Cytokine regulation of metalloproteinase gene expression

Matrix metalloproteinases belong to a family of zinc-dependent enzymes capable of degrading extracellular matrix and basement membrane components. Their expression is greatly modulated by cytokines and growth factors and involves the gene products of the Fos and Jun families of oncogenes. After extra(peri)cellular activation, their activity can be further controlled by specific tissue inhibitors of metalloproteinases. A correct balance between these regulatory mechanisms is necessary to ensure matrix remodeling in normal physiological processes such as embryonic development, but the overexpression of these enzymes may initiate or contribute to pathological situations such as cartilage degradation in rheumatoid arthritis or to tumor progression and metastasis. Delineation of the mechanisms of metalloproteinase and metalloproteinase inhibitors gene expression, understanding of their mode of interactions, and characterization of their patterns of expression in various tissues in normal and pathological states will lead to new therapeutic strategies to counteract the deleterious effects of matrix metalloproteinases in human disease.     

A tissue-culture model of cartilage breakdown in rheumatoid arthritis. Quantitative aspects of proteoglycan release

1. The destruction of articular cartilage in human rheumatoid and other arthritides is the result of diverse mechanical, inflammatory and local cellular factors. A tissue-culture model for studying cartilage-synovial interactions that may be involved in the final common pathway of joint destruction is described. 2. Matrix breakdown was studied in vitro by using bovine nasal-cartilage discs cultivated in contact with synovium. Synovia were obtained from human and animal sources. Human tissue came from patients with ;classical' rheumatoid arthritis, and animal tissue from rabbits with antigen-induced arthritis. 3. Cartilage discs increased their proteoglycan content 2-3-fold during 8 days in culture. Proteoglycan was also released into culture medium, approx. 70% arising from cartilage breakdown. 4. Synovial explants from human rheumatoid and rabbit antigen-induced arthritis produced equivalent stimulation of proteoglycan release. After an initial lag phase, the breakdown rate rose abruptly to a maximum, resulting in a 2-fold increase of proteoglycan accumulation in culture medium after 8-10 days. 5. High-molecular-weight products shed into culture media were characterized chromatographically and by differential enzymic digestion. Proteoglycan-chondroitin sulphate accounted for 90% of the released polyanion, and its partial degradation in the presence of synovial explants was consistent with limited proteolytic cleavage. 6. Rheumatoid synovium applied to dead cartilage increased the basal rate of proteoglycan release. Living cartilage was capable of more extensive autolysis, even in the absence of synovium. However, optimal proteoglycan release required the interaction of living synovium with live cartilage. These findings support the view that a significant component of cartilage breakdown may be chondrocyte-mediated.     

Integrins and their ligands in rheumatoid arthritis

Integrins play an important role in cell adhesion to the extracellular matrix and other cells. Upon ligand binding, signaling is initiated and several intracellular pathways are activated. This leads to a wide variety of effects, depending on cell type. Integrin activation has been linked to proliferation, secretion of matrix-degrading enzymes, cytokine production, migration, and invasion. Dysregulated integrin expression is often found in malignant disease. Tumors use integrins to evade apoptosis or metastasize, indicating that integrin signaling has to be tightly controlled. During the course of rheumatoid arthritis, the synovial tissue is infiltrated by immune cells that secrete large amounts of cytokines. This pro-inflammatory milieu leads to an upregulation of integrin receptors and their ligands in the synovial tissue. As a consequence, integrin signaling is enhanced, leading to enhanced production of matrix-degrading enzymes and cytokines. Furthermore, in analogy to invading tumors, synovial fibroblasts start invading and degrading cartilage, thereby generating extracellular matrix debris that can further activate integrins.     

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua)

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod (Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.     

ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein

Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.     

