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Keywords:

  • gelatinase B;
  • collagenase-3;
  • matrix metalloproteinases;
  • skeletal development;
  • cartilage lysis;
  • vascular invasion;
  • zymography

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In the transformation of the cartilaginous epiphysis into bone, the first indication of change in the surfaces destined for resorption is the cleavage of aggrecan core protein by unidentified matrix metalloproteinases (MMPs) (Lee et al., this issue). In cartilage areas undergoing resorption, the cleavage leaves as superficial, 6-μm-thick band of matrix, referred to as “pre-resorptive layer.” This layer harbors G1-fragments of the aggrecan core protein within a framework of collagen-rich fibrils exhibiting various stages of degeneration. Investigation of this layer in every resorption area by gelatin histozymography and TIMP-2 histochemistry demonstrates the presence of an MMP whose histozymographic activity is inhibited by such a low dose of the inhibitor CT1746 as to identify it as gelatinase A or B. Attempts at blocking the histozymographic reactions with neutralizing antibodies capable of inhibiting either gelatinase A or B reveals that only those against gelatinase B do so. Immunostaining of sections with anti–gelatinase B IgG confirms the presence of gelatinase B in every pre-resorptive layer, that is, at the blind end of excavated canals (stage I; 6-day-old rats), at sites along the walls of the forming marrow space (stage II; 7days), at sites within the walls of this space as it becomes the ossification center (stage III; 9 days) and along the wall of the maturing center (stage IV; 10–21 days). We also report the presence of collagenase-3 in precisely the same sites, possibly as active enzyme, but this remains to be proven. Because the results reveal that collagenase-3 is present beside gelatinase B in every pre-resorptive layer and, because these sites exhibit various signs of degradation including fibrillar debris, reduction in fibril number, or overt loss, we propose that gelatinase B and collagenase-3 mediate the lysis of this pre-resorptive layer—most likely through a cooperative attack leading to the disintegration of the collagen fibril framework. © 2001 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

During the transformation of a cartilage model into bone, the secondary ossification center is formed in the epiphysis as the result of complex excavations. Thus in the rat, canals are dug from the surface toward the center of the epiphysis, where their ends fuse to give rise to the initial marrow space which, with the acquisition of ossification units, becomes the secondary ossification center (Levene, 1964; Delgado-Baeza et al., 1991a,b). These excavations are the result of cartilage resorptions that have been localized in a series of sites and classified into two types, according to whether the resorption is associated with bone formation or not (Lee et al., 2001, this issue). The sites involved in bone formation are referred to as “ossification-coupled”, whereas those independent of it are called “free” (Table 1).

Table 1. Stages in the Development of the Rat Proximal Tibial Epiphysis
StageDescription of eventsResorption siteResorption modeOnset (range) in days of age
ICanals emergeCanal blind endFree6 (4–7)
IIMarrow space arisesAll marrow space wallsFree7 (6–8)
IIIHypertrophic chondrocytes appear on proximal wall of marrow spaceTransverse septaeOssification-coupled9 (8–10)
Marrow space expands mainly on the distal wallDistal wallFree
IVSecondary growth plate is delineated on the proximal wallTransverse septaeOssification-coupled10 (9–11)
Distal wall recedesEpiphyseal borderOssification-coupled (but some free segments)

The resorption of cartilage suggests the lysis of the two major cartilage components: the proteoglycan “aggrecan” and the “collagen” fibrils (Cole and Wezeman, 1985, 1987a,b; Cole and Cole, 1989). The lysis of one of the two, aggrecan, has been demonstrated in the proximal epiphysis of the rat by Lee et al. (2001, this issue). The enzyme responsible for the aggrecan lysis has not been directly identified; instead, its presence has been revealed by the detection of a neoepitope known to arise from the cleavage of the aggrecan core protein by a so far unidentified matrix metalloproteinase (MMPs). In the “free” sites, a space approximately 6 μm thick located behind the surface undergoing resorption and known as “pre-resorptive layer” contains the smaller of the two fragments arising from the aggrecan cleavage, the so-called G1-341 fragment. This fragment carries the neoepitope recognition site and is retained in the tissue. Meanwhile, the larger fragment that comprises the bulk of the core protein is lost from the tissue, so that the pre-resorptive layer is essentially composed of G1 fragments with some collagen fibrils. With the loss of the aggrecan mass, the pre-resorptive layer is weakened in preparation for the final resorption (Lee et al., 2001, this issue).

In the present article, the enzymes responsible for the lysis of the other major cartilage component, collagen, are being investigated. Because the cartilage collagen is in the form of heterogeneous fibrils (for review see Eyre and Wu, 1995), the goal is to find which enzymes are involved in the break-up of these collagen fibrils. The approach is based on the use of the histozymographic technique (Lee et al., 1999). This technique consists in applying unfixed frozen sections of a tissue onto slides bearing a target substrate that can be digested by the enzyme suspected of being present in the tissue, as initially done by Adams and Tuqan (1961) and Daoust (1965). In the present case, the slides have been coated with photographic emulsion that is then blackened by exposure to light, so that the target substrate is the emulsion gelatin, whose digestion is made visible by the loss of its black silver grains and the resulting lightening. The histozymographic technique has been used in three successive steps: (1) simple histozymography, (2) histozymography performed in the presence of enzyme inhibitors, and (3) histozymography performed in the presence of “neutralizing” antibodies.

In addition, a second approach has been taken to confirm the presence of an MMP. To do so, antibodies against the candidate enzyme have been used for immunostaining epiphyseal sections at the various stages in the hope of finding whether this enzyme is actually located in the areas inducing histozymographic reactions.

Lastly, TIMP-2, an inhibitor of MMPs, has been found to prevent the appearance of the histozymographic reactions. To find out whether or not this TIMP-2 binds to the tissue, and if so, to further delineate the sites in which it binds, it has been biotinylated, then applied to fresh tissue sections and detected through its biotin label.

By these various approaches, the conclusion has been reached that at least two enzymes are involved in lysing the cartilage collagen in the excavations required for the building of a secondary ossification center. These enzymes complete the lytic process that is initiated by the cleavage of aggrecan core protein.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Changes in the Tibial Head Architecture in Postnatal Rats

A few days before and up to 5 days after birth, the tibia is composed of an ossifying diaphysis between two solidly cartilaginous epiphyses (Fig. 1A). At approximately 6 days of age, a series of cartilage resorptions are initiated in the proximal epiphysis. These processes are divided into four stages, as described in Figure 2 of Lee et al. (2001, this issue) and summarized here in Table 1.

