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Tissue Inhibitors of Metalloproteinases (TIMPs): Their Biological Functions and Involvement in Oral Disease

Department of Orthodontics and Oral Biology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

^* corresponding author, h.vondenhoff{at}dent.umcn.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION TIMP EXPRESSION BIOLOGICAL FUNCTIONS OF TIMPs TIMPs IN ORAL DISEASE DIAGNOSTIC AND THERAPEUTIC... CONCLUSION REFERENCES

 
Several families of enzymes are responsible for the degradation of extracellular matrix (ECM) proteins during the remodeling of tissues. An important family of such enzymes is that of the matrix metalloproteinases (MMPs). To control MMP-mediated ECM breakdown, tissue inhibitors of metalloproteinases (TIMPs) are able to inhibit MMP activity. A disturbed balance of MMPs and TIMPs is found in various pathologic conditions, such as cancer, rheumatoid arthritis, and periodontitis. The role of MMPs in pathology has been extensively described in the literature. The main focus of this review lies in the biological functions of TIMPs and their occurrence in disease, especially in the head and neck area. Their biological functions and their role in diseases like oral cancers and periodontitis, and in the development of cleft palate, will be discussed. Finally, the diagnostic and therapeutical opportunities of TIMPs will be evaluated.

KEY WORDS: tissue inhibitor of metalloproteinase • TIMP • matrix metalloproteinase • MMP • biological functions • oral disease • extracellular matrix

	INTRODUCTION

TOP ABSTRACT INTRODUCTION TIMP EXPRESSION BIOLOGICAL FUNCTIONS OF TIMPs TIMPs IN ORAL DISEASE DIAGNOSTIC AND THERAPEUTIC... CONCLUSION REFERENCES

 
Proteins in the extracellular matrix (ECM) such as collagen are responsible for the coherence of connective tissues. Tissue remodeling during embryonic development, growth, or disease processes requires the degradation of these proteins to allow for changes in shape, cell migration, or tissue resorption. Matrix metalloproteinases are known to play an important role in these processes. However, other enzymes such as cathepsins, the plasminogen activator/plasmin system, and neutrophil elastase have recently gained more attention as ECM-degrading proteins (Uitto et al., 2003; Skrzydlewska et al., 2005). Matrix metalloproteinases (MMPs) are able to degrade most proteins of the ECM. MMPs are counteracted by the tissue inhibitors of metalloproteinases (TIMPs), which inhibit MMP activity and thereby restrict ECM breakdown. The balance between MMPs and TIMPs plays an important role in maintaining the integrity of healthy tissues. A disturbed balance of MMPs and TIMPs is found in various pathologic conditions, including rheumatoid arthritis, cancer, and periodontitis (Gomez et al., 1997; Lambert et al., 2004). In rheumatoid arthritis and malignant tumors, the imbalance is generally in favor of MMPs, and leads to cartilage destruction, or is associated with metastasis. Abundant information is available on the role of MMPs in health and disease, but information on TIMPs is limited, especially with respect to their role in oral diseases.

The first TIMP was described in 1975 as a protein, in culture medium of human fibroblasts and in human serum, which was able to inhibit collagenase activity (Bauer et al., 1975; Woolley et al., 1975). The molecular weight of this protein was later shown to be 28.5 kDa (Stricklin and Welgus, 1983). Since then, 3 new TIMPs have been discovered in different species, and have been designated TIMP-2, -3, and -4, respectively (Blenis and Hawkes, 1983; Herron et al., 1986; Staskus et al., 1991; Pavloff et al., 1992; Greene et al., 1996). The molecular weights of TIMP proteins vary between species.

All 4 currently known TIMPs are very well-conserved, since they have been identified in humans, other vertebrates, insects, and even in Caenorhabditis elegans, a nematode worm that is commonly used as a model organism for genetic and cell biological research (Pohar et al., 1999; Brew et al., 2000; Lambert et al., 2004).

The main goal of this review is to discuss the biological functions of TIMPs. First, the occurrence and biological functions of TIMPs in the human body will be discussed. Next, their role in pathology will be reviewed, with emphasis on oral cancer, periodontitis, and cleft palate. Finally, the diagnostic and therapeutic opportunities of TIMPs will be evaluated.

	TIMP EXPRESSION

TOP ABSTRACT INTRODUCTION TIMP EXPRESSION BIOLOGICAL FUNCTIONS OF TIMPs TIMPs IN ORAL DISEASE DIAGNOSTIC AND THERAPEUTIC... CONCLUSION REFERENCES

 
The genes that encode human TIMP-1 to -4 are mapped to the X-chromosome Xp11.3 – Xp11.23, chromosome 17q25, chromosome 22q12.1–q13.2, and chromosome 3p25, respectively (Willard et al., 1989; De Clerck et al., 1992; Apte et al., 1994; Olson et al., 1998). The size of the TIMP-1 mRNA transcript is 0.9 kb. For TIMP-2, 2 transcripts have been described, of 1.0 kb and 3.5 kb, respectively. The major TIMP-3 mRNA transcript is 5.0 kb, but 2 minor transcripts of 2.4 and 2.6 kb have also been found. Finally, the single transcript of TIMP-4 is 1.4 kb (Gomez et al., 1997).

