Tissue inhibitors of metalloproteinases
© BioMed Central Ltd. 2011
Published: 11 November 2011
Orchestration of the growth and remodeling of tissues and responses of cells to their extracellular environment is mediated by metalloproteinases of the Metzincin clan. This group of proteins comprises several families of endopeptidases in which a zinc atom is liganded at the catalytic site to three histidine residues and an invariant methionine residue. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinase (MMPs) family, and also of families such as the disintegrin metalloproteinases (ADAM and ADAMTS). TIMPs therefore have a pivotal role in determining the influence of the extracellular matrix, of cell adhesion molecules, and of many cytokines, chemokines and growth factors on cell phenotype. The TIMP family is an ancient one, with a single representative in lower eukaryotes and four members in mammals. Although much is known about their mechanism of action in proteinase regulation in mammalian cells, less is known about their functions in lower organisms. Recently, non-inhibitory functions of TIMPs have been identified in mammalian cells, including signaling roles downstream of specific receptors. There are clearly still questions to be answered with regard to their overall roles in biology.
Gene organization and evolutionary history
The naturally occurring inhibitory activities of the matrix metalloproteinases (MMPs) were initially identified in many cell and tissue culture studies, carried out over several decades. Between 1985 and 1996, however, four members of the tissue inhibitor of metalloproteinases (TIMP) family were definitively identified at the gene level in mammals. In fact, orthologs of the TIMPs are widely distributed across the animal kingdom and have now been identified in species as widely separated as Trichoplax, Hydra, molluscs, worms and insects, as well as in vertebrates such as fish and birds. Plants do have metzincins, but no plant TIMP ortholog has been identified.
TIMP1 was originally cloned in 1985 when it was found to have an erythroid potentiating activity  and to be an inhibitor of metalloproteinases . TIMP2 was cloned in 1990 by Stetler-Stevenson et al. , TIMP3 by Pavloff and colleagues in 1992 , and TIMP4 in 1996 . These proteins act as significant regulators of the activities of MMPs and, in some instances, of other metalloendopeptidases of the metzincin clan, namely the disintegrin metalloproteinases (ADAM) and the disintegrin metalloproteinases with thrombospondin motifs (ADAMTS). TIMPs inhibit with a 1:1 molar stoichiometry. Their importance in modulating the ability of a cell to control its extracellular environment, from the remodeling of the extracellular matrix to the interaction of cells via adhesion and signaling molecules such as growth factors has long been appreciated , but the significance of TIMPs as both proteinase inhibitors and signaling molecules in their own right is only just beginning to be documented .
Characteristic structural features and mechanism
Properties of human TIMPs, summarizing their activities in inhibiting metzincin metalloproteinases and in modulating phenotypic cell behavior 
Amino acid residues
24 or 27
Extracellular matrix/cell surface
MMPs poorly inhibited
MT1-MMP, MT3-MMP, MT5-MMP, MMP19
ADAM10, ADAM12, ADAM17, ADAM19, ADAM33, ADAMTS1, ADAMTS4, ADAMTS5, ADAMTS2 (weak)
EFEMP1, AngIIR, VEGFR
↑Smooth muscle cells and cancer cells
↑Mammary tumor cells
↓Wilm's tumor cells
↑Smooth muscle cells
↓Burkitt's lymphoma cells
↑Colorectal cancer cells
↑Retinal pigment epithelial cells
Angiogenesis in 3D collagen or fibrin gels
Sorsby's fundus dystrophy
The α-amino and carbonyl groups of the amino-terminal Cys1 of the human TIMPs have been shown in crystallographic analyses to chelate the Zn2+ of the enzyme active site; the OH group of Ser/Thr2 interacts with the nucleophilic Glu of the MMP catalytic cleft (Figure 3b). The latter interaction displaces a water molecule that is essential for peptide hydrolysis from the active site. TIMP1, TIMP2, TIMP3 and TIMP4 all inhibit all of the MMPs tested, but TIMP1 is a poor inhibitor of MT1-MMP, MT3-MMP, MT5-MMP and MMP19 [6, 8] (Table 1). TIMP2 is unique in that, in addition to inhibiting MMP activity, it selectively interacts with MT1-MMP to facilitate the cell-surface activation of proMMP2. Recent studies have shown that TIMPs can inhibit some of the disintegrin metalloproteinases (ADAM and ADAMTS). TIMP1 inhibits ADAM10 , whereas TIMP2 inhibits ADAM12 . TIMP3 has a much broader inhibition profile, including ADAM10 , ADAM12 , ADAM17  and several ADAMTSs (ADAMTS1, ADAMTS2, ADAMTS4 and ADAMTS5) [12–14]. The crystal structures of a number of TIMP-metalloproteinase complexes have shown that the mode of inhibition described above does not vary. The properties of the human TIMPs are summarized in Table 1.
