|Year : 2016 | Volume
| Issue : 1 | Page : 3-8
Matrix metalloproteinases: A double edge sword
Praveen Kumar Bali1, Dhanraj Kalaivanan2, Vijayalaksmi Divater3, Logarani4
1 Department of Pedodontics and Preventive Dentistry, College of Dental Sciences, Davangere, Karnataka, India
2 Department of Pedodontics and Preventive Dentistry, Karpaga Vinayaga Institute of Dental Science, Maduranthakam, Kancheepuram, India
3 Department of Periodontics, College of Dental Sciences, Davangere, Karnataka, India
4 Consultant Periodontist, ESI Hospital, Chennai, Tamil Nadu, India
|Date of Web Publication||15-Dec-2015|
Praveen Kumar Bali
Department of Pedodontics and Preventive Dentistry, College of Dental Sciences, Davangere - 577 004, Karnataka
Source of Support: None, Conflict of Interest: None
Dental caries is a dynamic process which results in demineralization of inorganic constituents and destruction of organic structure of the tooth. The basic mechanism of inorganic demineralization has been studied and documented well enough. However, the exact mechanisms and enzymes responsible for the organic matrix breakdown remain unknown. Matrix metalloproteinases (MMPs), a family of endopeptidases which are responsible for degrading all extracellular matrix components, which are expressed in normal dentin-pulp complex. MMP's are believed to act as double-edged sword since it causes progression of caries process and also helps in repair and defense mechanism initiated by caries in dentin-pulp complex. Several MMPs are also found in normal dentin-pulp complex cells and tissues, and they are considered to be involved in many physiological processes during the formation and maintenance of the dentin-pulp complex. This article gives a brief review of MMPs and its role in oral health.
Keywords: Adhesive restoration and chlorhexidine, dental caries, endopeptidases, extracellular matrix, matrix, metalloproteinases, periodontal disease
|How to cite this article:|
Bali PK, Kalaivanan D, Divater V, Logarani. Matrix metalloproteinases: A double edge sword. Dent Med Res 2016;4:3-8
| Introduction|| |
The most common problems in dentistry include dental caries and periodontal diseases both of which are caused by degradation of the organic matrix or extracellular matrix (ECM). Many theories have been proposed to explain the etiological factors behind these problems, but still exact mechanism with regard to its destruction of organic matrix is not known properly. Few of the proposed mechanism behind this include (i) the plasminogen-dependent pathway; (ii) the phagocytic pathway; and (iii) the matrix metalloproteinase (MMP)-dependent pathway. 
MMPs, collectively known as matrixins, form a multigene family within the metalloproteinase class of endopeptidases produced by leukocytes and ﬁbroblast-like cells that mediate the degradation of practically all ECM molecules, including native and denatured collagen and are also involved in some of physiological process of tissue remodeling. They can be differentiated from other class of endopeptidase by that they are dependence on cofactor (metal ion) for its action, specific DNA sequence and ability to degrade ECM.  The first report about an MMP was published in 1962 by Jerome Gross and Charles Lapière. All the MMP's are activated by cleavage of propeptide before which they are present in inactive proenzyme form. All MMPs contain zinc at the catalytic site and, in addition, require calcium for stability and activity. Until date, 23 MMP's are seen in human out of 24 MMP's cloned. 
| Basic Structure Of Matrix Metalloproteinase's|| |
All the members of the MMPs family basically have three basic, distinctive, and well-conserved domains based on structural considerations: Amino-terminal propeptide, a catalytic domain, carboxyl terminal hemopexin-like domain [Figure 1].  All the MMPs are initially present in inactive zymogens form with a pro-peptide domain. This pro-peptide domain contains 80-90 amino acid sequence containing cysteine residue which interacts with the zinc of the catalytic domain via its side chain thiol group preventing binding and cleavage of substrate, thus keeping propeptide domain in an inactive form. 
The catalytic domain consists of around 170 amino acid sequence in it along with the active site which is necessary for activation of all the MMPs. The basic structure of catalytic domain contain two zinc ions one of which is present in active site which is responsible for proteolytic activity of MMP's, at least one calcium ion coordinated to various residues and three histidine residues that coordinate with the catalytic zinc are conserved among all the MMPs. Only little is known about the function of another zinc and calcium ion and is thought to help in the stabilization of the MMP's.  The hemopexin domain is attached to the catalytic domain through a bridge referred as hinge region. It ranges in length from 0 to 75 residues and has no determinable structure. The longest hinge is found in MMP 9. Hemopexin domain contains around 210 amino acids with highly conserved sequence similar to the plasma protein, hemopexin. This domain helps in binding to substrate and also interacts with tissue inhibitors of matrix metalloproteinases (TIMP's), a specific MMP's protein inhibitor.
