Involvement of matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases and apoptosis in tissue remodelling in the sheep placenta

Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi.     

Matrix metalloproteinases 2 and 9 as the factors of head and neck tumor metastases

The levels of metalloproteinases (MMP-2,-9), their tissue inhibitors (TIMP-1,-2) and extracellular matrix metalloproteinase inducer (EMMPRIN) were studied in tumor tissue and blood serum from patients with head and neck squamous cell carcinoma. Immunohistochemical investigation showed much higher expression of MMP-9 and TIMP-1 in tumor tissue compared with MMP-2 and TIMP-2. There was different distribution of the investigated parameters (except TIMP-1) in cancer cells and stroma. Accumulation of MMP-2, MMP-9, and TIMP-2 was found mainly in cell elements (fibrocytes, leukocytes, etc.) and in stromal extracellular space. Expression of EMMPRIN was significantly higher in tumor cells than in stromal cells. It is possible that carcinoma cells express EMMPRIN, which may increase MMP production by surrounding cells. There was significant decrease of TIMP-1 expression in carcinoma cells with N1 grade of metastasis than in tumors without metastasis. The level of TIMP-1 in blood serum from patients with tumor metastases to regional lymph nodes was lower than in serum from patients without metastases. Thus, MMP-9 and TIMP-1 play an important role in the development of head and neck squamous cell carcinoma and the TIMP-1 level in blood serum and cancer tissues is linked to the first grade of regional lymph node metastasis.     

Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.     

Matrix metalloproteinases 2 and 9 mediate epidermal growth factor receptor transactivation by gonadotropin-releasing hormone

Rapid engagement of the extracellular signal-regulated kinase (ERK) cascade via the Gq/11-coupled GnRH receptor (GnRHR) is mediated by transactivation of the epidermal growth factor receptor (EGFR). Here we show that the cross-talk between GnRHR and EGFR in gonadotropic cells is accomplished via gelatinases A and B (matrix metalloproteinases (MMPs) 2 and 9), identifying gelatinases as the first distinct members of the MMP family mediating EGFR transactivation by G protein-coupled receptors. Using a specific MMP2 and MMP9 inhibitor, Ro28-2653, GnRH-dependent EGFR transactivation was abrogated. Proving the specificity of the effect, transient transfection of alphaT3-1 cells with ribozymes directed against MMP2 or MMP9 specifically blocked EGFR tyrosine phosphorylation in response to GnRH stimulation. GnRH challenge of alphaT3-1 cells furthered the release of active MMP2 and MMP9 and increased their gelatinolytic activities within 5 min. Rapid release of activated MMP2 or MMP9 was inhibited by ribozyme-targeted down-regulation of MT1-MMP or MMP2, respectively. We found that GnRH-induced Src, Ras, and ERK activation were also gelatinase-dependent. Thus, gelatinase-induced EGFR transactivation was required to engage the extracellular-signal regulated kinase cascade. Activation of c-Jun N-terminal kinase and p38 MAPK by GnRH was unaffected by EGFR or gelatinase inhibition that, however, suppressed GnRH induction of c-Jun and c-Fos. Our findings suggest a novel role for gelatinases in the endocrine regulation of pituitary gonadotropes.     

Matrix metalloproteinases in inflammation

Background

Matrix metalloproteinases (MMPs) are a family of ubiquitously expressed zinc-dependent endopeptidases with broad substrate specificity and strictly regulated tissue specific expression. They are expressed in physiological situations and pathological conditions involving inflammation. MMPs regulate several functions related to inflammation including bioavailability and activity of inflammatory cytokines and chemokines. There is also evidence that MMPs regulate inflammation in tumor microenvironment, which plays an important role in cancer progression.

Scope of review

Here, we discuss the current view on the role of MMPs in the regulation of inflammation.

Major conclusions

MMPs modulate inflammation by regulating bioavailability and activity of cytokines, chemokines, and growth factors, as well as integrity of physical tissue barriers. MMPs are also involved in immune evasion of tumor cells and in regulation of inflammation in tumor microenvironment.

General significance

There is increasing evidence for non-matrix substrates of MMPs that are related to regulation of inflammatory processes. New methods have been employed for identification of the substrates of MMPs in inflammatory processes in vivo. Detailed information on the substrates of MMPs may offer more specific and effective ways of inhibiting MMP function by blocking the cleavage site in substrate or by inhibition of the bioactivity of the substrate. It is expected, that more precise information on the MMP–substrate interaction may offer novel strategies for therapeutic intervention in inflammatory diseases and cancer without blocking beneficial actions of MMPs. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Dynamic in vivo changes in tissue inhibitors of metalloproteinases 1 and 2, and matrix metalloproteinases 2 and 9, during prostaglandin F(2alpha)-induced luteolysis in sheep