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Figure 1. Histozymographic reactions (B) produced by a section of the tibia from a prenatal rat at embryonic day 20 (E20). A: The structure of the tibia showing two white epiphyses and, between them, a dotted shading indicative of the extent of the primary ossification center. B: Two lightened areas are apparent in the blackened emulsion. These occur adjacent to the metaphyseal interface of the two epiphyses, hereafter referred to as metaphyseal borders. These borders are identified by arrows in 1A). Both are sites of active cartilage resorption. Scale bar = 250 μm in B (applies to A,B).

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Histozymographic Reactions

Exposure of epiphyseal sections from prenatal rats (E20) to blackened gelatin emulsion for 4-hr results in two histozymographic reactions seen as light bands, that correspond to the metaphyseal borders, where endochondral bone formation is taking place (Fig. 1B). At stage I, when the emergence of cartilage canals has commenced, exposure of sections for the same amount of time results in small, more or less intense histozymographic reactions (Fig. 2B) which, by comparison with the sections (Fig. 2A), are assigned to the blind ends of the canals. In addition, an intense band-like reaction at the base of the figure corresponds to the endochondral bone formation under way at the metaphyseal border, where active cartilage resorption has been shown to take place (Lee et al., 1998, 1999, 2001 [this issue]). At stage II, a histozymographic reaction is seen on all marrow space walls or only on part of them as shown by comparing Figure 2C and D. A similar situation is observed at stages III and IV when a histozymographic reaction is observed between as well as over groups of hypertrophic chondrocytes (Fig. 2E,F). The band-like reaction at the base of the figure, which is clear-cut in Figure 2D and distinguishable in Figure 2F, again corresponds to the area of endochondral bone formation at the metaphyseal border.

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Figure 2. Comparison between tibial head section at left, and the histozymograms produced when placed in contact for 4 hr with blackened emulsion slides, at right, shown at stage I (A,B), stage II (C,D), and early stage IV (E,F). A: The section separated from the emulsion and stained with toluidine blue exhibits a large canal and two small ones, all opening at the free surface of the cartilaginous epiphysis. B: The emulsion reveals three discrete histozymographic reactions (arrows), which can be assigned to the blind ends of the three canals, whereas the band-like reaction at the base of the figure (arrows, B) corresponds to the metaphyseal border of the epiphysis. C: The section exhibits one narrow canal, which opens above at the free surface of the cartilaginous epiphysis and is continuous below with the marrow space (MS). D: The emulsion reveals a histozymographic reaction (arrow) which is assigned to the lateral wall of the marrow space (MS) while the canal is unreactive. E: The section exhibits one canal and a marrow space around which groups of hypertrophic chondrocytes can be distinguished (larger arrows). F: The emulsion reveals a circular shaped lightening of the emulsion, which is assigned to the interface of the marrow space walls. In B, D, and F, the band-like reaction at the base of the figure, which is complete in the first two and interrupted in F, corresponds to the metaphyseal border of the epiphysis. The interpretation is that tissue sites facing the lightened emulsion contain a gelatinolytic enzyme. Scale bar = 500 μm in A (applies to A–F).

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When exposure time is reduced to 2 hr (Fig. 3A), the intensity of the histozymographic reactions is much less than in a serial section exposed 4 hr (Fig. 3B). Thus, it is concluded that a proteinase endowed with gelatinolytic activity is reacting at a time-related rate in the sites defined by histozymography.

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Figure 3. Histozymographic reactions produced by placing adjacent serial sections of stage I tibial head in contact with the blackened emulsion for either 2 hr (A) or 4 hr (B). After 2 hr, the reactions are approximately half or less than half as intense as after 4 hr, as shown by the degree of lightening produced at the blind end of the canals (large arrows) or the metaphyseal border of the epiphysis (small arrows) versus time. Scale bar = 500 μm in A (applies to A,B).

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Effect of Inhibitors on the Histozymographic Reactions

To identify the enzyme responsible for the gelatinolytic activity detected by histozymography, the first approach has been to repeat the procedure in the presence of an inhibitor of known specificity during the reaction incubation. In this way, inhibitors of cysteine, serine, and aspartic proteinases do not interfere with the production of histozymograms by sections at stages I–IV (data not shown), but the natural MMP inhibitor TIMP-2 does inhibit the histozymographic reaction (Table 2). Another MMP inhibitor, CT1746, induces a partial inhibition at 0.04 nM (Fig. 4B) and a complete one at 1 nM concentration (Table 2). Anderson et al. (1996) have shown the following inhibition constants (Ki): 0.04 nM for gelatinase A (MMP-2), 0.17 nM for gelatinase B (MMP-9), 10.9 nM for stromelysin-1 (MMP-3), 132 nM for interstitial collagenase (MMP-1), and 136 nM for matrilysin (MMP-7); therefore, the 0.04 nM range observed by histozymography with this inhibitor (Table 2) indicates that the reactivities are produced by gelatinase A or B.

Table 2. Assay of MMP Inhibitors by Histozymographya
InhibitorConcentrationInhibition
  • a

    MMP, matrix metalloproteinase.

CT17460.04 nMPartial
1 nMComplete
TIMP-20.023 μMNone
0.23 μMPartial
2.3 μMComplete
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Figure 4. Comparison of histograms prepared in the absence (A) and presence (B) of low concentration of the MMP inhibitor CT1746. In the absence of the inhibitor, the histozymogram shows reactions at the canal blind end and metaphyseal border (arrows in A), whereas the serial section exposed to the inhibitor shows absent or weak reactivity in these areas (arrows in B). (A higher concentration of CT1746 completely inhibits the reactions; Table 2). Scale bar = 500 μm in A (applies to A,B).

