of the skeleton

Two-day-old mice were eviscerated, fixed in ethanol, and stained with Alcian blue (Sigma, St Louis, MO, USA) using standard procedures. After clearing in 1% KOH, the skeletons were stained with Alizarin red (Sigma). The clearing process was completed in decreasing ratios of 2% KOH to glycerol.

Electron microscopy analysis

Metatarsals from 5-day-old mice were processed for electron microscopic examination as described previously.(28)

Biochemical analysis of bone

The long bones from 1-week-old mice were isolated and cleaned from adhesive tissue and bone marrow. They were subsequently defatted in trichloroethylene (100%), dehydrated in ether-ethanol 50:50 (vol/vol) followed by ether (100%), and dried in air. The bones were frozen in liquid nitrogen and powdered down to fine particles that were 40–160 μm in size. A part of the bone powder was weighed and analyzed for the mineral content, determined by microcolorimetry, and for type I collagen content, assessed by measuring the hydroxyproline concentration as described.(29) Data of the biochemical quantifications were normalized for dry weight.

The rest of the bone powder was weighed and extracted overnight at 4°C with guanidine HCl/EDTA (4 M/0.5 M, pH 8), in the presence of proteinase inhibitors (1 mM polymethylsulfonilfluoride, 10 μg/ml leupeptin, 1 μg/ml antipain, and 10 μg/ml trasylol; Sigma). After centrifugation at 10,000 rpm for 15 minutes at 4°C, the supernatant was desalted on Sephadex PD-10 columns (Pharmacia, Uppsala, Sweden). The total protein was measured by spectrophotometry (λ 280 nm) using bovine serum albumin (BSA) as standard. Osteocalcin was measured using an in-house radioimmunoassay (RIA) as described.(30) Fibronectin was measured by a competitive ELISA using a method previously described.(31)

Isolation of primary osteoblasts

To prepare primary osteoblasts, calvariae of newborn mice were stripped of the endosteum and periosteum and digested with 2 mg/ml collagenase A (Roche, Mannheim, Germany) in PBS (Gibco BRL, Paisley, Scotland) at 37°C during six 10-minute time intervals. Digestions 1 and 2 were discarded, and the cells harvested from digestions 3–6 were pooled and counted. The cells were cultured at a density of 2.5 × 104/cm2 in α-minimal essential medium (α-MEM; Gibco BRL) supplemented with 10% fetal calf serum (FCS; Gibco BRL) at 37°C in 5% CO2 in air.

Proliferation of primary osteoblasts

Proliferation of primary osteoblasts was studied by assessing the incorporation of3H-thymidine. Subconfluent osteoblasts were replated at 2 × 103 cells/well in a 96-well culture plate, incubated for 48 h in α-MEM with 10% FCS, and thereafter switched to α-MEM supplemented with either 1% FCS or 0.1% BSA (Stem Cell Technologies Inc., Vancouver, BC, Canada). At the indicated time points, cells were pulsed with 1 μCi [3H]-thymidine/well (Amersham International, Buckinghamshire, UK) for 5 h, and the amount of radioactivity incorporated was measured.

Differentiation of primary osteoblasts

Subconfluent primary osteoblasts were trypsinized and seeded at 2.5 × 105 cells/well onto 6-well culture plates in α-MEM supplemented with 10% FCS. At confluence, 25 μg/ml ascorbic acid (Sigma) and 10 mM β-glycerophosphate (Sigma) were added to the culture medium, and the cells were cultured for 4 weeks. At the indicated time points, the cells were lysed in Tris 100 mM, pH 7.6, in the presence of the proteinase inhibitors described above. The cell lysate was sonicated for 20 s and centrifuged at 10,000 rpm for 10 minutes at 4°C. The proteins were precipitated with 15% trichloroacetic acid and thereafter hydrolyzed in 6 N HCL at 120°C for 2 h. The amount of calcium and phosphorus in the hydrolysates was assessed by microcolorimetry (Sigma). The type I collagen content was calculated by measuring the hydroxyproline concentration as described above. The amount of osteocalcin secreted by the osteoblasts in the culture medium was quantified as described above.

Extracellular bone-like matrix preparation and degradation assay

A bone-like non-mineralized matrix was prepared as previously described.(15) Briefly, primary osteoblasts from wild-type (WT) mice were grown until confluency in 6-well culture plates in α-MEM with 10% FCS, whereafter they were cultured for 4 days in medium supplemented with 25 μg/ml ascorbic acid and 10 mM β-glycerophosphate. A radiolabeled matrix was obtained by culturing the cells during the last 3 days of the culture period in medium supplemented with either 0.5 μCi/ml [3H]-amino acid mixture (high specific activity; Amersham International), 0.5 μCi/ml l-[5-3H]-proline (specific activity 15–40 Ci/mmol; Amersham International), or 1 μCi/ml H235SO4 (specific activity 1050–1600 Ci/mmol; NEN Life Science Products, Boston, MA, USA). The cells were lyzed using 10 mM NaHPO4, 140 mM NaCl, pH 7.4 containing 0.5% Triton X-100, and the cytoskeleton was removed by 25 mM NH4OH treatment. Matrices were washed with H2O followed by 75% ethanol, dried, and stored at −20°C.