TIMP-1 and TIMP-3 expression is inducible, whereas TIMP-2 expression is largely constitutive (Table). In contrast to the other TIMPs, TIMP-4 mRNA expression is highly regulated and restricted to neural tissue, fetal testes, and Sertoli cells or ovaries, and cardiac, breast, and skeletal muscle tissues in mice, and this is partly confirmed for humans (Young et al., 2002; Lambert et al., 2004). This restriction might be due to specific binding sites for transcription factors in the TIMP-4 gene (Young et al., 2002). A distinguishing feature of TIMP-3 is its ability to bind tightly to the ECM (Leco et al., 1994; Yu et al., 2000). The primary protein structures of the TIMPs are shown in Fig. 1. Their amino acid sequences show about 40% homology (Lambert et al., 2004). The tertiary structures of TIMP-1 and -2 have been resolved by x-ray diffraction and NMR studies (Williamson et al., 1994; Gomis-Rüth et al., 1997). Due to their homology with these TIMPs, the tertiary structures of TIMP-3 and -4 have been suggested to be very similar (reviewed by Douglas et al., 1997). All currently known TIMP proteins contain 6 loops and have a junction between the N- and C-terminal domains. TIMPs are produced in many tissues, although not every tissue expresses all 4 TIMPs. In general, most mesenchymal and epidermal cells are able to produce TIMPs (Rowe et al., 1997). TIMP-1, -2, and -3 are also produced by white blood cells (Oelmann et al., 2002; Bjerkeli et al., 2004). TIMPs can be co-expressed with MMPs, but some studies have showed a reciprocal regulation of their expression, which may depend on endogenously expressed (growth) factors and cytokines (Gomez et al., 1997). In summary, the balance between MMPs and TIMPs is variable, both in physiological processes, such as growth and development, and in pathological conditions, such as cancer and periodontitis (Morris-Wiman et al., 2000; Chang et al., 2002; Kerkelä and Saarialho-Kere, 2003; Lambert et al., 2004).

View larger version (44K): [in this window] [in a new window]   Figure 1. The primary protein structure of TIMPs. In each TIMP, 6 pairs of cysteines are linked to each other to form 6 disulfide bridges. The arrows indicate the junctions between the N- and the C-terminal domains of the proteins. The conserved VIRAK sequence is indicated in yellow. Reprinted from Critical Reviews in Oncology/Hematology, 49, Lambert E, Dassé E, Haye B, Petitfrère E, TIMPs as multifacial proteins, 187–198, Copyright (2004), with permission from Elsevier.

TIMPs in Body Fluids
TIMPs and MMPs can be produced by many different cell types and are also found in all body fluids, such as saliva, gingival crevicular fluid (GCF), serum, and urine (Fujimoto et al., 1993; Ingman et al., 1996; Durkan et al., 2003; Asatsuma et al., 2004). MMP and TIMP levels change during physiological and pathological processes. Correlations between the progression of rheumatoid arthritis and different forms of cancer, and the levels of MMPs and TIMPs are found in urine and serum. The MMP-9/TIMP-1 ratio in urine is thought to predict the risk of bladder cancer, whereas the TIMP-2 levels in urine from individuals with urothelial carcinomas are significantly decreased (Monier et al., 2002; Durkan et al., 2003). In the serum of individuals with hepatocellular carcinoma and rheumatoid arthritis, TIMP-2 levels are significantly increased, whereas the serum TIMP-2 levels in individuals with gastric cancer and cancer of the uterus are significantly decreased (Fujimoto et al., 1993). This shows that MMPs and TIMPs occur throughout the entire body, including all body fluids. An imbalance between MMPs and TIMPs might be associated with disease. In general, the relevance of systemic levels of enzymes and inhibitors can be questioned, since the local balance of these proteins eventually determines matrix degradation.

	BIOLOGICAL FUNCTIONS OF TIMPs

TOP ABSTRACT INTRODUCTION TIMP EXPRESSION BIOLOGICAL FUNCTIONS OF TIMPs TIMPs IN ORAL DISEASE DIAGNOSTIC AND THERAPEUTIC... CONCLUSION REFERENCES

 
The number of known biological functions of TIMPs has been expanding rapidly in recent decades. These findings indicate that TIMPs are multifunctional proteins, not merely MMP inhibitors. In addition, it is becoming more evident that MMPs have several additional biological functions. It has even been questioned whether ECM breakdown should be considered as a primary function (Overall, 2004).