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Effect of Neutralizing Antibodies on the Histozymographic Reactions

The second approach to the gelatinolytic enzyme identification has been to expose stage I–IV tibial heads to neutralizing antibodies against gelatinase A or B before the histozymographic procedure in the hope that one of the two would prevent the reactions from appearing. To test for the presence of gelatinase A, three doses of sheep anti-human gelatinase A (0.5, 1.0, 5.0 mg/ml) have been applied to rat tibial head sections for 30 min at 37°C, then again during contact with blackened emulsion at room temperature, but the production of the histozymograms has not been prevented (Table 3). However, when two concentrations of sheep anti-pig gelatinase B (0.5 mg or 1.0 mg/ml of IgG) have been applied to sections under the same conditions, the lower dosage has partially prevented the reaction, whereas the higher one has inhibited it completely (Table 3), whether the tibial heads are of stage I (Fig. 5A–C), stage II (not shown), stage III (Fig. 5D–F), or stage IV (not shown). Because the gelatinolytic proteinase is neutralized by the anti–gelatinase B IgG, the reactions are attributed to gelatinase B. The suggestion is that gelatinase B is an enzyme located within the histozymographically reactive sites of the tissue.

Table 3. Assay of Neutralizing Antibodies by Histozymographya
TargetPurified IgGAmount (in mg/ml)Inhibition
  • a

    MMP, matrix metalloproteinase; IgG, immunoglobulin G.

Gelatinase A (MMP-2)Sheep anti-human MMP-20.5None
1.0None
5.0None
Gelatinase B (MMP-9)Sheep anti-pig (MMP-9)0.5Partial
1.0Complete
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Figure 5. Comparison of the histozymograms prepared in the absence (B,E) and presence of anti–gelatinase B immunoglobulin G (IgG) antibodies (C,F). At top are reference sections at stage I on the left, showing the blind end of a canal (arrow in A) and at stage III on the right, showing the marrow space wall, which is plain on one side (arrow 2 in D) but associated with hypertrophic chondrocytes on the other side (arrow 1). Below are the corresponding histozymogram controls showing at stage I reactions of canal blind end (arrow in B) and metaphyseal border, and at stage III reactions over the plain side of the marrow space wall (arrow 2 in E) and the hypertrophic chondrocyte associated side (arrow 1) as well as over the two canals and the metaphyseal border. Finally, after exposure to anti–gelatinase B IgG, the serial sections at stage I (C) and III (F) are unreactive. Scale bar = 500 μm in A (applies to A–F).

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Immunolocalization of Gelatinase B

In the hope of confirming the presence of gelatinase B in histozymographically reactive sites, the enzyme has been looked for by an immunohistochemical method. To do so, 8-μm-thick sections of stage I–IV rat tibial heads have been immunostained with anti–gelatinase B IgGs — an approach that detects not only the active enzyme but also its inactive proenzyme and the TIMP-inhibited form of the enzyme. With this reservation in mind, three immunoreactive sites have been identified: the canal walls with emphasis on the blind ends at stage I (Fig. 6B,C), segments of the wall in the forming marrow space at stage II (Fig. 6D), and along the edges of the wall containing groups of hypertrophic chondrocytes at stages III and IV (not shown). Hence, these sites include gelatinase B. Finally, because the immunoreactivity coincides with sites shown to have gelatinolytic activity by histozymography, it is concluded that these sites contain the active form of the enzyme.

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Figure 6. Tibial head sections immunostained by using either preimmune IgG for control (A) or anti–gelatinase B IgG (B,C,D) or, for comparison, collagenase-3 antiserum (E and F, being respectively serial sections of C and D). Although the control shows no reaction (A), the anti–gelatinase B IgG produces a reaction predominantly at the canal blind ends (at stage I), as indicated by the arrowheads in B and C and on the wall of the forming marrow space at stage II (D), thus confirming the presence of gelatinase B in sites to which it has been ascribed by histozymography. Finally, the panels of the lowest row show the presence of collagenase-3 in canal blind end at stage I (E) and marrow space walls at stage II (F). Comparisons with gelatinase B in serial sections (C and D, respectively) demonstrate some overlap of the two antibodies at the base of the canals (arrows, C and E) and the distal border of the marrow space (arrow, D and F). However, the collagenase-3 is restricted to the center of the canal blind end (E), whereas gelatinase B extends to the nearby portions of the canal side walls (C). Scale bar = 200 μm in A (applies to A–F).

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For the localization of gelatinase B at the electron microscopic (EM) level, thin sections of stage I epiphyses have been immunostained with 10 μg/ml of anti–gelatinase B IgGs. The blind ends of the canals display a reaction visualized as an accumulation of dark dots (Fig. 7A). The reaction in the depicted photomicrograph extends from the free surface of the canal down into the cartilage matrix to a depth of 4.0 ± 0.85 μm. This thickness varies in other sites. We consider that Figures 7A and B correspond to the region mentioned above under the name “pre-resorptive layer.” In contrast, the rest of the matrix is generally free of dark dots (lower right corner of Fig. 7A) and so are the sections exposed to preimmune IgG for control (Fig. 7B).

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Figure 7. Immunoelectron microscopy of the blind end of a stage I canal exposed to 10 μg/ml of either anti–gelatinase B IgG (A) or preimmune IgG for control (B). A: After exposure to the anti–gelatinase B IgG, the section appears gray due to the accumulation of dark dots indicative of reactivity. The dark dots extend from the marrow space at left (MS) through the pre-resorptive layer, which happens to include a degenerating chondrocyte (dCH). B: After exposure of a close-by section to preimmune IgG, the pre-resorptive layer appears pale due to the absence of the dark dots. C: For further comparison, a nonimmunostained, routine electron photomicrograph of the same tissue location has been stained with uranyl acetate and lead citrate. This photomicrograph shows the pre-resorptive layer (PRL). The proximal limit of the layer is the free surface of the canal blind end subjected to resorption, as indicated by the horizontal arrows at left. The distal limit of the layer is the normal cartilage matrix (CM) with the precise site suggested by the row of arrowheads. Within the layer type II collagen-rich fibrils (cf) are found at various stages of digestion. Thus, the anti–gelatinase B reactivity in A coincides with the site where the deterioration of the collagen fibrils takes place. E, the cytoplasm of an endothelial cell within the canal lumen. Scale bar = 2 μm in A (applies to A–C).

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To examine the structure of the pre-resorptive layer, EM sections of the canal blind end have been prepared by routine methods, as shown in Figure 7C, in which the lumen of the canal is seen at left. At the right extremity of the figure, normal cartilage matrix (CM) displays intact collagen fibrils between which fine proteoglycan particles are scattered. The rest of the figure exhibits a decreasing gradient of fibril length and staining density, whereas the fine proteoglycan particles are rare or absent. The matrix close to the free surface of the canal (indicated by three parallel arrows) appears pale with a few unidentifiable elements.

