- Tissue Inhibitors of MMPs (TIMPs)
- Diseases in which MMPs and TIMPs have been incriminated
Interstitial collagenase, the first MMP family member identified, was initially discovered in experiments designed to explain collagen remodeling in the metamorphosis of a tadpole into a frog1. Since collagens (numerous types identified) represent the major structural proteins of all tissues and the chief obstacle to cell migration, it was proposed three decades ago that collagenolytic enzymes play pivotal roles in numerous physiologic (fetal development) and pathologic conditions (cancer and arthritis). More recent scientific interest has expanded to include the role of MMPs in numerous disease states involving the cardiovascular, pulmonary, renal, gastrointestinal, musculoskeletal, visual, and hematopoietic systems.
Matrix metalloproteinase (MMPs) belong to the family of zinc endopeptidases collectively referred to as metzincins. The metzincin superfamily is distinguished by a highly conserved motif containing three histidines that bind to zinc at the catalytic site and a conserved methionine that sits beneath the active site 2. The metzincins are subdivided into four multigene families: seralysins, astacins, ADAMs/adamalysins, and MMPs. Although our knowledge of the metzincin biology is rapidly expanding, we still do not fully understand how these enzymes regulate biological functions.
The MMP family is comprised of more than 20 related zinc-dependent enzymes that share common functional domains. These enzymes have both a descriptive name generally based on a preferred substrate and a MMP numbering system based on order of discovery (Figure 1). MMPs were initially characterized by their extensive ability to degrade extracellular matrix proteins including collagens, laminin, fibronectin, vitronectin, aggrecan, enactin, tenascin, elastin, and proteoglycans3. More recently, it has been recognized that MMPs cleave many other types of peptides and proteins and have a myriad of other important functions that may be independent of proteolytic activity 4. MMPs have distinct but often overlapping substrate specificities, hence leading to the absence of distinct phenotypes in most genetically-engineered mice with knockdown of specific MMPs; MMP14 is the exception.
Domain Structure of MMPs
Figure 1: History and Domain Structure of MMPs.
Compilation of MMPs. Date of discovery of MMP and amino acid sequence listed in parentheses.
The basic structure of MMPs is made up of the following homologous domains: 1) signal peptide which directs MMPs to the secretory or plasma membrane insertion pathway; 2) prodomain that confers latency to the enzymes by occupying the active site zinc, making the catalytic enzyme inaccessible to substrates; 3) zinc containing catalytic domain; 4) hemopexin domain which mediates interactions with substrates and confers specificity of the enzymes; and 5) hinge region which links the catalytic and the hemopexin domain (Figure 1). The smallest MMP in size, MMP7 or matrilysin, lacks the hemopexin domain, yet displays specificity in substrate degradation. Additional structural domains and substrate specificities have led to the division of MMPs into subgroups. The membrane-type MMPs contain an additional 20 amino acid transmembrane domain and a small cytoplasmic domain (MMP14, MMP15, MMP16, and MMP24) or a glycosylphosphatidyl inositol linkage (MMP17 and MMP25), which attaches these proteins to the cell surface. MMP2 and MMP9 (referred to as gelatinases based on their substrate preference) contain fibronectin-like domain repeats which aid in substrate binding.
Two sequence motifs are highly conserved in the protein structure of MMPs. The consensus motif HExGHxxGxxH, found in the catalytic domain of all MMPs, contains 3 histidines that coordinate with the zinc ion (Zn) in the active center. The PRCGxPD motif is located in the C-terminal portion of the prodomain of MMPs; coordination of the cysteine residue (C) of this locus with the zinc atom of the active center confers latency to the proenzyme3,5.
In vivo activity of MMPs is under tight control at several levels. These enzymes are generally expressed in very low amounts and their transcription is tightly regulated either positively or negatively by cytokines and growth factors such as interleukins ( IL-1, IL-4, IL-6), transforming growth factors (EGF, HGF, TGFß), or tumor necrosis factor alpha (TNFα)6,7. Some of these regulatory molecules can be proteolytically activated or inactivated by MMPs (feedback effect). Post-transcrpitionally, MMP activity is restricted by the latency conferred by the propeptide located in the N-terminal end of the newly synthesized proenzymes. Activation of MMPs following secretion from cells depends on disruption of the prodomain interaction with the catalytic site, which may occur by conformational changes or proteolytic removal of the prodomain. MMPs that contain furin-like recognition domains in their propeptides (MMP11, MT-MMPs, MMP28) can be activated in the trans Golgi network by members of the subtilisin family of serine proteases. With the exception of MMP2, the mechanism for in vivo activation of secreted MMPs is not well understood. MMP14 plays an integral role in the activation of proMMP2 on the cell surface. Extracellular proteolytic activation of secreted MMPs can be mediated by serine proteases such as plasmin, which implies an interdependence of these two enzyme groups in ECM remodeling 7. Some active MMPs can activate other proMMPs e.g. MMP3 activation of MMP9 and MMP1. Once activated, MMPs are further regulated by endogenous inhibitors, autodegradation, and selective endocytosis. Endocytosis of MMP2,9, and 13 through a low density lipoprotein receptor-related protein (LRP) mechanism has been demonstrated8. Following secretion from the cell, MMP9 is able to bind to the cell surface where it is somewhat protected from local inhibitors.