Primary osteoblasts derived from WT mice were suspended in α-MEM with 10% FCS, counted, and seeded onto the labeled bone-like matrix. After 2 h, medium was changed to α-MEM supplemented with 0.1% BSA and 10 μg/ml human plasminogen. The medium was removed 36 h later, and the remaining matrix and cells were washed three times with PBS. The matrices were degraded with 2.5 mg/ml trypsin (Roche) and 1 mg/ml collagenase in PBS for 1 h at 37°C. Matrix degradation was expressed as the percentage of radioactivity released by the cells in the medium over the total amount of radioactivity calculated as the sum of the amount released in the medium added to the amount remaining in the matrix.

Analysis of gene regulation by real time quantitative reverse transcriptase-polymerase chain reaction

Total RNA was extracted from the long bones derived from newborn mice or from cultures of primary osteoblasts using TRIZOL (Gibco BRL). One to 5 μg RNA was reverse transcribed using Superscript II RT (Gibco BRL) at 42°C for 80 minutes in the presence of 5 μM Oligo(dT)16 (Perkin-Elmer/Applied Biosystems, Foster City, CA, USA). Real time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed according to the manufacturer's protocol using specific forward and reverse primers, and probes with fluorescent dye (FAM) and quencher (TAMRA) for collagen Iα1, osteopontin, and osteocalcin, as described.(26) The expression levels of these genes were normalized for the hypoxanthine transferase (HPRT) gene.

Statistical analysis

Statistical analysis was performed using a statistical software program (NCSS, Kaysville, UT, USA). Results are expressed as mean ± SEM. Data were analyzed by two-tailed Student's t-test, with differences considered significant at p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Morphological analysis of the skeleton

General inspection of the long bones showed that total bone length was increased in 5-day-old knockout mice, with both tibia and femora being longer compared with WT mice (p < 0.05; Fig. 1A). In addition, the ossification centers, measured in the mid-shaft region of long bones stained with Alcian blue and Alizarin red, were consistently longer in 2-day-old tPA−/−:uPA−/− mice compared with WT mice (Fig. 1B). One of the major determinants of longitudinal bone growth is the growth plate. Therefore, we examined the growth plate morphology in H&E stained sections of the proximal tibia. No difference was observed between the two genotypes in the length of the zones of proliferating, maturing, or hypertrophic chondrocytes in 2-, 5-, or 7-day-old mice (results not shown) nor in the morphological appearance of the cells, suggesting that plasminogen activator deficiency did not affect chondrocyte development at the ages investigated. In addition, cartilage calcification proceeded normally in the knockout mice, because the mineralized surface in the hypertrophic cartilage, expressed as percentage of the total zone of hypertrophic chondrocytes, was similar in plasminogen activator-deficient and WT mice (16.8 ± 1.4%, n = 5 versus 20.4 ± 1.9%, n = 6, respectively, NS).

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Figure FIG. 1.. Morphological analysis of the skeleton. (A) Bone length in 5-day-old tPA−/−:uPA−/− (empty bars, n = 8) and WT mice (filled bars, n = 6). Both tibias and femora were significantly longer (p < 0.05) in the knockout mice compared with WT mice. (B) Alcian blue/Alizarin red staining of the skeleton. Skeletal preparations of 2-day-old mice were stained with Alcian blue and Alizarin red as described in the Materials and Methods section. The ossification centers in the midshaft region of metacarpals were longer in tPA−/−:uPA−/− mice compared with WT mice (328 ± 16, n = 3 vs. 284 ± 15, n = 4, respectively, p < 0.05). Bar, 250 μm.

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Increased bone mass in plasminogen activator-deficient mice

Given the increase in the length of the ossification centers, we measured trabecular bone volume in the proximal tibial metaphysis and found a significant increase (25%) in tPA−/−:uPA−/− mice compared with WT mice (Fig. 2A). To confirm this observation, long bones derived from 1-week-old mice were analyzed biochemically. The calcium content in bone from tPA−/−:uPA−/− mice was increased by 14% (p < 0.01) compared with WT mice (Fig. 2B). Similarly, the percentage of phosphorus in bone was significantly increased in the knockout mice compared with WT mice (Fig. 2C). These biochemical data support the results obtained from the histomorphometric analysis that bone mass is increased in tPA−/−:uPA−/− mice.