The hemopexin domain is an absolute requirement for collagenases to cleave triple helical interstitial collagens, although the catalytic domains alone retain proteolytic activity toward other substrates. Apart from the basic structure, Some MMPs possess special structures such as furin cleavage site insert, membrane insertion extension, and fibronectin repeats which are necessary for its function. 
| Classification of Matrix Metalloproteinases|| |
Based on their substrate speciﬁcity, sequence similarity, and domain organization, MMPs can be divided into six subgroups: Collagenases, stromelysins, gelatinases or type IV collagenases, matrilysins, metalloelastase, and membrane type metalloproteinase. 
The specific action of collagenous is its ability to cleave interstitial collagen I, II, and III at a specific site and apart from this, it has also ability to degrade other ECM and non-ECM.  Examples include MMP-1, MMP-8, MMP-13, and MMP-18 (Xenopus).
The basic structure of gelatinase includes three repeats of a type II fibronectin domain inserted in the catalytic domain, which bind to gelatin, collagens, and laminin. Gelatinase helps in digesting denatured collagen and gelatins. Examples include MMP-2 (gelatinase A) and MMP 9 (gelatinase B). 
Stromelysin has similar substrate specificities, but stromeolysin 1 (MMP 3) differ from stromeolysin 2 (MMP 10) in there proteolytic efficiency with former having greater abiltiy to cleave. Apart from digesting ECM, it also helps in activating the pro-MMP into an active form. Stromeolysin 3 (MMP 3) is grouped with other MMPs because the sequence and substrate specificity diverge from those of MMP-3. 
The matrilysins are characterized by the lack of a hemopexin domain. Matrilysin 1 (MMP-7) and matrilysin 2 (MMP-26), also called endometase, are in this group. 
| Membrane-Type Matrix Metalloproteinases|| |
There are six membrane-type MMPs (MT-MMPs): Four are type I transmembrane proteins (MMP-14, MMP-15, MMP-16, and MMP-24), and two are glycosylphosphatidylinositol anchored proteins (MMP-17 and MMP-25). With the exception of MT4-MMP, they are all capable of activating pro-MMP-2. These enzymes can also digest a number of ECM molecules, and MT1-MMP has collagenolytic activity on type I, II, and III collagens. 
| Other Matrix Metalloproteinases|| |
Many MMPs which cannot be classified in different classes are included in other MMPs. Metalloelastase (MMP-12) is mainly expressed in macrophages and is essential for macrophage migration and also helps in digesting proteins apart from elastin.
For the normal mineralization and maturation of tooth, digestion of amelogenin an enamel protein is necessary which is done by enamelysin (i.e. MMP 20) which is found in tooth enamel. Amelogenin imperfecta, a genetic disorder caused by defective enamel formation, is due to mutations at MMP-20 cleavage sites.
The patient with rheumatoid arthritis (RASI) shows a high level of MMP 19 which can identify by cDNA cloning from liver and as a T-cell - derived autoantigen. MMP-22 was first cloned from chicken fibroblasts. Basis structure of cysteine array MMP (MMP 23) lacks cysteine-switch motif and hemopexin domain, but it has cysteine-rich domain followed by an immunoglobulin-like domain which are majorly seen in reproductive tissue.
Epilysin or MMP-28 mainly expressed in keratinocytes. It helps in tissue hemostasis and wound repair. 
Activation of matrix metalloproteinase's
All the MMPs are initially secreted as enzyme precursor zymogens in which cysteine of propeptide binds to zinc in catalytic domain which constitute cysteine-switch, thus keeping this enzyme in inactive form which needs to be activated for its action. MMPs can be activated by proteinases or in vitro by chemical agents, such as thiol-modifying agents (4 aminophenylmercuric acetate, HgCl 2 , and N-ethylmaleimide), oxidized glutathione, sodium dodecyl sulfate, chaotropic agents, and reactive oxygen.
Low pH and heat treatment can also lead to activation of MMPs which mainly act by modifying cysteine-zinc interaction. Activation of MMP's is stepwise mechanism in which initial cleavage occurs within the propeptide and that this reaction is intramolecular and remaining cleavage occurs through intermolecular reaction. Once the MMPs are activated by proteolytic activation it initially attacks the exposed propeptide looop region between 1 st and 2 nd helices and the specific cleavage site (bait region) for activation is assessed by sequence found in MMPs. Once the propeptide region in MMPs are destabilized then the rest of the reaction are carried out through cysteine-switch motif which allows intermolecular reaction through partially activated MMP's or other MMP's.
