The role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the development of esophageal cancer

Esophageal cancer (EC) is one of the most aggressive malignant tumors of the gastrointestinal tract. There are two distinct histological types of EC: esophageal squamous cell carcinoma and adenocarcinoma of the esophagus. Etiologic factors and the patterns of incidence of both subtypes are different. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in esophageal carcinogenesis. Gellatinases MMP-2 and MMP-9 are able to degrade collagen IV from basement membranes and extracellular matrix which is related to tumor progression, including invasion, metastasis, growth and angiogenesis. It has been shown that increased expression of MMPs plays a crucial role in the development of several human malignancies, including esophageal cancer. The activity of MMPs is regulated by their endogenous natural inhibitors (TIMPs). Among these, the roles of TIMP-1 and TIMP-2 in EC development, tumor progression and formation of metastases have been most extensively characterized and best recognized.     

Advances in assays of matrix metalloproteinases (MMPs) and their inhibitors

Matrix metalloproteinases (MMPs) play an important role in many physiological and pathological processes. To assay the activities of MMPs is important in diagnosis and therapy of the MMPs associated diseases, such as neoplastic, rheumatic and cardiovascular diseases. Several assay systems have been developed, which include bioassay, zymography assay, immunoassay, fluorimetric assay, radio isotopic assay, phage-displayed assay, multiple-enzyme/multiple-reagent assay and activity-based profiling assay. The principle, application, advantage and disadvantage of these assays have been reviewed in this article.     

Advances in assays of matrix metalloproteinases (MMPs) and their inhibitors

Matrix metalloproteinases (MMPs) play an important role in many physiological and pathological processes. To assay the activities of MMPs is important in diagnosis and therapy of the MMPs associated diseases, such as neoplastic, rheumatic and cardiovascular diseases. Several assay systems have been developed, which include bioassay, zymography assay, immunoassay, fluorimetric assay, radio isotopic assay, phage-displayed assay, multiple-enzyme/multiple-reagent assay and activity-based profiling assay. The principle, application, advantage and disadvantage of these assays have been reviewed in this article.     

Herbal medicine Sho-saiko-to (TJ-9) increases expression matrix metalloproteinases (MMPs) with reduced expression of tissue inhibitor of metalloproteinases (TIMPs) in rat stellate cell

We have reported that Sho-saiko-to (TJ-9) prevents liver fibrosis in vivo. To gain further insights into the effect of TJ-9, the matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) balance was examined. Hepatic stellate cells (HSCs) were isolated from male Wistar rats and cultured with TJ-9 (0-1000 microg/ml) on uncoated plastic dishes for 4 days. To elucidate the effects on the MMPs/TIMPs balance by TJ-9, quantitative analysis of type IV collagen-degrading activity, gelatin zymography and reverse zymography were carried out. Northern blot analysis was performed to determine the expression of MMP-2, 13 and TIMP-1 mRNAs. TJ-9 treatment resulted in dose-dependent upregulation of MMP-2, 13 mRNA and downregulation of TIMP-1 mRNA up to 500 microg/ml. Gelatin zymography, reverse zymography and quantitative analysis of type IV collagen-degrading activity confirmed that TJ-9 increased MMP-2 activity and prevented TIMP-1, 2 activities in a dose-dependent manner. SB203580 diminished the reduction of mRNA as well as the activity of TIMP-1 by TJ-9 and induction of mRNA as well as the activity of MMP-2. These results show that TJ-9 increased MMP-2, 13 activity with reduced TIMP-1, 2 activities on HSCs possibly via P38 pathway.     

Structural features of the reprolysin atrolysin C and tissue inhibitors of metalloproteinases (TIMPs) interaction

Atrolysin C is a P-I snake venom metalloproteinase (SVMP) from Crotalus atrox venom, which efficiently degrades capillary basement membranes, extracellular matrix, and cell surface proteins to produce hemorrhage. The tissue inhibitors of metalloproteinases (TIMPs) are effective inhibitors of matrix metalloproteinases which share some structural similarity with the SVMPs. In this work, we evaluated the inhibitory profile of TIMP-1, TIMP-2, and the N-terminal domain of TIMP-3 (N-TIMP-3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular modeling. While TIMP-1 and TIMP-2 had no inhibitory activity upon atrolysin C, the N-terminal domain of TIMP-3 (N-TIMP-3) was a potent inhibitor with a K(i) value of approximately 150nM. The predicted docking structures of atrolysin C and TIMPs were submitted to molecular dynamics simulations and the complex atrolysin C/N-TIMP-3 was the only one that maintained the inhibitory conformation. This study is the first to shed light on the structural determinants required for the interaction between a SVMP and a TIMP, and suggests a structural basis for TIMP-3 inhibitory action and related proteins such as the ADAMs.     

Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis

Tissue inhibitors of matrix metalloproteinases (TIMPs) regulate the breakdown of extracellular matrix components and play an important role in tissue remodelling and growth, in both physiological and pathological conditions. We studied the autoimmune response to TIMPs in patients with rheumatoid arthritis (RA). Eighty-nine paired blood and synovial fluid samples from patients with RA were assessed for their reactivity with recombinant tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 by an ELISA and were compared with blood from 62 healthy controls and 21 synovial fluid samples from patients with degenerative joint diseases. Presence of antibodies was established as the absorbance of the sample more than 2 standard deviations above the mean of the controls. In addition, immunoglobulin G (IgG) from blood samples of RA patients possessing TIMP antibodies was isolated on protein A–sepharose and tested for the in vitro ability to neutralize TIMP-2-dependent effects on metalloproteinase 9 (MMP9). Anti-TIMP antibodies were found in 56% of RA samples but in only 5% of the controls (P < 0.005). RA patients had high frequencies of antibodies against all TIMPs except TIMP-3. TIMP-2 antibodies were most frequently found (33%), being significantly more prevalent (P = 0.024) in patients with nonerosive than erosive RA. TIMP-1 antibodies were significantly more often found in synovial fluid samples than in the matched blood samples (P < 0.025). Importantly, the IgG fraction containing TIMP antibodies down-regulated the TIMP-2 inhibitory effect, thereby supporting MMP9 activity in vitro. In the present study, we show that RA patients frequently develop autoimmune response to TIMPs that may act as a functionally significant regulator of MMP activity and thereby of joint destruction.     

Dynamic in vivo changes in the activities of gelatinases, matrix metalloproteinases (MMPs), and tissue inhibitor of metalloproteinases (TIMPs) in buffalo (Bubalus bubalis) uterine luminal fluid during estrous cycle and early pregnancy

In ruminants, the phenomenon of endometrial tissue remodeling during the estrous cycle and early pregnancy is not fully understood. In this report, the occurrence of tissue remodeling, if any, in buffalo endometrium was studied by detecting gelatinases, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs); the key regulators of tissue remodeling, in uterine luminal fluids (ULF) of cycling and early pregnant (approx. 43–65 days) buffaloes. Each stage of the estrous cycle and pregnant ULF demonstrated a unique profile of gelatinase activities compared to serum/follicular fluid, with a major gelatinase band of 60 kDa with highest activity in early‐luteal stage. In addition to a 32 kDa uterus‐specific gelatinase band detected in both non‐pregnant and pregnant ULFs, the pregnant ULF displayed three new gelatinase bands of 86, 78, and 57 kDa. Western blot technique confirmed the presence of MMP‐2 (54 kDa), MMP‐9 (76/73 kDa), TIMP‐1 (32 kDa), TIMP‐2(20 kDa), and two molecular weight forms (31 and 22 kDa) of TIMP‐3 in buffalo ULF with varying band intensities. Highest MMP‐2 and MMP‐9 activities were observed in follicular and early‐luteal stage ULFs, respectively. Highest TIMP‐1 activity was observed in early‐luteal ULF. Interestingly, TIMP‐2 activity was only detected in mid‐luteal, late‐luteal, and follicular stage ULFs with significantly increasing intensities. Highest activities of 31 and 22 kDa TIMP‐3 were associated with late‐luteal and early‐luteal stage ULFs, respectively. The varied activities of MMPs and TIMPs in buffalo ULF during the estrous cycle and early pregnancy might be a reflection of dynamic structural remodeling of the endometrium and/or developing conceptus. Mol. Reprod. Dev. 77:944–953, 2010. © 2010 Wiley‐Liss, Inc.     

Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of secreted/cell-surface-anchored multidomain zinc endopeptidases, all of which exhibit a catalytic domain of a common metzincin-like topology, and which are involved in degradation of the extracellular matrix but also in a number of other biologic processes. Normally, the proteolytic activity of the MMPs is precisely regulated by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumor growth, and tumor metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and their associations with dysfunctions. Since the reports of the first atomic structures of MMPs and TIMPs in 1994, considerable structural information has become available about both of these families of substances. Many of the MMP structures have been determined as complexes with synthetic inhibitors, facilitating knowledge-based drug design. This review focuses on the currently available 3D structural information about MMPs and TIMPs.     

Production of tissue inhibitors of metalloproteinases (TIMPs) by pig ovarian cells in vivo and the effect of TIMP-1 on steroidogenesis in vitro

