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1.
Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.  相似文献   

2.
Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity. Also, mutations in the core of the MMP interaction surface of N-TIMP-3 dramatically reduce the binding affinity for MMPs but have little effect on the inhibitory activity for TACE. These results suggest that the mechanism of inhibition of ADAM-17 by TIMP-3 may be distinct from that for MMPs. The mutant proteins are also effective inhibitors of tumor necrosis factor alpha (TNF-alpha) release from phorbol ester-stimulated cells, indicating that they provide a lead for engineering TACE-specific inhibitors that may reduce side effects arising from MMP inhibition and are possibly useful for treatment of diseases associated with excessive TNF-alpha levels such as rheumatoid arthritis.  相似文献   

3.
Blockage of the metastasis process remains a significant clinical challenge, requiring innovative therapeutic approaches. For this purpose, molecules that inhibit matrix metalloproteinases activity or induce the expression of their natural inhibitor, the tissue inhibitor of metalloproteinases (TIMPs), are potentially interesting. In a previous study, we have shown that synthetic ligands binding to cell surface nucleolin/nucleophosmin and known as HB 19 for the lead compound and NucAnt 6L (N6L) for the most potent analog, inhibit both tumor growth and angiogenesis. Furthermore, they prevent metastasis in a RET transgenic mice model which develops melanoma. Here, we investigated the effect of N6L on the invasion capacity of MDA-MB-435 melanoma cells. Our results show that the multivalent pseudopeptide N6L inhibited Matrigel invasion of MDA-MB-435 cells in a modified Boyden chamber model. This was associated with an increase in TIMP-3 in the cell culture medium without a change in TIMP-3 mRNA expression suggesting its release from cell surface and/or extracellular matrix. This may be explained by our demonstrated N6L interaction with sulfated glycosaminoglycans and consequently the controlled bioavailability of glycosaminoglycan-bound TIMP-3. The implication of TIMP-3 in N6L-induced inhibition of cell invasion was evidenced by siRNA silencing experiments showing that the loss of TIMP-3 expression abrogated the effect of N6L. The inhibition of tumor cell invasion by N6L demonstrated in this study, in addition to its previously established inhibitory effect on tumor growth and angiogenesis, suggests that N6L represents a promising anticancer drug candidate warranting further investigation.  相似文献   

4.
We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better Ki value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.  相似文献   

5.
Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.  相似文献   

6.
7.
Decorin is a small leucine-rich proteoglycan that plays a role in control of cell proliferation, cell migration, collagen fibrillogenesis and modulation of the activity of TGF-beta. In the present study, we investigated the effects of decorin on the production of metalloproteinases (MMP-1, -2, -3, -9 and -13), tissue inhibitors of metalloproteinases (TIMP-1, -2) and cytokines (TGF-beta, IL-1beta, IL-4 and TNF-alpha). Decorin was overexpressed in cultured human gingival fibroblasts using adenovirus-mediated gene transfer. Decorin infection resulted in decreased protein levels of MMP-1 and MMP-3 whereas MMP-2 and TIMP-2 secretion was increased. MMP-9, MMP-13 and TIMP-1 were not affected by decorin infection. Cytokine measurements by ELISA showed that decorin overexpression reduced TGF-beta and IL-1beta. In contrast, IL-4 and TNF-alpha levels were markedly increased in decorin-infected cells. These results suggest that decorin could modulate the expression of certain metalloproteinases and their inhibitors, as well as the production of cytokines. Altogether, our data suggest that decorin might play a pivotal role in tissue remodeling by acting on the balance between extracellular matrix synthesis and degradation.  相似文献   

