Matrix metalloproteinases and their role in periodontal diseases

Introduction to matrix metalloproteinases

The matrix metalloproteinases (MMPs) are a family of structurally and functionally related endoproteinases that are collectively capable of degrading most of the components of the extracellular matrix (ECM) ^{1, 2}. They are calcium-dependent, zinc-containing endopeptidases, which are involved in tissue remodeling and degradation of the ECM, including collagens, elastins, gelatin, matrix glycoproteins, and proteoglycans. MMPs are secreted by a variety of connective tissue and pro-inflammatory cells including fibroblasts, osteoblasts, endothelial cells, macrophages, neutrophils, and lymphocytes. These enzymes are expressed as zymogens, which are subsequently processed by other proteolytic enzymes (such as serine proteases, furin, plasmin, and others) to generate the active forms. Under normal physiological conditions, the proteolytic activity of the MMPs is controlled primarily at any of the following three known stages: transcription, activation of the zymogens, and inhibition of the active forms by various tissue inhibitors of MMPs (TIMPs). These enzymes share common functional domains and activation mechanisms as they depend on Ca²⁺ and Zn²⁺ ions and are active at neutral pH.

Formation and activation of MMPs

The MMPs are secreted as latent enzymes and require activation. They are produced as zymogens, with a signal sequence and propeptide segment that must be removed during activation. Their proteolysis is tightly regulated to prevent tissue damage.

Structure

MMPs share a common structure that comprises of four main domains:

• A short signal sequence is followed in order by a propeptide which endows the virgin enzyme with catalytic latency,
• A catalytic domain which contains the active site and the catalytic machinery,
• A proline-rich hinge region, and
• A pexin-like COOH-terminal domain which plays a role in determining substrate specificity.

     The propeptide domain contains a cysteine residue that binds zinc in the active site to form the cysteine switch. The binding of cysteine in the catalytic domain blocks the active zinc site, maintaining the latent or inactive state ⁴. The structural organization of all MMPs presents a prepeptide sequence that directs their secretion in the extracellular environment and a propeptide domain that maintains them in their zymogenic form. The propeptide keeps the enzyme in an ……….Contents available in the book……….Contents available in the book……….Contents available in the book……….Contents available in the book………..

     MMP-2 and MMP-9 (also known as gelatinases based on their substrate preference) contain fibronectin-like domain repeats which aid in substrate binding. MMP-7 or matrilysins is smallest in size and lacks the hemopexin domain, yet it displays specificity in substrate degradation. Other MMPs, known as membrane-type MMPs (MT-MMP), are linked to the plasma membrane either by a transmembrane domain or by a glycosyl-phosphatidyl inositol linkage, attached to the hemopexin domain. So, all MMP’s may be regarded as derivatives of a 4-domain prototype structure, formed either by addition or deletion of regulatory domains.

Classification of MMPs

MMPs are able to degrade practically all ECM components and can be classified according to their substrate specificity as collagenases, gelatinases, stromelysins, and matrilysins 6 (Figure 10.2). Collagenases degrade triple-helical fibrillar collagens, which are the main components of bone and cartilage. Gelatinases degrade molecules in the basal lamina around capillaries, facilitate angiogenesis and neurogenesis, and contribute to instigating cell death. Stromelysins [MMP-3, MMP-10, MMP-11, and MMP-7 (also known as matrilysins)] are small proteases that degrade components of the ECM, although not the triple-helical fibrillar collagens. Another type of MMPs are called as the MT-MMPs (Membrane Type Matrix Metalloproteinases) that are localized to the cell surface. Four of the MT-MMPs contain hydrophobic transmembrane domains (MMP-14, MMP-15, MMP-16, MMP-24), followed by a cytoplasmic domain. Two other members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP) are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor.

Biological action of MMPs

The substrate specificity of the MMPs is not yet fully characterized. Known substrates include most of the ECM components (fibronectin, vitronectin, laminin, entactin, tenascin, aggrecan, myelin basic protein, etc.). The collagens (Types I, II, III, IV, V, VI, VII, VIII, IX, X, XIV) have all been shown to be the substrates for different MMPs, with greatly different efficacies. In addition to connective tissue and ECM components, proteinase inhibitors such as α1-proteinase inhibitor, antithrombin-III and α2-macroglobulin are selectively cleaved by MMPs.

Up regulators of MMP production

• IL-1β.
• Tumor necrosis factor- α.
• Epidermal growth factor.
• Platelet-derived growth factor (PDGF).
• Transforming growth factor – α (TGF-α).

Down regulators of MMP expression

• Interferon-γ.
• TNF-β.
• Glucocorticoids.

