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PubMed2002

Transport of insulin-like growth factor-I across endothelial cell monolayers and its binding to the subendothelial matrix.

Cultured human umbilical vein endothelial cells (HUVEC) were used as a model to study transendothelial IGF-I transport, and its deposition into the extracellular matrix (ECM). Specific binding of (125)I-IGF-I to HUVEC monolayers was demonstrated, which was inhibited by aIR-3, a specific antibody directed against the IGF-I receptor. ECM-associated (125)I-IGF-I was approximately 10% of cell-bound IGF-I at 22 degrees C, and increased 4.5-fold at 37 degrees C, indicating that endothelial metabolism is required for the transport. However, neither monensin and cytochalasin B, both of which block endocytosis, nor aIR-3 did inhibit transport of (125)I-IGF-I into the ECM. In order to characterize IGF-I binding to the subendothelial ECM, HUVEC were removed nonenzymatically by treatment with Triton X-100 and ammonia. Specific, saturable binding of (125)I-IGF-I to the isolated ECM was observed, which was protease-sensitive. Antibodies directed against vitronectin inhibited IGF-I binding to the matrix by 35%, while antibodies directed against other ECM proteins had no significant influence on IGF-I binding. Using radioimmunoassays the IGF binding protein-2 was detected in the ECM, while IGFBP-1 and IGFBP-3 were below the detection limits. In order to evaluate functional aspects of IGF-I binding to the matrix, HUVEC were incubated under serum-free conditions in the absence and presence of IGF-I. Under serum-free conditions 48% of cells rounded up and started to detach after 2 hours incubation, while only 23% of the cells started to detach in the presence of IGF-I. These data indicate that IGF-I is transported via a paracellular route across endothelial cells, and becomes bound to the subendothelial ECM. Vitronection seems to be involved in binding of IGF-I to the ECM. ECM-associated IGF-I might play a role in endothelial cell survival and stability.

Ammonia↗
PubMed1997

Integrin chatter and vascular function in diabetic retinopathy.

The interplay between adhesion receptors and proteolytic cascades is crucial for cell proliferation and migration in the (patho-)physiology of vascular function. Disregulated angiogenesis in diabetic retinopathy appears to be associated with the appearance of advanced glycation end products that disturb interactions between capillary cells and extracellular matrix. Vitronectin receptor-type integrins are expressed on angiogenic endothelial cells and contribute to unwanted vascular sprouting in the retinal tissue. In a hypoxia-induced retinal neovascularization model, intervention with low molecular weight integrin antagonist resulted in significant reduction of unwanted angiogenesis indicating that this angiostatic therapy appears to be promising also for late complications in diabetes.

Diabetic Retinopathy↗
PubMed2001

Characterization of pseudoxanthoma elasticum-like lesions in the skin of patients with beta-thalassemia.

BACKGROUND: Pseudoxanthoma elasticum (PXE), an inherited disorder of unknown pathogenesis, is characterized by elastic fiber mineralization, collagen fibril alterations, and accumulation of thread material in the extracellular space. PXE-like clinical lesions have been described in patients with beta-thalassemia. OBJECTIVE AND METHODS: Dermal lesions in these two genetic disorders were compared by light and electron microscopy and by immunocytochemistry. RESULTS: In both disorders, elastic fiber polymorphism, fragmentation, and mineralization were structurally identical. Elastic fiber mineralization in beta-thalassemia was associated with vitronectin, bone sialoprotein, and alkaline phosphatase, similar to what was observed in inherited PXE. Furthermore, abnormalities of collagen fibrils and filament aggregates were identical in both disorders. In both inherited and beta-thalassemia-associated PXE, unrelated gene defects seem to induce cell metabolic abnormalities that lead to identical clinical and structural phenotypes. CONCLUSION: Data indicate that patients with beta-thalassemia may undergo important alterations of connective tissues, a better understanding of which may help in preventing clinical complications.

Adult↗
PubMed2002

Eicosanoids in sickle cell disease: potential relevance of neutrophil leukotriene B4 to disease pathophysiology.

