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. (+info)
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. (+info)
Matrix metalloproteinases (MMPs) regulate fibrin-invasive activity via MT1-MMP-dependent and -independent processes.
Cross-linked fibrin is deposited in tissues surrounding wounds, inflammatory sites, or tumors and serves not only as a supporting substratum for trafficking cells, but also as a structural barrier to invasion. While the plasminogen activator-plasminogen axis provides cells with a powerful fibrinolytic system, plasminogen-deleted animals use alternate proteolytic processes that allow fibrin invasion to proceed normally. Using fibroblasts recovered from wild-type or gene-deleted mice, invasion of three-dimensional fibrin gels proceeded in a matrix metalloproteinase (MMP)-dependent fashion. Consistent with earlier studies supporting a singular role for the membrane-anchored MMP, MT1-MMP, in fibrin-invasive events, fibroblasts from MT1-MMP-null mice displayed an early defect in invasion. However, MT1-MMP-deleted fibroblasts circumvented this early deficiency and exhibited compensatory fibrin-invasive activity. The MT1-MMP-independent process was sensitive to MMP inhibitors that target membrane-anchored MMPs, and further studies identified MT2-MMP and MT3-MMP, but not MT4-MMP, as alternate pro-invasive factors. Given the widespread distribution of MT1-, 2-, and 3-MMP in normal and neoplastic cells, these data identify a subset of membrane-anchored MMPs that operate in an autonomous fashion to drive fibrin-invasive activity. (+info)
Malignant conversion of non-tumorigenic murine skin keratinocytes overexpressing PACE4.
Proprotein convertases (PCs) have been implicated in tumor cell invasion by processing a variety of substrates including matrix metalloproteinases (MMPs). PACE4, a member of the family of PCs was shown to enhance mouse skin carcinoma progression by increasing tumor cell invasiveness. However, the effects of PACE4 on malignant conversion have not been investigated. In the present study we address the possible role of PACE4 as a trigger of malignant conversion by transfecting with a full-length PACE4 cDNA, three keratinocyte cell lines with no or little tumorigenic potential, i.e. non-tumorigenic BALB/MK-2 cells, tumorigenic non-invasive MT1/2 cells and tumorigenic moderately invasive p117 mouse skin keratinocytes. Overexpression of PACE4 led to a significant increase in the processing of stromelysin-3, a well-characterized substrate of this PC. When assayed for invasive ability, the PACE4-transfected cells were invasive both in vitro and in vivo, whereas their control counterparts were not. In addition, an enhanced processing ability of MT2-MMP a known substrate of PCs was detected in the PACE4-transfected cells. This was accompanied by MMP-2 and MMP-9 activation in PACE4 transfectants. Invasion and MMP processing were remarkably reduced when PACE4 was inhibited with a specific antibody. By triggering the processing of crucial invasion-related proteases, PACE4 is not only able to enhance the invasive ability of malignant cells as demonstrated previously, but also played a significant role in converting non-invasive keratinocytes into malignant cells. (+info)
Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs).
Macro- and microvascular endothelial cells (EC) formed tubular structures when cultured within a 3D fibrin matrix, a process that was enhanced by vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), hepatocyte growth factor/scatter factor (HGF/SF) and an angiogenic cocktail composed of nine angiogenic factors. Endothelial tubulogenesis was also increased in co-culture with tumour cells such as U87 glioma cells, but not with non-tumorigenic cell types such as Madin-Darby canine kidney (MDCK) epithelial cells. VEGF/FGF-2-stimulated tube formation was dependent on metalloproteinase function [it is inhibited by the addition of tissue inhibitor of metalloproteinases-2 (TIMP-2)], whereas aprotinin, E64 [trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane] and pepstatin had no effect. In addition, TIMP-4 also inhibited tubulogenesis, but TIMP-1 or the C-terminal haemopexin domain of matrix metalloproteinase-2 (MMP-2) (PEX) and an anti-MMP-2 function-blocking antibody were unable to block tube formation. This suggests that MMP-2 and other soluble MMPs are not essential for tubulogenesis in fibrin gels, instead TIMP-1-insensitive MMPs, such as members of the membrane type-MMPs (MT-MMP) sub-group (MT1-, MT2-, MT3- or MT5-MMP), are required for this process. Further support for a role for MT1-MMP in endothelial tubulogenesis is that recombinant Y36G N-terminal TIMP-2 mutant protein, which retains an essentially unaltered apparent inhibition constant (K(i)(app)) for several MMPs compared to wild-type N-TIMP-2 but is a 40-fold poorer inhibitor of MT1-MMP, was unable to block tubulogenesis. Furthermore, when EC were cultured within fibrin gels, the mRNA levels of several MMPs (including MT1-MMP, MT2-MMP, MT3-MMP and MMP-2) increased during tubulogenesis. Therefore MT-MMPs and specifically MT1-MMP are likely candidates for involvement during endothelial tubulogenesis within a fibrin matrix, and thus their blockade may be a viable strategy for inhibition of angiogenesis. (+info)
The low density lipoprotein receptor-related protein LRP is regulated by membrane type-1 matrix metalloproteinase (MT1-MMP) proteolysis in malignant cells.