Immunolocalization of TIMP-2

A second approach has been to use TIMP histochemistry to more precisely pin down the location of active gelatinase B in the tissue. Since TIMP-2 was known to form complexes with active MMPs in vitro resulting in their inhibition (Butler et al., 1999), and since histozymography revealed that TIMP-2 can block the gelatinolytic activity of gelatinase B (Table 2), we have attempted to use TIMP-2 to localize the enzyme in tissues. To do so, recombinant TIMP-2 labeled with biotin has been applied to sections of stage I–IV rat tibial heads. The labeled TIMP-2 retained within the sections has then been detected by treating them first with the avidin/biotin horseradish peroxidase complex, then with 3,3′-diaminobenzidine (DAB). In practice, adjacent serial sections have been cut in pairs at 20 μm. The biotinylated TIMP-2 has been applied to one section of each pair for its localization, whereas histozymography has been used to detect gelatinolytic sites in the other. For the localization, control was provided by replacing the biotinylated TIMP-2 either with TIMP-2 carrying no biotin label (data not shown) or with biotinylated recombinant, noninhibitory human TIMP-1, produced as either a glycosylated or nonglycosylated form. The TIMP-1 control has allowed us to assess the amount of staining on the tissue sections that could be attributed to nonspecific protein binding, which occurs, but is low (Fig. 8C,G). In contrast, the biotinylated TIMP-2 is visualized as a brown reaction band. It is, thus, bound at stage I to canal blind ends (Fig. 8A,B) and metaphyseal border (Fig. 8A). At stage III, TIMP-2 is seen to bind to the walls of the marrow space (Fig. 8D) either in association with hypertrophic chondrocytes (Fig. 8E, small arrows) or in the sites lacking these cells (Fig. 8E, large arrows), as well as at the metaphyseal border (Fig. 8D). At stage IV, the proximal wall of the marrow space, associated with hypertrophic chondrocytes is still reactive, as are the epiphyseal (EB) and metaphyseal borders (MB) of the primary growth plate as shown in Figure 8F. Thus, the TIMP-2 bound to all of the pre-resorptive cartilage layers and, in so doing, confirmed the presence of active gelatinase B therein.

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Figure 8. Outcomes of the treatments of unfixed tissue sections from tibial heads at stages I, III, and IV with mutant noninhibitory TIMP-1 for control (C,G) or normal biotinylated TIMP-2 (A,B,D,E,F). Reactivity is visualized with avidin/biotin horseradish peroxidase, then 3,3′-diaminobenzidine (DAB). In A, the section exhibits a large central canal (arrow), which is enlarged in B to reveal a brown reactivity, assigned to the pre-resorptive layer underlying the surface of the canal blind end. C: After exposure of a serial section to mutant TIMP-1, the pre-resorptive layer appears pale due to a low insignificant brown reaction. D: A stage III tibial head, the marrow space has formed through the merger of two canals; the one at left is fully in view, the one at right, is only partially visible. E: The left canal and adjoining marrow space, are enlarged, where a brown reaction is observed along plain segments of the wall of the marrow space (MS) described as sites of “free” cartilage resorption (large arrows) and a weaker reaction on groups of hypertrophic chondrocytes associated with “ossification-coupled” resorption (small arrows). G: A neighboring section treated with mutant TIMP-1 lacks the reactivity revealed in E. F: In a stage IV tibial head, the primary (1°GP) and secondary (2°GP) growth plates are delineated and spots of reactivity, more intense than those found at stage III (D), are revealed in the forming secondary ossification center (small arrows). Moreover, the epiphyseal border (EB) of the primary growth plate (mostly a “free” mode of resorption) is intensely reactive. Furthermore, some reactivity is also observed at the bases of two emerging canals (arrows, upper left). Finally, in A, D, and F, the small arrows at the base of the photomicrographs identify a brown colored reaction line found along the metaphyseal border located between epiphysis and metaphysis. At stage IV (F), other reactivity also appears along the lateral (Lat B) and medial (Med B) borders of the primary growth plate where lysis was detected previously (Lee et al., 1998). The overall interpretation is that the TIMP-2 horseradish peroxidase DAB reactions coincide with the sites of cartilage lysis. Scale bars = 500 μm in A (applies to A,D,F), 200 μm in B (applies to B,C,E,G).

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The second section of each serial pair processed by histozymography showed reactions in exactly the same tissue locations as the staining produced with the biotinylated TIMP-2 (compare Fig. 9A with 8A or Fig. 9B with 8D). Briefly, the tissue sites of gelatinolytic activity coincide with those of TIMP-2 binding.

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Figure 9. Histozymographic reactions produced by sections from stage I (A) and III (B) tibial heads neighboring the sections exposed to biotinylated TIMP-2 depicted in Figure 8A and D, respectively). The histozymograms reveal a lightening of the emulsion at the same sites as those stained with the biotinylated TIMP-2 (compare the reactions indicated with large and small arrows in Fig. 8A and 9A at stage I, and in Figs. 8D and 9B at stage III). Thus, TIMP-2 binds at the sites that contain active gelatinase B. Scale bar = 500 μm in A (applies to A,B).

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Immunolocalization of Collagenase-3