The Tissue Inhibitors of MetalloProteinases comprise a four-member family of homologous MMP inhibitors (TIMP1, 2, 3, and 4) 3 (Figure 2). TIMP concentrations generally far exceed the concentration of MMPs in tissue and extracellular fluids, thereby limiting their proteolytic activity to focal pericellular sites. In contrast to the usual inhibitory role, low concentration of TIMP2 enhance MMP14 induced activation of MMP2 by forming a triplex with these proteins on the cell surface 7. In addition, TIMPs have been shown to have growth promoting activities which are independent of their MMP inhibitory function and apoptosis-inducing properties (TIMP3). The transcription of TIMPs is regulated by similar cytokines and growth factors that control MMP expression i.e. TGFß, TNFα, IL-1, IL-6, although often in distinctive ways. Other endogenous inhibitors include plasma protein α2macroglobulin, and a surface inhibitor of MMPs, the RECK inhibitor 9.
Figure 2: Schematic representation of TIMP-1 structure. TIMP1 contains 12 cysteine residues which form six loop structures through disulfide bonds. The N-terminus of TIMPs 1-4 binds to the catalytic domain of most activated MMPs and inhibits function. The C-terminus of TIMP1 and TIMP2 binds to the hemopexin domain of proMMP2 and proMMP9, respectively; this binding regulates MMP function.
Thousands of papers have been written on the subject of MMPs dealing with various aspects of their physiologic and pathologic roles in biologic processes. The simplicity of thinking about MMPs solely as extracellular matrix degrading enzymes and TIMPs solely as inhibitors of these processes has been eroded by the recognition of numerous other important roles for these proteins. Our understanding of the biology of MMPs and TIMPs has been expanded with the availability of specific MMP and TIMP knockout mice,10. Families with genetic mutations of MMP2 have demonstrated important differences between mice and men in terms of mutation phenotypes. The mutated MMP2 phenotype in man looks more like the MMP14 mouse knockout.
MMPs are collectively able to degrade virtually all ECM components. In cancer, special emphasis was initially placed on the degradation of type IV collagen, a major protein component of basement membranes by MMP2 and MMP9. Subsequently, it has been demonstrated that many non-ECM proteins can be cleaved by selected MMPs. Deciding which protein substrates are physiologically and pathologically relevant has been difficult to ascertain. MMP-induced release from the cell surface (shedding) of heparin binding epithelial growth factor, insulin-like growth factor, and fibroblast growth factor enhance cell proliferation. On the other hand, release and activation of ECM sequestered TGFß by MMPs can lead to inhibition of cell proliferation. MMP-induced shedding of Fas ligand from the cell surface can either enhance or interfere with cell survival. MMP14 and MMP1 have also been incriminated in enhancing cancer cell migration. MMPs are capable of inducing both positive and negative effects on angiogenesis and immunity. It has been observed that some MMPs readily cleave specific chemokines which may have profound effects on numerous biologic processes, not just limited to inflammation4. It has been proposed that cleavage of collagen type IV by MMP2/9 exposes a cryptic site which displays affinity for αvß3 integrin, thereby leading to enhancement of angiogenesis. The capacity of MMPs e.g. MMP12, to cleave plasminogen and generate angiostatin, a powerful inhibitor of tumor angiogenesis in mouse cancer models, reminds us that multi-purpose proteases can display cancer enhancement effects, as well as anti-tumor effects. Clinical experience has taught us that the myriad of MMP functions complicates our ability to predict the outcome of MMP inhibitor use in cancer patients 7.