8.
Tumor cells become malignant, in part, because of their activation of matrix metalloproteinases (MMPs) and inactivation of tissue inhibitor of metalloproteinases (TIMPs). Myocardial tumors are rarely malignant. This raises the possibility that the MMPs and TIMPs are differentially regulated in the heart compared to other tissues. Therefore, we hypothesized that a tissue specific tumor suppressor exists in the heart. To test this hypothesis we prepared cardiac tissue extracts from normal (n = 4), ischemic cardiomypathic (ICM) [n = 5], and dilated cardiomyopathic (DCM) [n = 8] human heart end-stage explants. The level of cardiospecific TIMP-4 was determined by SDS-PAGE and Western-blot analysis. The results suggested reduced levels of TIMP-4 in ICM and DCM as compared to normal heart. TIMP-4 was purified by reverse phase HPLC and gelatin-sepharose affinity chromatography. Collagenase inhibitory activity of chromatographic peaks was determined using fluorescein-conjugated collagen as substrate and fluorescence spectroscopy. The activity of TIMP-4 (27 kDa) was characterized by reverse zymography. The role of TIMP-4 in cardiac fibroblast cell migration was examined using Boyden chamber analysis. The results suggested that TIMP-4 inhibited cardiac fibroblast cells migration and collagen gel invasion. To test whether TIMP-4 induces apoptosis, we cultured cardiac normal and polyomavirus transformed fibroblast cells in the presence and absence of TIMP-4. The number of cells were measured and DNA laddering was determined. The results suggested that TIMP-4 controlled normal cardiac fibroblast transformation and induced apoptosis in transformed cells. Cardiospecific TIMP-4 plays a significant role in regulating the normal cell phenotype. The reduced levels of TIMP-4 elicit cellular transformation and may lead to adverse extracellular matrix degradation (remodeling), cardiac hypertrophy and failure. This study suggests a possible protective role of TIMP-4 in other organs which are susceptible to malignancy.  相似文献   

9.
In response to periodontal inflammation, host cells release matrix metalloproteinases (MMPs) that contribute to periodontal tissue breakdown unless the tissue inhibitors of metalloproteinases (TIMPs) neutralize their activity. In this study, the capacity of Porphyromonas gingivalis to inactivate TIMP-1 was investigated. Proteolytic digestion of TIMP-1 was monitored by SDS-PAGE and Western immunoblotting. Planktonic cells and biofilms of P. gingivalis degraded TIMP-1 with production of several lower molecular mass fragments. Incorporation of human serum in the assay mixture had no effect on the degradation of TIMP-1 by P. gingivalis, whereas a cysteine proteinase inhibitor caused a complete inhibition. Using a fluorogenic assay, it was found that TIMP-1 treated with P. gingivalis lost its capacity to inhibit MMP-9 activity. This study revealed the potential of P. gingivalis to inactivate TIMP-1 through proteolytic degradation. This phenomenon may contribute to increasing significantly the level of active MMPs in affected periodontal sites and subsequently favor tissue destruction.  相似文献   

10.
Tissue inhibitor of metalloproteinases (TIMPs; TIMP-1, -2, -3 and -4) are endogenous inhibitor for matrix metalloproteinases (MMPs) that are responsible for remodeling the extracellular matrix (ECM) and involved in migration, invasion and metastasis of tumor cells. Unlike under normal conditions, the imbalance between MMPs and TIMPs is associated with various diseased states. Among TIMPs, TIMP-1, a 184-residue protein, is the only N-linked glycoprotein with glycosylation sites at N30 and N78. The structural analysis of the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1 suggests new possibilities of the role of TIMP-1 glycan moieties as a tuner for the proteolytic activities by MMPs. Because the TIMP-1 glycosylation participate in the interaction, aberrant glycosylation of TIMP-1 presumably affects the interaction, thereby leading to pathogenic dysfunction in cancer cells. TIMP-1 has not only the cell proliferation activities but also anti-oncogenic properties. Cancer cells appear to utilize these bilateral aspects of TIMP-1 for cancer progression; an elevated TIMP-1 level exerts to cancer development via MMP-independent pathway during the early phase of tumor formation, whereas it is the aberrant glycosylation of TIMP-1 that overcome the high anti-proteolytic burden. The aberrant glycosylation of TIMP-1 can thus be used as staging and/or prognostic biomarker in colon cancer. [BMB Reports 2012; 45(11): 623-628]  相似文献   

11.
Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT1 receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT2 receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT1 receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.  相似文献   