By using antibodies specific for collagenase-3, the localization of this enzyme has been examined in young rat tibial heads. Although preimmune antibodies used for control yield negative results (not shown), the anti–collagenase-3 antibodies induce an immunostaining restricted to canal blind ends at stage I (Fig. 6E) and to segments of the wall of the marrow space, particularly the distal wall, at stage II (Fig. 6F). In addition, there is weak, diffuse immunostaining throughout the cartilage matrix of the epiphysis. When compared with similar sections immunostained for gelatinase B (Fig. 6C,D), the localizations are comparable, although the restriction to the canal blind end is more definite with anti–collagenase-3 than with anti–gelatinase B antibodies, while the weak matrix staining is lacking with the latter (compare Fig. 6E with C). Hence, only collagenase-3 is distributed throughout the matrix, yet this proteinase is concentrated at the distal cartilage wall belonging to the canal blind end and to the distal wall of the marrow space, that is, the very sites where the cartilage is resorbed.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The development of the skeleton is the result of a balance between two opposite processes: the deposition of new bone and cartilage components in specific sites and, in contrast, the lysis of these components at other sites. Indeed, it is the combination of these two processes that cause the small cartilage models of the fetus to gradually evolve into the long bones of the adult. As a continuation of our interest in lytic phenomena, we searched for activated enzymes during the transformation of the cartilaginous epiphysis into a secondary ossification center. Histozymography revealed that some gelatin-cleaving proteinase was present during the tunneling in progress at the blind end of the canals (stage I) and during the excavation of the marrow space wall (stages II, III, and IV). Three approaches were used to identify the proteinase: histozymographic reactions were completely ablated by the anti–gelatinase B neutralizing IgG; routine immunohistochemistry showed that gelatinase B resided where the activity of the enzyme was assigned by histozymography; and TIMP-2 histochemistry showed TIMP-2 (capable of blocking the histozymography reactions) to bind to the very sites in the tissue where gelatinase B was revealed by the other methods. Recently, a new application of neoepitope antibodies to the localization of “activated” gelatinase B confirmed its presence in the sites revealed by histozymography (Lee et al., 2000). Finally, the gelatinase B sites were of the two types defined by Lee et al. (2001, this issue): “ossification-coupled” and “free.” The prominent ossification coupled site was the epiphysis metaphyseal border which, between stages I and IV, became the growth plate metaphyseal border. A critical role for gelatinase B in this site was also reported by Vu et al. (1998) who, however, did not think that gelatinase B played a significant role in the development of the secondary ossification center, whereas our observations showed it in abundance at “free” sites. Indeed, the various gelatinase B sites comprised all the resorption sites defined in the epiphysis by Lee et al. (2001, this issue), including those in which aggrecan was cleaved and the one in which it was not, that is, the transverse cartilage partitions of the secondary growth plate.

Localization of Collagenase-3

Immunostaining also revealed that collagenase-3 was associated with gelatinase B in every pre-resorptive layer. The immunohistochemical approach did not indicate whether the detected collagenase-3 was functional, but because it was present in sites of cartilage resorption, it was possible that, like gelatinase B, it was in an active form and, therefore, played a role in these resorptions. Hence, the pre-resorptive layer included the two identified matrix metalloproteinases.

Collagen as a Likely Target of Gelatinase B and Collagenase-3 in the Pre-resorptive Layer

The pre-resorptive layer underlying the canal blind end was depicted by routine electron microscopy in Figure 7C. At the extreme right, normal cartilage matrix displayed abundant collagen fibrils, but in the rest of the picture, the collagen fibrils gradually decreased in width, length, and density to finally disintegrate as the lumen of the canal was approached. Thus, the compartment was the site of gradual deterioration of collagen fibrils culminating in their digestion. The presence of both gelatinase B and collagenase-3 in the pre-resorptive layer suggested that these enzymes were responsible for the demise of the collagen fibrils. What was known of the collagenolytic potential of these two enzymes will now be examined.

Mechanism of collagen lysis.

Of the various components of the collagen fibrils present in cartilage, helical type XI collagen was known to be degraded by gelatinase B (Murphy et al., 1981; Pourmotabbed et al., 1994; Niyibizi et al., 1994). As for the more abundant type II collagen (approximately 80% of total), gelatinase B was known to attack its C-telopeptide domain (Eyre, 2000; unpublished communication), but not to cleave its triple helix (Murphy et al., 1982). Cleavages of the helix could only be achieved by collagenases, such as collagenase-3 (MMP-13), the interstitial collagenase (MMP-1), and neutrophil collagenase (MMP-8), which all cleaved type II collagen in vitro at the G775-L776 bond, yielding two fragments whose length was three-quarters and one-quarter that of the whole molecule (Miller et al., 1976; Hasty et al., 1990; Mitchell et al., 1996). Thereafter, the fragments were known to be susceptible to degradation by gelatinases (Harris and Krane, 1972; Murphy et al., 1982). In the young rat tibial head, collagenase-3 was detected in every site where gelatinase B was present and, even though its activity had not been demonstrated, its association with gelatinase B in resorption sites made it likely that the two enzymes functioned in tandem to break down the collagen. Engsig et al. (2000) demonstrated that gelatinase B had a weak, and collagenase-3 a strong, ability to lyse type II collagen in vitro, and that in the presence of both enzymes, collagenolytic activity more than doubled that achieved by collagenase-3 alone. Hence, gelatinase B potentiated collagenase-3 activity.

However, because other components were present within the matrix under attack, the question naturally arose as to whether or not gelatinase B or collagenase-3 might also lyse the aggrecan core protein. Although gelatinase B was not efficient at cleaving either isolated aggrecan or aggrecan aggregates in vitro (Murphy et al., 1982, 1991a), collagenase-3 could not be discounted as a candidate for this lysis. Finally, other components were present in minor amounts, but their susceptibility to either gelatinase B or collagenase-3 was not known (Handler et al., 1997; Gerber et al., 1999; Aszodi et al., 2000; Carlevaro et al., 2000; Engsig et al., 2000).

Behavior of collagen and aggrecan in the pre-resorptive layer.

The events taking place in the pre-resorptive layer were summarized in Figure 10. Because there was a progressive deterioration of collagen fibrils throughout the layer (Fig. 7C), the amount of these fibrils, which was considered 100% in normal cartilage, was assumed to decrease regularly to 0% from the distal to the proximal limit, presumably under the joint action of the enzymes collagenase-3 and gelatinase B detected in the area. As for the aggrecan core protein, its amount was assigned to 100% in normal cartilage, but was believed to drop to none at the distal limit, as a result of cleavage at this site (Lee et al., 2001, this issue). Finally, this cleavage yielded G1-341 fragments, whose amount was thought to remain essentially unchanged from the time they arose at the distal limit to the time they were resorbed at the proximal limit.