MMP Inhibition
The first known biological function of TIMPs, which led to their discovery, is the inhibition of collagenases. At present, 25 MMPs have been identified, numbered consecutively from 1 to 28 (but MMP-4, -5, and -6 are missing). The MMPs can be divided into subgroups according to their primary substrates (Snoek-van Beurden and Von den Hoff, 2005).

In the ECM, TIMPs form non-covalent 1:1 stoichiometric complexes with MMPs. Almost all MMPs can be inhibited by all 4 TIMPs, although differences in binding affinity have been reported (Olson et al., 1997). The so-called ‘membrane-type’ (MT-) MMPs form a distinct group, since they are bound to the cell membrane and are rarely inhibited by TIMP-1 (English et al., 2001; Baker et al., 2002).

All TIMPs contain the NH₂-terminal domain and the conserved VIRAK amino acid consensus sequence, and both have been suggested to be essential for MMP binding and inhibition (Woessner, 1991; Gomez et al., 1997; Lambert et al., 2004). However, analysis of the tertiary structure of TIMP-2 by NMR measurements showed that the VIRAK sequence was sterically unable to be involved in binding interactions (Williamson et al., 1994). X-ray crystallographic studies have been performed to analyze TIMP-1·MMP-3 and TIMP-2·MT1-MMP complexes (Gomis-Rüth et al., 1997; Fernandez-Catalan et al., 1998). These studies showed that the TIMP-1 and -2 molecules have a wedge-like shape that fits into the active-site cleft of an MMP, as would a substrate molecule (Visse and Nagase, 2003). The conserved cysteine in the N-terminal domain, at least in TIMP-1, will subsequently chelate the active-zinc site and expel the water molecule, thereby inactivating the MMP protein (Gomis-Rüth et al., 1997). The x-ray structures of TIMP-3 and -4 and their complexes with MMPs have not yet been described (Maskos, 2005). Besides MMP inhibition, many other biological functions of TIMPs have been discovered. Some of these might be explained by the inhibition of MMP activity, but most of the alternative functions seem to be MMP-independent.

MMP Activation
In contrast to the inhibition of MMPs by TIMPs, some studies clearly showed that TIMP-2 is also involved in the activation of pro-MMP-2 (Fig. 2). The N-terminal region of TIMP-2 first binds to the active site of the membrane-bound MT1-MMP (MMP-14). The hemopexin-like domain of the secreted pro-MMP-2 subsequently binds to the C-terminal region of the complexed TIMP-2 (Zhao et al., 2004). The bound pro-MMP-2 is then activated by MT1-MMP in the pro-MMP-2·TIMP-2·MT1-MMP complex, or by uncomplexed MT1-MMP (Strongin et al., 1995; Imai et al., 1996; Zhao et al., 2004). A similar pro-MMP-2 activation is known to occur in complexes of TIMP-3 and MT3-MMP, but not in those of TIMP-3 and MT1-MMP. TIMP-4 is unable to activate pro-MMP-2 in a ternary complex with either MT1- or MT3-MMP (Zhao et al., 2004). Theoretical calculations show that a half-molar ratio of TIMP-2 to MT1-MMP is optimal for activation of pro-MMP-2 (Yoshizaki et al., 2001). This mechanism stimulates cell migration and processes such as tumor metastasis and invasion.

View larger version (21K): [in this window] [in a new window]   Figure 2. Schematic representation of pro-MMP-2 activation by MT1-MMP and TIMP-1. MT1-MMP is bound at the cell surface (A). TIMP-1 binds to MT1-MMP (B). Pro-MMP-2 binds to this complex, and becomes activated. Uncomplexed MT1-MMPs may be recruited and aid in the activation of pro-MMP-2 (C). Subsequently, the complex dissociates (D). The binding sites for the formation of this complex are found in the N-terminal and C-terminal domains of TIMP-2, the hemopexin-like domain of pro-MMP-2, and the active site of MT1-MMP, as described in the text.

In other cell types, such as aortic smooth-muscle cells and keratinocytes, TIMP-1 also has a mitogenic effect (Bertaux et al., 1991; Akahane et al., 2004). The mitogenic signal might be transduced through tyrosine-phosphorylation, Ras effector, ERK2, and MAPK pathways (Yamashita et al., 1996; Luparello et al., 1999; Akahane et al., 2004). Most of these intracellular effects of TIMP-1 are also found after TIMP-2 stimulation (Yamashita et al., 1996), but very little is known about the actual TIMP receptors. A few putative receptors for TIMP-1 have been found, which might mediate the mitogenic effects (Avalos et al., 1988; Bertaux et al., 1991). These receptors, including the 80-kDa transmembrane protein, have not yet been fully characterized (Lambert et al., 2004). Moreover, these receptor studies have all been performed in vitro, but their function in vivo is completely unknown. In contrast, the presence of TIMP-1 in the nuclei of fibroblasts in vitro strongly suggests the existence of a non-degrading transport mechanism, which might include a specific TIMP receptor.