Thus, the final step in the activation is conducted by an MMP.  Most of MMP's are activated extracellularly, several MMPs may also be activated intracellularly by furin or related proprotein convertases. 
Regulation of the activity of matrix metalloproteinases
All the MMPs are secreted in proenzymes form and must regulate the normal activities, loss of control in regulating leads to diseases.  The regulation of MMPs occurs through the various process it include proteolytic degradation and inactivation, nonspeciﬁc endogenous inhibitors such as α2-macroglobulin, and especially by speciﬁc tissue inhibitors of MMPs like TIMP's.  TIMPs are small (21-28 kDa), multi-functional secreted proteins that are found at the cell surface in association with membrane-bound proteins. These TIMPs are secreted in 1:1 stoichiometric enzyme inhibitor complexes thereby regulating the activity of MMP's. Until date, 4 TIMP's are identified in vertebrates which include TIPM's 1, 2, 3 and 4. Each TIMPs consisting of 184-194 amino acids which are subdivided into an N-terminal and a C-terminal sub-domain with three conserved disulfide bonds and the N-terminal domain folds as an independent unit with MMP inhibitory activity. The exact mechanism of inhibition is based on the crystal structure of the MMP-TIMP's complexes. The overall shape of TIMPs is wedge which slots into active site of pro-peptide domain of MMP's, thereby chelating catalytic zinc atom by the N-terminal amino group and the carbonyl group of cysteine, which expels the water molecule bound to the zinc atom.
Other possible mechanism by which synthetic inhibitors acts are by interaction with the propeptide fragment of an MMP, and some MMP inhibitors may act by coating the substrate and thereby preventing MMP access and activity. 
TIMP 1 - Inhibition of all known MMP family members. Associates with pro-MMP-9. Inhibits angiogenesis and erythroid-potentiating activity
TIMP 2 - Inhibitions all known MMP family members. Associates with MT1-MMP and MMP-2 at the cell surface and regulates MMP-2 activation
TIMP 3 - Inhibition of all known MMP family members
TIMP 4 - Inhibition of all known MMP family members. The restricted expression suggests tissue-specific TIMP function. 
Matrix metalloproteinases in normal tooth structure
Several evidences published suggest that MMPs have a potential role in normal development and remodeling of oral tissue. The process of tooth formation include epithelial-mesenchymal interaction, formation of ameloblasts and odontoblasts followed by root formation in which MMP's are thought to play major role for its completion which are found in hard and soft tissue compartment of dentin-pulp complex. MMPs are responsible majorly for epithelial-mesenchymal interaction. These interactions determine the early morphogenetic events in tooth development as well as the later processes that give rise to odontoblasts and ameloblast differentiation.  The possible role of MMPs may be it is involved in epithelia-mesenchymal interactions during the initial stages of odontogenesis and are likely to play a role in the turnover and degradation of basement membrane proteins in embryonic tooth germs during morphogenesis and cytodifferentiation.  During early tooth development, dental mesenchymal cells are observed to express at least MMP-1, -2, -3, -9, 14, and -20 as well as TIMP-1, -2, and -3. Another possible role of MMP's is it helps in onset of the mineralization in the developing tooth germ. The absence of MMP's activity prevents degradation of proteoglycan in predentin near the mineralization front which is a necessary step before mineral deposition.
TIMPs synthesized by mature human odontoblasts and pulp tissue could be secreted into a border of predentin which is necessary for the regulation of dentin mineralization by controlling MMP activities. 
Matrix metalloproteinases in dental caries and periodontal diseases
Dental caries results from demineralization of inorganic structures and destruction of the organic component of tooth structure. It is well-known that Streptococcus mutans is main bacteria responsible for caries which causes demineralization of inorganic structures but destruction of the triple helical collagen structures is not possible from the bacteria. Traditionally, microbial enzymes have been held responsible for this matrix degradation.
Recent evidence suggests that the MMPs present in dentin matrix or in saliva may be responsible for the dentin matrix degradation in dentinal caries lesions. MMPs found in dental caries are MMP 2, 8, 9, and 20.  There are two possible sources for the MMPs in caries lesions.
- Salivary and gingival crevicular fluid (GCF) MMPs are reserved in plaque, which is also the potential site for acid activation
- MMPs are also produced by odontoblasts. 
The active form of MMPs are short lived, and its presence suggests the active role in the destruction of the dentinal matrix. The activation of MMP's during the progression of the caries process is mainly because of the fall in pH due to sugar consumption. The activation and destruction of collagen during caries process can be explained through Stephen's curve. After sugar ingestion, the pH decreases below the critical pH in which demineralization occurs (pH 5.5) [Figure 2].
|Figure 2: Stephan curve showing demineralization and collagen breakdown after sugar ingestion|
Click here to view
During demineralization, the organic matrix collagen ﬁbrils are exposed. The latent puriﬁed forms of MMPs are activated at low pH (4.5), but their activity is still low. After process of neutralization, their activity subsequent increases leading to collagen breakdown. 
