Antihemostatic activity of human granzyme B mediated by cleavage of von Willebrand factor

The cytotoxic lymphocyte protease granzyme B (GrB) is elevated in the plasma of individuals with diseases that elicit a cytotoxic lymphocyte-mediated immune response. Given the recently recognized ability of GrB to cleave extracellular matrix proteins, we examined the effect of GrB on the pro-hemostatic molecule von Willebrand factor (VWF). GrB delays ristocetin-induced platelet aggregation and inhibits platelet adhesion and spreading on immobilized VWF under static conditions. It efficiently cleaves VWF at two sites within the A1-3 domains that are essential for the VWF-platelet interaction. Like the VWF regulatory proteinase ADAMTS-13, GrB-mediated cleavage is dependent upon VWF conformation. In vitro, GrB cannot cleave the VWF conformer found in solution, but cleavage is induced when VWF is artificially unfolded or presented as a matrix. GrB cleaves VWF with comparable efficiency to ADAMTS-13 and rapidly processes ultra-large VWF multimers released from activated endothelial cells under physiological shear. GrB also cleaves the matrix form of fibrinogen at several sites. These studies suggest extracellular GrB may help control localized coagulation during inflammation.     

Extracellular matrix remodelling in rat endometrium during early pregnancy: The role of fibronectin and laminin

The endometrial extracellular matrix (ECM) remodelling has a crucial role in the establishment of a successful pregnancy. In addition to its basic function such as regulation of cell function, differentiation, migration, proliferation, the substantial alterations in the endometrial ECM may play a specific role in the trophoblast invasion, placentation, cell death and formation of the proper and functional implantation chamber around the embryo. In the present study, immunolocalizations of fibronectin and laminin were determined using avidin-biotin complex-peroxidase in rat implantation sites during 7-10 days of pregnancy. Both proteins were present in the basal membrane of blood vessels and in decidual matrix whereas they were absent or had very weak reactivity in the primary decidual zone on day 7. When placentation has begun, the immunoreactivity of both proteins was increased in the placental bed and in the basal membrane of blood vessels of the mesometrial region. The immunolocalization of both proteins seemed to be decreased in the antimesometrial decidua, however, it was increased in the mesometrial decidual matrix on days 9 and 10. Therefore, it could be suggested laminin and fibronectin demonstrating dynamic expressions in relation with the morphological differentiation of endometrial stroma may play crucial roles in the control of trophoblast adhesion and invasion, in placentation and angiogenesis, in the determination of cell shape and fate thus contributing the endometrial receptivity and a successful pregnancy.     

Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.     

Coordinate regulation of fibronectin matrix assembly by the plasminogen activator system and vitronectin in human osteosarcoma cells

Background  

Plasminogen activators are known to play a key role in the remodeling of bone matrix which occurs during tumor progression, bone metastasis and bone growth. Dysfunctional remodeling of bone matrix gives rise to the osteoblastic and osteolytic lesions seen in association with metastatic cancers. The molecular mechanisms responsible for the development of these lesions are not well understood. Studies were undertaken to address the role of the plasminogen activator system in the regulation of fibronectin matrix assembly in the osteoblast-like cell line, MG-63.     

Inhibition of fibronectin matrix assembly by the heparin-binding domain of vitronectin.

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin's heparin-binding domain inhibited both beta(1) and non-beta(1) integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin-associated proteins alpha-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton.     

Expression of extracellular matrix components fibronectin and laminin in the human fetal heart.

It has been well documented that the extracellular matrix components fibronectin and laminin promote or regulate morphogenesis of the myocardial cells in mammalian heart. However, their chronological change of expression (or localization) in the human heart remains elusive. In this study, fibronectin and laminin in the left ventricle of forty-two human fetuses aged from 8 to 26 weeks gestation and left ventricular tissues obtained from a 2-week old infant and two adults were investigated by Western blot analyses and indirect immunofluorescence technique with monoclonal antibodies. In the fetal heart, fibronectins were present along the endocardium, epicardium, and linings of larger blood vessels. In 14-16 weeks gestation, fibronectin immunofluorescence became stronger but not evenly dispersed in the interstitium. After 24 weeks gestation, they were strongly positive only in the relatively larger blood vessels, as well as those in the infant and adult cardiac tissues. Laminins were strongly positive along the endocardium and basement membrane of the myocardial cells and fibroblasts during fetal life. After birth, laminins formed fine fibrillar network along the basement membrane in association with the transverse tubules of myocardial cell; these morphological characteristics remained in the adult cardiac tissues. These results indicate that fibronectin expression is relatively constant during fetal life but decreases after birth; in contrast, laminin expression is not age-dependent and constant throughout the life.     

Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3

Abstract

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells–fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α₅ integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α_5β1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.     