Neutrophil activation with the release of intracellular granule contents has been observed in sickle cell disease (SCD). Because leukotriene B(4) (LTB(4)), a 5-lipoxygenase metabolite of arachidonic acid in neutrophils, is a chemoattractant and enhances neutrophil adhesion to endothelium, we assessed plasma levels of this metabolite in controls (n = 9) and individuals with SCD, SS genotype, both in basal "steady state" (n = 37) and during episodes of vaso-occlusion (n = 10) and acute chest syndrome (n = 5). Thirteen patients with SCD, SC genotype, in steady state were also studied. Although no significant differences were noted between the control (136 +/- 32 fmol/mL) and SC genotype (177 +/- 83 fmol/mL, P >.15), LTB(4) levels were markedly increased in patients with SS genotype in basal steady state (207 +/- 64 fmol/mL, P <.003) compared with those in controls. Values were further increased during vaso-occlusion (264 +/- 94 fmol/mL) and acute chest syndrome (363 +/- 124 fmol/mL). These levels were significantly different from measurements taken during steady state (P <.04 and P <.0001, respectively). No correlation was noted between LTB(4) level and total white cell or neutrophil count. Additionally, the significant correlation noted in SCD between increased levels of plasma LTB(4) and soluble L-selectin (P <.03) reflects neutrophil activation. We also observed an effect of LTB(4) on red cell-endothelial adhesion at concentrations that appear clinically relevant (1-10 pmol/mL) with concomitant up-regulation of mRNA for the endothelial vitronectin receptor. These properties of LTB(4) are relevant to disease pathophysiology, providing further evidence of the contribution of the neutrophil to the proinflammatory and proadhesive phenotype in SCD.

Adolescent↗
PubMed2000

Inhibition of phosphatidylinositol 3-kinase and protein kinase C attenuates extracellular matrix protein-induced vascular smooth muscle cell chemotaxis.

PURPOSE: Intimal hyperplasia (IH), a significant cause of vascular reconstructive failure, is characterized by abnormal vascular smooth muscle cell (VSMC) migration, proliferation, and extracellular matrix (ECM) deposition. The ECM proteins, thrombospondin-1 (TSP-1), fibronectin (Fn), and vitronectin (Vn) can induce VSMC migration; however, the cellular signaling pathways involved are not identical for each ECM protein. Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) are two enzymes that have been associated with VSMC migration. We sought to elucidate the roles of these enzymes in TSP-1-, Fn-, and Vn-stimulated VSMC migration. METHODS: Chemotaxis assays were performed by using a modified Boyden Chamber. TSP-1, Fn, or Vn (20 microg/mL) or serum-free media (SFM) was placed in the bottom wells of the chamber. Quiescent bovine aortic VSMC were preincubated with LY 294002 (100 micromol/L), a PI3K inhibitor, bisindolylmaleimide I (GF 109203X, 1 micromol/L), a PKC inhibitor, or in SFM alone for 30 minutes. VSMCs (50,000 cells per well) were then placed in the top wells of the chamber, and the assay was conducted for 4 hours at 37 degrees C. Results were recorded as the number of cells migrated per five fields (400x) and analyzed by means of the paired t test, with P value less than.05 considered to be significant (n = 3). RESULTS: The VSMC migration was significantly increased by TSP-1, Fn, and Vn. LY 294002 inhibited TSP-1-, Fn-, and Vn-stimulated VSMC migration (85% to 89%, P <.05). GF 109203X inhibited only TSP-1-stimulated migration (65%, P <.05). CONCLUSION: These results suggest that TSP-1-, Fn-, and Vn-stimulated migration is at least partially dependent on PI3K. However, only TSP-1 stimulated migration is at least partially dependent on PKC.

Animals↗
PubMed2005

Angiostatic peptides use plasma fibronectin to home to angiogenic vasculature.

A group of angiogenesis inhibitors are derived from fragments of extracellular matrix or blood proteins. Endostatin, antithrombin, and anastellin are members of this group of substances. The plasma adhesion proteins fibronectin and vitronectin serve as cofactors for these three antiangiogenic proteins. Anginex is a synthetic 33-amino acid peptide that was originally modeled to reproduce the beta-sheet structure of antiangiogenic proteins. Here, we show that anginex initiates fibronectin polymerization and is inactive in mice that lack plasma fibronectin. Anginex shares these characteristics with anastellin. Fluorescein-labeled anginex and anastellin specifically localized in angiogenic vessels in vivo. This localization was dependent on plasma fibronectin and inhibited by an Arg-Gly-Asp peptide. Thus, anginex shares with several physiological angiogenesis inhibitors a dependence on plasma adhesion proteins. The role of the adhesion protein interaction apparently is to form integrin-binding complexes that deliver the antiangiogenic proteins to sites of angiogenesis. This functional convergence of several antiangiogenic factors has important implications for antiangiogenic therapies.

Angiogenesis Inhibitors↗
PubMed2003

Activated integrin alphavbeta3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells.