Degradation of the heparin matrix of mast cell granules by cultured fibroblasts

The ability of cultured rat fibroblasts to phagocytose rat peritoneal mast cell granules has been previously demonstrated by light and electron microscopy. To determine if the heparin matrix of ingested granules could be degraded by fibroblasts after phagocytosis, the heparin within peritoneal mast cells was labeled with [35S]sulfate in vivo. The 35S-labeled rat peritoneal mast cells were purified and their granules were isolated and shown to contain [35S]heparin proteoglycan. Incubation of [35S]heparin proteoglycan-containing granules with cultured rat fibroblasts revealed internalization of radioactivity by the fibroblasts over the first 24 hr consistent with phagocytosis of the granules by these fibroblasts. The [35S]heparin proteoglycan internalized by the fibroblasts was shown to decrease in size over 72 hr indicating that the fibroblasts were capable of degrading the heparin within the ingested granules. Degradation of [35S]heparin proteoglycan within the fibroblast was accompanied by the appearance of free [35S]sulfate in the extracellular compartment. Similar findings were obtained using cultured human fibroblasts. These data demonstrate for the first time that both rat and human fibroblasts are not only capable of ingesting mast cell granules but also of degrading mast cell granule heparin proteoglycan. This ingestion and degradation of mast cell granules by fibroblasts may represent an important mechanism in the regulation of the biologic expression of heparin and other granule-associated mediators in immediate hypersensitivity reactions.     

Influence of the pericellular environment on the cells. The role of mucopolysaccharides in the protection of cartilage cells against immune reactions.

Problems related to rheumatoid arthritis have been investigated by a group at Cambridge using the organ culture technique. Since auto-allergic reactions may be concerned in the chronicity of the disease, the effects of reactive complement-sufficient antisera (AS+C') on embryonic and post-foetal cartilage were examined. The cartilaginous limb bone rudiments enlarged to several times their original volume in control medium, but in the presence of AS+C' they gradually disintegrated, owing to the breakdown of the cartilage matrix; only the superficial cells of the enveloping soft connective tissue were killed, however. Provided breakdown had not advanced too far, the effects of AS+C' were reversible. It was not clear how AS+C' produced these changes, since cartilage matrix is impermeable to molecules as large as the immunoglobulins. To find whether there was a difference in permeability between embryonic and post-foetal cartilage, similar experiments were made on the articular cartilage of young pigs. AS+C' had no effect on pure articular cartilage, and it was shown immunohistochemically that IgG did not penetrate beyond the most superficial layer of cartilage. When, however, the explant was associated with soft connective tissue either as invading marrow or as an adjacent explant of synovium, the cartilage matrix was depleted of proteoglycan; IgG antibodies then entered the cartilage and reacted with the chondrocytes. After a lapse of 8-10 days, collagen also began to break down. If the degradation of collagen was not too extensive, the changes were reversible. Pure cartilage was depleted of proteoglycan by trypsinization and then cultivated in AS+C'. All the chondrocytes reacted with the IgG antibodies. The peripheral cells were killed, but those in the interior survived and rapidly secreted pericellular capsules rich in proteoglycan, which shielded them from further contact with antibodies. In other experiments, pure cartilage was associated with a synovial explant and cultivated in AS+C' for 10 days; this caused severe depletion of the matrix. The synovial tissue was then removed and the isolated cartilage cultured for a further 10 days in either AS+C' or control medium. If mainly proteoglycan had been lost during the primary culture period, breakdown did not continue in AS+C', and sometimes a little new matrix was regenerated, though less than in control medium; if, however, the collagen had been extensively degraded, breakdown continued even in control medium. It is suggested that in both the embryonic and post-foetal cartilage, degradation of the cartilage matrix was due to the enzymatic activity of the associated soft connective tissue which caused a loss first of proteoglycan, which enabled antibodies to reach the chondrocytes, and then of collagen. The possible relevance of these results to the pathogenesis of rheumatoid arthritis is discussed.     

Phytochemicals targeting matrix metalloproteinases regulating tissue degradation in inflammation and rheumatoid arthritis

PurposeMatrix metalloproteinases, zinc dependent proteolytic enzymes, have significant implications in extracellular matrix degradation associated with tissue damage in inflammation and Rheumatoid arthritis. Numerous orchestrated pathways affects instigation and blockade of metalloproteinases as well as various factors that increase the expression of MMPs including inflammatory cytokines, hormones and growth factors. Direct inhibition of these proteolytic enzymes or modulation of these pathways can provide protection against tissue destruction in inflammation and rheumatoid arthritis. Inclination towards use of plant derived phytochemicals to prevent tissue damage has been increasing day by day. Diversity of phytochemicals have been known to directly inhibit metalloproteinases. Hence, thorough knowledge of phytochemicals is very important in novel drug discovery.MethodsPresent communication evaluates various classes of phytochemicals, in effort to unveil the lead molecules as potential therapeutic agents, for prevention of MMPs mediated tissue damage in inflammation and rheumatoid arthritis. Data have been analyzed through different search engines.ResultsNumerous phytochemicals have been studied for their role as MMPs inhibitors which can be processed further to develop into useful drugs for the treatment of inflammation and rheumatoid arthritis.ConclusionIn search of new drugs, phytochemicals like flavonoids, glycosides, alkaloids, lignans & terpenes offer a wide canvas to develop into valuable forthcoming medicaments.     

