Matrix metalloproteinases (MMPs) are a family of enzymes which, in concert, are capable of degrading collagen. We investigated whether human MMPs could participate in the degradation of dentin organic matrix after demineralization. We performed Western blot analyses using MMP-specific antibodies to identify MMPs in human dental caries lesions. Enzymography and functional activity assays, with 125I-labeled gelatin as substrate or quantitating the degradation of type I collagen, were used to determine the activity of purified and salivary gelatinolytic (MMP-2 and MMP-9) and collagenolytic (MMP-8) enzymes with and without acid-activation in pHs relevant to caries. Respective analyses were done with caries-related bacteria. We performed electron microscope analyses to assess the degradative activity of sterilized salivary host MMPs on demineralized human dentin. Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The latent purified forms of these enzymes were activated at low pH (4.5), followed by neutralization, mimicking the conditions during caries progression. Incubation of human saliva at low pH followed by neutralization resulted in a four-fold increase in the gelatinolytic activity. No gelatinolytic or collagenolytic activity was observed in bacterial samples. The activated enzymes in saliva degraded demineralized dentin organic matrix in vitro. These results demonstrate the pH-dependent activation mechanism of MMPs, which may have a distinct role in different physiological and pathological conditions. They further demonstrate that host MMPs, activated by bacterial acids, have a crucial role in the destruction of dentin by caries.

No abstract

Sorsa T, Uitto V-J, Suomalainen K, Vauhkonen M, Lindy S.: Comparison of interstitial coliagenases from human gingiva, sulcular fluid and polymorphonuclear leukocytes. J Periodont Res 1988: 23: 386-393. Mammalian coliagenases (EC 3.4,24,7) have been suggested as playing an essential role in the initiation of the collagen degradation in periodontal diseases. Two distinct types of interstitial coliagenases have been characterized in vertebrate tissues. These enzymes, the fibrobiast-and the neutrophil-type coliagenases,, differ in molecular weight and antigenic properties, as well as substrate specificity and mechanism of activation. In order to determine the cellular origin and mode of action of coliagenase in periodontal tissue, we studied the molecular size, the substrate specificity and the activation of coliagenases partially purified from inflamed human gingival extracts, sulcular fluid, gingival expiant culture medium and polymorphonuclear leukocytes (PMN). Types I, II and III collagens used as substrates were purifted from bovine tendon, cartilage and amnion membrane, respectively. Apparent molecular weights of 70-75 k were obtained for gingival extract, sulcular fluid and PMN coliagenases and 45 k for gingival expiant culture collagenase by gel filtration technique. The gingival extract and sulcular fiuid coliagenases as well as PMN collagenase could be activated by gold thioglucose and gold thiomalate; no activation of gingival expiant culture collagenase was noted. The gingival extract collagenase. sulcular fluid collagenase and PMN collagenase degraded preferentially types I and 11 collagens relative to type-Ill collagen. In contrast, gingival expiant culture coliagenase degraded preferentially types I and III collagens relative to type-II collagen. The results indicate that collagenase in extracts of inflamed human gingiva and in sulcular fluid during inflammation is mostly derived from PMN cells. On the other hand, collagenase produced by gingival explants in culture is probably synthesized by fibrobiasts.

No abstract

Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supraand subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were PrevotelUa intermedia (ATCC 25261), PrevoteUla buccae (ES 57), PrevoteUa oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nukeatum (ATCC 10953), Mitsuokela dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub-and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supraand subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.

Periodontitis is a bacterium-induced chronic inflammation that destroys tissues that attach teeth to jaw bone. Pathologically excessive matrix metalloproteinase 8 (MMP-8) is among the key players in periodontal destruction by initiating type I collagen degradation. We studied MMP-8 in Porphyromonas gingivalis-induced periodontitis by using MMP-8-deficient (MMP8 ؊/؊ ) and wild-type (WT) mice. Alveolar bone loss, inflammatory mediator expression, serum immunoglobulin, and lipoprotein responses were investigated to clarify the role of MMP-8 in periodontitis and systemic inflammatory responses. P. gingivalis infection induced accelerated site-specific alveolar bone loss in both MMP8؊/؊ and WT mice relative to uninfected mice. The most extensive bone degradation took place in the P. gingivalis-infected MMP8 ؊/؊ group. Surprisingly, MMP-8 significantly attenuated (P < 0.05) P. gingivalis-induced site-specific alveolar bone loss. Increased alveolar bone loss in P. gingivalis-infected MMP8 ؊/؊ and WT mice was associated with increase in gingival neutrophil elastase production. Serum lipoprotein analysis demonstrated changes in the distribution of high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL) particles; unlike the WT mice, the MMP8 ؊/؊ mice underwent a shift toward a smaller HDL/VLDL particle sizes. P. gingivalis infection increased the HDL/VLDL particle size in the MMP8 ؊/؊ mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total lipopolysaccharide activity and the immunoglobulin G-class antibody level in response to P. gingivalis were significantly elevated in both infected mice groups. Thus, MMP-8 appears to act in a protective manner inhibiting the development of bacterium-induced periodontal tissue destruction, possibly through the processing anti-inflammatory cytokines and chemokines. Bacterium-induced periodontitis, especially in MMP8 ؊/؊ mice, is associated with systemic inflammatory and lipoprotein changes that are likely involved in early atherosclerosis.

A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, al-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50°C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(O-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction. * Corresponding author.35405 that has the ability to hydrolyze many functionally important serum and tissue proteins. MATERIALS AND METHODSBacterial strains and culture conditions. T. denticola ATCC

Proteolytic enzymes released by the host cells are associated with the tissue destruction in periodontal diseases. Matrix metalloproteinases (MMPs) have the primary role in this process, since, in concert, they can degrade most of the extracellular matrix components. In the present study, we investigated MMP-2 and MMP-9 in oral fluids of healthy subjects and periodontitis patients and the contributions of different oral cells to the enzyme production. The enzymograms revealed that the main gelatinase in oral rinses, crevicular fluid, and whole saliva migrated at 92 kDa. Activity was also detected at 200 kDa and 130 kDa and minor activity at 86 kDa, 72 kDa, and 40 kDa. Traces of gelatinolytic activity were also detected in pure parotid secretions. The 92-kDa enzyme was identified to MMP-9 and the 200-kDa gelatinase to MMP-2, by means of specific anti-72-kDa antiserum. Gingival keratinocytes produced mainly MMP-9, while gingival and granulation tissue fibroblasts expressed MMP-2. Glandular tissue contained mainly MMP-9, and mRNA for MMP-9 was also found in acinar epithelial cells. Periodontitis patients had significantly higher levels of MMP-9 than healthy subjects. Also, MMP-2 was elevated in periodontitis patients. Periodontal treatment reduced the amount of gelatinases dramatically. This study shows that gelatinases are produced by various cells in the oral cavity. The amount of gelatinases is elevated during periodontal disease, while conventional periodontal treatment efficiently reduces the levels these enzymes. We suggest that MMP-2 and MMP-9 could participate in tissue destruction in periodontitis.