Veli-Jukka Uitto

The Activation and Function of Host Matrix Metalloproteinases in Dentin Matrix Breakdown in Caries Lesions

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.

Matrix Metalloproteinases (MMP-2 and MMP-9) of the Oral Cavity: Cellular Origin and Relationship to Periodontal Status

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.

Local and systemic responses in matrix metalloproteinase 8-deficient mice during Porphyromonas gingivalis-induced periodontitis.

ABSTRACT 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.

Doxycycline and Chemically Modified Tetracyclines Inhibit Gelatinase A (MMP‐2) Gene Expression in Human Skin Keratinocytes

The mechanism of tetracycline-induced inhibition of matrix metalloproteinases (MMP) was studied by measuring the MMP secretion and MMP-2 mRNA levels in unkeratinizing periodontal ligament epithelial cells and skin keratinocytes cultured in the presence of doxycycline or chemically modified tetracyclines (CMT) lacking antimicrobial activity. Doxycycline, CMT-1, and CMT-8 exerted a direct dose-dependent inhibition of porcine periodontal ligament epithelial cell medium MMP activity as assayed by gelatin enzymography. Both the 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases were inhibited by the tetracyclines added to the conditioned medium. Culturing the cells in the presence of the tetracyclines required considerably smaller concentrations to reduce the secreted MMP activity. The drugs were not toxic to the epithelial cells at concentrations from 4 to 250 micrograms/mL up to 24 h of culture. Tetracycline effects on the MMP-2 mRNA levels were studied in human skin keratinocytes using Northern hybridization analysis with a specific cDNA probe. A marked inhibition in the MMP-2 gene expression was observed by 6 h with 5 micrograms/mL of doxycycline, CMT-1 or CMT-8. Doxycycline inhibition was somewhat stronger than the two other tetracyclines. After 24 h of culture with 50 micrograms/mL of the drugs, the total RNA levels also decreased by 33 to 40%. The 72-kDa gelatinase activity in culture medium of the keratinocytes followed roughly the pattern of inhibition of the gene expression. We conclude that doxycycline and the chemically modified tetracyclines, in addition to inhibiting the MMP activity may also reduce the enzyme expression at the transcriptional level.

Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9), and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer.

Squamous cell carcinoma (SCC) of the oral cavity is a highly invasive tumour of stratified squamous epithelium that spreads through degradation of the basement membrane (BM) and extracellular matrix (ECM). There are currently no reliable tissue or serum markers to predict whether the tumour has metastasized at the time of diagnosis. Verrucous carcinoma (VC) of the oral cavity is a rare low‐grade variant of oral SCC that penetrates into the subepithelial connective tissue. Many matrix metalloproteinases (MMPs), such as MMP‐1, ‐2, ‐7, ‐9, ‐13, and ‐14, as well as integrin receptors have been implicated in cancer invasion. Integrin αvβ6 is induced in SCC and appears to be involved in up‐regulation of MMP‐9 expression by oral keratinocytes and promotion of their migration. The aim of this study was to investigate whether the pattern of MMP expression or that of αvβ6 integrin contributes to the differences in the biological behaviour of oral SCC and VC. The results show that the less aggressive nature of oral VC may be connected to its MMP expression profile. Typically, VCs were devoid of epithelial MMP‐3, ‐7, ‐9, ‐12 and ‐13 expression, compared with SCCs. MMP‐19 was expressed by epithelial keratinocytes in hyperproliferative areas of verrucous hyperplasia, VC, and SCC, but was absent in the invasive cancer cell nests of SCC. MMP‐26 was expressed by hyperproliferative keratinocytes in VC as well as by invasive cancer cells in SCCs. MMP‐10 was expressed widely in the epithelium of all SCC specimens. αvβ6 integrin expression was also detected in some cases of epithelial hyperplasia but was significantly more abundant in cancers at the invasive front. The absence of MMP‐7, ‐9 and ‐12 from epithelial cells may serve as a good prognostic marker of non‐invasive oral carcinoma. Blocking the activity of invasion‐specific MMPs or αvβ6 integrin might offer novel therapeutic modalities in early‐stage oral carcinoma. Copyright

Membrane Components of Treponema denticola Trigger Proteinase Release from Human Polymorphonuclear Leukocytes

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.

The role of gingival crevicular fluid and salivary interstitial collagenases in human periodontal diseases.

Interstitial collagenases (matrix metalloproteinase-1, EC 3.4.24.7), isolated from extracts of inflamed human gingiva, gingival crevicular fluid and saliva were characterized for their molecular weight, proteolytic and non-proteolytic activation and substrate specificity against soluble collagen types I, II and III. All three collagenases had Mr of 70 K. The enzymes existed predominantly in a latent form that could be activated by aminophenylmercuric acetate, gold thioglucose and hypochlorous acid. Among serine proteases tested, trypsin, chymotrypsin, neutrophil cathepsin G and a combination of trypsin and human gingival fibroblast prostromelysin activated gingival and salivary interstitial collagenases. Plasmin and plasma kallikrein, however, were relatively ineffective activators. The collagenases degraded soluble type I and II collagens at apparently equal rates but considerably faster than they did type III collagen. These findings suggest that the characteristics of interstitial collagenases found in inflamed human gingiva, gingival crevicular fluid and saliva are consistent with those of human neutrophil interstitial collagenase rather than the fibroblast-type interstitial collagenase. Thus, neutrophils are suggested to be the main source of such enzymes in inflamed human gingiva, crevicular fluid and saliva during adult periodontitis.

Human leukocyte collagenase: Characterization of enzyme kinetics by a new method

Abstract Human collagenase was partially purified from polymorphonuclear leukocytes, and a new method for assay of the collagenase activity was developed. The assay employs native radioactive collagen in soluble form as a substrate. The enzyme incubations are performed at 25°C which is below the melting temperatures of the cleavage products TCA and TCB, and these peptides are quantitatively recovered by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Employing this method, an apparent K m value of 1.04 × 10 −6 m for human leukocyte collagenase using type I collagen as a substrate was measured.

Activation of Latent Collagenase of Human Leukocytes and Gingival Fluid by Bacterial Plaque

Collagenase activity was studied in human leukocytes, gingival crevicular fluid and bacterial plaque, with soluble radioactive collagen as substrate. Inflamed gingiva liberated vertebrate type collagenase into the crevicular fluid in active form. Healthy gingiva, in contrast, released collagenase in a latent form that could be activated by trypsin or plaque. Plaque also stimulated leukocytes to release collagenase, and activated the latent enzyme.

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.