Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MMP should exist as oligomers on the cell surface. To better understand the functions of MT1-MMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP. Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface. The presence of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells into the mature proteinase capable of activating pro-MMP-2 and stimulating cell locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydroxamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independent, alternative pathway of MT1-MMP activation in breast carcinoma cells. The expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part of MT1-MMP and the intracellular milieu.     

Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis

Since their introduction almost a century ago, chick embryo model systems involving the technique of chorioallantoic grafting have proved invaluable in the in vivo studies of tumor development and angiogenesis and tumor cell dissemination. The ability of the chick embryo’s chorioallantoic membrane (CAM) to efficiently support the growth of inoculated xenogenic tumor cells greatly facilitates analysis of human tumor cell metastasis. During spontaneous metastasis, the highly vascularized CAM sustains rapid tumor formation within several days following cell grafting. The dense capillary network of the CAM also serves as a repository of aggressive tumor cells that escaped from the primary tumor and intravasated into the host vasculature. This spontaneous metastasis setting provides a unique experimental model to study in vivo the intravasation step of the metastatic cascade. During experimental metastasis when tumor cells are inoculated intravenously, the CAM capillary system serves as a place for initial arrest and then, for tumor cell extravasation and colonization. The tissue composition and accessibility of the CAM for experimental interventions makes chick embryo CAM systems attractive models to follow the fate and visualize microscopically the behavior of grafted tumor cells in both spontaneous and experimental metastasis settings.     

Processing of integrin alpha(v) subunit by membrane type 1 matrix metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase.

Recently, we have shown that membrane type 1 matrix metalloproteinase (MT1-MMP) exhibits integrin convertase activity. Similar to furin-like proprotein convertases, MT1-MMP directly processes a single chain precursor of alpha(v) integrin subunit (pro-alpha(v)) into the heavy and light alpha-chains connected by a disulfide bridge. To evaluate functionality of MT1-MMP-processed integrins, we examined breast carcinoma MCF7 cells co-expressing alpha(v)beta(3) integrin with either the wild type or mutant MT1-MMP in a variety of migration and adhesion tests. Specific inhibitors of proprotein convertases and MMP were employed in our cell system to attenuate the individual pathways of pro-alpha(v) maturation. We present evidence that MT1-MMP cleavage of pro-alpha(v) in the cells did not affect RGD-ligand binding of the resulting alpha(v)beta(3) integrin but enhanced outside-in signal transduction through a focal adhesion kinase pathway. Enhanced tyrosine phosphorylation of focal adhesion kinase in cells co-expressing MT1-MMP and alpha(v)beta(3) integrin contributed to efficient adhesion and, especially, migration of cells on vitronectin, a ligand of alpha(v)beta(3) integrin. These mechanisms underscore the significance of a coordinated interplay between MT1-MMP and alpha(v)beta(3) integrin in tumor cells and identify downstream signaling pathways resulting from their interactions. Regulation of integrin maturation and functionality may be an important role of MT1-MMP in tumor cells.     

Changes in the electrophoretic mobility of erythrocytes under the action of low-intensity pulsed magnetic field

Exposure of intact rats and human erythrocytes to low-intensity pulsed magnetic field leads to similar biphasic changes in the electrophoretic mobility of erythrocytes; this is accompanied by modification of their membrane and cytoskeletal protein spectrum.     

Correction of Metabolic Indicators of Erythrocytes and Myocardium Structure with Ozonized Red Blood-Cell Mass

The early posttransfusion period after acute blood loss using erythromass without ozone was characterized by decreased electrophoretic mobility of erythrocytes (EPME), ATP, 2,3-diphosphoglycerate (2,3- DPG) concentration, Na⁺-K⁺-ATPase activity, and increased concentration of malonic dialdehyde (MDA) and catalase activity in erythrocytes. Most parameters gradually were restored over 5 days, but a reduced concentration of 2,3-DPG was observed throughout the entire experiment with transfusion of erythromass without ozone. Transfusion of the ozonized erythrocyte mass 1 h after its administration caused an increase in 2,3- DPG concentration in erythrocytes circulating in the vascular bed. Twenty-four hours later, increased catalase activity, EPPE, and ATP concentration were registered, and, after 5 days, enhanced Na⁺-K⁺-ATPase activity was registered. Their values were maintained until the end of the experiment at a higher level than in animals that had undergone transfusion of the nonozonized erythromass. Ozonized erythrocyte mass improves the functional metabolic state of erythrocytes, promotes an earlier recovery of the oxygen transport in blood, and limits the damage to the microcirculatory bed of the myocardium and cardiomyocytes during blood loss.     