We demonstrate that the presentation of LRP and the subsequent uptake of its ligands by malignant cells are both strongly regulated by MT1-MMP. Because LRP is essential for the clearance of multiple ligands, these findings have important implications for many pathophysiological processes including the pericellular proteolysis in neoplastic cells as well as the fate of the soluble matrix-degrading proteases such as MMP-2. MT1-MMP is a key protease in cell invasion and a physiological activator of MMP-2. Cellular LRP consists of a non-covalently associated 515-kDa extracellular alpha-chain (LRP-515) and an 85-kDa membrane-spanning beta-chain, and plays a dual role as a multifunctional endocytic receptor and a signaling molecule. Through the capture and uptake of several soluble proteases, LRP is involved in the regulation of matrix proteolysis. LRP-515 associates with the MT1-MMP catalytic domain and is highly susceptible to MT1-MMP proteolysis in vitro. Similar to MT1-MMP, the metalloproteinases MT2-MMP, MT3-MMP and MT4-MMP also degrade LRP. The N-terminal and C-terminal parts of the LRP-515 subunit are resistant and susceptible, respectively, to MT1-MMP proteolysis. In cells co-expressing LRP and MT1-MMP, the proteolytically competent protease decreases the levels of cellular LRP and releases its N-terminal portion in the extracellular milieu while the catalytically inert protease co-precipitates with LRP. These events implicate MT1-MMP, not only in the activation of MMP-2, but also in the mechanisms that control the subsequent fate of MMP-2 in cells and tissues. (+info)
The shedding of betaglycan is regulated by pervanadate and mediated by membrane type matrix metalloprotease-1.
Betaglycan is a membrane-anchored proteoglycan that binds transforming growth factor-beta (TGF-beta) via its core protein. A soluble form of betaglycan can be released by proteolytic cleavage (also known as shedding) of the membrane-bound form, yielding soluble betaglycan. The mechanism leading to the generation of soluble betaglycan is poorly understood. Because the membrane and soluble forms of betaglycan have opposite effects regulating the availability of TGF-beta, it is important to characterize the shedding of betaglycan further. Here we present evidence showing that in certain cell types, pervanadate, a general tyrosine phosphatase inhibitor, induces the release of the previously described fragment that encompasses almost the entire extracellular domain of betaglycan (sBG-120). In addition, treatment with pervanadate unveils the existence of a novel 90-kDa fragment (sBG-90). Noticeably, the cleavage that generates sBG-90 is mediated by a tissue inhibitor of metalloprotease-2-sensitive protease. Overexpression of all membrane type matrix metalloproteases (MT-MMPs) described to date indicates that MT1-MMP and MT3-MMP are endowed with ability to generate sBG-90. Furthermore, the patterns of expression of different MT-MMPs in the cell lines used in this study suggest that MT1-MMP is the protease involved in the shedding of sBG-90. Overexpression of MT1-MMP in COS-1 cells, which do not express detectable levels of this metalloprotease, confirms the feasibility of this hypothesis. Unexpectedly, during the course of these experiments, we observed that MT2-MMP decreases the levels of MT1-MMP and betaglycan. Finally, binding competition experiments indicate that, similar to the wild type membrane betaglycan, sBG-90 binds the TGF-beta2 isoform with greater affinity than TGF-beta1, suggesting that once released, it could affect the cellular availability of TGF-beta. (+info)
Expression of matrix metalloproteinases and their inhibitors in medulloblastomas and their prognostic relevance.
PURPOSE AND EXPERIMENTAL DESIGN: The cellular mechanisms leading to metastatic disease in medulloblastoma (MB), the most common malignant brain tumor in childhood, are mainly unknown. Recently, however, the involvement of matrix metalloproteinases (MMPs) has been suggested. We examined the expression and localization of four MMPs-MMP-2 and -9, membrane-type 1 and 2 MMP (MT1- and MT2-MMP)-and correlated the data with those for their main inhibitors, tissue inhibitors of metalloproteinases (TIMP-1, -2, and -3), in 83 classical and 18 desmoplastic MBs. RESULTS: Independent of the histological subtype, MMP-2 expression was found in a small percentage of tumors, whereas MMP-9 and MT1- or MT2-MMP were expressed in >75% of tumor samples. The expression of TIMP-1, -2, and -3, on the other hand, was found to depend on the histological subtype: TIMP-3 was often found in classical MB, whereas TIMP-2 was often expressed in desmoplastic MB (P = 0.007-0.001). In addition, both TIMP-3 and -2 correlated significantly with the expression of all studied metalloproteinases except MMP-2. TIMP-1, detected only in classical MB in a low percentage, was the only TIMP that correlated with the expression of MMP-2. Kaplan-Meier estimation revealed significantly reduced long-term survival of patients with strong MMP expression in tumor samples. In multivariate logistic regression analysis, however, the prognosis was significantly determined only by clinical parameters. CONCLUSIONS: TIMP-3 and -2 expression is highly correlated with histological subtypes of MBs and strongly associated with the expression of certain MMPs. The expression of TIMPs and MMPs, however, does not determine prognosis independently of clinical parameters. (+info)