TIMP-2 also stimulates the proliferation of a wide range of other cells, such as dental pulp fibroblast-like cells and gingival fibroblasts, as well as leukocytes and epithelial cells (Hayakawa et al., 1994). Most of these cells are TIMP-2 producers themselves, suggesting an autocrine mechanism of action. In contrast to their mitogenic effects, TIMPs can also inhibit cell growth. The overexpression of TIMP-2 inhibits tumor growth, metastasis, and invasion in vivo (Imren et al., 1996). The latter two effects can be explained by the inhibition of matrix degradation by MMPs (Montgomery et al., 1994). TIMP-2 is also able to inhibit angiogenesis in vivo, probably by an MMP-independent mechanism. This might be the cause of the inhibition of tumor growth. Moreover, angiogenic responses to the vascular endothelial growth factor and fibroblast growth factor 2 are also inhibited by TIMP-2 in an MMP-independent way (Seo et al., 2003). In vitro, TIMP-2, but not TIMP-1, inhibits the proliferation of human microvascular endothelial cells (Murphy et al., 1993). This might explain the inhibition of angiogenesis by TIMP-2 in vivo.

Apoptosis
TIMPs seem to play a role in the regulation of apoptosis. In epithelial breast cells and in B-lymphocytes, apoptosis is reduced by TIMP-1, but not by TIMP-2 (Guedez et al., 1998a,b; Li et al., 1999). Alternatively, TIMP-2 inhibits apoptosis in melanoma cell lines (Valente et al., 1998). It has been suggested that an increased expression of TIMP-3 stimulates apoptosis in retinas affected by simplex retinitis pigmentosa, an autosomal-dominant mutation that disrupts the retina (Jones et al., 1994; Jomary et al., 1995; Schwartz et al., 2003). In addition, TIMP-3 may also have indirect pro-apoptotic effects by protection of tumor necrosis factor (TNF)- receptors on human colon carcinoma cells from degradation by MMPs. Consequently, these cells are more sensitive to apoptosis induction by TNF- (Smith et al., 1997). The overexpression of human TIMP-4 in rat vascular smooth-muscle cells and in transformed, but not wild-type, cardiac fibroblasts also induces apoptosis (Tummalapalli et al., 2001; Guo et al., 2004). The mechanism behind this process remains to be elucidated. Analysis of these data shows that TIMP-1 and TIMP-2 seem to have anti-apoptotic effects, whereas TIMP-3 and TIMP-4 seem to be pro-apoptotic. Furthermore, the direct or indirect effects of TIMPs on apoptosis are cell-type-specific.

Immunological Interactions
In the immune system, TIMP-1 is primarily produced by B-cells, whereas TIMP-2 expression is restricted to T-cells (Oelmann et al., 2002). Other lymphocytes—such as mononuclear phagocytes, neutrophils, and dendritic cells—are also able to produce TIMPs and MMPs (Lacraz et al., 1995; Osman et al., 2002; Kerkelä and Saarialho-Kere, 2003). Neutrophils are also able to store pro-MMP-8 and -9, and to release them upon stimulation (Ding et al., 1995). Dendritic cells patrol the body by migrating through the extracellular matrix, and sample the local ‘antigenic’ environment. They are able to produce, store, and secrete MMP-1, -2, -3, -9, and TIMP-1, -2, and -3. The balance between MMPs and TIMPs determines the migratory capacity of these cells (Kouwenhoven et al., 2002; Osman et al., 2002). MMPs are able to cleave all 4 known human monocyte chemoattractant proteins (MCPs). Upon cleavage, the MCPs are inactivated and may even function as receptor antagonists (McQuibban et al., 2002).

Many cytokines are produced by the immune system and are involved in cell signaling. Some cytokines are able to interact with the transcription of MMP and TIMP genes, or to alter their expression. This makes the effects of cytokines on MMPs and TIMPs very complex. Depending on the cell type, the same cytokine can either stimulate or inhibit MMP or TIMP expression. Some important effects of specific cytokines on MMP and TIMP expression are discussed below. These cytokines also play a role in some of the pathologies we will discuss later.

In general, TGF-ß down-regulates MMP expression and up-regulates TIMP expression (Overall et al., 1989; Birkedal-Hansen, 1993). However, the expression of several MMPs in fibroblasts and keratinocytes in vitro is stimulated by TGF-ßs (Blavier and DeClerck, 1997; Blavier et al., 2001; Chang et al., 2001; Shimizu et al., 2005). The expression of TIMP-2 in vivo seems to be unaffected by TGF-ß in dermal fibroblasts, but IL-13 and TGF-ß synergistically increase TIMP-3 expression in airway fibroblasts (Ihn et al., 2002; Qureshi et al., 2005; Zhou et al., 2005). IL-10 inhibits MMP-9 expression in vitro, but induces TIMP-1 expression (Lacraz et al., 1995). In contrast, IL-10 is able to inhibit the expression of other cytokines, and thus may counteract its own effects on MMP and TIMP expression (de Waal Malefyt et al., 1991; Birkedal-Hansen, 1993; Silacci et al., 1998; Shimizu et al., 2005). TNF- stimulates the expression of several MMPs, and thus contributes to tissue degradation in inflammatory conditions (Shimizu et al., 2005). Thus, in general, changes in cytokine expression may affect the MMP/TIMP balance in a cell-type-dependent manner.