12.
Tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, have been shown to possess biological functions that are independent of their ability to inhibit matrix metalloproteinases. We have previously shown that the C-terminal domain of TIMP-2 and, in particular, Loop 6 inhibit capillary endothelial cell proliferation and angiogenesis both in vitro and in vivo. To elucidate the mechanism by which Loop 6 inhibits angiogenesis, we sought to determine whether its biological effects were the result of a known TIMP-2 protein-protein interaction or of a receptor-mediated event. In this study, we identify insulin-like growth factor-1 receptor as a binding partner of Loop 6/TIMP-2 and characterize this interaction on the endothelial cell surface and the consequences of this interaction on downstream receptor signaling.  相似文献   

13.
Binding of tissue inhibitor of metalloproteinase-2 (TIMP-2) to pro-MMP-2 and mature membrane type-1 MMP (MT1-MMP) on the cell surface is required for activation of MMP-2. It has been reported that following binding to cell surface receptors, TIMP-2 undergoes endocytosis and extensive degradation in lysosomes. The purpose of this study was to reexamine the fate of TIMP-2 following binding to transfected HT1080 cell surface MT1-MMP at 4 degrees C. Following 37 degrees C incubation, 125I-TIMP-2 release, endocytosis, and degradation were characterized under varying conditions. More than 85% of the total 125I-TIMP-2 bound to cells was released as intact functional molecules; <15% was degraded. Transfection of HT1080 cells with dominant negative mutant dynamin cDNA resulted in delayed endocytosis and release of 125I-TIMP-2 from cells. Pharmacologic agents that induce clustering of cell surface receptors (concanavalin A) and interfere with endosomal/lysosomal function (bafilomycin A(1)) resulted in enhanced binding of 125I-TIMP-2 to cell surface receptors. Abrogation of activation of proMT1-MMP with a furin inhibitor prevented binding and endocytosis of 125I-TIMP-2. Biotinylation of cell surface MT1-MMP followed by Western blotting confirmed the presence of mature MT1-MMP on the cell surface and degraded MT1-MMP in the intracellular compartment. In conclusion, these studies demonstrate that TIMP-2 is released from cells primarily as an intact functional molecule following binding to MT1-MMP on the cell surface.  相似文献   

14.
Matrix metalloproteinases are proteolytic enzymes which play a major role in resorption of collagen and other components of the extracellular matrix. They are controlled by specific inhibitors, so-called tissue inhibitors of metalloproteinases (TIMPs). The balance between matrix metalloproteinases and TIMPs seems to play a major role in controlling extracellular matrix homeostasis and cell migration. The influence of TIMP-1 on migration behaviour was explored in human hepatoma cells transiently and stably transfected with mouse TIMP-1, and incubated with biologically active TIMP-1. Transfection and biosynthesis were verified by Northern blotting, Western blotting, metabolic labeling, and reverse zymography. Overexpression of and incubation with TIMP-1 resulted in suppressed migration and seemed to enhance cell-cell contact. Using gelatin zymography and Western blotting we measured a significant increase of matrix metalloproteinases-2 and matrix metalloproteinases-9 in cells transfected with TIMP-1. This new phenomenon may be of important physiological significance in modulating TIMP and MMP expression. Our results indicate a functional involvement of TIMP-1 in matrix homeostasis and some automatic control in matrix turnover.  相似文献   

15.
Chan CY  Chen YS  Lee HH  Huang HS  Lai YL  Chen CF  Ma MC 《Life sciences》2007,81(9):717-723
Factors predisposing to extracellular matrix degradation associated with myocardial ischemia/reperfusion (IR) usually cause cell death. Recombinant human erythropoietin (EPO) protects the myocardium from IR, but whether it affects extracellular matrix (ECM) degradation is not known. This study examined the effect of the Jak2-ERK pathway, which is triggered by EPO, on the expression of matrix metalloproteinases (MMPs), tissue inhibitor of MMP 4 (TIMP-4), and collagen in post-ischemic hearts. Rat hearts were isolated and perfused in a Langendorff apparatus. IR was induced by 40 min of stopped flow and 120 min of aerobic reperfusion; EPO was added immediately before reperfusion. Compared to untreated controls, poor recovery of the left ventricular developed pressure (LVDP) was seen in IR hearts. IR resulted in myocyte injury measured by creatine kinase MB release and infarction. Western blot analysis showed increased levels of MMP-2 and MMP-9 and reduced levels of TIMP-4 and collagen III. IR rats given 5 IU/ml of EPO showed improved LVDP with reduced injury. EPO increased Jak2 and ERK activity, decreased MMP expression, increased TIMP-4 expression, and prevented collagen degradation in IR hearts. All these effects were blocked by the upstream ERK inhibitor, U0126 (5 microM). These observations show that EPO attenuates extracellular matrix degradation following IR and this may be the basis of the protection from cell death. Jak2-ERK phosphorylation may be an important signal in this process.  相似文献   