Prostaglandin F(2alpha) (PGF(2alpha)) typically initiates a cascade of events that leads to the functional and structural demise of the corpus luteum. A sheep model was used in which a 1-h, systemic infusion of PGF(2alpha) (20 microg/min) is given at midcycle. Such an infusion mimics the onset of spontaneous luteolysis by causing a transient decrease in peripheral plasma progesterone, which reaches a nadir ( approximately 60% of controls) at 8 h but returns to control levels by 16-24 h. We investigated whether PGF(2alpha) also influenced the endogenous protein levels of tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and matrix metalloproteinases, MMP-2 and MMP-9, all of which have been implicated in remodeling of the extracellular matrix (ECM). Corpora lutea (Day 11) were collected at 0 h and at 1, 8, 16, and 24 h post-PGF(2alpha) infusion (n = 3 sheep at each time). Immunoblot analysis revealed an immediate and precipitous decline in TIMP-1 (30 kDa) and TIMP-2 (19 kDa) protein levels (60% and 90%, respectively; P < 0.05) at the 1-h time point and remained depressed at 8 h (P < 0.05). Gelatin zymography and other procedures identified three MMPs (85, 70, and 64 kDa), which were shown to be the latent form of MMP-9 and the active and latent forms of MMP-2, respectively. In contrast to the rapid decrease in TIMP-1 and -2 levels, an increase in MMP-2 activity (165% of controls, P < 0.05) occurred at 8 h, which corresponded to the nadir in plasma progesterone. These early changes in TIMPs and MMPs indicate that alterations in the structure of the ECM by PGF(2alpha) may play a hitherto unsuspected role in the subsequent process of functional luteolysis.     

Matrix metalloproteinases and angiogenesis

Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissue. MMPs participate in this remodeling of basement membranes and ECM. However, it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM-bound angiogenic growth factors, by exposing cryptic proangiogenic integrin binding sites in the ECM, by generating promigratory ECM component fragments, and by cleaving endothelial cell-cell adhesions. MMPs can also contribute negatively to angiogenesis through the generation of endogenous angiogenesis inhibitors by proteolytic cleavage of certain collagen chains and plasminogen and by modulating cell receptor signaling by cleaving off their ligand-binding domains. A number of inhibitors of MMPs that show antiangiogenic activity are already in early stages of clinical trials, primarily to treat cancer and cancer-associated angiogenesis. However, because of the multiple effects of MMPs on angiogenesis, careful testing of these MMP inhibitors is necessary to show that these compounds do not actually enhance angiogenesis.     

Matrix metalloproteinases in development and disease

Matrix metalloproteinases (MMPs) are key modulators of many biological processes during pathophysiological events, such as skeletal formation, angiogenesis, cellular migration, inflammation, wound healing, coagulation, lung and cardiovascular diseases, arthritis, and cancer. Twenty-four members of the MMP family have been identified in humans, degrading many components of the extracellular matrix, cellular receptors, and cytokines. This review describes the molecular structure, activation and inhibition, and substrate specificity of MMPs, and their biological function in development and disease.     

Matrix metalloproteinases in lung biology

Despite much information on their catalytic properties and gene regulation, we actually know very little of what matrix metalloproteinases (MMPs) do in tissues. The catalytic activity of these enzymes has been implicated to function in normal lung biology by participating in branching morphogenesis, homeostasis, and repair, among other events. Overexpression of MMPs, however, has also been blamed for much of the tissue destruction associated with lung inflammation and disease. Beyond their role in the turnover and degradation of extracellular matrix proteins, MMPs also process, activate, and deactivate a variety of soluble factors, and seldom is it readily apparent by presence alone if a specific proteinase in an inflammatory setting is contributing to a reparative or disease process. An important goal of MMP research will be to identify the actual substrates upon which specific enzymes act. This information, in turn, will lead to a clearer understanding of how these extracellular proteinases function in lung development, repair, and disease.     

Matrix metalloproteinases in arthritic disease

The role of matrix metalloproteinases in the degradative events invoked in the cartilage and bone of arthritic joints has long been appreciated and attempts at the development of proteinase inhibitors as potential therapeutic agents have been made. However, the spectrum of these enzymes orchestrating connective tissue turnover and general biology is much larger than anticipated. Biochemical studies of the individual members of the matrix metalloproteinase family are now underway, ultimately leading to a more detailed understanding of the function of their domain structures and to defining their specific role in cellular systems and the way that they are regulated. Coupled with a more comprehensive and detailed study of proteinase expression in different cells of joint tissues during the progress of arthritic diseases, it will be possible for the future development and application of highly specific proteinase inhibitors to be directed at specific key cellular events.     

