Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases.

The site of action of cysteine-proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP-inhibitor trans-epoxy-succinyl-L-leucylamido (4-guanidino)-butane (E-64) and the MMP-inhibitor N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide (Cl-1) in an in vitro model system of PTH-stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor-treated explants than in controls. Moreover the percentage of osteoclasts in close contact with such demineralized bone areas appeared to be significantly higher in inhibitor-treated explants than in control specimens (60% and 5%, respectively). These effects were not apparent when the osteoclastic activity was inhibited with calcitonin. No significant differences were found between the effects of the two inhibitors, E-64 and Cl-1. Our observations indicate that under the influence of inhibitors of MMPs and CPs demineralization of bone by osteoclasts proceeded up to a certain point whereas matrix degradation was strongly inhibited. It is concluded that within the osteoclastic resorption lacuna both CPs and MMPs participate in the degradation of the collagenous bone matrix.     

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects.Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Mc. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.     

Selective inhibition of collagen synthesis by the Ca2+ ionophore A23187 in cultured human fibroblasts

The question of whether the Ca2+ ionophore A23187 affects collagen production relative to total protein synthesis or has possible effects on collagen degradation was investigated. Cultured normal human fibroblasts were incubated with radioactive proline, and the radioactivity of collagenase-sensitive and -resistant proteins was used to calculate the rates of protein production. The net production of collagen relative to total proteins was inhibited by A23187 in a dose-related manner, and 50% inhibition of basal collagen production was achieved with 0.6 microM A23187. There was a 70% decrease in the absolute rate of collagen production in the presence of 0.6 microM A23187 which represented a 4-fold greater inhibition of collagen production than of noncollagen protein production. The major mechanism for the decreased net production of collagen was decreased synthesis, rather than increased degradation. Ca2+ mobilization induced by cholecystokinin octapeptide was also associated with selective inhibition of collagen production in normal human fibroblasts. These studies establish that the Ca2+ ionophore A23187 induces a selective decrease in collagen polypeptide synthesis by normal human fibroblasts and suggest a modulatory role of Ca2+ on collagen metabolism.     

Collagen degradation in the rabbit skin during short-term tissue culture

Full thickness rabbit skin explants were cultured on plastic dish for 1 week and the sequential morphological changes were examined daily by light and electron microscopy. During the cultured period, bundles of dermal collagen fibres gradually loosened and were removed from the upper dermis and from the cut margin of the explant, which was covered by a sheet of migrating epidermal cells. In these areas, cells containing phagocytosed collagen fibrils were observed from the 3rd day to the end of the culture period. These cells containing phagocytosed collagen fibrils included dermal fibroblasts and macrophages, epidermal keratinocytes and endothelial cells lining blood vessels. The presence of acid phosphatase activity in vacuoles containing the collagen fibrils suggested that intracellular degradation of collagen was occurring. In addition, extracellular collagen degradation was recognized around fibroblasts and beneath the migrating epidermis by the high collagenolytic activity at these sites. These findings suggest that both intra- and extracellular collagen degradation may participate in collagen removal from dermal connective tissue in cultured skin explants.     

Expression of cathepsin K mRNA and protein in odontoclasts after experimental tooth movement in the mouse maxilla by in situ hybridization and immunoelectron microscopy

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacuane, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.     

Collagen breakdown in soft connective tissue explants is associated with the level of active gelatinase A (MMP-2) but not with collagenase.

Recent data suggest that gelatinase A (matrix metalloproteinase-2, MMP-2) plays an important role in the degradation of collagen of soft connective tissues. In an attempt to investigate its participation in more detail we assessed the digestion of collagen in cultured rabbit periosteal explants and compared this with the level of active MMP-2 and collagenases. The data demonstrated that both collagen degradation and MMP activity increased with time. Conditioned medium obtained from explants cultured for 72 h showed that the level of active MMP-2 correlated with collagen degradation (r = 0.80, d.f. = 23, P < 0.0001). Such a relationship was not found with collagenase activity (r = -0.08, d.f. = 21, NS). The possible involvement of MMP-2 in collagen degradation was investigated further by incubating explants with selective gelatinase inhibitors (CT1166, CT1399 and CT1746). In the presence of these compounds breakdown of collagen was almost completely abolished (approximately 80%). Finally we assessed whether periosteal fibroblasts had the capacity to degrade collagen type I that conferred resistance to collagenase activity. Breakdown of this collagen did not differ from degradation of normal collagen. Taken together, our data provide support for the view that MMP-2 plays a crucial role in collagen degradation of soft connective tissue.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