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Figure FIG. 2.. Bone mass in perinatal tPA−/−:uPA−/− (empty bars, n = 17) and WT mice (filled bars, n = 11). (A) The trabecular bone volume in the proximal tibial metaphysis and (B) calcium and (C) phosphorus content in long bones were significantly increased in plasminogen activator-deficient mice. *p < 0.05; **p < 0.01 vs. WT.

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The increased bone mass was also reflected in the disturbed alignment of osteocytes relative to surrounding matrix as observed by electron microscopy. As shown in Fig. 3, the osteoid layer between the osteocytes and the mineralized bone was thinner in the knockout bones. Semiquantitative analysis of electron microscopic images of 30 randomly chosen osteocytes of each genotype showed that, in tPA−/−:uPA−/− bones, the number of osteocytes lacking non-mineralized matrix was increased (37% versus 26% in WT), whereas the number of osteocytes surrounded by a thick layer of osteoid was decreased (by 9%). In addition, an increased number of osteocytes were surrounded by a fully mineralized matrix in the knockout bones (46% versus 16% in WT bones). These data suggest that either matrix mineralization is increased or osteocyte-mediated non-mineralized matrix degradation is increased in plasminogen activator-deficient mice.

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Figure FIG. 3.. Ultrastructural analysis of bone matrix surrounding the osteocytes in metatarsals of 5-day-old mice. Note the presence of a thick osteoid layer (asterisks) between the osteocytes and the mineralized bone (B) in WT bones (left). In contrast, non-mineralized bone matrix is hardly visible in tPA−/−:uPA−/− bones (right), compatible with an increased mineralization in the latter. N, cell nucleus. Magnification ×19,000.

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The plasminogen activators have been implicated in angiogenesis,(32) and because bone vascularization and endochondral ossification are tightly coupled, we studied vascularization in the proximal tibial metaphysis. Immunohistochemical staining for the endothelial cell marker CD-34 allowed the assessment of the vascularized bone surface, which was calculated as the percentage of bone surface covered by blood vessels. Although this parameter was somewhat lower in tPA−/−:uPA−/− mice compared with WT mice (15.6 ± 1.4%, n = 4 versus 20.2 ± 3.5%, n = 5, respectively), this difference was not statistically significant.

The plasminogen activators may also exert their effect by participating in the remodeling of the extracellular matrix.(33) We therefore determined biochemically the different components of bone matrix in 7-day-old mice. No difference was observed between tPA−/−:uPA−/− and WT bones in the amount of hydroxyproline, which was used as an indicator of type I collagen content (Fig. 4A). In contrast, the osteocalcin content was increased by 45% in bones from the knockout mice compared with WT mice (Fig. 4B). Similarly, the fibronectin content was significantly increased in tPA−/−:uPA−/− bones (Fig. 4C). Finally, we assessed the amount of proteoglycans by staining bone sections from the proximal tibial metaphysis with Safranin O and measuring the bone surface staining positively for proteoglycans. This surface was significantly increased in tPA−/−:uPA−/− bones (Fig. 4D).

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Figure FIG. 4.. Extracellular matrix composition in long bones of 1-week-old tPA−/−:uPA−/− (empty bars, n = 17) and WT mice (filled bars, n = 11). (A) The OH-proline concentration, used as an indicator of type I collagen content, was not different between the two genotypes. In contrast, (B) osteocalcin and (C) fibronectin content, as well as (D) the amount of proteoglycans in the proximal tibial metaphysis, was significantly increased in plasminogen activator-deficient mice. **p < 0.01; ***p < 0.001 vs. WT.

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Role of the plasminogen system in the degradation of bone matrix

A possible explanation for the increase in mineralized matrix and modification of bone matrix composition in plasminogen activator-deficient mice is that bone matrix resorption is altered when plasminogen activators are lacking. We have previously shown, using a coculture of osteoclasts and osteoblasts, that the plasminogen system is not required for mineral resorption, but affects the degradation of non-mineralized bone matrix.(15) Because osteoblasts are considered to remove non-mineralized matrix from the bone surface, the contribution of plasminogen activators produced by primary osteoblasts was investigated. Primary WT osteoblasts degraded the extracellular matrix metabolically labeled with a [3H]-amino acid mixture more efficiently in the presence of plasminogen (p < 0.0001), and this effect was completely inhibited by trasylol (Fig. 5). In contrast, the amount of matrix degraded by tPA−/−:uPA−/− osteoblasts remained low even in the presence of plasminogen (Fig. 5). These results indicate that the plasminogen activators are involved in osteoblast-mediated extracellular matrix degradation through a plasminogen/plasmin-dependent pathway.