Activity-based protein profiling implicates urokinase activation as a key step in human fibrosarcoma intravasation

Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. This study utilizes activity-based protein profiling to compare the active serine hydrolase repertoire in high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line to determine which enzyme(s) play a role in intravasation. Activity-based protein profiling revealed multiple serine hydrolases with altered activity between HT-hi/diss and HT-lo/diss cells, with the largest difference being the activity of urokinase-type plasminogen activator (uPA). Levels of inactive uPA zymogen were similar between the two cell variants, but only HT-hi/diss conditioned medium contained active uPA, suggesting that uPA activation may contribute to the enhanced intravasation of HT-hi/diss cells. To analyze the role of uPA activity specifically in the process of intravasation, we grafted cells from the two HT-1080 variants onto the chorioallantoic membrane of chick embryos and measured levels of tumor cell intravasation in the distal chorioallantoic membrane using quantitative human-specific Alu PCR. Inhibition of uPA activity with natural (plasminogen activator inhibitor-1) or synthetic (amiloride) inhibitors diminished HT-hi/diss Matrigel invasion in vitro and intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression.     

A Novel Mode of Intervention with Serine Protease Activity: TARGETING ZYMOGEN ACTIVATION*

Serine proteases are secreted from cells as single-chain zymogens, typically having activities orders of magnitude lower than those of the mature two-chain enzymes. Activation occurs by a conformational change initiated by cleavage of a specific peptide bond. We have derived a monoclonal antibody (mAb-112) which binds with subnanomolar affinity to pro-uPA, the zymogen form of urokinase-type plasminogen activator (uPA). We mapped the epitope of the antibody to the autolysis loop, one of the structural elements known to change conformation during zymogen activation. A mechanistic evaluation with biophysical methods elucidated a novel bifunctional inhibitory mechanism whereby mAb-112 not only delays the proteolytic conversion of single-chain pro-uPA into the two-chain form but also subsequently averts the conformational transition to a mature protease by sequestering the two-chain form in a zymogen-like, noncatalytic state. Functional studies employing two variants of human HT-1080 cells, exhibiting high and low levels of dissemination in a chorioallantoic membrane assay, demonstrate that mAb-112 is an effective inhibitor of tumor cell intravasation. These findings show that pharmacological interference with zymogen activation is a plausible and robust means to regulate uPA activity and the downstream effects of plasminogen activation in the spread of cancer and other processes of pathological tissue remodeling. A strategy that targets regions related to pro-enzyme activation likely provide a unique inhibitor-protease interaction surface and is, thus, expected to enhance the chances of engineering high inhibitor specificity. Our results provide new information about the structural flexibility underlying the equilibrium between active and inactive forms of serine proteases.In nature a key mechanism for regulation of serine proteases with a trypsin-like fold is the activation of secreted zymogens or proenzymes, which typically have activities orders of magnitude lower than the mature enzymes. Zymogen activation is the central step in natural protease cascade regulation, allowing for rapid amplification of the activation signal. The catalytic activity of a zymogen relative to the mature protease can generally be thought of as a problem of equilibrium between active and inactive conformational states of the protease domain. Zymogen activation generally occurs by cleavage of the bond between amino acid residues 15 and 16.² The liberated amino terminus inserts into a hydrophobic binding cleft of the catalytic domain forming, in addition to hydrophobic interactions, a salt bridge to the side chain of Asp¹⁹⁴ which stabilizes the substrate binding pocket and oxyanion hole in a catalytically productive conformation. Conformational changes after cleavage involves four disordered regions of the activation domain, including the activation loop (residues 16-21), the autolysis loop (residues 142-152), the oxyanion stabilizing loop (residues 184-194), and the S1 entrance frame (residues 216-223) (Fig. 1A) (for reviews, see Refs. 1-3).Open in a separate windowFIGURE 1.Three-dimensional structure of uPA. A, overview of the three-dimensional structure of the serine protease domain of active uPA, displayed as ribbons. Depicted as sticks are the residues Ile¹⁶, Asp¹⁹⁴, and Ser¹⁹⁵. The activation domain, i.e. the activation loop (residues 16-21), the autolysis loop (residues 