Inhibition of Other Metalloproteinases
TIMPs are also able to inhibit other metalloproteinases. The transmembrane proteins known as "a disintegrin and metalloproteinase" (ADAMs) are widely expressed in mammalian tissues. As an exception, ADAMs with thrombospondin repeats (ADAM-TS) are soluble factors that do not contain a transmembrane domain. Members of this family may be involved in a range of cellular processes, such as fertilization, myogenesis, neurogenesis, and angiogenesis. All ADAMs have a metalloproteinase-like domain, but some lack the zinc-binding catalytic consensus sequence HEXXH, and therefore lack proteolytic activity. Members of this last group of proteins are expressed in specific tissues and have specialized functions (reviewed by Handsley and Edwards, 2005), such as cell adhesion in the testes (Andreini et al., 2005). TIMPs are able to inhibit both the ADAM and ADAM-TS proteins (Handsley and Edwards, 2005). There is no evidence, up to now, that TIMPs are able to inhibit aspartic, serine, and cysteine proteinases (reviewed by Uitto et al., 2003).

Indirect Systemic Effects of TIMPs
Besides the direct effects on a (peri)cellular level, TIMPs are also indirectly involved in the regulation of blood pressure through their effects on MMPs (reviewed by Overall, 2004). Blood pressure can be increased by MMP-2 through cleaving big endothelin-1 to the active protein endothelin-1, a potent vasoconstrictor (Fernandez-Patron et al., 1999). MMP-2, but not MMP-9, is also able to control blood pressure by cleaving the vasodilator peptide adrenomedullin (Martínez et al., 2004). Available TIMPs are able to inhibit MMP activity and thereby also control their indirect systemic effects.

Taken together, these studies show that TIMPs have a broad range of biological functions extending far outside their ability to inhibit metalloproteinases. Just like TIMPs, MMPs have biological effects other than ECM breakdown. To our knowledge, no biological TIMP inhibitors have been described up to now, although MMPs might be considered as such.

	TIMPs IN ORAL DISEASE

TOP ABSTRACT INTRODUCTION TIMP EXPRESSION BIOLOGICAL FUNCTIONS OF TIMPs TIMPs IN ORAL DISEASE DIAGNOSTIC AND THERAPEUTIC... CONCLUSION REFERENCES

 
A disturbed balance between MMPs and TIMPs might contribute to the disease process in degenerative diseases. Similar patterns of MMP and TIMP expression can be found in different diseases involving matrix degradation. In some cases, the occurrence of MMPs and TIMPs in body fluids such as saliva, gingival crevicular fluid (GCF), or serum provides additional information about the progression of the disease.

Periodontitis
Periodontitis is an infection that results from the interactions of many bacteria. Chronic periodontitis has been defined by the American Academy of Periodontology as "an infectious disease resulting in inflammation within the supporting tissues of the teeth, progressive attachment and bone loss...characterized by pocket formation and/or recession of the gingiva. The inflammatory reactions over time lead to tissue destruction within the periodontium" (Baelum and Lopez, 2003). Bacteria that are implicated in periodontitis are mainly anaerobic, Gram-negative organisms, and they carry lipopolysaccharide (LPS) in their outer membrane. They are also referred to as putative periodontal pathogens (Eley and Cox, 2003).

In healthy periodontal tissue, TIMP levels are generally higher than in inflamed periodontal tissue, in which MMP levels exceed TIMP levels. The more severe the inflammation, the higher the concentrations of active MMPs (Page, 1991). In GCF and in gingiva from humans, MMP-1, -2, -3, and -9 are significantly increased, whereas TIMP-1 and -2 are significantly decreased, compared with samples from healthy controls (Soell et al., 2002). Tissue destruction might be reduced if this balance were restored.

