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Cathepsin C, Matrix Metalloproteinases, and their Tissue Inhibitors in Gingiva and Gingival Crevicular Fluid from Periodontitis-affected Patients

¹ Unité INSERM U 424, 11 rue Humann, 67085 Strasbourg CEDEX, France; and
² Parogène, Strasbourg, France;

*corresponding author, h.tenenbaum{at}odonto3.u-strasbg.fr

	ABSTRACT

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Successive active phases observed in periodontal diseases may be explained either by a sudden activation of the pro-forms of tissue-stored degradative enzymes such as metalloproteinases (MMPs) or by an imbalance between metalloproteinases and their tissue inhibitors (TIMPs). To discriminate between these two hypotheses, we quantified the levels, the percentage of active form, and the activities of four metalloproteinases (MMPs -1, -2, -3, and -9), as well as the levels of two tissue inhibitors of metalloproteinases (TIMP-1 and -2) and the activity of cathepsin C in tissue extract supernatants and their corresponding gingival crevicular fluid samples collected from periodontitis-affected and healthy patients. Our results supported evidence that tissue destruction results from an imbalance of metalloproteinases over their tissue inhibitors rather than from a sudden activation of the pro-forms of these enzymes. A significant reduction in the activity of cathepsin C also contributed to the degradative process.

KEY WORDS: cathepsin C • matrix metalloproteinases • tissue inhibitors of metalloproteinases • periodontitis

	INTRODUCTION

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Periodontitis is an infectious disease characterized by periodontal attachment loss, bone destruction, and eventually tooth loss (Socransky and Haffajee, 1992; Genco et al., 1998). Matrix metalloproteinases (MMPs) contribute significantly to tissue destruction and remodeling events in this disease (Wolley and Davies, 1981), and numerous in vitro and in vivo studies have substantiated the relationship between MMPs and periodontal diseases. The presence of interstitial collagenase (MMP-1), gelatinase-A (MMP-2), stromelysin-1 (MMP-3), neutrophil collagenase (MMP-8), and gelatinase-B (MMP-9) has been identified in gingival tissues and gingival crevicular fluid samples from periodontitis-affected patients (Tonetti et al., 1993; Aiba et al., 1996). The cellular origin of some of these enzymes is beginning to be better known. MMP-2, MMP-8, and MMP-13 originate from inflamed gingival sulcular epithelium (Tervahartiala et al., 2000). The polymorphonuclear leukocyte MMP-9 can be found in connective tissue but not in the sulcular and oral epithelium (Westerlund et al., 1996), and MMP-1 originates from fibroblastic-type spindle-shaped cells in close contact with inflammatory cells (Dahan et al., 2001).

Other enzymes may also contribute to the tissue destruction in periodontitis. Indeed, several studies have shown increases in both level and activity of cathepsins B, D, G, and L in periodontal diseases (Trabandt et al., 1995; Tervahartiala et al., 1996; Chen et al., 1998). More recently, Toomes et al. (1999) have emphasized that mutations within the cathepsin C gene lead to a loss-of-enzyme activity in patients with periodontal disease and palmoplantar keratosis (Papillon-Lefèvre syndrome).

Overall et al. (1987) have explained the cyclical progression of periodontitis by the "burst hypothesis". The activation of the tissue-stored pro-form of degradative enzymes such as MMPs may induce a sudden period of activity. During the period of remission, a balance is re-introduced via an increase of metalloproteinase tissue inhibitors (TIMPs). When the inflammatory response takes over again, a reservoir of pro-enzymes is gradually built up and stored in the extracellular matrix, and, following activation, a new "burst" of disease activity occurs. Little is known about the activity and percentage of the active form of any particular enzyme, especially during active phases of the disease. The aim of our study was to compare the levels, the activity, the percentages of active forms of MMP-1, MMP-2, MMP-3, and MMP-9, the levels of TIMP-1 and TIMP-2, and the activity of cathepsin C in the same samples of tissue extract supernatants and their corresponding gingival crevicular fluid samples taken from healthy and periodontitis-affected patients.