Prostaglandin E(2) inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling

Uncontrolled fibroblast activation is one of the hallmarks of fibrotic lung disease. Prostaglandin E(2) (PGE(2)) has been shown to inhibit fibroblast migration, proliferation, collagen deposition, and myofibroblast differentiation in the lung. Understanding the mechanisms for these effects may provide insight into the pathogenesis of fibrotic lung disease. Previous work has focused on commercially available fibroblast cell lines derived from tissue whose precise origin and histopathology are often unknown. Here, we sought to define the mechanism of PGE(2) inhibition in patient-derived fibroblasts from peripheral lung verified to be histologically normal. Fibroblasts were grown from explants of resected lung, and proliferation and collagen I expression was determined following treatment with PGE(2) or modulators of its receptors and downstream signaling components. PGE(2) inhibited fibroblast proliferation by 33% and collagen I expression by 62%. PGE(2) resulted in a 15-fold increase in intracellular cAMP; other cAMP-elevating agents inhibited collagen I in a manner similar to PGE(2). These effects were reproduced by butaprost, a PGE(2) analog selective for the cAMP-coupled E prostanoid (EP) 2 receptor, but not by selective EP3 or EP4 agonists. Fibroblasts expressed both major cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1), but only a selective PKA agonist was able to appreciably inhibit collagen I expression. Treatment with okadaic acid, a phosphatase inhibitor, potentiated the effects of PGE(2). Our data indicate that PGE(2) inhibits fibroblast activation in primary lung fibroblasts via binding of EP2 receptor and production of cAMP; inhibition of collagen I proceeds via activation of PKA.     

Aggrecan protects cartilage collagen from proteolytic cleavage

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6-8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.     

Reversible inhibition of osteoclastic activity by bone-bound gallium (III).

Gallium(III) is a new therapeutic agent for hypercalcemia. Ga3+ reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7-12 microM Ga(III) reduces calcium release from bone, but surprisingly, 10-100 microM Ga3+ added to isolated avian osteoclasts did not reduce their degradation of L-(5-3H)-proline bone. 3H-proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III) greater than 100 microM inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured 67Ga distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1 microM gallium from 500 microliters of tissue culture medium, with steady state at greater than 24 h. Osteoclasts on bone inhibited gallium binding capacity approximately 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum-containing medium than in phosphate-buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre-bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100 micrograms of bone pre-incubated with 1 ml of 1 microM Ga3+, with 10 pmoles Ga3+/micrograms bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/micrograms bone, resorption was essentially zero. In contrast, pre-treatment of bone with [Ga3+] as high as 15 microM had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below approximately 150 pg/micrograms bone acts for a limited time and does not permanently damage the cells. We conclude that bone-bound Ga(III) from medium concentrations less than 15 microM inhibits osteoclasts reversibly, while irreversible toxicity occurs at solution [Ga3+] greater than 50 microM.     

Influence of vanadate on migrating fibroblast orientation within a fibrin matrix

Treating rats with vanadate, a nonspecific inhibitor of protein tyrosine phosphatases, optimizes the uniform packing of collagen fiber bundles in wound granulation tissue and doubles wound breaking strength in rat incisional wounds. The speculation is vanadate optimizes the packing of collagen fiber bundles through the orientation of newly arrived wound fibroblasts in the fibrin clot filling the defect. Segments of 14 day chick embryo tendons were placed on fibrin clots and maintained in organ culture with and without 30 microM vanadate. On day 7 explants were examined histologically and biochemically. Tendon fibroblast outgrowth from untreated explants migrated in a random fashion, while fibroblasts from vanadate-treated explants migrated out in linear arrays. Fibroblasts were elongated by 20% form vanadate treated explant compared to controls. Myosin ATPase, required for optimal cell motility, is optimized by the phosphorylation of its myosin light chain (MLC). Western blot analysis of lysates from the fibroblasts that migrated into the fibrin showed vanadate increased MLC-P levles. These findings support the notion that vanadate promotes the deposition of regular, parallel collagen fiber bundles by advancing the orientation of fibroblasts in parallel linear arrays early in the wound repair process.     

