Osteoclasts form an acidic compartment at their attachment site in which bone demineralization and matrix degradation occur. Although both the cysteine proteinases and neutral collagenases participate in bone resorption, their roles have remained unclear. Here we show that interstitial collagenase has an essential role in initiating bone resorption, distinct from that of the cysteine proteinases. Treatment of osteoclasts with cysteine proteinase inhibitors did not affect the number of resorption lacunae ("pits") formed on the surface of dentine slices, but it generated abnormal pits that were demineralized but filled with undegraded matrix. Treatment with metalloproteinase inhibitors did not alter the qualitative features of lacunae, but it greatly reduced the number of pits and surface area resorbed. Treatment of bone cells with an inhibitory anti-rat interstitial collagenase antiserum reduced bone resorption markedly. In the presence of collagenase inhibitors, resorption was restored by pretreatment of dentine slices with rat interstitial collagenase or by precoating the dentine slices with collagenase-derived gelatin peptides or heatgelatinized collagen. Immunostaining revealed that interstitial collagenase is produced at high levels by stromal cells and osteoblasts adjacent to osteoclasts. These results indicate that interstitial collagenase can function as a "coupling factor," allowing osteoblasts to initiate bone resorption by generating collagen fragments that activate osteoclasts.Normal bone turnover is highly regulated. Osteoclasts, the cells that degrade bone, require activation to trigger their boneresorptive capacity. Once activated, osteoclasts secrete both protons and proteinases at their attachment site, resulting in dissolution of bone mineral and degradation of the matrix (1). Osteoclasts produce several cysteine proteinases (2-4), enzymes with acidic pH optima, of which cathepsin K appears to be essential for normal bone resorption (5, 6). Studies indicate that the major function of secreted cysteine proteinases is matrix degradation (3, 7).The role of neutral metalloproteinases, a second class of proteinase produced in bone tissue (3,8), is less clear. Members of the metalloproteinase family have neutral pH optima, are secreted as proenzymes, and contain a zinc atom at the active site (9, 10). Although some prior studies suggested that neutral metalloproteinases contribute to osteoclast matrix degradation (11), recent evidence indicates that osteoclasts do not produce collagenase (12). Collagenase is produced by cells of osteoblastic lineage and may be required for resorption of intact bone tissue (13)(14)(15)(16). Observations that isolated osteoclasts that had no detectable collagenase activity were able to resorb bone prompted the suggestion that collagenase promotes resorption by removing unmineralized matrix from the bone surface, facilitating osteoclast attachment (8,17,18).In this study we have examined the qualitative and quantitative roles of acid cysteine proteinases and interstitial...
Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. Elastin is an extracellular matrix protein composed of highly cross-linked, hydrophobic tropoelastin monomers which provides resilience to elastic fibers. The hydrophobicity and extensive cross-linking of tropoelastin monomers result in an insoluble elastic fiber which is highly resistant to proteolysis (1). Thus, under normal physiologic conditions, elastin undergoes minimal turnover (2). However, certain pathologic situations, including pulmonary emphysema (3) and abdominal aortic aneurysm (4), are characterized by proteolytic destruction of elastic fibers. The involvement of serine proteases in such pathologies has long been suspected. More recently, participation of cysteine proteinases (5) and matrix metalloproteinases (MMPs, 1 6 -8) in these diseases has been proposed. The MMPs comprise a gene family that collectively is capable of degrading all components of extracellular matrix in physiologic and pathologic states (9, 10). As presently recognized, this family consists of fibroblast (11), neutrophil (12), and breast carcinoma-derived (13) collagenases, three stromelysins, 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME, MMP-12), matrilysin, and a recently described 66-kDa membrane-type metalloproteinase (14). These enzymes are organized into homologous structural domains, with some differences in domain composition and number. All members share a zymogen domain, containing a conserved PRCGXPD motif involved in enzyme latency, and a zinc-binding CD. Most members also contain a hemopexin-like domain at their C terminus, the exception being matrilysin, which lacks this domain completely. Unique to the 72-kDa and 92-kDa gelatinases is an additional domain composed of three fibronectin type II-like repeats inserted in tandem within the zinc-binding CD. The 92-kDa gelatinase also contains an ␣2(V) collagen-like domain not found in any of the other family members.The issue of substrate specificity has received considerable attention recently in MMP biology. The determinants which confer substrate specificity to these enzymes appear to be localized within discrete structural domains. For example, the ability of the collagenases to degrade triple-helical collagen requires the presence of the C-terminal hemopexin-like domain (15)(16)(17). In contrast, the stromelysins degrade a variety of substrates in a manner which is independent of the C-terminal hemopexin-like dom...