Localization and function
The TIMPs show tissue-specific, constitutive, or inducible expression, which is regulated at the transcriptional level by various cytokines and growth factors. TIMP1 is widely expressed in many mammalian tissues, notably in the reproductive organs. In the central nervous system, TIMP1 expression is restricted to regions of persistent neuronal plasticity, such as the hippocampus, olfactory bulb, and cerebellum . TIMP2 is constitutively expressed in most tissues but is not inducible by common growth factors. TIMP3 is expressed in many tissues and is often found as a significant matrix protein in the basement membranes of the eye and kidney. The relatively restricted tissue distribution of TIMP4 (in the heart, kidney, ovary, pancreas, colon, testes, brain and adipose tissue) suggests that it plays a role in tissue-specific physiological functions , but the role of this protein is relatively understudied.
The roles of TIMPs in both normal physiology and in pathological processes have been investigated by gene ablation studies in mice, and these have indicated that there is some functional redundancy. Timp1-null mice do not exhibit many inherent abnormalities, apart from alterations in the reproductive cycle and impaired learning and memory, unless the animals are challenged pathologically. Timp2-null mice have neurological and motor-function abnormalities, but again they only manifest phenotypes that are associated with matrix remodeling upon challenge (reviewed in [6, 15]). Timp3 ablation causes lung emphysema-like alveolar damage  and faster apoptosis of mammary epithelial cells after weaning . This suggests that TIMP3 is a major regulator of MMP activities in vivo. Furthermore, TIMP3 plays a key role in innate immunity by regulating the processing of tumor necrosis factor α by ADAM17 .
The individual TIMP genes appear to be regulated rather differently: TIMP1 and TIMP3 are upregulated in vitro in response to phorbol myrsistate acetate and transforming growth factor β, whereas TIMP2 is downregulated. All the TIMP promoters have sites for binding the transcription factor SP1. The TIMP1, TIMP2 and TIMP3 promoters have additional common elements, such as AP1 and PEA3, that are also present in several MMP promoters [19, 20]. Otherwise, there is little organizational conservation of the common motifs in the TIMP promoters. Like many 'housekeeping genes' - that is, genes that are always expressed because they encode proteins that are constantly required - TIMP1, TIMP2 and TIMP4 lack obvious TATA boxes. TIMP1 has multiple transcription start sites and GC boxes. TIMP2, which has a TATA box and multiple transcription start sites, contains MEF2 and NF-IL6 binding sites in its promoter. The TIMP3 promoter uniquely has binding sites for the transcription factors NF-κB, c-MYC and p53. TIMP3 has a promoter-proximal GC-rich region that mediates its basal expression, and regions further upstream that mediate serum induction. TIMP3 is notably regulated by gene methylation, which can lead to its downregulation in many cancer cells. The TIMP4 promoter has no TATA box but does contain an initiator sequence and several transcription start points. TIMP4 also contains a myogenin-binding site, which is likely to be important for its skeletal muscle-specific expression, and an AP4 transcription factor binding site .