The characterisation and function of the polysaccharidases of human synovial fluid in rheumatoid and osteoarthritis.

A potential enzymic mechanism for the degradation of glycosaminogly cans was characterised using enzymes found in rheumatoid synovial fluid from the knee joint. This mechanism involves a true hyluronidase together with the concerted action of beta-glucuronidase and beta-N-acetylhexosaminidase. The contribution of the exopolysaccharidases to hyaluronate degradation was demonstrated by the use of specific inhibitors, while the distinct identity of a true hyaluronidase was shown by ammonium sulphate and agarose gel column fractionations. Only the hyluronidase fraction was capable of degrading high molecular weight hyaluronate. The exopolysaccharidase activities were shown to be markedly elevated in rheumatoid as compared to osteoarthritic synovial fluid and also normal serum. On the other hand, hyluronidase was similarly active in rheumatoid and osteoarthritic synovial fluids; both these levels were lower than that of normal human serum. Hyaluronidase in synovial fluid may thus be derived by diffusion from serum, since it is of relatively low molecular weight (60 000). The pH requirements of this enzyme system and the strong inhibition of hyaluronidase by synovial fluid make it unlikely that the mechanism operates extracellularly. It is proposed that as a lysosomal mechanism, however, it is an important contributing factor in the chronic erosion process characteristic of rheumatoid arthritis.     

Human mast cell beta-tryptase is a gelatinase

Remodeling of extracellular matrix is an important component in a variety of inflammatory disorders as well as in normal physiological processes such as wound healing and angiogenesis. Previous investigations have identified the various matrix metalloproteases, e.g., gelatinases A and B, as key players in the degradation of extracellular matrix under such conditions. Here we show that an additional enzyme, human mast cell beta-tryptase, has potent gelatin-degrading properties, indicating a potential contribution of this protease to matrix degradation. Human beta-tryptase was shown to degrade gelatin both in solution and during gelatin zymographic analysis. Further, beta-tryptase was shown to degrade partially denatured collagen type I. beta-Tryptase bound strongly to gelatin, forming high molecular weight complexes that were stable during SDS-PAGE. Mast cells store large amounts of preformed, active tryptase in their secretory granules. Considering the location of mast cells in connective tissues and the recently recognized role of mast cells in disorders in which connective tissue degradation is a key event, e.g., rheumatoid arthritis, it is thus likely that tryptase may contribute to extracellular matrix-degrading processes in vivo.     

Schistosoma mansoni: degradation of host extracellular matrix by eggs and miracidia

A radioactively labeled in vitro model of the extracellular matrix of the mammalian intestinal wall and of snail tissue was used to determine whether proteolytic enzymes released by eggs and miracidia of Schistosoma mansoni could degrade connective tissue macromolecules in the type of interactive framework found in vivo. Eggs were collected and miracidia hatched in the presence of antibiotics to eliminate bacterial contamination. Uninfected livers were used as controls to ensure that the tissue dissociation and egg collection procedures did not produce proteolytic activity. One thousand live eggs incubated with the extracellular matrix for 72 hr at 37 C degraded 31% of the glycoprotein in the matrix; there was no degradation of elastin or collagen. Medium conditioned by incubation with eggs degraded 60% as much of the matrix as the live eggs themselves. The proteolytic activity of the egg-conditioned medium was greater in the presence of dithiothreitol. Miracidia incubated with the extracellular matrix in tissue culture medium at 27 or 37 C rapidly transformed to living sporocysts. This transformation was accompanied by a release of proteolytic activity, resulting in the degradation of 49 to 58% of the glycoprotein in the extracellular matrix by 1000 miracidia. Again, no elastin or collagen was degraded. The time course of degradation by miracidia was rapid over 24 hr and thus similar to that previously reported for cercariae. Degradation by eggs occurred more slowly over 72 hr. These data confirm that both eggs and miracidia secrete proteinases which are capable of degrading at least the glycoprotein components of extracellular matrix to facilitate their migration through intestinal wall or penetration of snail tissue.     