Insoluble elastin was used as a substrate to characterize the peptide bond specificities of human (HME) and mouse macrophage elastase (MME) and to compare these enzymes with other mammalian metalloproteinases and serine elastases. New amino termini detected by protein sequence analysis in insoluble elastin following proteolytic digestion reveal the P 1 residues in the carboxyl-terminal direction from the scissile bond. The relative proportion of each amino acid in this position reflects the proteolytic preference of the elastolytic enzyme. The predominant amino acids detected by protein sequence analysis following cleavage of insoluble elastin with HME, MME, and 92-kDa gelatinase were Leu, Ile, Ala, Gly, and Val. HME and MME were similar in their substrate specificity and showed a stronger preference for Leu/Ile than did the 92-kDa enzyme. Fibroblast collagenase showed no activity toward elastin. The amino acid residues detected in insoluble elastin following hydrolysis with porcine pancreatic elastase and human neutrophil elastase were predominantly Gly and Ala, with lesser amounts of Val, Phe, Ile, and Leu. There were interesting specificity differences between the two enzymes, however. For both the serine and matrix metalloproteinases, catalysis of peptide bond cleavage in insoluble elastin was characterized by temperature effects and water requirements typical of common enzyme-catalyzed reactions, even those involving soluble substrates. In contrast to what has been observed for collagen, the energy requirements for elastolysis were not extraordinary, consistent with cleavage sites in elastin being readily accessible to enzymatic attack.Elastin is the extracellular matrix protein that imparts elastic recoil to tissues. Its cross-linked nature and extreme hydrophobicity make it one of the most stable proteins in the body (1-3). A contributing factor to elastin's longevity is its relative resistance to proteolysis by all but a limited number of proteinases that are capable of degrading the mature, insoluble protein under physiological conditions. These enzymes, referred to as elastases, have a wide distribution in nature and are found in animals as well as in plants and bacteria (4, 5). Elastases are heterogeneous with differing substrate specificities and catalytic mechanisms. In fact, enzymes with elastolytic activity can be found in most of the major proteolytic families, including serine, thiol, aspartic, and metallo enzymes (4). Despite the differences in catalytic mechanisms, all of these elastases share a common specificity for cleaving peptide bonds associated with hydrophobic or aromatic amino acids.The most widely studied elastases, human neutrophil elastase (HLE) 1 and pancreatic elastase (PPE), belong to the serine proteinase family of enzymes. Neutrophil elastase is found in the azurophil granules of polymorphonuclear leukocytes and is essential for phagocytosis and defense against infection. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestine wh...
The matrix metalloproteinase 92-kDa gelatinase is a major product of inflammatory cells. Macrophages synthesize and secrete this proteinase as a proenzyme in association with tissue inhibitor of metalloproteinases (TIMP) (92TIMP), whereas neutrophils store and release it from secondary granules as a TIMP-free proenzyme (92TIMP-free). Metalloproteinase proenzymes can be activated in vitro by a variety of agents, including organomercurials and proteinases, resulting in loss of an 8-10-kDa NH2-terminal domain which disrupts the interaction of a conserved cysteine residue with the catalytic zinc molecule. We report that the activation and processing of 92-kDa gelatinase differs depending on its association with TIMP and the nature of the activating agent. We observed that 92TIMP undergoes classic activation to 82 kDa by stromelysin, whereas exposure to 4-aminophenylmercuric acetate (APMA) results in a final product of 83 kDa that still contains the "prodomain" cysteine. Association with TIMP appears to stabilize the COOH-terminal domain, whereas 92TIMP-free is converted by APMA to a final product of 67 kDa lacking the COOH-terminal portion. In the continued presence of APMA, which maintains cysteine-zinc disruption, the 67-kDa species is at least as active as the classic 82 kDa. In contrast, activation of 92TIMP-free by stromelysin initially generates the 82-kDa form which is followed by final conversion to a 50-kDa species that lacks the catalytic domain of the parent molecule. Therefore, although stromelysin activation of 92TIMP-free is initially efficient, the active 82-kDa form is short-lived and is replaced by an inactive 50-kDa product. This complex pattern of activation of the 92-kDa gelatinase may serve to restrict its proteolytic capacity following exposure to stromelysin and may serve to regulate proteinase activity in vivo.
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