Year : 2006 | Volume
: 17 | Issue : 4 | Page : 161--166
Expression of matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in oral submucous fibrosis
R Rajendran1, PK Rajeesh Mohammed1, Saleem Shaikh1, Shanthi2, MR Pillai1,
1 Department of Oral Pathology and Microbiology, Govt Dental College, Kerala, India
2 Department of Molecular Medicine, Regional Cancer Centre, Trivandrum 695 011, Kerala, India
Department of Oral Pathology and Microbiology, Govt Dental College, Kerala
Immunohistochemical staining of formalin fixed, paraffin embedded tissue sections of OSF for MMPs-1,2,9 and their tissue inhibitors TIMP-1and 2 was performed using monospecific antibodies coupled with gelatin zymography (MMP-2 and 9) for measuring enzymatic activity quantitatively and for distinguishing the active from the inactive variants of enzymes. The present study, contrary to earlier reports, recorded statistically significant increase in the levels of stromal expression of MMP-1, MMP-2 and MMP-9 and TIMP-1 and TIMP-2 using monospecific antibodies reacting against tissue antigens.The simultaneous increase in reactivity of MMPs and TIMPs poise difficulty in interpretingthe results of this study. The possible reasons for this result, against the backdrop of existing knowledge, were attempted in this study.
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Rajendran R, Rajeesh Mohammed P K, Shaikh S, Shanthi, Pillai M R. Expression of matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in oral submucous fibrosis.Indian J Dent Res 2006;17:161-166
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Rajendran R, Rajeesh Mohammed P K, Shaikh S, Shanthi, Pillai M R. Expression of matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in oral submucous fibrosis. Indian J Dent Res [serial online] 2006 [cited 2021 May 11 ];17:161-166
Available from: https://www.ijdr.in/text.asp?2006/17/4/161/29870
Matrix metalloproteinases (MMPs) are a group of enzymes which together can degrade all the known protein components of the extracellular matrix (ECM). Presently, the MMP gene family consists of 21 members and the family is still growing. MMPs are products of different genes dispersed in the genome, although there is an MMP gene cluster on chromosome 11 . MMPs differ structurally and that each MMP has the ability to degrade a particular subset of matrix proteins. The protein products are, however, classified by shared functional and structural characteristics. MMPs can be divided into the following subgroups 1) collagenases, 2) gelatinases 3) stromelysins 4) stromelysin like MMPs, 5) membrane type (MT) MMPs, and 6) other MMPs according to their structure and substrate specificity. It is important to remember that most of the MMP substrates defined till date have been demonstrated in-vitro and their importance in biological context is not known .
MMPs are highly regulated, with both secretion and activity levels under tight control. In general, in normal tissue, MMPs are expressed at very low levels, if at all, but their production and activation is rapidly induced when active tissue remodeling is needed. Regulation occurs at multiple levels, including transcription, modulation of mRNA half-life, secretion, localization, zymogen activation, and inhibition of proteolytic activity. MMPs have natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), and certain nonspecific proteinase inhibitors, such as macroglobulin .
Oral submucous fibrosis (OSF) is a prototype ofpathological fibrosis having a loco- regional predisposition to its occurrence . The exact cause of this stromal aberration with consequent deposition of fibrous tissue in the submucosa still remains enigmatic, though significant progress had been made recently in elucidating the pathologic pathways. Conceptually, fibrosis results from an imbalance in the normal processes of synthesis and degradation of the extracellular matrix components. The extent to which alterations in the matrix proteolysis play a role in the development and regression of fibrosis is poorly understood.
Immunohistochemical staining of formalin fixed, paraffin embedded tissue sections of OSF for MMPs-1, 2, 9 and their tissue inhibitors TIMP-1 and 2 was performed using monospecific antibodies coupled with gelatin zymography (MMP-2 and MMP-9) for measuring enzymatic activity qualitatively and for distinguishing the active from the inactive variants of enzymes.
MATERIALS AND METHODS
This study was reviewed and approved by the review board of the institution and informed consent was obtained from all the patients for their inclusion in the study. The study was designed to assess semiquantitatively the expression of MMP-1, 2 and 9 and their inhibitors TIMP-1 and 2 in tissue samples of OSF using immunohistochemical and zymographical analyses.
Twenty (20) cases each of clinically advanced OSF and age and sex matched healthy adults prospectively screened constituted the test and control samples. Five (5) millimeter size punch biopsies collected from identical oral site (left buccal mucosa close to occlusal line) were immediately fixed in phosphate buffered 10% formalin, embedded in parafn, and sectioned at a thickness of 5µm. Formalin fixed tissues were processed using standard methods of paraffin embedding and staining with haematoxylin and eosin.
Antigen retrieval was performed by immersing the slides in 0.0 1M citrate buffer, cooked in a pressure cooker for 10 minutes at full pressure. The slides were washed in distilled water after the treatment.