Expression of adhesion receptor integrin alphavbeta3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that alphavbeta3 cooperates with matrix metalloproteinase type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated by the activation state of the integrin. Expression of activated alphavbeta3 in metastatic variants of MDA-MB 435 human breast cancer cells and primary metastatic cells from breast cancer patients strongly enhanced migration toward vitronectin and fibrinogen. This enhancement was mediated by a soluble factor produced by breast cancer cells expressing activated alphavbeta3. When transferred, this factor also up-regulated alphavbeta3-dependent migration of breast cancer cells that express the nonactivated integrin. The factor was identified as metalloproteinase MMP-9. Whereas all tested breast cancer cell variants produced latent MMP-9, only those with activated alphavbeta3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced alphavbeta3-dependent breast cancer cell migration. The migratory response was inhibited by tissue inhibitors of metalloproteinase or when MMP-9 was depleted from the inducing supernatants. The results indicate a causal relationship between the expression of activated integrin alphavbeta3 and production of enzymatically active MMP-9 in metastatic breast cancer cells. These molecules cooperate to enhance breast cancer cell migration toward specific matrix proteins, and this may contribute to the strongly enhanced metastatic capacity of breast cancer cells that express activated alphavbeta3.

Breast Neoplasms↗
PubMed1986

The effect of hexapeptides on attachment and outgrowth of mouse blastocysts cultured in vitro: evidence for the involvement of the cell recognition tripeptide Arg-Gly-Asp.

Differentiation of the nonadherent trophectoderm cells of the mammalian embryo into attachment-competent trophoblast cells appears to be a prerequisite to invasion of the uterine stroma. To investigate the molecular basis of trophoblast differentiation free of maternal environmental constraints, we used a model system in which attachment and outgrowth of trophoblast cells occurs in vitro. Recently, it was found that either fibronectin or laminin, both of which are extracellular matrix glycoproteins of the uterine stroma, will support trophoblast outgrowth in vitro. In this study we report that the outgrowth of blastocysts on fibronectin-coated dishes is inhibited in a dose-dependent manner by the presence of a hexapeptide containing the sequence Arg-Gly-Asp, which has been shown previously to be recognized by the fibronectin receptor. This peptide had no effect on laminin-mediated trophoblast outgrowth, suggesting that the trophoblasts contain different cell surface receptors for fibronectin and laminin. Trophoblast attachment and limited outgrowth also could be obtained on dishes to which the hexapeptide Gly-Arg-Gly-Asp-Ser-Pro was coupled. Under these conditions, however, outward migration of the trophoblast cells appeared to be reduced. Vitronectin, another adhesion molecule that apparently binds to cells via a cell surface receptor that recognizes Arg-Gly-Asp sequences, also was capable of supporting trophoblast outgrowth. These findings suggest that differentiation of cells of the trophectoderm into trophoblast cells with an invasive phenotype may involve the production of cell surface receptors for fibronectin and possibly for other proteins that contain the Arg-Gly-Asp recognition sequence.

Amino Acid Sequence↗
PubMed1986

T-lymphocyte differentiation and the extracellular matrix: identification of a thymocyte subset that attaches specifically to fibronectin.

A population of murine thymocytes adheres specifically to fibronectin but not to vitronectin, laminin, or collagen type I. The interaction of these thymocytes with fibronectin could be inhibited by the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which comprises the previously identified cell-attachment determinant of the molecule, suggesting that the cell attachment site on fibronectin is recognized by these cells. A similar peptide, in which the aspartate residue had been replaced with glutamate, had no effect on this adhesion. The fibronectin-adherent thymocytes were found to be cortisone-sensitive; to bind peanut agglutinin; to have a Thy-1.2+, Ia- surface phenotype; and to express H-2 antigen only weakly on their surface. In addition, approximately 80% of the fibronectin-adherent cells expressed L3T4 and 80% expressed Ly-1 on their surface, whereas greater than 95% were positive for Ly-2. The data suggest that these cells, which constitute 10% of all thymic lymphocytes, are cortical thymocytes. We propose that their adhesion to fibronectin may be important for their differentiation. The binding to fibronectin provides a means to selectively isolate these cells for study.

Animals↗
PubMed1992

T-cell activation molecule 4-1BB binds to extracellular matrix proteins.