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Figure 10. Semigraphic depiction of the lytic events occurring within the pre-resorptive layer, within which the gelatinase B is located. The X-axis denotes the position of the proximal limit of the pre-resorptive layer on the left and the position of the distal limit on the right. The Y-axis indicates how the percentage of the substances present varies with the location. The distal limit marks the transition from the pre-resorptive layer into the normal cartilage where the content of collagen and aggrecan is complete (100%). At the junction between the distal limit and the normal cartilage, aggrecan core protein is cleaved at the Asn341-Phe342 (Lee et al., 2001), as indicated by the arrow. Thus, the solid black line depicts a rapid decline in overall aggrecan content. So, although the G1-341 content increases rapidly to 100% in the pre-resorptive layer, the bulk of the aggrecan molecule, that is the G2-G3 fragments containing the attached glycosaminoglycan chains, have essentially been lost. In contrast, the dotted line depicts a steady loss of fibrils beginning at the distal limit of the layer and continuing throughout the pre-resorptive layer until completion toward the free surface undergoing resorption (the proximal limit). Thus, in contrast to aggrecan cleavage, the lysis of the fibrils is perceived to take place in all parts of the pre-resorptive layer.

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Thus, the pre-resorptive layer was a dynamic entity. Moreover, that the lysis of either collagen or aggrecan was observed whenever looked for by the available methods suggested that the lysis of both substances was taking place continuously. This conclusion suggested the existence of a steady state not only for collagen and aggrecan, but also for the whole compartment, that is, for instance during the digging of the canals, the loss of substance by resorption at the proximal limit should be balanced by expansion of the distal limit into normal cartilage. Lee et al. (2001, this issue) had estimated, for example, that the turnover time of the compartment was 42 min at the canal blind end. Hence, the events occurring at the distal limit of the layer preceded those occurring at the proximal limit by 42 min and, therefore, Figure 10 represented events developing in time from right to left. Accordingly, the G1-341 fragments produced at the distal limit spent 42 min in the layer before being lost at the proximal limit. Finally, the time taken for the degradation of collagen fibrils from normal to complete disintegration should also be of the order of 42 min.

Sequence of Lytic Events in the Cartilage Resorption Required for the Formation of the Secondary Ossification Center

Lee et al. (1999) have previously proposed that cartilage resorptions take place in three main steps, namely, aggrecan lysis, collagen II cleavage by collagenase-3, collagen fragment lysis by gelatinase B. Although the present results and those of Lee et al. (2001, this issue) essentially support this sequence, findings that have recently come to light suggest modifications. Our tentative proposal for the role of the three involved enzymes, i.e., the unidentified MMP, gelatinase B, and collagenase-3, in the lysis of the pre-resorptive layer is as follows.

  • 1
    It is first proposed that these enzymes originate in the cells facing the surface about to be resorbed and then migrate from there to their site of actions. Besides recent evidence pointing to the origin of gelatinase B and collagenase-3 in canal lumen cells (Lee E.R., unpublished data), indirect support for this view has been derived from what is known of the enzyme MT1-MMP. This enzyme, which is present at the surface of canal cells (Lee E.R., McQuibban G.A., and Overall C.M., unpublished) where it is presumably anchored in the plasma membrane (Sato et al., 1997), has been investigated by knocking-out the controlling gene, in which case the formation of canals in the epiphysis is prevented (Holmbeck et al., 1999; Zhou et al., 2000). The likely mechanism of this prevention is that the canals cell lack MT1-MMP—a potent activator of other MMPs (for review, see Murphy and Knäuper, 1997). As a result, the three enzymes involved in cartilage resorption would not be activated, and canals would not form. It is tentatively concluded that the first step in the cartilage resorption sequence was the production of the unidentified MMP, gelatinase B, and collagenase-3 within the cells facing the resorbed areas, followed by the MT1-MMP activation of these enzymes and their migration to the site of action.
  • 2
    Because, as indicated in Figure 10, aggrecan lysis precedes collagen lysis, the next step is the cleavage of aggrecan core protein by a so far unidentified MMP, as shown by the presence of G1 fragments terminating in the amino acid residues …FVDIPEN in the walls and blind end of the canals, the walls of the marrow space, and the metaphyseal border (Lee et al., 2001, this issue). In addition, aggrecanase participates in the cleavage of the aggrecan on the epiphyseal border of the primary growth plate.
  • 3
    The third step in the overall lysis is the initiation of collagen fibril digestion by a collagenase. The evidence indicates that gelatinase B only attacks helical type II collagen after it has been cleaved by collagenase (Murphy et al., 1982). We have detected collagenase-3 in the resorption sites, although the activity of the collagenase has not been demonstrated so far. We nevertheless postulate the cleavage of type II collagen by collagenase-3 as the third step in the sequence.
  • 4
    The fourth step is the completion of collagen fibril digestion by gelatinase B. The role of this enzyme is the degradation of the type II fragments produced by collagenase (for review see, Murphy and Crabbe, 1995). In addition, the type XI collagen present in cartilage can be directly attacked by gelatinase B (Murphy et al., 1981, 1982; Pourmotabbed et al., 1994; Niyibizi et al., 1994). Moreover, digestion may be enhanced by the joint action of collagenases and gelatinases, which together have been shown to facilitate the collagenolytic process (Harris and Krane, 1972; Murphy et al., 1982; Engsig et al., 2000).
  • 5
    What is the final step in the resorption of the wall present at the proximal limit of the compartment? A priori, the demise of collagen would seem to be sufficient to insure the collapse of the hyaluronate-attached G1341 fragments and other cartilage remnants making up the wall. This does not seem to be the case, however, because examination of the proximal limit wall in the electron microscope (as seen in Fig. 5C,D of Lee et al., 2001, this issue) indicates that the cytoplasmic expansions from intracanal cells are seen breaking through the wall. Presumably, enzymes on the cell surface, perhaps MT1-MMP attached to the cell membrane, make possible the final cell invasion, which culminates in the complete resorption of the proximal limit of the pre-resorptive layer.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Animals and Preparation of Tissue

Male Sprague-Dawley rats, from birth to 12 days of age, housed and handled according to the recommendations made by the Canadian Council on Animal Care, have been anesthetized with sodium pentobarbital (Somnotol, MTC Pharmaceuticals, Cambridge, Ontario; I.P., 50 mg/kg) before proximal tibiae were harvested and prepared for gelatin histozymography, peroxidase immunohistochemistry, or TIMP histochemistry.