Of considerable interest are the cell types responsible for producing MMPs in cancer. Contrary to expectations, most MMPs in tumors are produced by stromal cells rather than the cancer cells (Figure 3). One explanation for this phenomenon is that cancer cells produce Extracellular Matrix Metalloproteinase Inducer (EMMPRIN), a cell surface glycoprotein, which directly stimulates fibroblasts (through direct cell contact) to produce MMP1, 2, 3, and MMP14 (Figure 2) 11. EMMPRIN is also up regulated in inflammatory cells and has been implicated in tissue injury. The importance of cytokines such as TNF-α, interleukin (IL)-1, and IL-6 in stimulating production of MMPs in disease has been emphasized. Increased levels of TIMP1 and TIMP2 have been identified in malignant stromal tissue.
MMP and Cancer
Figure 3: Schematic diagram of interaction between cancer cells and stromal within a tumor with a focus on
role of MMPs.
Based on the important role of MMPs in cancer and successful drug trials in mice, numerous clinical trials were initiated in the 1990’s to test the effectiveness of hydroxamic acid-derived MMP inhibitors (MMPIs) in patients with cancer12. Based on the lack of effectiveness of several broad spectrum MMPIs in advanced cancer types, these drugs have languished. Retrospective assessment of the design of these clinical trials has led to the recognition that specific MMPIs used in conjunction with cytotoxic chemotherapy in early stage, rather than late stage cancer, needs future consideration.
Many reports have implicated MMP1,MMP3, and MMP9 in rheumatoid and osteoarthritis. An important role for aggrecanase, a member of the ADAM family of metalloproteinases, in articular damage has been proposed. A clinical trial of an MMPI in arthritis failed to achieve its goal.
There has been a long-standing interest in the role of MMPs in cardiovascular disease. Numerous studies have demonstrated increased levels of MMPs, especially MMP9, at sites of atherosclerosis and aneurysm formation13. The concept that the inflammatory process may play a leading role in the development of atherosclerotic plaques, has led to the suggestion that secretion and activation of MMPs by macrophages induces degradation of extracellular matrix in the atherosclerotic plaque and plaque rupture. Based on these concepts, MMPs have been proposed to represent sensitive markers of inflammation in patients with coronary artery disease. The importance of collecting plasma rather than serum specimens for measuring MMP9 in diagnostic testing has been stressed 14.
MMP tissue levels are increased in the heart in congestive heart failure. Although inhibitors of MMPs have shown value in experimental models of heart disease, a recent clinical trial demonstrated a lack of efficacy of a broad-spectrum MMP inhibitor in patients with congestive heart failure. The results of this trial emphasize the importance of segregating patients into different pathophysiologic categories that are more likely to respond to a specific drug.
Elevated levels of MMPs have been implicated in the pathophysiology of various lung diseases, including acute respiratory distress syndrome, asthma, bronchiectasis, and cystic fibrosis. MMPs, EMMPRIN, and TIMPs are produced by many of the resident cells in the lung, hence complicating the analysis of their role in disease15. Potential use of MMP inhibitors for treatment of these disorders in patients remains to be explored.
Following observations of the critical role of MMP9 in animal models resembling multiple sclerosis and Guillain-Barre’s syndrome, MMPs has been implicated in several different types of neurologic diseases13,16. Treatment with synthetic inhibitors of MMPs has reversed some of the pathology in animal models of brain injury, especially stroke.
MMP8 and MMP9 are stored in the granules of polymorphonuclear leukocytes. These cells are key effectors in inflammatory and infectious processes. A role for these MMPs in shock is supported by studies in MMP9 deficient mice that were shown to be resistant to endotoxic shock. Dubois et al.17 proposed that specific MMP9 inhibition constitutes a potential approach for the treatment of septic shock syndromes.
Acute and chronic wounds are associated with high levels of MMP2 and MMP9. These observations have led to the suggestion that nonhealing ulcers develop an environment containing high levels of activated MMPs, which results in chronic tissue turnover and failure of wound closure13. MMP9 has been implicated in blistering skin diseases and contact hypersensitivity13,18. MMPs have long been implicated in periodontal disease19 and more recently, in inflammatory bowel diseases. Tetracyclines and chemically-modified tetracyclines, which have inhibitory functions against MMPs, have been shown to be useful in treatment of periodontal disease19.
The involvement of MMPs in disease processes and normal embryologic development continues to be of major interest to scientists from many fields. At present, the pharmaceutical industry remains reluctant to further test MMPIs in human disease, but this might change with the development of more specific inhibitors that lack the troublesome side-effects demonstrated with broad-spectrum MMPIs and a better understanding of the basic role of MMPs in pathophysiology.
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