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Figure FIG. 5.. Plasmin(ogen)-dependent degradation of non-mineralized bone-like matrix by primary osteoblasts. WT (filled bars) and tPA−/−:uPA−/− (open bars) cells were cultured on bone-like matrix labeled with a [3H]-amino acid mixture in serum-free medium in the absence (−) or presence (+) of plasminogen (10 μg/ml) or trasylol (10 U/ml). Matrix degradation was calculated as the percentage of the radioactivity released in the medium over the total amount of radioactivity. The data represent the mean ± SE of three separate experiments. *p < 0.0001 vs. WT with plasminogen without trasylol.

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The extracellular matrix produced by cultured osteoblasts is composed mainly of type I collagen and noncollagenous proteins. To investigate the role of the plasminogen system in the degradation of the major components of the extracellular matrix, we used matrices labeled with [3H]-proline, to assess type I collagen degradation, or with [35S]-sulfate, to assess degradation of proteoglycans. Although type I collagen degradation by WT osteoblasts increased in the presence of plasminogen (Fig. 6A), the actual amount of type I collagen degraded by WT cells with plasminogen was low (7.0 ± 0.8% of the type I collagen content in the extracellular matrix). In contrast, degradation of proteoglycans by WT osteoblasts was strongly enhanced in the presence of plasminogen (62 ± 1% versus 14 ± 2%; Fig. 6B). These data suggest that the plasminogen activator/plasmin pathway is primarily involved in the osteoblast-mediated degradation of proteoglycans present in the non-mineralized bone matrix. Moreover, they provide an explanation for the increased amount of proteoglycans in bones of plasminogen activator-deficient mice.

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Figure FIG. 6.. Plasmin(ogen)-dependent degradation of type I collagen and proteoglycans present in the matrix. WT primary osteoblasts were cultured on WT matrix labeled with (A) [3H]-proline or (B) [35S]-sulfate in serum-free medium in the absence (−) or presence (+) of plasminogen (10 μg/ml). Cell-independent matrix degradation was assessed by isotope release in medium in the absence of cells. The percentage of the radioactivity released in the medium relative to the total amount of radioactivity is given. Similar results were obtained in three separate experiments and the data show the results of a representative experiment. *p < 0.01 vs. WT with plasminogen.

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Osteoblast characteristics in tPA−/−:uPA−/− mice

Alternatively, the increase in mineralized matrix in the knockout bones may be related to alterations in osteoblastic bone formation. To examine whether the variations in bone matrix proteins reflect changes in gene expression levels, we performed qRT-PCR analysis of mRNA isolated from long bones of newborn mice. Collagen I-α1 mRNA level was 50% higher in tPA−/−:uPA−/− bones compared with WT bones (92 ± 14, n = 4 versus 61 ± 8, n = 4, respectively, NS). Furthermore, mRNA levels for osteopontin and osteocalcin, two proteins associated with the mature osteoblast phenotype, were increased by 83% (p < 0.01) and 76% (p < 0.05), respectively, in tPA−/−:uPA−/− bones compared with WT bones. In contrast, mRNA levels of osteoblast-related proteins involved in osteoclastogenesis such as RANKL or osteoprotegerin (OPG) were not different between the two genotypes (results not shown).

To investigate whether this increase in matrix production is related to altered osteoblast proliferation and/or differentiation, these processes were studied in vitro using primary cultures. Plasminogen activator deficiency resulted in increased osteoblast proliferation, as indicated by a 35% increase in [3H]-thymidine incorporation in cultures of tPA−/−:uPA−/− osteoblasts compared with WT osteoblasts (8504 ± 283, n = 5 versus 6297 ± 292, n = 5, respectively, p < 0.001). This effect was apparent after 24 h of culture and persisted at the additional time points studied (48, 72, and 96 h; results not shown) independently of the presence or absence of serum.

Osteoblast differentiation was studied in long-term cultures, starting from identical numbers of primary osteoblasts from tPA−/−:uPA−/− and WT mice. Deposition of type I collagen in the extracellular matrix, assessed by measuring hydroxyproline content in matrix, was consistently higher in tPA−/−:uPA−/− cultures compared with WT cultures, and reached statistical significance at the second week of culture (Fig. 7A). The osteocalcin secretion in medium, a specific marker of the differentiated osteoblasts, increased significantly after the second week of culture in osteoblasts of both genotypes and corresponded with the initiation of mineralization. The osteocalcin concentration was, however, consistently higher in media from tPA−/−:uPA−/− osteoblasts compared with WT osteoblasts (Fig. 7B). Similarly to osteocalcin production, significantly more calcium (Fig. 7C) and phosphorus (results not shown) were deposited in the matrix produced by plasminogen activator-deficient osteoblasts compared with WT osteoblasts.