The potential role of human kidney cortex cysteine proteinases in glomerular basement membrane degradation

(1) The degradation of glomerular basement membrane and some of its constituent macromolecules by human kidney lysosomal cysteine proteinases has been investigated. Three cysteine proteinases were extracted from human renal cortex and purified to apparent homogeneity. These proteinases were identified as cathepsins B, H and L principally by their specific activities towards Z-Arg-Arg-NHMec, Leu-NNap and Z-Phe-Arg-NHMec, respectively, and their Mr on SDS-polyacrylamide gel electrophoresis under reducing conditions. (2) Cathepsins B and L, at acid pH, readily hydrolysed azocasein and degraded both soluble and basement membrane type IV and V collagen, laminin and proteoglycans. Their action on the collagens was temperature-dependent, suggesting that they are only active towards denatured collagen. Cathepsin L was more active in degrading basement membrane collagens than was cathepsin B but qualitatively the action of both proteinases were similar, i.e., at below 32 degrees C the release of an Mr 400,000 hydroxyproline product which at 37 degrees C was readily hydrolysed to small peptides. (3) In contrast, cathepsin H had no action on soluble or insoluble collagens or laminin but did, however, hydrolyse the protein core of 35S-labelled glomerular heparan sulphate-rich proteoglycan. (4) Thus renal cysteine proteinases form a family of enzymes which together are capable of degrading the major macromolecules of the glomerular extracellular matrix.     

Localizing matrix metalloproteinase activities in the pericellular environment

Matrix metalloproteinases (MMPs) are a group of structurally related proteolytic enzymes containing a zinc ion in the active site. They are secreted from cells or bound to the plasma membrane and hydrolyze extracellular matrix (ECM) and cell surface-bound molecules. They therefore play key roles in morphogenesis, wound healing, tissue repair and remodeling in diseases such as cancer and arthritis. Although the cell anchored membrane-type MMPs (MT-MMPs) function pericellularly, the secreted MMPs have been considered to act within the ECM, away from the cells from which they are synthesized. However, recent studies have shown that secreted MMPs bind to specific cell surface receptors, membrane-anchored proteins or cell-associated ECM molecules and function pericellularly at focussed locations. This minireview describes examples of cell surface and pericellular partners of MMPs, as well as how they alter enzyme function and cellular behaviour.     

MT1-MMP: a potent modifier of pericellular microenvironment

Cells are regulated by many different means, and there is more and more evidence emerging that changes in the microenvironment greatly affect cell function. MT1-MMP is a type I transmembrane proteinase which participates in pericellular proteolysis of extracellular matrix (ECM) macromolecules. The enzyme is cellular collagenase essential for skeletal development, cancer invasion, growth, and angiogenesis. MT1-MMP promotes cell invasion and motility by pericellular ECM degradation, shedding of CD44 and syndecan1, and by activating ERK. Thus MT1-MMP is one of the factors that influence the cellular microenvironment and thereby affect cell-signaling pathways and eventually alters cellular behavior. As a proteinase, MT1-MMP is regulated by inhibitors, but it also requires formation of a homo-oligomer complex, localization to migration front of the cells, and internalization to become a "functionally active" cell function modifier. Developing new means to inhibit "functional activity" of MT1-MMP may be a new direction to establish treatments for the diseases that MT1-MMP mediates such as cancer and rheumatoid arthritis.     

Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.     

The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis

Matrix metalloproteinases (MMPs) are a large group of enzymes responsible for matrix degradation. Among them, the family of gelatinases (MMP-2/gelatinase A and MMP-9/gelatinase B) is overproduced in the joints of patients with rheumatoid arthritis. Because of their degradative effects on the extracellular matrix, gelatinases have been believed to play an important role in progression and cartilage degradation in this disease, although their precise roles are yet to be defined. To clarify these roles, we investigated the development of Ab-induced arthritis, one of the murine models of rheumatoid arthritis, in MMP-2 or MMP-9 knockout (KO) mice. Surprisingly, the MMP-2 KO mice exhibited severe clinical and histologic arthritis than wild-type mice. The MMP-9 KO mice displayed milder arthritis. Recovery from exacerbated arthritis in the MMP-2 KO mice was possible by injection of wild-type fibroblasts. These results indicated a suppressive role of MMP-2 and a pivotal role of MMP-9 in the development of inflammatory joint disease.     

Expression and cDNA sequence of matrix metalloproteinase-2 (MMP-2) in a mammalian model of human disease processes: Tupaia belangeri.