Gelatin Histozymography

The first step is the preparation of blackened emulsion slides in the dark room, when clean glass slides are dipped in NTB2 radioautographic emulsion (Eastman Kodak Company, Rochester, NY) and, after drying, are exposed to room light for 5 min, developed in Kodak D-170 for 7 min, rinsed, and fixed in 24% sodium thiosulfate for 10 min. They are then stored at 4°C until use. For histozymography, 20-μm-thick sections of unfixed tibiae are cut in a Bright cryostat and collected on tape. A 3-μl drop of PBS is placed on the tape-mounted sections, which are then inverted and placed in contact with a blackened emulsion slide. The slide emulsion complex is incubated for 4 hr at room temperature in a sealed humidified Petri dish. After peeling the section off the emulsion, it is stained in 1% aqueous toluidine blue, and both section and emulsion are then photographed (see Lee et al., 1999, for further details).

Inhibitors.

To identify the proteinases responsible for the emulsion digestion, inhibitors are applied on sections destined to histozymography. A 3-μl volume of each solution at the concentration indicated in Table 2 is added to a section before it is placed on the emulsion. For control, the same volume but lacking the inhibitor, is added to a neighbouring section before applying it to the emulsion. The results reported in Tables 2 and 3 are the outcome of six trials per inhibitor tested. The effects of the inhibitors used to characterize proteinase activity are summarized in Table 2. The recombinant human TIMP-2 has been expressed in NSO myeloma cells and purified as described previously (Murphy et al., 1991b; Willenbrock et al., 1993). The concentration-dependant gelatinase hydroxysuccinamide, CT1746, has also been described previously (Anderson et al., 1996). The neutralizing immunoglobulins, which have been purified from anti-human gelatinase A (MMP-2) and anti-pig gelatinase B (MMP-9) sheep antisera, have been described before (Murphy et al., 1989; Hipps et al., 1991, Lelongt et al., 1997). Cross-reactivity of these sheep IgGs with rat gelatinases has been shown previously by Lee et al. (1999). Although the specificity of the IgGs is an integral part of targeting the appropriate proteinase, their ability to bind and inactivate, that is neutralize, the mature (active) proteinase has also been shown by Lelongt et al. (1997). Thus, these IgGs are used to neutralize enzymatic activity of gelatinase A and gelatinase B, respectively.

Peroxidase Immunohistochemistry

Tibial sections processed as described below are prepared for staining with sheep anti-pig gelatinase B IgG or rabbit anti-rat collagenase-3 antiserum. The latter is an anti-peptide antiserum prepared according to the method described in the preceding article (Lee et al., 2001, this issue). Briefly, three peptides have been synthesized corresponding to the residues 127-136 (CGSEVEKAFRKA), 267-278 (DPNPKHPKTPEKC), and 369-379 (CGFPKEVKRLSA) found on rat collagenase-3; each includes a G and C residue added to the N or the C-terminus (Quinn et al., 1990). The peptide is coupled to ovalbumin through the terminal cysteine residue, and a mixture of the three conjugated peptides has been injected into a New Zealand White rabbit. Antiserum specificity has been tested against a partially activated recombinant human collagenase-3 prepared in transfected NSO mouse myeloma cells (Knauper et al., 1996). Reactivity of the antiserum with the recombinant proteinase is demonstrated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques in Figure 11.

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Figure 11. Immunoblot of recombinant human collagenase-3 immunostained with anti–collagenase-3 antiserum (A) or preimmune serum (B). The recombinant proteinase (0.5 μg per lane) was electrophoresed on a reduced 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel before transfer to nitrocellulose paper. The immunoblot in lane A reveals reactivity of the antiserum with the 60-kDa proenzyme form and the processed 48-kDa active form (Knauper et al., 1996). No reactions are observed in lane B stained with the preimmune serum. Reactivity was visualized by exposing the membrane to alkaline phosphatase conjugated anti-rabbit immunoglobulin (Promega, Madison, WI) and to NitroBlue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega). The position of two molecular weight (MW) standards is indicated at left.

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Immunostaining for Light Microscopy

Proximal tibial epiphyses are immersed in a periodate-lysine-paraformaldehyde fixative (including 2% formaldehyde; McLean and Nakane, 1974) and, then, placed at 4°C overnight. After rinsing in azide-free phosphate buffered saline (PBS), the tibial epiphyses are split in half along the sagittal plane and decalcified in 10% EDTA in 0.1 M Tris, pH 7.4, at 4°C. The tissue is infiltrated first with 20% sucrose in PBS and then in two parts of this solution and one part of OCT compound (VWR-Canlab, Ville Mont-Royal, Quebec). Embedding is done in this final mixture by using standard specimen cryomolds (Miles, Elkhart, IN), and tissue is frozen within the block over dry ice. Eight-micrometer sections are cut in a Bright's OTF cryostat, collected on gelatin-coated slides, and stored at −20°C.

For immunolocalization of gelatinase B with the sheep anti-pig gelatinase B immunoglobulin, the fixed, slide-mounted sections are taken to room temperature, immersed in 4% formaldehyde, and rinsed in PBS before a 1 hr digestion at 37°C with 0.25 U/ml of chondroitinase ABC (Proteus vulgaris; ICN Biomedical, Costa Mesa, CA) in the presence of proteinase inhibitors. Thereafter, the slide-mounted sections are exposed to 0.3% H2O2 (v/v in methanol) to quench endogenous peroxidase reactivity before blocking in 1.5% normal goat serum in PBS for 20 min and incubation with anti–gelatinase B immunoglobulins (IgGs) for 30 min. After two PBS washes, the bound IgG is localized by using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Briefly, the tissue-bound IgG recognizes an avidin-biotin-horseradish peroxidase complex, which is detected by a 5-min exposure to DAB, which, over the areas containing gelatinase B, yields a precipitate that appears brown in the light microscope. After rinsing, the sections are dried and counterstained with Gill's hematoxylin for 1 min on a hot plate, rinsed in three changes of distilled water, and mounted under a coverslip.

Immunoelectron Microscopy

The procedure is similar to the one described for light microscopy with several differences. First, the sections are cut at a 25 μm thickness. Second, after immunostaining through the same steps as for light microscopy, the DAB-exposed sections are stored overnight in PBS at 4°C and, the next morning, are post-fixed by using a droplet of 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. The sections are then dehydrated in increasing gradients of acetone and then acetone-Epon up to pure Epon, a drop of which is finally placed on the section to be cured overnight at 60°C. A polymerized Epon block is then inverted onto the section, and the two are held together by a fresh drop of resin. While the two are held together, they are cured overnight at 60°C. The slide is then immersed in liquid nitrogen, causing the block to contract and snap away from it. Thin sections are prepared and examined in a Philips 400 EM in the absence of counterstaining.