16.
Activation of matrix metalloproteinase 2 (MMP-2) has been shown to play a significant role in the behavior of cancer cells, affecting both migration and invasion. The activation process requires multimolecular complex formation involving pro-MMP-2, membrane type 1-MMP (MT1-MMP), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because calcium is an important regulator of keratinocyte function, we evaluated the effect of calcium on MMP regulation in an oral squamous cell carcinoma line (SCC25). Increasing extracellular calcium (0.09-1.2 mm) resulted in a dose-dependent increase in MT1-MMP-dependent pro-MMP-2 activation. Despite the requirement for MT1-MMP in the activation process, no changes in MT1-MMP expression, cell surface localization, or endocytosis were apparent. However, increased generation of the catalytically inactive 43-kDa MT1-MMP autolysis product and decline in the TIMP-2 levels in conditioned media were observed. The decrease in TIMP-2 levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that calcium promotes recruitment of TIMP-2 to MT1-MMP on the cell surface. Despite the decline in soluble TIMP-2, no accumulation of TIMP-2 in cell lysates was seen. Blocking TIMP-2 degradation with bafilomycin A1 significantly increased cell-associated TIMP-2 levels in the presence of high calcium. These data suggest that the decline in TIMP-2 is because of increased calcium-mediated MT1-MMP-dependent degradation of TIMP-2. In functional studies, increasing calcium enhanced MMP-dependent cellular migration on laminin-5-rich matrix using an in vitro colony dispersion assay. Taken together, these results suggest that changes in extracellular calcium can regulate post-translational MMP dynamics and thus affect the cellular behavior of oral squamous cell carcinoma.  相似文献   

17.
Glucocorticoids (GCs) are used in the treatment of neuroinflammatory diseases such as multiple sclerosis. Several studies have demonstrated the beneficial effect of GCs on the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). We could demonstrate that all four known TIMPs are present at the blood-brain barrier (BBB) endothelium. Hydrocortisone (HC) selectively upregulates TIMP-3 while TIMP-1, TIMP-2 and TIMP-4 were downregulated on the mRNA-level. This effect could be completely reversed by the glucocorticoid receptor inhibitor mifepristone (Mife). On the protein-level all TIMPs could be detected in the apical supernatants whereas in the isolated extracellular matrix (ECM) only TIMP-3 was found. The application of HC led to a strong enrichment of TIMP-3 in the ECM. Our findings demonstrate that HC directly targets TIMP-3 at the BBB assuming a protective role against matrix disruption and thus to guarantee the barrier integrity.  相似文献   

18.
The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.  相似文献   

19.
Matrix metalloproteinases (MMPs) and adamalysins (ADAMs) cleave many extracellular proteins, including matrix, growth factors, and receptors. We profiled the RNA levels of every MMP, several ADAMs, and inhibitors of metalloproteinases (TIMPs and RECK) in numerous mouse tissues during development and in the uterus during pregnancy. Observations include: most secreted MMPs are expressed at low to undetectable levels in tissues, whereas membrane-bound MMPs, ADAMs and inhibitors are abundant; almost every proteinase and inhibitor is present in the uterus or placenta at some time during gestation; the mouse collagenases mColA and mColB are found exclusively in the uterus and testis; and each tissue has its unique signature of proteinase and inhibitor expression.  相似文献   

20.
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