Matrix metalloproteinases and collagen catabolism

The matrix metalloproteinase (MMP)/matrixin family has been implicated in both normal tissue remodeling and a variety of diseases associated with abnormal turnover of extracellular matrix components. The mechanism by which MMPs catabolize collagen (collagenolysis) is still largely unknown. Substrate flexibility, MMP active sites, and MMP exosites all contribute to collagen degradation. It has recently been demonstrated that the ability to cleave a triple helix (triple-helical peptidase activity) can be distinguished from the ability to cleave collagen (collagenolytic activity). This suggests that the ability to cleave a triple helix is not the limiting factor for collagenolytic activity-the ability to properly orient and potentially destabilize collagen is. For the MMP family, the catalytic domain can unwind and cleave a triple-helical structure, while the C-terminal hemopexin-like domain appears to be responsible for properly orienting collagen and destabilizing it to some degree. It is also possible that exosites within the catalytic and/or C-terminal hemopexin-like domain may exclude some MMPs from cleaving collagen. Overall, it appears that many proteases of distinct mechanisms possess triple-helical peptidase activity, and that convergent evolution led to a few proteases possessing collagenolytic activity. Proper orientation and distortion of the triple helix may be the key factor for collagenolysis.     

基质金属蛋白酶与类风湿性关节炎

类风湿性关节炎是一种慢性炎症性自身免疫性疾病,其特点是软骨和骨骼的不可逆损伤。基质金属蛋白酶参与结缔组织重塑,在关节环境炎症级联中起着重要作用,或可成为类风湿性关节炎治疗的潜在新靶点。本文就其在类风湿性关节炎发生发展和治疗中的研究进展作一综述。     

Comparative permeability of canine visceral and parietal pleura

To determine the permeability of canine pleural mesothelium, visceral and intercostal parietal pleura from mongrel dogs was carefully stripped from the underlying tissue and mounted as a planar sheet in a Ussing-type chamber. The hydraulic conductivity (Lp) was determined from the rate of volume flux in response to hydrostatic pressure gradients applied to either the mucosal or serosal surface of the pleural membrane. The diffusional permeability (Pd) of radiolabeled water, sucrose, inulin, and albumin was determined under equilibrium conditions from the unidirectional tracer flux. The Lp of the visceral pleura was 0.39 +/- 0.032 (SE) X 10(-4) ml.s-1.cmH2O-1.cm-2 and that Lp of parietal pleura was 1.93 +/- 0.93 X 10(-4) ml.s-1.cmH2O-1.cm-2 (P less than 0.001). The Pd of the visceral pleura ranged from 12.21 +/- 0.45 X 10(-4) cm/s for 3H2O to 0.34 +/- 0.03 X 10(-4) cm/s for [3H]albumin. The Pd of the parietal pleura for water and sucrose was similar to that of the visceral membrane, whereas its Pd for the larger inulin and albumin molecules was greater than that of visceral pleura (P less than 0.01). A spontaneous potential difference could not be detected across either membrane. The relatively higher parietal pleural Lp and Pd for larger solutes is probably due to the presence of stomata in this membrane. These results indicate that both the parietal and the visceral pleura are extremely permeable tissues which offer little resistance to water and solute flux.     

Matrix metalloproteinases MMP-2 and MMP-9 of wound and burn exudates and their effect on extracellular matrix proteins

The dynamics of matrix metalloproteinases (MMP), as well as of fibronectin concentration in wound and burn fluids was traced. The wound fluid proteolytic activity was studied by gelatin zymography method. The data on degradation of fibronectin and various laminin isoforms by wound fluid proteases show that laminin-1, laminin-2/4 and fibronectin were degraded by wound fluid into small fragments. Remodelling of extracellular matrix proteins occurs. Dynamics of MMP-2 and MMP-9 content in wound or burn fluids as well as that of adhesive protein fibronectin content could be used as a base for development of method of controlling the extracellular matrix remodelling process.     

Matrix metalloproteinase-9 in homocysteine-induced intestinal microvascular endothelial paracellular and transcellular permeability

Although elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with inflammatory bowel disease (IBD), the mechanism of Hcy action is unclear. In the present study, we tested the hypothesis that HHcy activates matrix metalloproteinase-9 (MMP-9), which in turn enhances permeability of human intestinal microvascular endothelial cell (HIMEC) layer by decreasing expression of endothelial junction proteins and increasing caveolae formation. HIMECs were grown in Transwells and treated with 500 μM Hcy in the presence or absence of MMP-9 activity inhibitor. Hcy-induced permeability to FITC-conjugated bovine serum albumin (FITC-BSA) was assessed by measuring fluorescence intensity of solutes in the Transwells' lower chambers. The cell-cell interaction and cell barrier function was estimated by measuring trans-endothelial electrical impedance. Confocal microscopy and flow cytometry were used to study cell junction protein expressions. Hcy-induced changes in transcellular transport of HIMECs were estimated by observing formation of functional caveolae defined as caveolae labeled by cholera toxin and antibody against caveolin-1 and one that have taken up FITC-BSA. Hcy instigated HIMEC monolayer permeability through activation of MMP-9. The increased paracellular permeability was associated with degradation of vascular endothelial cadherin and zona occludin-1 and transcellular permeability through increased caveolae formation in HIMECs. Elevation of Hcy content increases permeability of HIMEC layer affecting both paracellular and transcellular transport pathways, and this increased permeability was alleviated by inhibition of MMP-9 activity. These findings contribute to clarification of mechanisms of IBD development.