Periodontal disease are caused by the bacterial plaque which results in the formation of deep periodontal pockets which is mainly due to the destruction of ECM and alveolar bone loss. There are different pathways for metabolic degradation of ECM. One pathway is thought to be due to activation of MMPs. They are expressed by inflammatory cells (monocytes, macrophages, lymphocytes, and polymorphonuclear cells) and by resident cells (fibroblasts, epithelial cells, and endothelial cells).
MMPs involved in the progression of periodontal disease is MMP 2, 8, 9, 13, and 14. MMP play an important role in bone resorption. The resorption activity is carried out by MMP's by allowing the osteoclasts to access to resortion lacunae, the inhibition of which prevents cell migration.MMP-14 is located in the ruffled border of osteoclasts, possibly contributing to the osteoclast matrix interaction that controls osteoclast attachment and detachment to the bone. MMP-13 is present in resorption lacunae, and it is essential for removal of collagen remnants left over by osteoclasts.
MMPs may also contribute to osteoclastic bone resorption by regulating osteoclast recruitment and activity by, for example, releasing cytokines and growth factors (such as transforming growth factor-b by MMP-9 or RANKL by MMP-14) from bone matrix, or by regulating the messenger binding to the receptors. MMP-3 is effective in degrading proteoglycans and fibronectin. Diagnosis of periodontitis can be done on the chair side by detecting the level of MMP 8 in GCF using dipstick test. The reduction of MMP-8 levels in GCF indicates that this enzyme may be useful as an indicator of current disease status and as a predictor of future disease. 
Matrix metalloproteinase's role in adhesive dentistry
Prevention for extension holds true in the modern concept of dentistry with the advanced material sciences which requires only minimal cavity preparation. However, the problem with the adhesive material is the long-term stability of the material since it has been demonstrated that hydrophilic dentin adhesives deteriorate over time. The collagen fibers exposed during acid etching procedure are more susceptible for degradation leading to loss of adhesion.
The possible explanation for the loss of adhesion is the MMPs from mineralized dentin matrix might have been activated during acid etching and were probably responsible for collagen matrix degradation in the aqueous environment. Therefore, they considered that preventing the degradation of incompletely resin infiltrated collagen fibrils by MMPs in the hybrid layers would be an important procedure. The MMP inhibitors like chlorhexidine are used from preventing the activation of MMP and thus maintaining long-term stability of the restoration. ,
| Conclusion And Future Perspective|| |
In the recent concepts, basics of the host related factors play a key role in understanding the disease process. Most of the oral disease is mainly due to microorganism which causes progression of the disease by various pathways leading to the breakdown of ECM. In the recent years, MMP's dependent pathway for a breakdown of ECM has gained more attention.
A wealth of knowledge about MMP's which are involved in both physiological and pathological processes are not known but considerable advancement have been made to understand biochemical and structural's aspect of MMP's including its activation and inhibition of MMP's, substrate specificity and mechanism of action. X-ray diffraction crystal structures research is lacking which permit us to see the active center, the location of the zinc atom, the origin of latency, and the mode of binding to TIMP and more important, it will facilitate the design of inhibitors. 
Currently, 23 MMPs and more than 30 ADAM metalloproteinases are known in humans, the inhibition of MMPs during the pathologic process by generating its specific inhibitors are critical in future prospective. Several studies have been done based on which various MMP inhibitors are designed and synthesized which have been used in clinical trials but the long-term clinical efficacy still remains unclear as the failure may be attributed to limited knowledge of the function of MMP's and lack of selective inhibitors and also some inhibitors have been applied at end stage of disease.  Hence, a detailed understanding of MMP expression patterns, in terms of both cellular origins and timing is a necessity. Furthermore, some knowledge of the precise role of an individual MMP within each context is important. This needs to be coupled with the ability to generate specific inhibitors, either targeting the active site or other interaction.
With more advances in research such as magnetic resonance imaging, the development of selective MMP inhibitors still remains a possibility.  In future; the use of MMP inhibitor in combination with the biological restorative material could alter the disease progression. In addition, further development of diagnostic technology may also allow the use of one or more MMP inhibitors; TIMPs which are used in combination with chairside diagnostic test or mouth rinse screening test could be a landmark in diagnosis and monitoring the progression of disease process.
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[Figure 1], [Figure 2]