Routine Electron Microscopy

Tibiae are dissected from rats that have been perfused through the heart with a mixture containing 2.5% glutaraldehyde (highly purified, J.B. EM Services; Pointe Claire, Quebec) and 2.0% formaldehyde (freshly prepared from paraformaldehyde), both in 0.1 M cacodylate buffer (pH 7.3). All samples are post-fixed in potassium ferrocyanide–reduced osmium tetroxide, dehydrated in acetone, and embedded in JEMbed epoxy resin before examination in a Philips 400 Electron Microscope at 80 kV.

TIMP Histochemistry

Biotinylation of the TIMPs.

Although recombinant human TIMP-2 is used as such in inhibition tests (Table 2) as described before, a biotinylated form is used to localize its binding sites in epiphyseal sections and an inactive biotinylated recombinant human TIMP-1 is used for control. Biotinylation of the two TIMPs has been achieved with EZ Link Sulfo-NHS-LC-Biotin, according to the directions of the manufacturer (Pierce Chemical Co., Rockford, IL). Briefly, the recombinant TIMPs (0.2 mg/ml in 20 mM Tris-HCl, pH 7.6) are dialyzed overnight at 4°C against a 50-mM sodium bicarbonate buffer (pH 8.5), before EZ Link Sulfo-NHS-LC biotin is added at a 4 M excess. The reaction is allowed to continue for 3 hr at room temperature after which the biotinylated TIMPs are dialyzed into azide-free PBS and stored at −20°C until needed. To confirm that the proteins are adequately labeled with biotin, they have been exposed to standard SDS-PAGE and blotting analysis as shown in Figure 12. For the inactivation of TIMP-1 (which has been expressed in Pichia pastoris either as glycosylated or nonglycosylated form), both forms have been rendered inactive by the addition of a four–amino acid residue extension at the N-terminus. To test whether or not the biotinylated TIMP-2 still retains its ability to inhibit active enzyme after biotinylation, the biotinylated TIMP-2 has been examined in a gelatinase A reverse zymogram. The recombinant human gelatinase A is mixed with porcine gelatin and both are polymerized into the gel as described by Oliver et al. (1997). EDTA fully prevents the digestion of the gelatin by the enzyme (Fig. 13A), as does the biotinylated TIMP-2 (Fig. 13B), showing that the biotinylated form of the inhibitor has retained the capacity to inhibit an active MMP.

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Figure 12. Blot of active TIMP-2 in (A) (lanes 1 and 2) and inactive TIMP-1 in (B) (lanes 3–6). TIMP-1 is the glycosylated form in lanes 3 and 5 and the nonglycosylated form in lanes 4 and 6. The TIMP in lanes 2, 5, and 6 was reacted with sulfosuccinimidobiotin before electrophoresis whereas the TIMP in lanes 1, 3, and 4 was not. The samples, electrophoresed on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels before transfer to nitrocellulose paper, were then stained with streptavidin-alkaline phosphatase and visualized by exposing the membrane to NitroBlue Tetazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega). All TIMPS treated with sulfosuccinimidobiotin are stained. Molecular weight (MW) standards are indicated to the right of each panel.

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Figure 13. Reverse zymograms in which biotinylated TIMP-2 has been electrophoresed in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing porcine gelatin (2.25 mg/ml) and recombinant human gelatinase A (640 ng/ml). Thereafter, the gel has been incubated in Triton X-100 buffer either in the presence (A) or absence of EDTA (B). After the incubation is completed, the gels have been stained with Coomassie blue (G-250) to reveal the digestion of the gelatin by gelatinase A, which is indicated by a lightening of the gel. When gelatinase A is inhibited and unable to degrade the gelatin (as occurs in the presence of EDTA), the gel is darkly stained with Coomassie blue. Note the increased inhibition of gelatinase A (indicated by arrow) as the concentration of the biotinylated TIMP-2 is increased in the lanes from left to right (200 pg, 830 pg, 3 ng, 7 ng, and 13 ng, respectively). Therefore, it is concluded that the biotinylated TIMP-2 has retained its inhibitor properties after biotinylation.

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TIMP peroxidase histochemistry.

The TIMPs have been applied to frozen tape-mounted sections prepared in the same way as described under histozymography. Obviously, when MMPs are active, they can bind TIMP and be blocked by it. Hence, it is likely that the TIMPs will be bound to histologic sections at sites containing MMPs. Therefore, to visualize active MMPs in the sections, TIMPs are applied as a 3-μl droplet (40 μg/ml in azide-free PBS), which is rapidly spread by cover-slipping, before the slide-section complex is transferred to a humidified chamber for 1 hr at room temperature. The complex is then disassembled and, to wash away the unbound TIMP, the section is transferred (tissue side down) successively on the surface of four wells filled with azide-free PBS. Bound TIMP is revealed by exposing the section for 30 min to a preformed avidin-biotin horseradish peroxidase complex made according to the directions of the manufacturer (Vector Laboratories, Burlington, Ontario). After washing away all unbound ABC complex, a reddish brown stain is produced at reactive sites by exposing the section for 2.5 min to the chromogen 3,3′-diaminobenzidine (DAB) in the presence of H2O2. The tape-attached section is then rinsed in distilled water and wet mounted on a glass slide with coverslip by using a saturated aqueous solution of polyvinyl pyrrolidone (Sigma) and photographed with a Zeiss Axiovert-35 inverted microscope.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors thank Dr. Mohamed El-Alfy, currently at the Medical Research Group in Molecular Endocrinology, CHUL Research Centre, Laval, Quebec, for teaching M.A. Davoli the technique of histozymography. We also thank Dr. Anneliese Recklies (Joint Diseases Laboratory, Shriners Hospital for Children) for her suggestion to use recombinant TIMP to localize active MMPs in tissue prepared for histology. The peptides in this study were made by Ms. Elisa de Miguel in the Core Biotechnology Facility of the Shriners Hospital, and the figures and art work were prepared by Mr. Mark Lepik and Ms. Guylaine Bedard. Dr. Gillian Murphy is a Senior Fellow of the Arthritis Research Campaign, U.K.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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