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Figure FIG. 7.. Differentiation and mineralization of tPA−/−:uPA−/− (empty circles) and WT (filled circles) primary osteoblasts during long-term culture in the presence of ascorbic acid and β-glycerophosphate. (B) Osteocalcin secretion in medium, (A) type I collagen content, and (C) mineral deposition in the extracellular matrix were enhanced in osteoblast cultures from plasminogen activator-deficient mice. Similar results were obtained in three independent experiments, and the data represent the results of a representative one. *p < 0.05; **p < 0.01 vs. WT cells at the same time point.

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To investigate whether this increased matrix production reflected altered gene expression levels, qRT-PCR was performed on mRNA from mutant and WT osteoblast cultures. While collagen I-α1 mRNA level was consistently higher in tPA−/−:uPA−/− cultures, osteopontin mRNA level fluctuated during the culture period but tended to be higher in the knockout cultures (Figs. 8A and 8B). Gene expression level for osteocalcin was higher in tPA−/−:uPA−/− osteoblasts compared with WT osteoblasts at all the time points studied, and this difference became significant at the end of the culture period (Fig. 8C).

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Figure FIG. 8.. Gene expression in tPA−/−:uPA−/− (empty circles) and WT (filled circles) primary osteoblasts during long-term culture in the presence of ascorbic acid and β-glycerophosphate. The levels of (A) collagen Iα1, (B) osteopontin, and (C) osteocalcin mRNA were corrected for the HPRT message level. The data represent the mean ± SE of three separate experiments and are expressed as percentage of mRNA levels expressed by WT osteoblasts at the end of the culture period, which were arbitrarily set to 100%. *p < 0.05 vs. WT cells at the same time point.

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Taken together, these in vitro data support the increased bone mass in plasminogen activator-deficient mice, and moreover, identify the enhanced osteoblast proliferation and differentiation as a possible underlying mechanism for the in vivo observations.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We studied the role of the plasminogen activators in endochondral bone formation and report here that inactivation of these proteases in vivo is associated with increased bone mass and altered composition of extracellular bone matrix. Defects in degradation of the noncollagenous components of the extracellular bone matrix, together with accelerated development of osteoblasts, emerge as mechanisms that contribute to the bone phenotype of plasminogen activator-deficient mice.

Endochondral bone formation is a complex process requiring careful regulation of the activities of different cell types. The plasminogen activators seem, however, not to be involved in chondrocyte development in neonatal mice, because at the investigated ages, the morphology of the growth plate was normal in plasminogen activator-deficient mice, despite the fact that bone length was increased. Nevertheless, the increase in the length of the ossification center, in the trabecular bone volume, and in the mineral fraction all point to an increased bone mass in the knockout mice. Electron microscopic analysis revealed a thinner unmineralized matrix layer between the osteocytes and mineralized matrix, compatible with an accelerated mineralization and making the contribution of impaired osteoclast activity less likely. In agreement with the latter, we have previously shown that osteoclast formation and activity to resorb mineral are not affected by plasminogen activator deficiency.(15) Interestingly, specific components of the extracellular matrix were regulated differentially in the knockout bones, with a strong accumulation of noncollagenous proteins such as osteocalcin, fibronectin, and proteoglycans. These data indicate that in the absence of the plasminogen activators, bone matrix is abnormally remodeled during endochondral ossification, resulting in an increased bone mass.

The plasminogen system is implicated in extracellular matrix proteolysis and tissue remodeling in a variety of biological processes, including ovulation, angiogenesis, tumor growth and metastasis, and inflammatory response.(32) Several mechanisms contribute to this role: direct degradation of extracellular matrix components by plasmin,(33) activation by uPA-generated plasmin of other MMPs,(13) and release and activation of growth factors from the extracellular matrix.(34,35) In line with its role in extracellular matrix remodeling in nonbone tissues, we and others have previously shown that the plasminogen activator/plasmin pathway is involved in degradation of extracellular bone-like matrix.(9,14,15) In the present study, we extend these observations and provide evidence that the plasminogen activators produced by osteoblasts play a restricted but definitive role in the degradation of non-mineralized bone matrix. This finding correlates with recent morphological data showing that the serine proteases participate in digestion of demineralized bone matrix by bone-lining cells.(16) Our data also show that the plasminogen system is specifically required for degradation of the noncollagenous components of the extracellular bone matrix. This finding is consistent with the well-defined role of plasmin to degrade the protein core of proteoglycans,(11,35) fibronectin,(11) and osteocalcin.(12) The in vitro data showing that the plasminogen activator/plasmin pathway is required for the removal of noncollagenous components of bone matrix may provide an explanation for the altered bone composition and accumulation of proteoglycans, osteocalcin, and fibronectin in bones of plasminogen activator-deficient mice. Moreover, all these extracellular matrix components have been implicated in bone mineralization,(36–39) suggesting a link between their accumulation in bone tissue and the increased bone formation in tPA−/−:uPA−/− mice. Although plasmin is an activator of MMPs,(13) immunoprecipitation studies did not provide evidence for the participation of active MMPs in plasmin-dependent matrix degradation (our unpublished observations). Compatible with this observation, type I collagen—whose degradation cannot be accomplished by plasmin, but requires active MMPs—was only minimally degraded during plasmin-dependent extracellular matrix breakdown.