	MATERIALS & METHODS

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Patient Selection
Sixteen patients (ages 15-71 yrs; mean, 39 yrs), eight males and eight females, who attended the Department of Periodontology (Dental Faculty, Université Louis Pasteur, Strasbourg, France) were selected for the study after informed consent was obtained according to the Helsinki Declaration. The protocol was approved by the institutional ethic committee. Eleven of these patients (ages 36-71 yrs; mean, 49 yrs), five males and six females, were diagnosed as being affected with advanced periodontitis, for which flap surgery was indicated. The remaining five patients (ages 15-18 yrs; mean, 16 yrs), three males and two females, with clinically healthy periodontium, were used as controls. The patients had impacted third molar extraction. The 5 biopsy samples included soft tissues directly in contact with the distal surface of the second molar.

Gingival Biopsies
Gingival biopsies were collected during surgery and included pocket epithelium and adjacent connective tissues. The detailed procedure was established from a protocol previously described (Golub et al., 1995) and appears in the Appendix (www.dentalresearch.org).

Gingival Crevicular Fluid Sample Collection
From the periodontitis-affected patients, gingival crevicular fluid samples were collected from periodontal pockets over 4 mm in probing depth; samples were collected from the gingival sulcus of healthy patients immediately before surgical treatment. The surface of each tooth was dried gently with air and kept dry with cotton rolls. Three paper points were placed into the pocket or the sulcus for 10 sec, and the absorbed fluid was eluted as described by Ingman et al. (1994). The procedure is detailed in the Appendix (www.dentalresearch.org).

Antibodies
Antibodies used for ELISA, zymography, and Western blots are indicated in the Appendix (www.dentalresearch.org).

Quantification of MMPs and TIMPs
The quantification of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 was performed in tissue extract supernatants and gingival crevicular fluid samples by means of a sandwich ELISA assay based on a procedure described by Cooksley et al. (1990) and is detailed in the Appendix (www.dentalresearch.org). The results are expressed as µg of antigen per mg of total proteins.

The quantification of cathepsin C has not been performed, because no specific antibody or commercial ELISA test was available at the time of the experiments.

Percentage of Active Forms of MMPs
The visualization of active forms and pro-forms of MMPs in tissue extract supernatants and gingival crevicular fluid samples was performed by zymography for MMP-2 and MMP-9 and by Western blot experiments for MMP-1 and MMP-3. Both types of experiments are detailed in the Appendix (www.dentalresearch.org).

Zymograms were performed as described by Overall et al. (1989), and Western blot experiments were performed as described by Towbin et al. (1979).

The intensities of bands corresponding either to the active form or the pro-form were quantified directly on zymograms or chemiluminescent detection films, by means of a KODAK Digital Science 1D image analyzer (Eastman Kodak Company, Rochester, NY, USA). For each MMP, the sizes of pro-forms and active-forms are detailed in the Appendix (www.dentalresearch.org). The level of the active form for a given MMP was calculated as a percentage, with 100% considered as the total obtained when the intensity values of active and pro-form bands were added. The results are expressed as mean ± SE.

Proteolytic Activities of MMPs
The proteolytic activities of MMPs were determined in tissue extract supernatants and gingival crevicular fluid samples by quantification of the degradation of a biotinylated substrate: human collagen type-1 for MMP-1, casein for MMP-3, and gelatin for the combined MMP-2 and MMP-9 activities. All these substrates were purchased from Sigma (St. Louis, MO, USA) and biotinylated with NHS-LC-Biotin (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. A 1-µg quantity of each purified human MMP served as positive control. Assays performed are detailed in the Appendix (www.dentalresearch.org). Results are expressed as mean ± SE percentage of biotinylated substrate degraded/1 µg of total protein/1 hr.

Activity of Cathepsin C
We determined cathepsin C activity on 1 µg of total proteins from tissue extract supernatants and gingival crevicular fluid samples by measuring the amount of 7-amido-4-methyl-coumarin (AMC) (Bachem, Heidelberg, Germany) released by hydrolysis of the synthetic substrate glycyl-L-arginine-7-amido-4-methyl-coumarin (Bachem, Heidelberg, Germany), using a modified protocol described by Smyth and O'Cuinn (1994). The AMC formed was monitored by means of a VersaFluor Multiple-Wavelength fluorometer (Bio Rad, Hercules, CA, USA) at 370-nm excitation and 460-nm emission. A 1-µg quantity of purified human cathepsin C served as positive control. The cathepsin C specific activity was expressed as µMoles of AMC produced/min/mg of total proteins.