Description of individual MMPs ⁷:

MMP-1

MMP-1, also known as interstitial collagenase, is produced by fibroblasts, chondrocytes, macrophages, keratinocytes, endothelial cells, and osteoblasts. It can cleave collagen Types I, II, III, VII, VIII, and X helices to yield characteristic one-quarter-three-quarter products. MMP-1 is secreted as a latent pro-enzyme (57 kDa) that is subsequently processed to a MMP-1 (41/42 kDa) active forms. MMP-1 can be activated in vivo by proteinases (e.g., plasmin, trypsin). MMP-3 is also an important activator of MMP-1 in vivo. Although MMP sequences are conserved across species and there is >50% homology among MMP-1, MMP-8, and MMP-13, they can be distinguished from other MMPs by their domain structure and substrate specificity.

MMP-2

MMP-2, also known as the 72 kDa gelatinase/ Type IV collagenase and gelatinase A, cleaves a number of substrates including gelatins, collagen Type I, III, IV, V, VII, and X, fibronectin, elastin, and proteoglycan. The MMP-2 gene is located on chromosome 16 at position 12.2. It is synthesized as a 72 kDa pro-enzyme that is proteolytically processed to the 66 kDa active form. Mutations in the MMP-2 gene are associated with Torg-Winchester syndrome, multicentric osteolysis, arthritis syndrome, and possibly keloids.

MMP-3

MMP-3, also known as stromelysin-1 and transin-1, cleaves a number of substrates including, cartilage proteoglycan, collagen Type ……….Contents available in the book……….Contents available in the book……….Contents available in the book……….Contents available in the book………..

MMP-7

MMP-7, also known as matrilysin and PUMP-1 cleaves a number of substrates including collagen Type IV and X, elastin, fibronectin, gelatin, laminin, and proteoglycans. It is the smallest of all the MMPs, consisting of only a propeptide domain and a catalytic domain. It lacks the hemopexin-like domain common to other members of the MMPs. It is secreted as a 28 kDa pro-enzyme that can be activated by MMP-3 to form a 19 kDa active MMP-7. The active MMP-7 can activate pro-MMP-1 and pro-MMP-9.

MMP-8

MMP-8, also known as collagenase-2 and neutrophil collagenase, preferentially cleaves collagen Type I over Type II and III. In addition, it can cleave natural proteins such as fibronectin, cartilage aggrecan and serpins and peptides such as angiotensin and substance P. It is found in specific granules in neutrophils but is also expressed by diverse cell types, including epithelial cells, fibroblasts, macrophages, and endothelial cells. It is closely related to the fibroblast collagenases (47% homology). MMP-8 is secreted as an 85 kDa pro-enzyme that can be activated by trypsin, but not by cell-bound plasmin, in a two-step process. Both steps are inhibited by TIMP-1 and TIMP-2, which bind to the catalytic domain of the enzyme. The active enzyme is generally unstable at 37°C, breaking down into two fragments of 40 kDa and ~27 kDa. The 40 kDa fragment has been identified as the biologically active catalytic domain.

MMP-9

Matrix metalloproteinase-9 (MMP-9), also known as gelatinase B and 92 kDa gelatinase/Type IV collagenase, exhibits a broad range of substrate specificity for native collagens including Types IV, V, VII, and X as well as gelatin, proteoglycans, and elastin. MMP-9 is secreted by a wide number of cell types, including neutrophils, macrophages, and fibroblasts. In neutrophils, MMP-9 is synthesized during granulocyte differentiation in the bone marrow. It is secreted as a 92 kDa pro-enzyme and can be activated by cathepsin G and MMP-3. MMP-9 can be inhibited by TIMP-1 which binds exclusively to pro-MMP-9.

MMP-10

Matrix metalloproteinase-10, also known as stromelysin-2 and transin-2, is a member of the stromelysin subfamily with similar substrate specificities as MMP-3. It is secreted as a 56 kDa pro-enzyme which is activated by proteolytic cleavage to active 47 kDa form. The active MMP-10 can activate pro-MMP-1, pro-MMP-7, pro-MMP-8, and pro-MMP-9. MMP-10 is expressed in a number of tumors, including oral squamous carcinoma, and head and neck squamous cell carcinomas.

MMP-11

MMP-11 or Stromelysin-3 is also known as STMY3, SL-3 and ST-3. Unlike other MMPs, human MMP-11 does not cleave ECM proteins, such as collagen and elastin. MMP-11 has been shown to cleave a variety of non-matrix substrates, including alpha-1-protease inhibitor.