The recently isolated 4-1BB cDNA clone encodes a cell surface protein expressed by activated T cells. Its extracellular domain is homologous to members of the nerve growth factor receptor super family and its cytoplasmic domain contains a sequence homologous to the binding site for the T-cell-specific tyrosine kinase p56lck found in the cytoplasmic domains of CD4 and CD8 alpha. At present the function of 4-1BB is not known. We prepared a 4-1BB-immunoglobulin fusion protein (4-1BB Rg). This protein was used in immunohistochemical studies to identify tissues that express the 4-1BB ligand. 4-1BB Rg bound to virtually all tissues examined, suggesting that extracellular components might function as its ligands. To explore this possibility, 4-1BB was expressed in COS cells and found to mediate the binding of fibronectin, vitronectin, laminin, and collagen VI but not of collagen I. The binding of extracellular matrix proteins to 4-1BB was not mediated by Arg-Gly-Asp (RGD) or CS-1 amino acid sequences. Experiments with overlapping proteolytic fragments of fibronectin showed that 4-1BB interacts with multiple regions of fibronectin. The interaction between extracellular matrix proteins and 4-1BB was completely blocked by the anionic carbohydrate polymer fucoidan and was partially blocked by the anionic carbohydrate polymer dextran sulfate and the glycosaminoglycan heparin sulfate but was unaffected by desulfated heparin. These results suggest that carbohydrates may play a role in mediating the 4-1BB-extracellular matrix protein adhesion.

Animals↗
PubMed1993

Pattern formation and handedness in the cytoskeleton of human platelets.

The cytoskeletal patterns of human platelets spread on a glass surface are analyzed. F-actin is arranged in patterns of parallel microfilaments, microfilaments forming triangles, or microfilaments radiating tangentially from a central ellipse or circle. Vinculin, a cytoskeletal protein, is located at both ends of the filaments. In platelets with tangentially radiating microfilaments, vinculin patches are aligned on the branches of a two-armed spiral. The spirals are always left-handed. Talin and two integrins (gpIIb-IIIa, vitronectin receptor), proteins usually associated with focal contacts in tissue culture cells, are not concentrated at the ends of microfilaments in human platelets. It is suggested that the distribution of vinculin is due to competitive aggregation of vinculin close to the inner leaflet of the ventral plasma membrane and that sites of cytoskeleton-membrane linkage are important for generating supramolecular asymmetries of biological systems.

Actin Cytoskeleton↗
PubMed1996

Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells.

Osteoclastogenesis is a complex process that is facilitated by bone marrow stromal cells (SCs). To determine if SCs are an absolute requirement for the differentiation of human hematopoietic precursors into fully mature, osteoclasts (OCs), CD34+ cells were mobilized into the peripheral circulation with granulocyte colony-stimulating factor, harvested by leukapheresis, and purified by magnetic-activated cell sorting. This procedure yields a population of CD34+ cells that does not contain SC precursors, as assessed by the lack of expression of the SC antigen Stro-1, and that differentiates only into hematopoietic cells. We found that CD34+, Stro-1- cells cultured with a combination of granulocyte/macrophage colony-stimulating factor, interleukin 1, and interleukin 3 generated cells that fulfill current criteria for the characterization of OCs, including multinucleation, presence of tartrate-resistant acid phosphatase, and expression of the calcitonin and vitronectin receptors and of pp60c-src tyrosine kinase. These OCs also expressed mRNA for the noninserted isoform of the calcitonin receptor and excavated characteristic resorption pits in devitalized bone slices. These data demonstrate that accessory SCs are not essential for human osteoclastogenesis and that granulocyte colony-stimulating factor treatment mobilizes OC precursors into the peripheral circulation.

Acid Phosphatase↗
PubMed1999

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins alphavbeta3 and alphaIIbbeta3.

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin alphavbeta3 (also known as the vitronectin receptor) or alphaIIbbeta3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin alphaIIbbeta3. Defined beta3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing alphavbeta3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.

Alleles↗
PubMed1995

Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs.