A range of cell types produce interleukins, such as IL-6, during inflammation. The level of this cytokine is also increased during periodontitis (Lee et al., 2005). In turn, IL-6 will increase MMP-1 and -9, and TIMP-1 expression. In addition, stimulated neutrophils will release stored pro-MMP-8 and -9, which are activated by bacterial proteases (Ding et al., 1995). Other pro-MMPs present in the tissue are also activated by bacterial proteases. LPS-stimulated macrophages will express TNF- and MMPs. Subsequently, TNF- will also increase MMP expression by other cell types. TIMP-1 expression is not affected by bacterial proteases (Sorsa et al., 1992; Birkedal-Hansen, 1993). Furthermore, fibroblasts in inflamed periodontal tissue will also become activated and start to produce latent metalloproteinases, plasminogen activator, and TIMPs. Plasminogen activator activates plasmin, which in turn activates metalloproteinases (Page, 1991). All these processes will contribute to the destruction of the diseased tissue. Although some studies showed increased TIMP levels in periodontitis, these levels were probably insufficient to inhibit all MMPs (Garlet et al., 2004). Thus, bacterial inflammation will lead to a cascade of expression and activation of MMPs by different cell types, resulting in degradation of the ECM.

Apoptotic neutrophils are able to interact with LPS-activated monocytes through cell-cell contact. Subsequently, the monocytes will mainly express IL-10 and TGF-ß, instead of the proinflammotary cytokines (Byrne and Reen, 2002). TGF-ß seems to reduce inflammatory progression in periodontal diseases (Chang et al., 2001). One of the many effects of this cytokine is the up-regulation of TIMP expression and the down-regulation of certain MMPs (Overall et al., 1989; Chang et al., 2001). In contrast, TGF-ß also induces the synthesis of MMP-2, but the overall result is an overexpression of TIMPs relative to MMPs (Overall et al., 1989).

The amount of TIMPs and MMPs in GCF also changes during periodontitis. In chronic periodontitis, MMP-1, -8, and -9, and TIMP-1 levels are elevated (Ingman et al., 1996). In saliva, TIMP-1 and MMP-1 levels are not increased (Ingman et al., 1996). Taken together, the tissue and bone destruction in periodontitis reflects a relative overexpression of MMPs in relation to TIMPs. The inhibition of MMP expression or activity, or increased TIMP expression, might reduce tissue destruction in periodontitis.

Tumors
In general, different tumors show various patterns of MMP and TIMP expression. In serum from cancer patients, both MMP and TIMP levels are increased. If the MMP/TIMP ratio is in favor of MMPs, the prognosis for the patient seems to be poor (Larsen et al., 2005). However, there is no clear correlation between the serum level of MMPs and TIMPs and the metastatic potential of the tumor. Possibly, the serum levels of MMPs and TIMPs do not reflect the local tissue levels, which eventually contribute to the metastatic potential. Nevertheless, overexpression of TIMP-1 and -2 in tissues generally decreases tumor growth and metastasis, whereas down-regulation increases the invasiveness (Lambert et al., 2004). In the head and neck region, different types of tumors have been described, but relatively little is known about their MMP and TIMP expression.

    Oral Squamous Cell Carcinoma
In general, several MMPs and TIMPs are expressed in squamous cell carcinoma (SCC) of different tissues, such as the epidermis of skin, the airways, the uterine cervix, the vulva, the esophagus, and the mouth. SCC of the skin is a very common malignant tumor. In contrast to oral SCC, no TIMP expression is found in skin SCC (Yoshizaki et al., 2001; Kerkelä and Saarialho-Kere, 2003). In oral SCC, the correlation of MMP and TIMP expression with clinical features is still not fully understood (Katayama et al., 2004). In addition, the expression of MMP-1, -2, and -9 is increased in oral SCC, and pro-MMP-2 levels are strongly increased after lymph node metastasis (Kusukawa et al., 1993; Kurahara et al., 1999). The expression of MMP-2, MT1-MMP, and TIMP-2 correlates with local recurrence of tongue SCC (Yoshizaki et al., 2001). It has even been suggested that MMP-2 can be used as a predictive marker for metastasis (Kawamata et al., 1998). Moreover, the correlation between the levels of MMP-1, MMP-9, and TIMP-1, and, in contrast, the size of the tumor is significant, but no association has been found between these markers and the histological grade (O-Charoenrat et al., 2001). This shows that the levels of MMPs increase during oral SCC progression and invasion, and that TIMP-1 and -2 levels are correlated with tumor growth and recurrence.

The prognosis of SCC seems to depend on the cellular origin of MMPs. In one cell type, MMP expression may lead to increased invasion and metastasis, whereas, in another, the same MMP may lead to the inhibition of vascularization of the tumor, and thus limit its growth (Kerkelä and Saarialho-Kere, 2003).

    Salivary Gland Tumors
Salivary gland tumors are rare neoplasms of the head and neck region. In healthy salivary glands, MMP-2 and -9 and TIMP-1 and -2 seem to be expressed mainly by duct cells, but not by acinar cells (Azuma et al., 1996; Nagel et al., 2004). In contrast, acinar cells strongly express TIMP-3, whereas duct cells mostly have a low expression level of TIMP-3 (Nagel et al., 2004). Homogenized tumor tissue revealed a significantly higher expression of MMP-1, -2, -13, and -14 and TIMP-1 than did non-neoplastic tissue. The expression levels of MMP-7, -8, and -9 and TIMP-2 showed no significant difference in homogenized tumor tissue, but, in the acinar cells, MMP-2 and -9 and TIMP-1 and -2 levels were significantly higher (Kayano et al., 2004; Nagel et al., 2004). No correlation with pathological parameters, such as lymph node metastasis, has been found (Kayano et al., 2004). However, TIMP-1 expression is reduced in malignant salivary gland tumor cell lines compared with a benign cell line (Azuma et al., 1993), and so there is a clear difference between the in vitro and in vivo situation.