Matrix metalloproteinase-2 (MMP-2) is one of a family of proteolytic enzymes that are involved in the remodelling of tissue during normal growth processes and is capable of degrading structural components of the extracellular matrix. Increases in MMP-2 expression and activity have been reported in diseases that involve degradation of the extracellular matrix. Reported here for the first time are the relative levels of expression of MMP-2 in tissues of the tree shrew along with 2587 bases of the mRNA sequence. Translation of this sequence predicts a protein 660 amino acids in length, containing all of the features expected of mammalian MMP-2. The tree shrew is a species close to the primate line and is an emerging animal model for a variety of human diseases, including hepatitis and myopia that feature MMP-2 mediated remodelling of the extracellular matrix.     

Immunohistochemical demonstration of collagenase and tissue inhibitor of metalloproteinases (TIMP) in synovial lining cells of rheumatoid synovium

Degradation of fibrillar collagens is a central process in joint destruction in rheumatoid arthritis. Collagenase responsible for the collagenolysis has been immunolocalized on the extracellular matrix components at the cartilage/pannus junction in the rheumatoid joint, but very little is known about cellular source of the proteinase. In this paper monospecific antibodies against collagenase and tissue inhibitor of metalloproteinases (TIMP) were applied to rheumatoid and normal synovium to identify cells synthesizing and secreting the enzyme and its inhibitor. By treating the specimens with the monovalent ionophore, monensin, both collagenase and TIMP could be immunolocalized in hyperplastic synovial lining cells in rheumatoid synovium, but not in the cells of normal synovium. Dual immunolocalization studies demonstrated that the majority of the lining cells (approximately 64%) produce both collagenase and TIMP, while approximately 3% of the cells were positive only for collagenase, and 11% only for TIMP. Neither collagenase nor TIMP was immunolocalized on the extracellular matrix components in the synovia examined. These data suggest that synovial lining cells in rheumatoid arthritis secrete both collagenase and TIMP into the joint cavity. The role of collagenase in joint destruction in rheumatoid arthritis is discussed with reference to the regulation of the activity by TIMP.     

Further evidence for secretion of matrix metalloproteinase-1 by Meckel's chondrocytes during degradation of the extracellular matrix

We examined the possibility that chondrocytes in Meckel's cartilage might secrete matrix metalloproteinase-1 (MMP-1) during degradation of the extracellular matrix. Evidence for the secretion of MMP-1 was obtained by immunohistochemical staining and immunoelectron microscopy, in addition to general histochemical staining for proteoglycans. Not only staining with toluidine blue and alcian blue but also immunostaining for chondroitin sulfate proteoglycan (CSPG) revealed that levels of glycoproteins are rapidly reduced at the late stage of degradation. MMP-1 was detected continuously in cells from chondrocytes at the early stage to hypertrophic chondrocytes at the late stage. Immunoelectron microscopy revealed that the deposition of colloidal golds shifted from an intracellular localization in chondrocytes at the early stage to pericellular spaces at the late stage. The localization of tissue inhibitor of the metalloproteinase-1 (TIMP-1) at the early stage was similar to that of MMP-1, but the level of TIMP-1 decreased significantly in hypertrophic cartilage. These findings suggest that MMP-1 is present continuously in Meckel's chondrocytes but that the active form, which degrades the extracellular matrix, is the MMP-1 that accumulates in the pericellular spaces around hypertrophic chondrocytes.     

Metalloproteinases in endochondral bone formation: appearance of tissue inhibitor-resistant metalloproteinases

Dissected embryonic chick limbs release neutral metalloproteinases during endochondral bone development. These enzymes degrade cartilage proteoglycan and gelatin in culture medium. We found the enzymes active in the medium conditioned by explants of the region adjacent to the bone marrow cavity (cavity-surround). These enzymes degrade proteoglycan (PG) and/or gelatin. These spontaneously active enzymes are resistant to serum and tissue proteinase inhibitors, alpha 2-macroglobulin, and cartilage metalloproteinase inhibitor (TIMP). The other enzymes secreted from tarsus and bone marrow explants are mostly latent in the culture medium. Activated tarsus enzymes (PG degrading and gelatinolytic) are blocked by the above inhibitors. Activated marrow enzyme does not degrade PG but is resistant to those inhibitors. Cavity-surround enzymes may play an important role in embryonic osteogenesis of long bones because of their resistance to tissue and serum inhibitors. The in vivo mechanisms by which cavity-surround enzymes are activated are yet to be determined.