The present study also suggests that the increased bone mass in plasminogen activator-deficient mice is at least partly related to changes in osteoblast function. First, we found that deficiency of the plasminogen activators results in increased osteoblast proliferation. These data do not support a role for the growth factor domain of uPA in stimulating osteoblast proliferation, as previously suggested.(19) A possible explanation for this contradiction may be the difference in the experimental system used: our results reflect proliferation of primary osteoblasts, whereas the data reported by Rabbani et al. have been obtained from cultures of human osteosarcoma cells (SaOS-2). Second, an enhanced expression of osteoblast markers and accelerated mineralization rate in osteoblast cultures derived from the knockout mice was found. This is further supported by the increased mRNA levels for type I collagen, osteocalcin, and osteopontin, observed both in vivo and in vitro, indicating that plasminogen activator deficiency is associated with increased matrix production by the osteoblasts. However, the alterations in osteoblast function seem to be restricted to their role in formation of new bone matrix, without affecting other osteoblast functions such as their ability to support osteoclastic bone resorption by producing RANKL and OPG. This may reflect a direct effect of the plasminogen activators to modulate osteoblast function. Alternatively, it may be caused by the deficient proteolytic activity in the absence of plasmin, resulting in impaired degradation and accumulation of noncollagenous proteins, which may in turn stimulate osteoblast activity. Indeed, several studies have shown that the extracellular bone matrix and its specific components play a critical role in regulating osteoblast gene expression, differentiation, and survival.(40–44) Proteoglycans may play an additional role in regulating osteoblast function based on the interaction of their glycosaminoglycans with growth factors. For instance, heparan sulfate proteoglycans bind basic fibroblast growth factor (bFGF)(45) and granulocyte-macrophage colony-stimulating factor (GM-CSF),(46) and decorin and biglycan bind TGF-β.(47) In this way proteoglycans participate in the storage of growth factors in the extracellular matrix, and digestion of their core proteins by plasmin may represent a mechanism for the release and activation of matrix-bound growth factors. Such a pathway has been shown to result in the release of biologically active bFGF from the extracellular structures.(35) Although several studies have evoked the role of plasmin in releasing and activating TGF-β from bone matrix,(20,48) the potential involvement of plasmin in releasing decorin- and biglycan-bound TGF-β from bone matrix remains to be determined. Recent studies suggest also additional roles for heparan sulfate proteoglycans, such as syndecan-2, in mediating the mitogenic activity and intracellular signaling induced by GM-CSF binding in osteoblasts.(46) Taken together, alterations in the specific components of extracellular bone matrix and their interactions with different ligands are likely to have functional biological implications in osteoblasts, resulting in fine tuning of osteoblast growth and differentiation. Consistent with this view, deficiency of the plasminogen activators is associated with altered matrix composition, increased osteoblast proliferation, and differentiation and enhanced bone formation. Whether a causal link exists between the modifications in bone matrix composition in plasminogen activator deficient bones and the enhanced osteoblast activity is at present unclear.