p130Cas (Cas) has been recently identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues and is stably associated with p47v-crk (v-Crk) and p60v-src (v-Src) oncogene products in cells transformed by the respective genes. Cas is a novel signaling molecule having a single Src homology (SH) 3 domain and a cluster of multiple SH2-binding motifs. While the tight association of Cas with v-Crk and v-Src is strongly suggestive of a significant role in regulating cellular transformation, the function of Cas in normal untransformed cells is totally unknown. We report here that cell adhesion to fibronectin rapidly promotes tyrosine phosphorylation of Cas in human and rat fibroblast cell lines. The response was equally induced by cell adhesion to plates coated with vitronectin, laminin, and collagen but not by cell attachment to nonspecific substrate poly-L-lysine. The kinetic profile of Cas phosphorylation was almost identical with that of tyrosine phosphorylation of focal adhesion kinase pp125FAK (Fak), which is well known to be activated subsequent to integrin-mediated cell adhesion. Adhesion-dependent Cas phosphorylation was completely inhibited by treating cells with cytochalasin D, an agent that disrupts polymerization of actin stress fibers. These results suggest that tyrosine phosphorylation of Cas is stimulated by normal cell adhesion in close association with Fak phosphorylation and the formation of actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the tyrosine phosphorylation of Cas is markedly increased in an adhesion-independent manner that is insensitive to treatment with cytochalasin D. Thus, Cas plays a role in signaling pathways mediated by cell adhesion as well as by transformation. We propose that Cas may amplify and propagate integrin-mediated signals by interacting with SH2-containing molecule(s).

Animals↗
PubMed1995

Interaction of single-chain urokinase and plasminogen activator inhibitor type 1.

Urokinase (u-PA) is synthesized and secreted as a single-chain polypeptide (single-chain u-PA, scu-PA), which has such little enzymatic activity in solution that it has been considered essentially enzymatically inert. We found that plasminogen activator inhibitor type 1 (PAI-1), the major PAI in plasma, demonstrated concentration-dependent inhibition of this solution-phase scu-PA enzymatic activity. 125I-scu-PA formed complexes with PAI-1 in a concentration- and time-dependent manner, as detected by SDS-polyacrylamide gel electrophoresis under reducing conditions. Among a given population of scu-PA molecules, all measurable enzymatic activity was inhibited by a 10-fold molar excess of PAI-1. However, at this stoichiometry, only a minority of 125I-scu-PA molecules formed SDS-stable complexes with PAI-1 (i.e. complexes that formed a covalent bond upon denaturation), even though the uncomplexed PAI-1 molecules remained competent to inhibit u-PA enzymatic activity. Neither the extent nor the time course of complex formation was altered by using PAI-1 that had been pre-incubated with native human vitronectin, compared with native PAI-1 alone. 125I-scu-PA.PAI-1 complexes that would form a covalent bond if denatured were reversible and existed in equilibrium with either non-complexed or loosely complexed reactants. These data suggest that scu-PA has more enzyme-like properties than previously appreciated and raises the possibility that it resembles single-chain tissue type-plasminogen activator in lacking a complete zymogen conformation.

Glycoproteins↗
PubMed1996

Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.

Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.

Amino Acid Sequence↗
PubMed1996

Regulation of plasminogen receptor expression on monocytoid cells by beta1-integrin-dependent cellular adherence to extracellular matrix proteins.

Plasminogen binding sites function to arm cell surfaces with the proteolytic activity of plasmin, critical for degradation of extracellular matrices. We have assessed the effects of adhesion of the representative monocytoid cell lines, THP-1 and U937, to purified extracellular matrix proteins on their expression of plasminogen receptors. After adhesion to immobilized fibronectin, adherent and nonadherent subpopulations of cells were separated. Plasminogen binding to the nonadherent population of cells increased 3-fold, whereas binding to the adherent population decreased by 60%. These changes were due to differences in the plasminogen binding capacities of the cells, while the affinities of the cells for plasminogen were unchanged. The up-regulation of receptor expression in the nonadherent cell population was: 1) induced rapidly and reversibly, 2) independent of new protein synthesis, 3) required an interaction between adherent and nonadherent cell populations, and 4) associated with an enhanced ability of the cells to promote plasminogen activation and to degrade fibronectin. Other immobilized adhesive proteins, laminin and vitronectin, also supported up-regulation of plasminogen receptors in the nonadherent cells. Carboxypeptidase B treatment eliminated the increment in the plasminogen binding capacity of the nonadherent cells, suggesting that the increase in binding was due to exposure of new carboxyl-terminal lysyl residues on the cell surfaces. Furthermore, both the adherence of the cells and up-regulation of plasminogen binding sites was abolished by beta1-integrin monoclonal antibodies. These results suggest that proteins found in extracellular matrices have the capacity to modulate the expression of plasminogen binding sites, thus regulating local proteolysis and cell migration.

Carboxypeptidase B↗
PubMed1997

Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for alpha1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha2(I) chains. DeltaMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of DeltaMT1 and MMP-1 indicate that DeltaMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of DeltaMT1 is 8-fold higher than that of MMP-1. DeltaMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha1-proteinase inhibitor and alpha2-macroglobulin. The activity of DeltaMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.

Collagen↗