Immunohistochemical staining was performed on fixed sections using avidin-biotin peroxidase technique. Briefly, unstained sections mounted on polylysine coated slides were deparaf inized with xylene and rehydrated with decreasing concentrations of ethanol. Non-specific binding was blocked with 1.5% normal horse serum. Avidin and biotin binding sites contained in the samples were blocked using a commercial avidin-biotin blocking kit (Vector laboratories Inc. Burlingame, CA). Sections were then incubated for 30 minutes at room temperature with the following mouse anti-human monoclonal antibodies diluted in phosphate buffered saline (PBS) containing normal horse serum; anti- MW-1 (1:40 dilution), anti MMP-2 (1:30 dilution) and anti-MMP-9 (1:50 dilution), incubated at 4°C for overnight (Calbiochem, Oncogene Science Inc. Cambridge, MA). The tissue sections were washed in ice-cold saline and incubated with a secondary biotinylated anti-mouse IgG. Endogenous peroxidase activity was blocked using 0.3% H 2 0 2 in horseradish peroxidase (Vector laboratories Inc). Peroxidase activity was visualized using diaminobenzidine (Vector laboratories Inc). This technique uses unlabelled primary antibody, biotinylated secondary antibody, and a preformed avidin and biotinylated horseradish peroxidase macromolecular complex (PC). The slides were then rinsed in water and lightly counterstained with haematoxylin.
Control procedures: Negative controls included omission of the primary antibody from the described staining protocol and its replacement with PBS, plus normal horse serum. To rule out the possibility that no artifacts were distorting the findings, two antibodies unrelated to the MMP family or their inhibitors were used. For this control, the primary antibodies were replaced with anti-cathepsin B (5µ/ml) or anti-cathepsin D (5µ/ml) (Oncogene Science).
Gelatin zymography (5): Approximately 100mg of tissue was pulverized in liquid nitrogen and homogenized in buffer (0.5M Tris, pH 7.6, 0.2M NaCl, 10mM CaCl 27 1 % triton X 100) using an ultraturrax T8 (IKA labortechnik, Germany) homogenizer. Total protein concentration of the supernatants were determined by Bradford's assay and l50µ1 of total protein loaded per sample well.
Inactive pro MMP-2 is 72 KDa protein while active MMP-2 is a 66KDa protein.
Inactive pro NOT-9 is 92 KDa protein while active MMP-9 is a 84KDa protein.
Homogenate supernatants were electrophoresed on 10% denaturing sodium dodecyl sulphate (SDS) polyacrylamide gels containing l mg/ml of gelatin. Gels were washed overnight in washing buffers and incubated for 24 hrs at 37°C in the above buffer without Triton X 100, so that renaturation of the enzyme could occur. Gels were stained for 30 minutes with 30% methanol or 10% acetic acid containing 0.5% coomassie blue and then destained in the same above solution without the dye. Gelatinolytic activity was visualized as clear band against a blue background. The intensity of each band present was analyzed using computer assisted image analysis (Kodak Digital Sciences, USA). The relative mobility will be determined using standard protein molecular weight markers (Bangalore Gene, Bangalore, India). The intensity of bands ofMMP-2 and MMP-9 in OSF samples were compared to that of controls.
Statistical analysis was carried out using Chi-Square test, which is a non- parametric test of statistical significance for bivariate analysis.
Immunoreactivity of OSF and control sections were scored semiquantitatively based on established criteria . The staining intensity was scored as mild, moderate, and intense with comparison to site specific healthy oral mucosa; intense when increased number of cell layers are positive and mild when irregular positivity in reduced number of cell layers was recorded.
Immunoreactivity of MMP-1
[Table 1] shows positive stromal expression in 95% (19/20) cases of which 65% (13/20) showed mild immunoreactivity, 20% (4/20) moderate and 10% (2/20) intense. Negative stromal expression was shown by 5% (1/20) of OSF samples. This observation is statistically significant (p=0.006).
[Table 2] shows 40% (8/20) moderate staining with MMP1 antibody, 5% intense (1/20) and 5% (1/20) mild reactivity. This observation is not statistically significant (p=O.79).
Immunoreactivity of MMP-2
[Table 3] shows positive stromal expression for MMP-2 in 85% (17/20) cases of advanced OSF, of which 55% (11/20) showed mild immunoreactivity, 30% (6/20) moderate to intense staining. Negative stromal expression was shown by 15% (3/20) of cases. This observation is statistically significant.
[Table 4] shows epithelial reactivity of MMP-2 which showed 25% each (5/20) of mild and more intense staining, while 50% (10/20) recorded negative expression for the antigen. This observation is statistically not significant (p=0.435).
Immunoreactivity of MMP-9
[Table 5] shows positive stromal expression in 100% (20/20) of cases.
[Table 6] shows 20% (4/20) mild, 5% (1/20) moderate to intense reactivity. 75% (15/20 recorded negative expression). This observation is not statistically significant (p=0.565).
Immunoreactivity of TIMP -1
[Table 7] shows positive stromal expression in 100% (20/20) of cases; of which 70% (14/20) recorded moderate to intense activity.
[Table 8] shows 65% (13/20) of cases of OSF stained positively for epithelial TIMP antigen. This observation is not statistically significant (p=0.442).