In conclusion, the plasminogen activators are involved in endochondral bone formation because their deficiency results in altered bone composition and increased bone formation. The plasminogen activators also play a critical role in degradation of noncollagenous proteins of bone matrix in vitro. We hypothesize that the cleavage of these components and bone matrix reorganization in vivo may affect osteoblast function, suggesting an indirect role for the plasminogen activators in modulating bone formation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by a grant from Fonds voor Wetenschappelijk Onderzoek (FWO, Grant G.022500). The authors are grateful to Dr P Carmeliet and Dr D Collen for providing the knockout animals, and thank K Moermans and R Van Looveren for technical assistance in bone histomorphometry.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM, Birkedal-Hansen H 1999 MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99:8192.
  • 2
    Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z 1998 MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411422.
  • 3
    Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaisse JM 2000 Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151:879889.
  • 4
    Carmeliet P, Collen D 1995 Gene targeting and gene transfer studies of the plasminogen/plasmin system: Implications in thrombosis, hemostasis, neointima formation, and atherosclerosis. FASEB J 9:934938.
  • 5
    Hackel C, Radig K, Rose I, Roessner A 1995 The urokinase plasminogen activator (u-PA) and its inhibitor (PAI-1) in embryo-fetal bone formation in the human: An immunohistochemical study. Anat Embryol (Berl) 192:363368.
  • 6
    Allan EH, Zeheb R, Gelehrter TD, Heaton JH, Fukumoto S, Yee JA, Martin TJ 1991 Transforming growth factor beta inhibits plasminogen activator (PA) activity and stimulates production of urokinase-type PA, PA inhibitor-1 mRNA, and protein in rat osteoblast-like cells. J Cell Physiol 149:3443.
  • 7
    Allan EH, Martin TJ 1995 Prostaglandin E2 regulates production of plasminogen activator izoenzymes, urokinase receptor, and plasminogen activator inhibitor-1 in primary cultures of rat calvarial osteoblasts. J Cell Physiol 165:521529.
  • 8
    Hoekman K, Löwik CWGM, Van De Ruit M, Bijvoet OLM, Verheijen JH, Papapoulos SE 1991 Regulation of the production of plasminogen activators by bone resorption enhancing and inhibiting factors in three types of osteoblast-like cells. Bone Miner 14:189204.
  • 9
    De Bart ACW, Quax PHA, Löwik CWGM, Verheijen JH 1995 Regulation of plasminogen activation, matrix metalloproteinases and urokinase-type plasminogen activator-mediated extracellular matrix degradation in human osteosarcoma cell line MG63 by interleukin-1 alpha. J Bone Miner Res 10:13741384.
  • 10
    Yang JN, Allan EH, Anderson GI, Martin TJ, Minkin C 1997 Plasminogen activator system in osteoclasts. J Bone Miner Res 12:761768.
  • 11
    Mignatti P, Rifkin DB 1993 Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 73:161195.
  • 12
    Novak JF, Hayes JD, Nishimoto SK 1997 Plasmin-mediated proteolysis of osteocalcin. J Bone Miner Res 12:10351042.
  • 13
    Carmeliet P, Moons L, Lijnen R, Baes M, Lemaitre V, Tipping P, Drew A, Eeckhout Y, Shapiro S, Lupu F, Collen D 1997 Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet 17:439444.
  • 14
    Ronday HK, Smits HH, Quax PHA, Van Der Pluijm G, Löwik CWGM, Breedveld FC, Verheijen JH 1997 Bone matrix degradation by the plasminogen activation system. Possible mechanism of bone destruction in arthritis. Br J Rheumatol 36:915.
  • 15
    Daci E, Udagawa N, Martin TJ, Bouillon R, Carmeliet G 1999 The role of the plasminogen system in bone resorption in vitro. J Bone Miner Res 14:946952.
  • 16
    Everts V, Delaisse JM, Korper W, Jansen DC, Tigchelaar-Gutter W, Saftig P, Beertsen W 2002 The bone lining cell: Its role in cleaning Howship's lacunae and initiating bone formation. J Bone Miner Res 17:7790.
  • 17
    Leloup G, Delaissé JM, Vaes G 1994 Relationship of the plasminogen activator/plasmin cascade to osteoclast invasion and mineral resorption in explanted fetal metatarsal bones. J Bone Miner Res 9:891902.
  • 18
    Leloup G, Lemoine P, Carmeliet P, Vaes G 1996 Bone resorption and response to calcium-regulating hormones in the absence of tissue or urokinase plasminogen activator or of their type 1 inhibitor. J Bone Miner Res 11:11461157.
  • 19
    Rabbani SA, Mazar AP, Bernier SM, Haq M, Bolivar I, Henkin J, Goltzman D 1992 Structural requirements for the growth factor activity of the amino-terminal domain of urokinase. J Biol Chem 267:1415114156.
  • 20
    Yee JA, Yan LY, Dominguez JC, Allan EH, Martin TJ 1993 Plasminogen-dependent activation of latent transforming growth factor beta (TGF-β) by growing cultures of osteoblast-like cells. J Cell Physiol 157:528534.
  • 21
    Campbell PG, Novak JF, Yanosick TB, McMaster JH 1992 Involvement of the plasmin system in dissociation of the insulin-like growth factor-binding protein complex. Endocrinology 130:14011412.