Analysis of these data shows that, in salivary gland carcinomas, some MMP levels are increased and seem to exceed TIMP levels.

Orofacial Clefts
During embryonic craniofacial development, MMP and TIMP expression is highly regulated to control tissue remodeling. If their balance is disrupted, malformations, such as cleft lip and palate, can occur (Blavier et al., 2001). Normally, the secondary palate develops from 2 lateral shelves in mammalian embryos. In the human fetus, the secondary palate is formed between the 6th and 8th weeks after implantation in the uterus (reviewed by Moxham, 2003). After a process of elevation and elongation, the opposing shelves fuse in the midline (Moxham, 2003; Chou et al., 2004) (Fig. 3). The midline seam will finally be degraded to allow for merging of the shelves.

View larger version (14K): [in this window] [in a new window]   Figure 3. Palatal fusion during embryonic development. Initially, the palatal shelves (ps) are positioned next to the developing tongue (A). Subsequently, the tongue descends, and the palatal shelves rotate toward each other (B). Finally, the palatal shelves fuse, and the midline seam (ms) is degraded by MMPs (C). The nasal septum (ns) simultanously merges with the secondary palate.

The epithelio-mesenchymal transdifferentiation is a process that normally occurs simultaneously with degradation of the midline seam, and is necessary for a complete fusion of the palatal shelves. This process seems to be MMP-dependent. The inactivation of MMPs by TIMP-2 or a synthetic MMP inhibitor in vitro leads to a failure of fusion of mouse palatal shelves (Blavier et al., 2001). Therefore, strict regulation of MMP activity is required for palatal fusion, and an imbalance in favor of TIMP-2 might increase the risk of a palatal cleft.

During palatal fusion in the mouse, not only the expression of MMPs and TIMPs but also the expression of TGF-ßs is tightly regulated (Moxham, 2003). As described earlier, TGF-ßs are able to regulate MMP and TIMP expression. TGF-ß3 expression is restricted to the medial edge epithelium of the palatal shelves (Fitzpatrick et al., 1990; Blavier et al., 2001). After fusion, TGF-ß1 and -2 are also detected in the medial edge epithelium and the mesenchymal cells, respectively (Moxham, 2003). TGF-ß3 knockout mice develop a palatal cleft. Moreover, palatal fusion in vitro can be restored by TGF-ß3 in these mice (Blavier et al., 2001). The expression of MMP-13 in the degrading midline seam in mice seems to be regulated by TGF-ß3 (Blavier et al., 2001). This shows that MMPs and TIMPs play a crucial role in palatal fusion, and also that TGF-ßs seem to be important for the regulation of this process. Further elucidation of these mechanisms might help to develop strategies to prevent this type of disorder in the future.

	DIAGNOSTIC AND THERAPEUTIC OPPORTUNITIES

TOP ABSTRACT INTRODUCTION TIMP EXPRESSION BIOLOGICAL FUNCTIONS OF TIMPs TIMPs IN ORAL DISEASE DIAGNOSTIC AND THERAPEUTIC... CONCLUSION REFERENCES

 
In degenerative diseases, the balance between MMPs and TIMPs is often disturbed. Relative overactivity of MMPs leads to tissue destruction, such as in periodontitis and rheumatoid arthritis, and facilitates the invasion of tumors, leading to metastases (Kerkelä and Saarialho-Kere, 2003). The general hypothesis for cancer is that an overexpression of MMP-2 relative to TIMP-1 leads to an increased metastasis of tumors. Metastasis is facilitated by increased ECM breakdown, in which MMPs play an important role. Since TIMP-1 has mitogenic properties and can protect cells from apoptosis, we think that a tumor-specific expression pattern of TIMP-1 might exist. Initially, TIMP-1 is overexpressed in relation to MMPs, which may favor tumor expansion and prevent apoptosis. Subsequently, MMPs are overexpressed, which could facilitate migration and metastasis by matrix degradation. A similar concept has been described for the role of MMPs in tumor progression (Folgueras et al., 2004). At present, MMP-9 and TIMP-1 are suggested to be suitable as clinical prognostic markers in different forms of cancer, such as oral SCC, non-small-cell lung carcinoma, and primary breast cancer (Gouyer et al., 2005; Würtz et al., 2005a; Ruokolainen et al., 2005a,b). In the development of palatal clefts, the opposite seems to occur. Overexpression of TIMPs impairs palatal fusion and increases the risk of a cleft palate. Although the exact relative levels of MMPs and TIMPs are not fully characterized in different tissues, therapeutic opportunities may be found in restoring this balance. Obviously, this problem can be addressed from the MMP perspective as well as from the TIMP perspective. For instance, the activity of MMP-8 and -13 can be reduced by low doses of doxycycline, which is already used clinically in the treatment of periodontitis (Golub et al., 1997). However, it may be more complex than suggested here.

