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.     

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.     

Membrane type-1 matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 RNA levels mimic each other during Xenopus laevis metamorphosis

Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.     

Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.     

Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium.

Matrix metalloproteinases (MMPs) are zinc-requiring enzymes that can degrade components of the extracellular matrix and that are implicated in tissue remodeling. Their role in the onset of menstruation in vivo has been proven; however, the expression and functions of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in vascular structures are poorly understood. We determined by immunocytochemistry, using characterized monoclonal antibodies, the distribution of MMPs and of their inhibitors TIMP-1 and TIMP-2 in the endometrium during the menstrual cycle. MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 had differing distributions and patterns of expression. In addition to the localization of MMP-9 in the epithelium and of MMP-2, MMP-3, and MMP-1 in the stromal tissue, these MMPs were detected in the vascular structures. MMP-2 (72-kDa gelatinase) and tissue inhibitors TIMP-1 and TIMP-2 were detectable in vessels throughout the cycle. In contrast, MMP-3 (stromelysin-1) was detected only in late-secretory and menstrual endometrial vessels, while MMP-9 (92-kDa gelatinase) was detected in spiral arteries during the secretory phase and in vascular structures during the midfollicular and menstrual phases. The expression of MMP-2 and MMP-9 in endometrial vessels during the proliferative and secretory periods suggests their relationship to vascular growth and angiogenesis. The pronounced expression of MMP-3 (stromelysin-1) in the vessels situated in the superficial endometrial layer during menses suggests that this metalloproteinase initiates damage in the vascular wall during menstrual breakdown. The finding of an intense expression of TIMP-1 and TIMP-2 in the vessels delineating necrotic from non-necrotic areas during menses also suggests that they could limit tissue damage, allowing regeneration of the endometrium after menses. These data indicate that, in addition to expression in epithelial cells and stromal tissue, MMPs are expressed in endometrial vascular cells in a cycle-specific pattern, consistent with regulation by steroid hormones and with specific roles in the vascular remodeling processes occurring in the endometrium during the cycle.     

Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system

Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.     

Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway

The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from a Timp2-/- mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 by Timp2-/- cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3-27 nm TIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (k(on)) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 x 10(5) m(-1) s(-1) and 6.52 x 10(5) m(-1) s(-1), respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.     

Individual Timp deficiencies differentially impact pro-MMP-2 activation

Membrane-type matrix metalloproteinases (MT-MMPs) have emerged as key enzymes in tumor cell biology. The importance of MT1-MMP, in particular, is highlighted by its ability to activate pro-MMP-2 at the cell surface through the formation of a trimolecular complex comprised of MT1-MMP/tissue inhibitor of metalloproteinase-2 (TIMP-2)/pro-MMP-2. TIMPs 1-4 are physiological MMP inhibitors with distinct roles in the regulation of pro-MMP-2 processing. Here, we have shown that individual Timp deficiencies differentially affect MMP-2 processing using primary mouse embryonic fibroblasts (MEFs). Timp-3 deficiency accelerated pro-MMP-2 activation in response to both cytochalasin D and concanavalin A. Exogenous TIMP-2 and N-TIMP-3 inhibited this activation, whereas TIMP-3 containing matrix from wild-type MEFs did not rescue the enhanced MMP-2 activation in Timp-3(-/-) cells. Increased processing of MMP-2 did not arise from increased expression of MT1-MMP, MT2-MMP, or MT3-MMP or altered expression of TIMP-2 and MMP-2. To test whether increased MMP-2 processing in Timp-3(-/-) MEFs is dependent on TIMP-2, double deficient Timp-2(-/-)/-3(-/-) MEFs were used. In these double deficient cells, the cleavage of pro-MMP-2 to its intermediate form was substantially increased, but the subsequent cleavage of intermediate-MMP-2 to fully active form, although absent in Timp-2(-/-) MEFs, was detectable with combined Timp-2(-/-)/-3(-/-) deficiency. TIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.     

Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis

Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. In the acute phases of liver injury and as liver fibrosis progresses, there is increased expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Among the changes described, striking features include increased expression of gelatinase A (MMP-2) and membrane type 1-MMP (MT(1)-MMP; MMP-14) as well as TIMP-1 and TIMP-2. These molecules and other family members are involved in regulating degradation of both normal and fibrotic liver matrix. This article outlines recent progress in this field and discusses the mechanisms by which MMPs and TIMPs may contribute to the progression and regression of liver fibrosis. Recently described properties of MMPs and TIMPs of relevance to the pathogenesis of liver fibrosis are outlined. The proposal that regression of liver fibrosis is mediated by decreased expression of TIMPs and involves degradation of fibrillar collagens by a combination of MT(1)-MMP and gelatinase A, in addition to interstitial collagenase, is explored.     

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

Background

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Methods

We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20).

Results

We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2.

Conclusion

These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.     

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP-2 and membrane-type-1 MMP (MT1-MMP) forms a cell surface located ''receptor'' involved in pro-MMP-2 activation. We have solved the 2.75 A crystal structure of the complex between the catalytic domain of human MT1-MMP (cdMT1-MMP) and bovine TIMP-2. In comparison with our previously determined MMP-3-TIMP-1 complex, both proteins are considerably tilted to one another and show new features. CdMT1-MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active-site cleft that might be important for interaction with macromolecular substrates. The TIMP-2 polypeptide chain, as in TIMP-1, folds into a continuous wedge; the A-B edge loop is much more elongated and tilted, however, wrapping around the S-loop and the beta-sheet rim of the MT1-MMP. In addition, both C-terminal edge loops make more interactions with the target enzyme. The C-terminal acidic tail of TIMP-2 is disordered but might adopt a defined structure upon binding to pro-MMP-2; the Ser2 side-chain of TIMP-2 extends into the voluminous S1'' specificity pocket of cdMT1-MMP, with its Ogamma pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP-1 for MT1-MMP compared with TIMP-2 might be explained by a reduced number of favourable interactions.     