MMP-12

MMP-12, also known as macrophage metalloelastase, is expressed as a 53 kDa pro-enzyme that is processed to a mature ~22 kDA enzyme. MMP-12 is produced initially as an inactive proform that requires proteolytic activation. Main substrates of this MMP are elastin, fibronectin, gelatin and laminin.

MMP-13

Matrix metalloproteinase-13 (MMP-13), also known as collagenase-3, is secreted as a 60 kDa pro-enzyme that is proteolytically processed to the 48 kDa active MMP-13 form. It shows specificity toward the interstitial collagens Type I, II, III, IV and gelatin. It exhibits greater specificity towards collagen Type II and cleaves collagen Type I with comparable efficiency to MMP-1 and MMP-8. MMP-13 can be inhibited by TIMP-1, TIMP-2, and TIMP-3.

MMP-18, MMP-19, MMP-20 and MMP-23

MMP-18, also known as Xenopus collagenase-4 or xCol4, is an interstitial collagenase and is known as a Xenopus enzyme. Sequence comparison of MMP-18 with other MMPs suggests that MMP-18 is not a homolog of any known collagenase. It is highly expressed in amphibian metamorphosis, suggesting a developmental role in the larval tissue degeneration and adult organogenesis.

MMP-19 (RASI) was initially named as MMP-181. This MMP has a wide variety of substrate range including aggrecan, fibronectin, type I gelatin, and basement membrane components such as laminin, nidogen, and collagen Type IV.

MMP-20 is also known as enamelysin. By virtue of its broad substrate specificity, MMP-20 has a wide range of targets including amelogenin, aggrecan, dentin, and collagen Type V. It must be noted that MMP-20 is expressed almost exclusively by developing teeth, thus playing an important role in tooth development.

MMP-23 is a recently described MMP having gelatinase activity. Its secretion and activation are regulated by a single cleavage at the furin site.

Membrane-Type Matrix Metalloproteinases (MT-MMPs)

MT-MMPs are a unique subclass of MMPs, four of which have transmembrane and cytosolic domains at the C-terminus. The other two MT-MMPs do not have a cytosolic domain and are thought to be glycosylphosphatidylinositol anchored to the cell surface. MT-MMPs contain a unique insertion of 11 amino acids between the propeptide and catalytic domains that is cleaved by furin-like enzymes to yield activated metalloproteinases. Activated MT1-, MT2-, and MT3-MMP cleave proMMP-2 and proMMP-13 to their active forms, and exhibit the ability to directly cleave ECM proteins such as collagen Type I and fibronectin.

Regulation of MMP activity

MMP activity can be controlled at various levels: transcription (by cytokines), proteolytic activation of the zymogen form (via plasmin-dependent or MMP-dependent pathway), and inhibition of the active enzyme ⁸.

Regulation at the transcriptional level

This task is performed by cytokines. Cytokines are the chemical messengers that affect the surrounding or distant cells by up-regulating or down-regulating the protein synthesis by these cells. As already discussed, the genes for MMP synthesis are activated when remodeling of ECM is required or under pathological conditions during inflammation. However, MMP-2 is an exception here, which is synthesized and secreted in low quantity in tissues and is ……….Contents available in the book……….Contents available in the book……….Contents available in the book……….Contents available in the book………..

     Similarly, the synthesis of MMP-13 by osteoblasts is up-regulated when stimulated by IL-1β and TNF-1α ^{11, 12}. Synthesis of MMP-13 by osteoblasts is also up-regulated by IL-6 ¹³. Transforming growth factor-β (TGF-β) has been shown to up-regulate the expression of MMP-13 but down-regulate the expression of MMP-1 and MMP-3 genes ¹⁴. IL-8 which is a potent chemoattractant for neutrophils, stimulates the release of MMP-9 stored in the tertiary granules of the neutrophils in the extracellular environment ¹⁵.

Regulation at the level of proteolytic activation of the zymogen form

Regulation at the level of proteolytic activation of the zymogen form to an active enzyme makes comparatively a smaller component of the regulatory mechanism of MMPs. The major regulatory mechanisms include regulation of MMP formation at the transcriptional level and the inhibition of the active enzyme.

Regulation of the active enzyme

α-Macroglobulins:
α-Macroglobulins are potent inhibitors of MMP activity. Active MMPs are captured by α-macroglobulins, by a unique venus-fly-trap mechanism acti­vated by cleavage of a bond in the “bait region.” This cleavage leads to the hydrolysis of a labile internal thiol-ester bond and covalent cross-linking of a nascent glutamyl residue to lysyl side chain exposed on the surface of the attacking proteinase. The rapid capture rates, particularly with collagenases, suggest that α-Macro-globulins, particularly α2- M, play an important role in the regulation of MMP activity in human periodontal diseases ¹⁶.