As in many complex biological systems, the modulation of a single factor may have unexpected side-effects. For example, broad-range MMP inhibitors are unable to block metastasis in patients with advanced cancer (reviewed by Folgueras et al., 2004; Overall, 2004). Several explanations have been suggested for this phenomenon. First, MMPs have multiple biological effects, such as the inhibition of angiogenesis (Hiraoka et al., 1998). Second, the broad-range MMP inhibitors affect not only MMPs, but also other proteases, such as ADAMs and anti-angiogenic ADAM-TSs (Sawa et al., 2002; Folgueras et al., 2004). Finally, other matrix proteinases, such as the cathepsins, which are not affected by TIMPs, seem to be involved in tumor metastasis as well (Nomura and Katunuma, 2005; Skrzydlewska et al., 2005). In addition, several polymorphisms and haplotypes of TIMPs are known to be involved in specific diseases. Other polymorphisms may also contribute to the disease process in conditions such as cancer and inflammatory diseases.

Although more research is needed to unravel all actions and interactions of MMPs and TIMPs, some diagnostic tools are already being developed. TIMP-1 might be a marker for the recurrence of non-small-cell lung carcinoma after surgery (Gouyer et al., 2005). Evidence is accumulating that TIMP-1 and MMP levels can be used as prognostic markers in serum of primary breast cancer patients (Würtz et al., 2005a). In oral SCC, MMP-9 levels in serum can be correlated to survival, and TIMP-1 levels also seem to have prognostic value (Ruokolainen et al., 2005a,b). In the serum of patients with other tumors, such as hepatocellular cancer and stage IV breast cancer, the levels of MMPs and TIMPs have also been shown to change (Fujimoto et al., 1993; Larsen et al., 2005). This may be helpful as a diagnostic tool in the future. However, TIMP-2 levels have already been shown to have only limited value as a prognostic marker in colorectal cancer (Larsen et al., 2005). MMP and TIMP levels in GCF change during periodontitis. MMP-8 in GCF may be suitable as a marker to monitor periodontal health (Ingman et al., 1996). This shows that several options are available for the diagnostic and therapeutic use of MMPs and TIMPs, but also that more research is required, especially with respect to the role of other matrix proteinases.

	CONCLUSION

TOP ABSTRACT INTRODUCTION TIMP EXPRESSION BIOLOGICAL FUNCTIONS OF TIMPs TIMPs IN ORAL DISEASE DIAGNOSTIC AND THERAPEUTIC... CONCLUSION REFERENCES

 
Degradation of the ECM depends on a delicate interplay between proteolytic enzymes and their inhibitors. Apart from MMPs, other ECM-degrading enzymes, such as cathepsins, are gaining more attention in this field. The biological effect of a shift in MMP and/or TIMP levels depends on their relative balance. Overactivity of MMPs results from their overexpression or from reduced expression of TIMPs, and vice versa. This sensitive balance is continuously controlled, from fertilization of the ovum to death. TIMPs have been found in all body fluids, and aberrant expression levels can be associated with disease. Therefore, they may have a diagnostic value as disease markers. However, the complete spectrum of actions and interactions of MMPs and TIMPs is still not fully known. In recent years, several additional biological functions of MMPs and TIMPs have been discovered, varying from local to systemic. Among these are the mitogenic functions of TIMPs and their effect on apoptosis, but also the regulation of blood pressure by MMPs. It is likely that more biological functions of MMPs and TIMPs will be found in the future. If all currently known biological effects of MMPs and TIMPs are taken into account, it is already nearly impossible to predict the exact result of changes in their levels. For example, in cancer treatment, it is difficult to use MMPs and TIMPs as targets, because a clear picture of the kinetics of MMP and TIMP interaction is still lacking. Moreover, the shift in MMP and TIMP levels in serum does not necessarily reflect the local balance in the tissue.

At present, we are just starting to discover the diagnostic and therapeutic opportunities of MMPs and TIMPs. In periodontitis, irreversible damage to the alveolar bone can be reduced by exogenous MMP inhibitors such as doxycycline. In cancer treatment, no successful therapy targeting MMPs or TIMPs has evolved until now. In conclusion, much is already known about the MMP and TIMP system, but more research is needed for full exploitation of their possible diagnostic and therapeutic possibilities.

Received November 15, 2005; Accepted March 15, 2006

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