TIMP-mediated inhibition of MMP activity is an important determinant of cell function, but the concept of MMP-independent TIMP regulation of cell behavior is now supported by the identification of both specific cell-binding partners for TIMP family members and specific signaling events that involve these proteins . Cell signaling mechanisms mediated by TIMP1 and TIMP2 have been shown to promote cell proliferation, although the receptor-mediated events that are involved are not well understood . TIMP1 has anti-apoptotic effects on some cell types, possibly acting by enhancing the activities of survival and differentiation factors in a signaling network that is mediated by the tetraspanin CD63 and by focal adhesion kinase, phosphoinositide 3-kinase and ERK. TIMP2 binds to α3β1 integrin, leading to G1 phase growth arrest and enhanced de novo expression of the cyclin-dependent kinase inhibitor p27Kip1. TIMP3 has conflicting activities according to cell type. In some cells, it appears to promote the development of a transformed phenotype: TIMP3 promotes apoptosis in several tumor cell lines and in smooth muscle cells, but this appears to involve the modulation of metalloproteinase activities. Work with timp3-null mice suggests that TIMP3 can either promote or inhibit apoptosis depending on the model system examined. TIMP3 can be sequestered in the extracellular matrix in some tissues, but secreted TIMP3 can be rapidly endocytosed by cells such as chondrocytes through a mechanism that involves the LDL-receptor-related protein (LRP) [6, 21].
Knowledge of the precise functions and spatio-temporal expression patterns of metalloproteinases are still somewhat limited. In vitro, the metalloproteinases have markedly overlapping substrate specificities and their in vivo functions will take some time to elucidate in detail (reviewed in ). Studies of function (for example, using transgenic mouse models) should be coupled with studies of the metalloproteinases' localization in tissues relative to the physiological or pathological status of those tissues. The importance of TIMPs in regulating metalloproteinases is consequently also less than clear. Nevertheless, gene-ablation studies in mice have indicated some key roles for TIMPs, with TIMP3 clearly being of some importance in both extracellular matrix remodeling and in inflammation. TIMP3 seems to function largely as a metalloproteinase inhibitor, but other TIMPs directly mediate signaling pathways that lead to the regulation of growth, apoptosis and other activities. TIMP1 concentrations are increased in cancer patients, particularly in those with breast or colorectal carcinomas, and this increase is negatively associated with patient outcome. Studies have shown the clinical utility of TIMP1 as a biomarker and independent prognostic factor in breast, colorectal, and several hematological cancers. There has been a growing awareness of the activity of TIMPs as modulators of nervous system physiology and pathology, and further elucidation of their roles alongside the activities of the metalloproteinases will be of interest in this context. Various polymorphisms of all the TIMP genes have been associated with predisposition to many different cancers. The function of TIMPs in lower organisms is not so well elucidated and might provide fascinating insights into the roles of metalloproteases in development.
Although several structures of TIMP-proteinase complexes have been solved, there are outstanding questions as to the importance of the flexibilities of both inhibitor and enzyme during binding. This could be key to future engineering of more specific forms of TIMPs as potential therapeutic agents. The regulation of TIMP function by extracellular interaction sites and by endocytosis is still to be studied fully . The role of TIMPs in the regulation of proteinase clearance (for example, by endocytosis) is also not well understood. The MMP-independent, cytokine-like functions of the TIMPs are now well recognized, and future work will involve the identification of cell surface receptors and the elucidation of downstream signaling . The pleiotropic activities of the TIMP family members are complex and depend upon subtle interactions with other extracellular components, as well as on direct interactions with cell-binding partners . Understanding these processes and how they are modulated during disease progression will be helpful to our understanding of disease pathology. TIMPs have rarely been considered as the basis for novel therapeutic interventions, but their use in model systems gives vital clues about the efficacy of metalloproteinase inhibitors in the abrogation of disease [9, 18].
a disintegrin metalloproteinase
a disintegrin metalloproteinase with thrombospondin motif
- DDC8 :
Differential display clone 8
tissue inhibitor of metalloproteinases.
Thanks to Hideaki Nagase, Keith Brew and Ian Clark for invaluable advice on the manuscript, and to my 'TIMP' colleagues for their hard work over the years. TIMP studies in the author's laboratory have been supported by Arthritis Research UK, the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), The Wellcome Trust and Cancer Research UK.
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