Statistical Analysis
The antigen levels, the proteolytic activities, and the percentages of active forms in periodontitis-affected tissue extract supernatants and gingival crevicular fluid samples were compared with those from healthy patients by means of the non-parametric Mann-Whitney Rank Sum Test. Differences between the two populations were considered significant when confidence intervals were > 95% (p < 0.05). Data represent the average of 3 different experiments performed under identical conditions.

	RESULTS

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Quantification of MMPs and TIMPs
We observed a significant increase (p < 0.03) in all MMP levels in tissue extract supernatants from periodontitis-affected patients as compared with healthy controls (450% for MMP-1, 200% for MMP-2, 500% for MMP-3, and 600% for MMP-9) (Fig. 1a).

View larger version (31K): [in this window] [in a new window]   Figure 1. Quantification of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 in tissue extract supernatants and gingival crevicular fluid samples from healthy and diseased patients. Evaluations were performed by means of a sandwich ELISA assay in tissue extract supernatants (TES/A) and gingival crevicular fluid samples (GCFs/B). Results were obtained from three different experiments (n = 3) and expressed as mean ± SE of µg of antigen/mg of total proteins. (*) indicated that the Mann-Whitney Rank Sum Test showed a statistically significant difference between diseased and healthy tissue extract supernatants (p < 0.003) and between diseased and healthy gingival crevicular fluid samples (p < 0.003).

We observed a significant decrease in the TIMP-1 level in tissue extract supernatants (p < 0.002) (Fig. 1a) and gingival crevicular fluid samples (p < 0.02) from periodontitis-affected patients compared with control values (Fig. 1b). The decrease in TIMP-1 was almost similar in tissue extract supernatants and gingival crevicular fluid samples (79% and 83%, respectively). A significant decrease in TIMP-2 level (p < 0.002) in tissue extract supernatants (80%) and gingival crevicular fluid samples (96%) from periodontitis-affected patients was observed compared with healthy controls.

Activities of MMPs and Cathepsin C
The activity of a particular MMP was calculated in tissue extract supernatants from diseased and healthy patients based on its ability to degrade a specific biotinylated substrate (Fig. 2a). The combined activities of MMP-2 and MMP-9, measured by gelatin degradation, were significantly increased (193%; p < 0.002) in tissue extract supernatants from periodontitis-affected patients compared with healthy controls. The MMP-3 activity, measured by casein degradation, and the MMP-1 activity, measured by collagen I degradation, were also significantly increased (p < 0.002) in diseased tissue extract supernatants compared with healthy controls (218% for MMP-3 and 500% for MMP-1).

View larger version (23K): [in this window] [in a new window]   Figure 2. Proteolytic activities of MMPs in tissue extract supernatants and gingival crevicular fluid samples from healthy and diseased patients. The proteolytic activities of MMPs were determined in tissue extract supernatants (TES/A) and gingival crevicular fluid samples (GCFs/B) from healthy and diseased patients by quantification of the percentage of degradation of a biotinylated substrate: human collagen type-1 for MMP-1, casein for MMP-3, and gelatin for both MMP-2 and MMP-9. Controls in TES/A indicate the results obtained when 1 µg of each purified human MMP was used in the assay. Results were obtained from three different experiments (n = 3), and activity was expressed as mean ± SE percentage of biotinylated substrate degraded/mg of total proteins/hr. (*) indicated that the Mann-Whitney Rank Sum Test showed a statistically significant difference between diseased and healthy tissue extract supernatants (p < 0.002) and between diseased and healthy gingival crevicular fluid samples (p < 0.001).

Cathepsin C activity was significantly lower in all diseased compared with healthy samples for tissue extract supernatants (p < 0.006) and gingival crevicular fluid samples (p < 0.033) (Fig. 3). In tissue extract supernatants from periodontitis-affected patients, cathepsin C activity (31 x 10^-5 µMoles/mg/min) was reduced to 25% of the activity found in healthy samples (122 x 10^-5 µMoles/mg/min). In gingival crevicular fluid samples from periodontitis-affected patients, the activity (183 x 10^-5 µMoles/mg/min) was reduced to 41% of that in healthy controls (441 x 10^-5 µMoles/mg/min).