  • 22
    Carmeliet P, Schoonjans L, Kieckens L, Ream B, Degen J, Bronson R, De Vos R, Van Den Oord JJ, Collen D, Mulligan RC 1994 Physiological consequences of loss of plasminogen activator gene function in mice. Nature 368:419424.
  • 23
    Daci E, Verstuyf A, Moermans K, Bouillon R, Carmeliet G 2000 Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency. J Bone Miner Res 15:15101516.
  • 24
    Ohshima S, Saeki Y, Mima T, Sasai M, Nishioka K, Nomura S, Kopf M, Katada Y, Tanaka T, Suemura M, Kishimoto T 1998 Interleukin 6 plays a key role in the development of antigen-induced arthritis. Proc Natl Acad Sci USA 95:82228226.
  • 25
    Ekholm E, Hankenson KD, Uusitalo H, Hiltunen A, Gardner H, Heino J, Penttinnen R 2002 Diminished callus size and cartilage synthesis in α1β1 integrin-deficient mice during bone fracture healing. Am J Pathol 160:17791785.
  • 26
    Maes C, Carmeliet P, Moermans K, Stockmans I, Smets N, Collen D, Bouillon R, Carmeliet G 2002 Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev 111:6173.
  • 27
    Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry: Standardization of nomenclature, symbols, and units. J Bone Miner Res 2:595610.
  • 28
    Everts V, Delaisse JM, Korper W, Niehof A, Vaes G, Beertsen W 1992 Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases. J Cell Physiol 150:221231.
  • 29
    Lammens J, Liu Z, Aerssens J, Dequeker J, Fabry G 1998 Distraction bone healing versus osteotomy healing: A comparative biochemical analysis. J Bone Miner Res 13:279286.
  • 30
    Bouillon R, Vanderschueren D, Van Herck E, Nielsen HK, Bex M, Heyns W, Van Baelen H 1992 Homologous radioimmunoassay of human osteocalcin. Clin Chem 38:20552060.
  • 31
    Hoeben E, Briers T, Vanderstichele H, De Smet W, Heyns W, Deboel L, Vanderhoydonck F, Verhoeven G 1995 Characterization of newly established testicular peritubular and prostatic stromal cell lines: Potential use in the study of mesenchymal-epithelial interactions. Endocrinology 136:28622873.
  • 32
    Carmeliet P, Collen D 1998 Development and disease in proteinase-deficient mice: Role of the plasminogen, matrix metalloproteinase and coagulation system. Thromb Res 91:255285.
  • 33
    Clark IM, Murphy G 1999 Matrix proteinases. In: SeibelMJ, RobinsSP, BilezikianJP (eds.) Dynamics of Bone and Cartilage Metabolism. Academic Press, San Diego, CA, USA, pp. 137150.
  • 34
    Saksela O, Rifkin DB 1988 Cell-associated plasminogen activation: Regulation and physiological functions. Annu Rev Cell Biol 4:93126.
  • 35
    Saksela O, Rifkin DB 1990 Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity. J Cell Biol 110:767775.
  • 36
    Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P, Karsenty G 1998 Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone 23:187196.
  • 37
    Boskey AL 1999 Mineralization, structure, and function of bone. In: SeibelMJ, RobinsSP, BilezikianJP (eds.) Dynamics of Bone and Cartilage Metabolism. Academic Press, San Diego, CA, USA, pp. 153164.
  • 38
    Globus RK, Doty SB, Lull JC, Holmuhamedov E, Humphries MJ, Damsky CH 1998 Fibronectin is a survival factor for differentiated osteoblasts. J Cell Sci 111:13851393.
  • 39
    Daculsi G, Pilet P, Cottrel M, Guicheux G 1999 Role of fibronectin during biological apatite crystal nucleation: Ultrastructural characterization. J Biomed Mater Res 47:228233.
  • 40
    Moursi AM, Globus RK, Damsky CH 1997 Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro. J Cell Sci 110:21872196.
  • 41
    Damsky CH 1999 Extracellular matrix-integrin interactions in osteoblast function and tissue remodeling. Bone 25:9596.
  • 42
    Lian JB, Stein GS 1999 The cells of bone. In: SeibelMJ, RobinsSP, BilezikianJP (eds.) Dynamics of Bone and Cartilage Metabolism. Academic Press, San Diego, CA, USA, pp. 165185.
  • 43
    Chen XD, Shi S, Xu T, Robey PG, Young MF 2002 Age-related osteoporosis in biglycan-deficient mice is related to defects in bone marrow stromal cells. J Bone Miner Res 17:331340.
  • 44
    Miao D, Bai X, Panda D, McKee MD, Karaplis AC, Goltzman D 2001 Osteomalacia in Hyp mice is associated with abnormal Phex expression and with altered bone matrix protein expression and deposition. Endocrinology 142:926939.
  • 45
    Saksela O, Moscatelli D, Sommer A, Rifkin DB 1988 Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol 107:743751.
  • 46
    Modrowski D, Basle M, Lomri A, Marie PJ 2000 Syndecan-2 is involved in the mitogenic activity and signaling of granulocyte-macrophage colony-stimulating factor in osteoblasts. J Biol Chem 275:91789185.
  • 47
    Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E 1994 Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J 302:527534.
  • 48
    Dallas SL, Rosser JL, Mundy GR, Bonewald LF 2002 Proteolysis of latent transforming growth factor-β (TGF-β)-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-β from bone matrix. J Biol Chem 277:2135221360.