Matrix metalloproteinases and lysosomal cysteine proteases in osteoclasts contribute to bone resorption through distinct modes of action

The effects of inhibitors of matrix metalloproteinases (MMPs) and lysosomal cysteine proteases on osteoclastic pit formation in dentine slices were investigated. A nonspecific cysteine protease inhibitor, E-64, inhibited pit formation on naked slices in a concentration-dependent manner, and at 10 microM E-64 reduced the pit volume by 70%. However, up to 10 microM of the MMP inhibitor, BB-94, did not show any inhibition of pit formation. On the other hand, on slices coated with reconstituted basement membrane, both BB-94 and E-64 at 10 microM showed a marked decrease in pit volume by 73% and 68%, respectively. By a combination of treatment with both BB-94 and E-64, pit formation could be completely suppressed. These results suggest that MMPs are necessary for the migration of precursor and/or immature osteoclasts to bone surface through basement membranes, while cysteine proteases are essential for the osteoclastic degradation of bone collagen.     

Role of discoidin domain receptor 1 in dysregulation of collagen remodeling by cyclosporin A

The anti-transplant rejection drug cyclosporin A (CsA) causes loss of collagen homeostasis in rapidly remodeling connective tissues, such as human gingiva. As a result of CsA treatment, collagen degradation by fibroblasts is inhibited, which leads to a net increase of tissue collagen and gingival overgrowth. Since fibrillar collagen is the primary ligand for the discoidin domain receptor 1 (DDR1), we hypothesized that CsA perturbs DDR1-associated functions that affect collagen homeostasis. For these experiments, human fibroblasts obtained from gingival explants or mouse 3T3 fibroblasts (wild type, over-expressing DDR1 or DDR1 knockdown) or mouse GD25 cells (expressing DDR1 but null for β1 integrin), were treated with vehicle (dimethyl sulfoxide) or with CsA. The effect of CsA on cell binding to collagen was examined by flow cytometry; cell-mediated collagen remodeling was analyzed with contraction, compaction and migration assays. We found that CsA inhibited cell binding to collagen, internalization of collagen, contraction of collagen gels and cell migration over collagen in a DDR1-dependent manner. CsA also enhanced collagen compaction around cell extensions. Treatment with CsA strongly reduced surface levels of β1 integrins in wild type and DDR1 over-expressing 3T3 cells but did not affect β1 integrin activation or focal adhesion formation. We conclude that CsA inhibition of collagen remodeling is mediated through its effects on both DDR1 and cell surface levels of the β1 integrin.     

Transcytosis of calcium from bone by osteoclast-like cells evidenced by direct visualization of calcium in cells

'Transcytosis' of calcium (Ca) from bone by osteoclasts was identified by using a newly developed method that uses fixed or living osteoclast-like cells previously differentiated in vitro, a Ca-specific cell-membrane-impermeable fluorescent dye, and confocal laser scanning microscopy. This method, called the cell-membrane-impermeable dye method, revealed that in fixed osteoclast-like cells, a large quantity of Ca was confined within vacuoles and transported toward the apical cell membrane in the cells. These Ca-confined vacuoles were co-localized with marker proteins of both ruffled border and lysosome. The vacuoles were disrupted when treated with an inhibitor of ruffled border ATPase. In living osteoclast-like cells, Ca-confined vacuoles were again preferentially located at the central region and near the apical cell membrane. These results suggest actual transcytosis of Ca from bone by osteoclasts, and are the first direct evidence of the significant role of osteoclasts in the entire process of Ca metabolism in bone.     

The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture

The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.     

Modulation of fibroblast functions by interleukin 1: increased steady- state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.     

Localization of acid phosphatase activity in collagen-secreting and collagen-resorbing fibroblasts

The ultrastructural localization of acid phosphatase (ACPase) activity was examined in cultured human gingival fibroblasts in the formative and resorptive phases. In the collagen-secreting fibroblasts, weak ACPase activity was demonstrated in the lysosomes, inner Golgi cisternae, and condensing vacuoles, and none was found in the Golgi-associated endoplasmic reticulum-lysosome system (GERL), presecretory granules, or secretory granules. On the contrary, collagen phagocytosis induced strong ACPase activity in the GERL, which was in addition to the weaker activity found in the same sites as those in the collagen-secreting cells. At the same time, collagen secretion was suppressed, and dense elongated secretory bodies associated with ACPase activity accumulated within the cells. When collagen fibrils had been interiorized in whole or in part within the phagosomes, primary lysosomes derived from the Golgi-GERL complex then fused with them to form phagolysosomes. Collagen degradation occurred within these bodies. The observations indicate significant differences in ACPase activity used as a marker for lysosomal enzyme activities in the different functional phases of fibroblasts. These results suggest that fibroblasts work only one way at a given time, viz., collagen synthesis or collagen degradation.