View larger version (14K): [in this window] [in a new window]   Figure 3. Proteolytic activities of cathepsin C in tissue extract supernatants and gingival crevicular fluid samples from healthy and diseased patients. Activity of cathepsin C in tissue extract supernatants (TES) and gingival crevicular fluid samples (GCFs) from healthy and diseased patients was determined by measurement of the amount of 7-amido-4 methyl-coumarin (AMC) released by hydrolysis of the synthetic substrate H-Gly-Arg-AMC. The AMC formed was monitored by means of a VersaFluor Multiple-Wavelength fluorometer luminescence spectrometer at 370-nm excitation and 460-nm emission. Results were obtained from three different experiments (n = 3) and expressed as mean ± SE of µMoles of AMC produced/min/mg of total proteins in the sample. (*) indicated that the Mann-Whitney Rank Sum Test showed a statistically significant difference between diseased and healthy tissue extract supernatants (p < 0.006) and between diseased and healthy gingival crevicular fluid samples (p < 0.003).

	DISCUSSION

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The degradation of collagen fibers and other extracellular matrix components in inflammatory diseases such as periodontitis (Fuller and Gibson, 1996) resulted from the activity of matrix metalloproteinases (MMPs) which originate from various cellular types (Westerlund et al., 1996; Tervahartiala et al., 2000). Periodontitis is characterized by cyclical phases of active tissue destruction, but the biological events which could counter the tissue destruction initiated by MMPs are poorly understood. One hypothesis relies on an increase of tissue inhibitors of metalloproteinases (TIMPs) (Ingman et al., 1994). Evidence that tissue destruction in diseased processes might result from an imbalance of MMPs over TIMPs came from findings in human osteoarthritic cartilage (Dean et al., 1989), in synovial explants from a rabbit arthritis model (Murphy and Reynolds, 1993), and from human broncho-pulmonary carcinomas (Nawrocki et al., 1997). Another hypothesis stipulated that pro-forms of MMPs are suddenly converted to their active forms during active phases of tissue destruction. We cannot conclude which of these two biological events appears to be predominant.

In the present study, we tried to determine if extracellular matrix destruction was resulting from an activation of pro-forms of MMPs rather than from an imbalance of MMPs over TIMPs. Considering that all the measurements were performed on the same tissue extract supernatants and gingival crevicular fluid samples, our results indicate a significant increase in MMP-1, MMP-2, MMP-3, and MMP-9 levels and a significant decrease in TIMP-1 and TIMP-2 levels in tissue extract supernatants and gingival crevicular fluid samples obtained from periodontitis-affected patients compared with healthy controls. The increases in MMPs levels are in agreement with results reported from previous studies (Villela et al., 1987; Sorsa et al., 1994; Aiba et al., 1996; Ingman et al., 1994) and were also associated with an increase of all MMP activities in tissue extract supernatants and their corresponding gingival crevicular fluid samples from periodontitis-affected patients. The significant decreases in TIMP-1 and TIMP-2 levels in diseased samples were in conflict with results reported in other studies. An increase of TIMP levels was found in gingival crevicular fluid samples from localized juvenile periodontitis-affected patients when compared with adult periodontitis-affected or control patients (Ingman et al., 1994). Enhanced levels of TIMPs were observed in healthy sites and support the hypothesis that tissue destruction resulted from an imbalance of MMPs over TIMPs due both to an increase in MMP levels and to a decrease in TIMP levels (Alexander and Damoulis, 1994). Our results, which showed no significant increase in the percentage of active form for a given MMP between healthy and diseased samples in either tissue extract supernatants or gingival crevicular fluid samples, are consistent with the latter hypothesis. These findings rule out the idea that tissue destruction in pathological situations resulted from sudden activation of tissue-stored pro-forms of MMPs.

In periodontitis-affected patients, other types of degradative enzymes—among them cathepsins B, D, G, and L—showed a significant increase (Trabandt et al., 1995; Tervahartiala et al., 1996; Chen et al., 1998). On the other hand, cathepsin C activity showed a significant decrease in Papillon-Lefèvre syndrome (Toomes et al., 1999). Our results supported evidence that cathepsin C might also be involved in adult periodontitis patients not suffering from this syndrome. This observation is of interest, since cathepsin C is, to date, the first enzyme whose activity decreases in periodontitis. Mutations of the gene encoding cathepsin C observed in patients suffering from Papillon-Lefèvre syndrome as well as the function of cathepsin C in periodontitis remain to be elucidated.

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	ACKNOWLEDGMENTS

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received November 15, 2000; Last revision October 29, 2001; Accepted January 15, 2002

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