Tissue inhibitors of metalloproteinases (TIMPs):
TIMPs are important regulators of MMP activity. The TIMP family consists of TIMP-1, TIMP-2, TIMP-3, and TIMP-4. The human TIMPs comprise 184 to 194 amino acids that form an N-domain and a C-sub-domain that are stabilized by six disulfide bonds. The TIMPs are about 40% identical and have overlapping abilities to inhibit individual MMPs. Binding of the TIMPs to their specific MMPs results in the inhibition of MMP activity. Their amnio-terminal domain is the inhibitory domain and it binds to the active site of the MMPs. TIMPs share a high degree of homology to each other, however, they may be either secreted extracellularly in soluble form (TIMP-1, TIMP-2, and TIMP-4) or bind to extracellular matrix components (TIMP-3). TIMPs have been shown to bind to the pro-enzyme forms of MMP-2 and MMP-9 with a high degree of specificity. This interaction provides an extra level of regulation by preventing activation. TIMP-1 binds to the pro-MMP-9, whereas TIMP-2 binds to the pro-MMP-2. TIMP-1, TIMP-2, TIMP-3 and TIMP-4 all inhibit all of the MMPs tested, but TIMP-1 is a poor inhibitor of MT1-MMP, MT3-MMP, MT5-MMP and MMP-19.

TIMP-1:
It is a 184-amino acid glycoprotein of 28.5 kDa, more widely distributed than the other TIMPs and inhibits the activity of all the active MMPs (except MT1-MMP, MT3-MMP, MT5-MMP and MMP19). Its binding to MMP-9 and MMP-1 occurs via a reversible non-covalent binding to the catalytic domain of the MMP protein. Among all TIMPs, TIMP-1 is the inducible form and is up-regulated by factors such as interleukin (IL)-1β, transforming growth factor (TGF)-β1, epithelial growth factor (EGF) and IL-6.

TIMP-2:
It is a 194-amino acid unglycosylated protein of 21.5 kDa, which has 41% and 44% sequence homology to TIMP-1 and TIMP-3, respectively. It inhibits the activity of all active MMPs and regulates the activation of pro-MMP-2 by binding to its C-terminal region.

TIMP-3:
It is a 27 kDa glycoprotein, expressed by a variety of cells. It has an affinity for the components of the ECM and as a consequence, it is largely sequestered there. It forms a non-covalent, stoichiometric complex with both latent and active MMPs. TIMP-3 prevents the activation of pro-MMP-2 by MT2-MMP.

TIMP-4:
The chromosomal location for the gene coding for TIMP-4 is 3p25. It functions in a tissue-specific manner in extracellular matrix homeostasis.

Biological functions of TIMPs:

Along with metalloproteinase-inhibiting activities, TIMPs have other biological functions. These primarily include,

• TIMP-1 and TIMP-2 have erythroid-potentiating activity and cell growth-promoting activities.
• TIMP-3 has pro-apoptotic activity, possibly through the stabilization of TNF-
  cell receptor 1, Fas, and TNF-related apoptosis, inducing ligand receptor-1, as shown for some tumor cells. It is in contrast to TIMP-1 and TIMP-2 which have anti-apoptotic activity.

Role of MMPs in periodontal diseases

The microbiological etiology of periodontal diseases is well defined. Lipopolysaccharides (LPS) derived from the bacteria have the capacity to activate junctional epithelial cells to release potent cytokines, such as interleukin-1, interleukin-8, tumor necrosis factor-α, prostaglandins, and proteases. These chemoattractant signals lead to the transmigration of leukocytes and monocytes/ macrophages. Further, these inflammatory cells stimulate the resident ligament cells, such as fibroblasts, macrophages, osteoblasts, keratinocytes, and endothelial cells to produce cytokines and MMPs.

     As already stated, under normal physiological conditions, the activities of MMPs are precisely regulated at the level of transcription, activation of the precursor zymogens, interaction with specific ECM components, and inhibition by endogenous inhibitors ^{17, 18}. However, under pathological conditions they play an important role in connective tissue degradation. Birkedal-Hansen et al. (1993) ¹⁹ have provided the following evidence to prove the involvement of MMPs in periodontal diseases,

1. Cells isolated from normal and inflamed gingiva are capable of expressing a wide variety of MMPs in culture,
2. Several MMPs can be detected from the gingiva in vivo and,
3. MMP-8 and MMP-3 are readily detected, in gingival crevicular fluid (GCF) from gingivitis and periodontitis patients.