Immunoreactivity of TIMP -2
[Table 9] shows positive stromal expression in 85% (17/20) cases of OSF samples, of which 40% (8/20) showed mild reactivity, and 45% (9/20) moderate to intense staining. Negative stromal expression was shown by 15% (3/20) of cases. This observation is statistically highly significant.
[Table 10] shows positive reactivity in 70% (14/20) of cases which is statistically significant (p=0.004).
[Table 11] shows a decreased intensity of bands for MMP-2 (active and inactive enzyme - gelatinase) and MMP-9 (active and inactive enzyme - gelatinase) in OSF samples when compared to normal mucosa.
Pathological fibrosis, irrespective of its site of occurrence, results from an imbalance in the normal processes of synthesis and degradation of extracellular matrix components. The extent to which such alterations in matrix turnover play a role in the initiation and progression of tissue fibrosis is poorly understood.
Oral submucous fibrosis is a prototype of pathological fibrosis, where the type of collagen deposited has been characterized as normal . What is intriguing is the cause of this excessive accumulation which seems to be due to homeostatic disequilibrium of native collagen metabolism. Areca nut alkaloids with possibly tannins (polyphenols) have been repeatedly incriminated as a possible causative agent to this condition . The epidemiological evidence in this direction is rather compelling. It has been described that areca quid as well as its components affect fibroblast proliferation, increase collagen synthesis, and promote an accumulation of ECM components in the oral mucosa. It has been speculated that areca quid ingredients could take part in the fibrotic process by abnormal accumulation of stromal components .
Many researchers have reported the etiology of submucous fibrosis as an increase of collagen deposition resultant to a decrease of collagen degradation . A decrease of the phagocytic activity of fibroblasts in OSF patients when compared with the normal persons has been reported . The number of phagocytic cells and phagocytic activity in cultured human oral fibroblasts from OSF site was lower than the fibroblasts from the normal regions of the same person. This result demonstrated that the fibroblast assumes heterogeneity in function in different areas from the same patient. This aspect has been discussed much further in one of our earlier publications and partly explains the loco-regional nature ofOSF .
TIMPs play an important role in various physiological processes that involve tissue remodeling. They help maintain a delicate balance between physiological degradation and synthesis of the extracellular matrix. TIMPs are widely distributed in the tissues and fluids and are expressed by many cells including fibroblasts . TIMPs play an important role in MMP inhibition and ECM turnover. In addition, TIMP-1 and TIMP-2 was reported to block collagen degradation in cartilage remodeling . We found in this report an upregulation of TIMP-1 and TIMP-2 activity in the stroma of OSF cases which were statistically significant alterations. This was in agreement with previous isolated reports on TIMP activity in OSF samples , where the upregulation was interpreted to be the reason for an excess increase of collagen in OSF. The pathogenesis of this disease could be explained perhaps due to increased synthesis and deposition of ECM proteins or alternatively due to altered fibrinolysis or both.
The present study, contrary to earlier reports, recorded statistically significant increase in the levels of stromal expression of MMP-1, MMP-2 and MMP-9 and TIMP-1 and TIMP-2 using monospecific antibodies reacting against tissue antigens. The simultaneous increase in reactivity of MMPs and TIMPs pose difficulty in interpreting the results ofthis study.
One of the inherent difficulties encountered in interpreting immunohistochemical data on tissue expression of enzyme levels is its inability to distinguish active from inactive forms of enzymes in question. This is relevant in the present situation more so because of the enzymes failed to be expressed qualitatively (active/inactive) in the stained sections. The quantitative expression of the enzymes is in no way reflecting the functional integrity of it. This problem was tried to be addressed in this study by using gelatin zymography to distinguish the active from inactive forms of the enzymes. The results of the gelatinise zymography showed that there was a decreased expression of active forms of MMP-2 and MMP-9 in OSF cases when compared to the controls. This test points to the fallacy of interpreting immunostaining data atleast with regard to tissue expression of metabolic enzymes. The role played by TIvIPs in this regard seems to be contributory in downregulating the MMP action at tissue levels. This investigation helps in elucidating further the pathogenesis of OSF which was attributed due to a decrease in the amount of collagen degradation due to defunct (inactive) tissue proteinases.
The excessive collagenisation taking place in the submucosa of OSF could set in to motion a regulatory reflex mechanism causing an upregulation in the matrix enzyme expression. This seems imperative, given the excessive collagen deposition noticed in the disease as part of the tissue homeostatic disequilibrium, the degradative enzyme levels could be upregulated to tackle the excessive stromal elements. This probably explains the increased immunostaining noticed of the matrix metalloproteinases but due to functional impairment (sterric conformational change?) it fails to degrade the excess tissue collagen. The increased cross-linkage of mature collagen bundles in OSF, caused due to an upregulation of tissue lysil oxidases, needs retrospection in this context. The mechanism(s) which cause down regulation of the activity of these enzymes in vivo seems rather speculative at present, which needs further research. The defunct tissue proteinases (MMPs) could be envisaged to be refractory to the regulatory influence of TIMPs, which further may lead to excessive fibrosis at tissue sites. A study of the role played by areca-alkaloids and other putative aetiological agents in this regard, in modulating tissue enzyme functions, seems promising in elucidating the primary cause of this prototype of pathological fibrosis.
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