142-152), the oxyanion stabilizing loop (residues 184-193), and the S1 entrance frame (residues 216-223) are colored green. B, the epitope of mAb-112, displayed on a surface presentation of the serine protease domain of active uPA. Alanine substitution of residues depicted in red resulted in a significant change in the affinity to mAb-112, whereas alanine substitution of residues depicted in blue did not. C, a close up view of the autolysis loop (residues Gly¹⁴¹ to Lys¹⁵⁶) and residues implicated in the binding of mAb-112. All figures were constructed with Pymol on the basis of the coordinates given in the PDB entry 1C5W.Several proteases contribute to a variety of pathophysiological states, thus stimulating considerable interest in the design of specific inhibitors for pharmacological intervention. Nonetheless, classical development of small molecule inhibitors of serine proteases has proved a daunting task, with only limited success in engineering inhibitors with high affinity and specificity for related proteases possessing conserved active site architecture and P1³ specificity (4, 5). Thus far targeting zymogen activation instead of the mature protease has been a greatly under-exploited strategy in therapeutic regulation of protease activity. This approach provides an opportunity to target more unique interaction surfaces, thereby increasing inhibitor specificity, and ultimately offering novel inhibitory mechanisms. In addition, a more efficient inhibition is expected by targeting enzymes functioning high up in a catalytic cascade.A serine protease of particular relevance for pursuing therapeutic intervention is urokinase-type plasminogen activator (uPA),⁴ which catalyzes the conversion of plasminogen to the active protease plasmin, which in turn acts directly on the degradation of extracellular matrix proteins (6). Abnormal expression of uPA is implicated in tissue remodeling in several pathological conditions, including rheumatoid arthritis, allergic vasculitis, and xeroderma pigmentosum. In particular, uPA is central to the invasive capacity of malignant tumors (6). As with all trypsin-like proteases, uPA has a catalytic serine protease domain with surface-exposed loops around residues 37, 60, 97, 110, 170, and 185. Besides the catalytic domain, uPA has an amino-terminal extension consisting of a kringle domain and an epidermal growth factor domain. The latter domain functions in binding to the cell surface-anchored uPA receptor (uPAR) (6). Several proteases including plasmin (6), glandular kallikrein (7), matriptase (8), and hepsin (9) can catalyze the activation of the zymogen, pro-uPA.A number of inhibitors targeting the proteolytic activity of uPA have been developed, such as small organochemical molecules, peptides, and monoclonal antibodies, with a perspective on their use for elucidating the pathophysiological functions of its various molecular interactions and generating leads during drug development. The most specific inhibitors to date appear to be those derived from antibodies and peptidyl inhibitors, which utilize binding sites involving surface loops of uPA and extended exosite interactions to drive selectivity and specificity (for reviews, see Refs. 4 and 5).Here we present evidence that targeting zymogen activation is an effective means to regulate protease activity. This conclusion was realized through the development and biochemical analysis of an inhibitory monoclonal antibody, referred to as monoclonal antibody (mAb)-112, which not only delays cleavage of pro-uPA but acts to stabilize the activated two-chain protease in a non-catalytic conformation by restricting the conformational mobility of the activation domain. Characterization of mAb-112 further provides new insights into the flexibility of protease domains and uPA zymogen activation mechanisms. Moreover, mAb-112 was shown to efficiently inhibit human tumor cell intravasation, a step in the metastatic cascade in which activation of pro-uPA was previously implicated as a key event (10).     

Influence of combined DC and AC magnetic fields on rat behavior.

The action of combined parallel static (DC) and alternating (AC) magnetic fields at the cyclotron frequencies for different biologically active ions, specifically, calcium, sodium, potassium, chlorine, magnesium and lithium, on rat behavior in the "open field" were investigated. It was shown that the DC and AC fields at the calcium cyclotron frequency lower the locomotor and exploratory activity of the rats, whereas action of the fields at the magnesium cyclotron frequency enhances these forms of behavioral activity. The effects were qualitatively alike at the weak (50 microT) and relatively strong (500 microT) DC fields with proportional changes in the frequencies and amplitudes of the AC fields. Statistically significant effects of cyclotron frequencies for other ions studied were not observed.