Matrix metalloproteinase expression in vein grafts: role of inflammatory mediators and extracellular signal-regulated kinases-1 and -2

Matrix metalloproteinases (MMPs) play key roles in vascular remodeling. We characterized the role of inflammatory mediators and extracellular signal-regulated kinases (ERKs) in the control of arterialized vein graft expression of MMP-9, MMP-2, and membrane-type 1-MMP (MT1-MMP) and of the tissue inhibitor of metalloproteinases-2 (TIMP-2). For this purpose we used a canine model of jugular vein to carotid artery interposition graft and analyzed the vein grafts at various postoperative times (30 min to 28 days) using the contralateral vein as a control. To study the role of ERK-1/2, veins were incubated with the mitogen-activated protein kinase kinase (MEK-1/2) inhibitor UO126 for 30 min before being grafted. Vein graft extracts were analyzed for MMPs, TIMP-2, tumor necrosis factor-alpha (TNF-alpha), polymorphonuclear neutrophil (PMN) infiltration, myeloperoxidase (MPO), and thrombin activity, and for ERK-1/2 activation. Vein graft arterialization resulted in rapid and sustained (8 h to 28 days) upregulation of vein graft-associated MMP-9, MMP-2, MT1-MMP, thrombin activity, and TNF-alpha levels with concomitant TIMP-2 downregulation. MMP-2 activation preceded MT1-MMP upregulation. PMN infiltration and vein graft-associated MPO activity increased within hours after arterialization, indicating a prompt, local inflammatory response. In cultured smooth muscle cells, both thrombin and TNF-alpha upregulated MT1-MMP expression; however, only thrombin activated MMP-2. Inhibition of ERK-1/2 activation blocked arterialization-induced upregulation of MMP-2, MMP-9, and MT1-MMP. Thus, thrombin, inflammatory mediators, and activation of the ERK-1/2 pathway control MMP and TIMP-2 expression in arterialized vein grafts.     

Mechanical forces-induced human osteoblasts differentiation involves MMP-2/MMP-13/MT1-MMP proteolytic cascade

Matrix metalloproteinase (MMP) family proteins play diverse roles in many aspects of cellular processes such as osteoblastic differentiation. Besides, mechanical forces that occur in 3D collagen gel promote the osteoblastic phenotype and accelerate matrix mineralization. Although MMPs have been involved in bone differentiation, the proteolytic cascades triggered by mechanical forces are still not well characterized. In this study, we have investigated the contribution of both proteolytic cascades, MMP-3/MMP-1 and MMP-2/MMP-13/MT1-MMP in the differentiation of human osteoblasts cultured in a floating type I collagen lattice (FL) versus an attached collagen lattice (AL). Compared to AL, contraction of human osteoblasts-populated FL led to a fast (1 day) induction of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteoprotegerin (OPG), and Runx-2 expression. At day 4, osteocalcin (OC) overexpression preceded the formation of calcium-containing nodule formation as assessed by X-ray analyses. MMP-1 and MMP-3 were produced to similar extent by cells cultured in FL and AL, whereas contraction of collagen lattices triggered both mRNA overexpression of MMP-2, MMP-13, and MT1-MMP (i.e., MMP-14), and their activation as evidenced by Western blotting or zymographic analyses. Down-regulating MT1-MMP expression or activity either by siRNA transfection or supplementation of culture medium with TIMP-1 or TIMP-2 highlighted the contribution of that enzyme in OC, ALP, and OPG expression. MMP-2 and MMP-13 were more directly involved in BSP expression. So, these results suggest that the main proteolytic cascade, MMP-2/MMP-13/MT1-MMP, and more particularly, its initial regulator MT1-MMP is involved in osteoblast differentiation through mechanical forces.     

Differential roles of TIMP-4 and TIMP-2 in pro-MMP-2 activation by MT1-MMP

The tissue inhibitors of metalloproteinases (TIMPs) are specific inhibitors of MMP enzymatic activity. However, TIMP-2 can promote the activation of pro-MMP-2 by MT1-MMP. This process is mediated by the formation of a complex between MT1-MMP, TIMP-2, and pro-MMP-2. Binding of TIMP-2 to active MT1-MMP also inhibits the autocatalytic turnover of MT1-MMP on the cell surface. Thus, under certain conditions, TIMP-2 is a positive regulator of MMP activity. TIMP-4, a close homologue of TIMP-2 also binds to pro-MMP-2 and can potentially participate in pro-MMP-2 activation. We coexpressed MT1-MMP with TIMP-4 and investigated its ability to support pro-MMP-2 activation. TIMP-4, unlike TIMP-2, does not promote pro-MMP-2 activation by MT1-MMP. However, TIMP-4 binds to MT1-MMP inhibiting its autocatalytic processing. When coexpressed with TIMP-2, TIMP-4 competitively reduced pro-MMP-2 activation by MT1-MMP. A balance between TIMP-2 and TIMP-4 may be a critical factor in determining the degradative potential of cells in normal and pathological conditions.