Precisely which ovarian cells produce tissue inhibitors of metalloproteinases (TIMPs) is unclear. Although granulosa cells are reported to produce TIMPs, thecal TIMP production has not been investigated nor has the influence of TIMPs on theca cells. Furthermore, although periovulatory follicles have been examined, little is known about smaller ovarian follicles. Follicles >/= 2 mm in diameter were collected from Large White hybrid gilts on the day before predicted oestrus (n = 3) or after hCG treatment (n = 3) and divided into 1 mm size classes. Small (2 to < 5 mm) follicles were kept intact, whereas follicles >/= 5 mm were separated into follicular fluid, granulosa and theca cell compartments. After homogenization, TIMP-1, -2 and -3 were detected by reverse zymography. Theca cells (50 x 10(3) per well) were cultured with TIMP-1 (10, 100 or 200 ng ml(-1) with or without long-R3 insulin-like growth factor I (IGF-I)) in a serum-free system to investigate the effect on steroidogenesis and the number of cells. Both large and small pig follicles produced TIMPs and TIMP-1, -2 and -3 were detected in follicular fluid, granulosa and theca cell samples. There was a phase x tissue type interaction for the presence of both TIMP-1 and -2 (P < 0.03, P < 0.05, respectively), and TIMPs were detected in more granulosa and theca cell samples after hCG than during the follicular phase. The concentrations were influenced by the type of tissue (TIMP-1, P < 0.005; TIMP-2, P < 0.005, TIMP-3, P > 0.05), and the highest concentrations occurred in the theca tissue. There were tissue type x follicle size interactions for the presence of both TIMP-1 and -2 (P < 0.001). In vitro, TIMP-1 increased thecal steroidogenesis after 144 h (oestradiol, P < 0.05, progesterone, P < 0.001) but reduced the number of viable cells (P < 0.001). In conclusion, TIMP-1, -2 and -3 were present in large and small pig follicles and were produced by both granulosa and theca cells, although concentrations differed with the type of tissue. Production was regulated by factors including follicle size and phase of the oestrous cycle. In addition to controlling tissue remodelling, TIMP-1 may also regulate steroidogenesis.     

Assays of matrix metalloproteinases (MMPs) activities: a review

Measurement of matrix metalloproteinase (MMP) activity often remains a challenge, mainly in complex media. Two sets of methods are currently used. The first one measures the hydrolysis of natural protein substrates (labeled or not) and includes the popular zymography. These techniques which are quite sensitive, cannot generally be carried out on a continuous basis. The second one takes mainly advantage of the increase of fluorescence, which is associated to the hydrolysis of initially quenched fluorogenic peptide substrates. Quite recently, another group, which is a compromise between the other two, has been developed. It measures the hydrolysis of synthetic triple-helical peptide substrates. These different methods are described and discussed.     

Role of TIMPs (tissue inhibitors of metalloproteinases) in pericellular proteolysis: the specificity is in the detail

Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor alpha-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Functional role of matrix metalloproteinases (MMPs) in mammary epithelial cell development

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell (MEC) function and is remodeled by matrix metalloproteinases (MMPs). To investigate the significance and regulation of MMP activity in normal MEC, we utilized a primary culture model in which rat MEC were grown three dimensionally within a reconstituted basement membrane (RBM) in defined serum-free medium. Zymograms of culture medium demonstrated that five major gelatinases of 97, 80, 74, 69, and 65 kDa were secreted by MEC and were distinct from gelatinases of RBM origin. Based on molecular weight, p-aminophenylmercuric acid activation, immunoblotting with MMP-specific antibodies, inhibition by EDTA, a peptide containing the prodomain sequence of MMP (TMRKPRCGNPDVAN) and two synthetic MMP inhibitors (BB-94 and CGS 27023A), these were classified as inactive and active forms of MMP-9 and MMP-2. The maximal MMP activities occurred when MEC were in a rapid proliferation and branching phase and declined after they underwent functional differentiation. Known regulators of MEC growth and differentiation were evaluated for their ability to modulate gelatinase activity in primary culture. Secretion of one or both MMPs was inhibited by EGF, TGFalpha, prolactin, and hydrocortisone and stimulated by progesterone. Furthermore, the functional significance of MMPs was demonstrated since three MMP inhibitors blocked branching morphogenesis elicited by the absence of hydrocortisone. Additionally, two synthetic MMP inhibitors not only inhibited epithelial cell growth but also inhibited normal alveolar development of the MEC. Finally, these drugs were found to enhance MMP secretion from MEC, although the activity of the secreted MMPs was inhibited as long as the drug was present.     

Regulation of matrix biology by matrix metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that contribute to growth, development and wound healing as well as to pathologies such as arthritis and cancer. Until recently, it has been thought that MMPs participate in these processes simply by degrading extracellular matrix (ECM) molecules. However, it is now clear that MMP activity is much more directed and causes the release of cryptic information from the ECM. By precisely cleaving large insoluble ECM components and ECM-associated molecules, MMPs liberate bioactive fragments and growth factors and change ECM architecture, all of which influence cellular behavior. Thus, MMPs have become a focal point for understanding matrix biology.     

Structural basis of the matrix metalloproteinases and their physiological inhibitors,the tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases with a metzincin-like catalytic domain, which are involved in extracellular matrix degradation but also in a number of other important biological processes. Under healthy conditions, their proteolytic activity is precisely regulated by their main endogenous protein inhibitors, the tissue inhibitors of metalloproteinases. Disruption of this balance results in pathophysiological processes such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and for rational drug design. Since the first appearance of atomic MMP structures in 1994, a large amount of structural information has become available on the catalytic domains of MMPs and their substrate specificity, interaction with synthetic inhibitors and the TIMPs, the domain organization, and on complex formation with other proteins. This review will outline our current structural knowledge of the MMPs and the TIMPs.     

Dynamic interdomain interactions contribute to the inhibition of matrix metalloproteinases by tissue inhibitors of metalloproteinases

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.