     Furthermore, the authors proposed the following mechanisms responsible for periodontal destruction based on MMPs and TIMPs interactions,

• Imbalance between MMPs and TIMPs causes irreversible connective tissue breakdown.
• MMP-8 (neutrophil collagenase) exists in elevated amounts in the GCF collected from inflamed periodontal pockets and is converted to the active form by the plaque, host and microbial-derived proteases.
• Periodontal ligament collagen fibers attached to the root surface are destroyed by MMPs, which favors the apical migration and lateral extension of the pocket epithelium i.e. attachment loss.

     Studies have shown that high concentrations of the natural tissue inhibitor of MMPs (TIMPs) are found in the gingival crevicular fluid of healthy gingiva ²⁰. As we know that collagen Type I is the main ECM component in the soft (gingiva and periodontal ligament) and hard (alveolar bone) periodontal tissues and thus, collagen Type I degradation is regarded as one of the key factors in the uncontrolled destructive lesion ^{21, 22}. The major collagenolytic MMPs associated with the severity of periodontal inflammation and disease are collagenase-2 (MMP-8) and collagenase-3 (MMP-13), whereas collagenase-1 (MMP-1) is related to the physiological periodontal tissue turnover ^{23, 24}. Experiments on MMP-8 deficient mice have confirmed the concept that MMP-8 is a central mediator in chronic infection-induced inflammatory conditions, and can also exert, in addition to the classical surrogate tissue destructive properties, the anti-inflammatory and defensive properties ^25-29. MMP-8 is the major type of interstitial collagenase present in human periodontitis-affected gingival tissue, GCF and peri-implant sulcular fluid ³⁰.

Matrix metalloproteinases and bone resorption

Although, the role of MMPs in bone resorption is not completely clear, but present information suggests that they do have an important role in this process. Various MMPs, including MMP-2, -3, -9, -11, -12, -13, and -14, are expressed in the osteoblasts. Bone resorption factors, such as PTH, 1α,25(OH)2D3, IL-1, IL-6, TNF-α, and PGE₂, regulate the production of various MMPs in the osteoblasts. Bone resorption first requires the osteoblasts to release collagenase to remove the nonmineralized organic matrix that covers the bone surfaces. Osteoclasts are then chemotactically attracted to the resorption site, where they settle onto the calcified matrix

     The mineralized bone matrix is covered by an ……….Contents available in the book……….Contents available in the book……….Contents available in the book……….Contents available in the book………..

     Further, research done on chemically modified tetracyclines clearly indicates that MMPs have an important role in periodontal bone resorption 37. The inhibition of the MMP activity is an important strategy in host response modulation. A detailed description of agents used to modulate the MMP activity has been given in, “Host response modulation therapeutic agents in periodontics”.

Conclusion

MMPs are important components in many biological and pathological processes because of their ability to degrade ECM components. Considerable advancements have been made in the understanding of biochemical and structural aspects of MMPs, including their activation and catalytic mechanisms, substrate specificity, and the mechanism of inhibition by TIMPs. The role of MMP’s in the progression of periodontal diseases has been well investigated. They can act as biomarkers for active periodontal disease. Studies have shown that collagenase-2 or MMP-8 can be used as a clinically useful oral fluid (GCF, mouthrinse or saliva) biomarker in periodontal and peri-implant diseases ^{38, 39}. Modulation of the host response is an important field of research in the treatment of periodontal diseases. Modification of host response via the use of MMP inhibitors, along with the use of bisphosphonates as blockers of periodontal tissue destruction, has shown promises in the treatment of these diseased states. Further research work is required to understand the role of MMPs in periodontal diseases and how it can be controlled or modified to prevent connective tissue destruction.

References

References are available in the hard-copy of the website.

Suggested reading

1. Ryan ME, Ramamurthy S, Golub LM. Matrix metalloproteinases and their inhibition in periodontal treatment. Current opinion in periodontology. 1996;3:85-96.
2. Birkedal‐Hansen H. Role of matrix metalloproteinases in human periodontal diseases. Journal of periodontology. 1993 May;64:474-84.
3. Sorsa T, Tjäderhane L, Salo T. Matrix metalloproteinases (MMPs) in oral diseases. Oral diseases. 2004 Nov;10(6):311-8.
4. Makela M, Salo T, Uitto VJ, Larjava H. Matrix metalloproteinases (MMP-2 and MMP-9) of the oral cavity: cellular origin and relationship to periodontal status. Journal of dental research. 1994 Aug;73(8):1397-406.
5. Min-hua S. Relation between MMPs and periodontal diseases [J]. Chinese Journal of Conservative Dentistry. 2007;1.