Distribution of the extracellular matrix proteins tenascin, fibronectin, and vitronectin in fetal, infant, and adult human spleens.

Using immunohistochemistry, we investigated the distribution of the extracellular matrix (ECM) glycoproteins tenascin, fibronectin, and vitronectin in fetal [16-24 gestational weeks (GW)], infant (40 GW), and adult human spleens to clarify the presence of these proteins during different phases of maturation. In the red and white pulp, tenascin and fibronectin were constant components of the reticular fibers from the age of 18 GW onwards, whereas vitronectin was seen only in adult spleens. The immunohistochemical staining patterns of tenascin and fibronectin remained unchanged at different fetal ages. Ring fibers, which are modified basement membranes around venous sinuses, became visible relatively late, and in adult spleens they contained both tenascin and vitronectin but lacked fibronectin. The composition of the ring fibers is therefore clearly different from that of ordinary basement membranes, which have not been reported to contain tenascin or vitronectin. The rapidly increasing number of reticular fibers in the spleen at the age of approximately 18 GW corresponds with the beginning of lymphatic colonization. Reticular fibers, rich in ECM glycoproteins, form a framework to which cells can migrate and attach. We suggest that the composition of these fibers might be important for lymphatic colonization and function of the spleen.     

Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes

We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co- localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.     

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.     

Extracellular matrix remodeling in canine and mouse myocardial infarcts

Extracellular matrix proteins not only provide structural support, but also modulate cellular behavior by activating signaling pathways. Healing of myocardial infarcts is associated with dynamic changes in the composition of the extracellular matrix; these changes may play an important role in regulating cellular phenotype and gene expression. We examined the time course of extracellular matrix deposition in a canine and mouse model of reperfused infarction. In both models, myocardial infarction resulted in fragmentation and destruction of the cardiac extracellular matrix, extravasation of plasma proteins, such as fibrinogen and fibronectin, and formation of a fibrin-based provisional matrix providing the scaffold for the infiltration of granulation tissue cells. Lysis of the plasma-derived provisional matrix was followed by the formation of a cell-derived network of provisional matrix composed of cellular fibronectin, laminin, and hyaluronic acid and containing matricellular proteins, such as osteopontin and osteonectin/SPARC. Finally, collagen was deposited in the infarct, and the wound matured into a collagen-based scar with low cellular content. Although the canine and mouse infarcts exhibited a similar pattern of extracellular matrix deposition, deposition of the provisional matrix was more transient in the mouse infarct and was followed by earlier formation of a mature collagen-based scar after 7-14 days of reperfusion; at the same timepoint, the canine infarct was highly cellular and evolving. In addition, mature mouse infarcts showed limited collagen deposition and significant tissue loss leading to the formation of a thin scar. In contrast, dogs exhibited extensive collagen accumulation in the infarcted area. These species-specific differences in infarct wound healing should be taken into account when interpreting experimental infarction studies and when attempting to extrapolate the findings to the human pathological process.     

Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.     

Expression, refolding, and purification of recombinant human granzyme B

Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates. To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor Xa (FXa) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli. Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies. These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni2+-NTA agarose column, and refolded by application of a cyclic refolding protocol. The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FXa or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step. A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared. Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.     

Growth conditions for the expression of fibronectin,collagen type I,vitronectin, and laminin binding toEscherichia coli strain NG7C

The ability ofEscherichia coli strains isolated from various human infections to bind fibronectin (Fn), collagen type I (Cn), laminin (Ln), and vitronectin (Vn) was studied. Binding of the proteins was shown to be specific and to be inhibited after protease treatment and heating of bacterial cells at 80°C. Binding of Fn, Cn type I, and Ln was not expressed in CFA broth and Voccani's medium with sodium chloride concentration above 0.5%. Fn binding was specifically enhanced for cells grown in CFA broth and Voccani's medium containing CaCl₂ (up to 10 mM final concentration), whereas binding of Cn type I was decreased. Growth in liquid medium yielded cells with maximal binding of Fn, Cn type I, Vn, and Ln after 20–22 h of growth. The binding of Fn, Cn type I, and Vn to cells of strain NG7C was inhibited for cells preincubated with antisera raised to the homologous strain;E. coli NG7C cells seem to have specific binding sites for Fn, Cn type I, Vn, and possibly also Ln.     

Cysteine cathepsins in extracellular matrix remodeling: Extracellular matrix degradation and beyond

Cysteine cathepsins have been for a long time considered to execute mainly nonspecific bulk proteolysis in the endolysosomal system. However, this view has been changing profoundly over the last decade as cathepsins were found in the cytoplasm, nucleus and in the extracellular milieu. Cathepsins are currently gaining increased attention largely because of their extracellular roles associated with disease development and progression. While kept under tight control under physiological conditions, their dysregulated and elevated activity in the extracellular milieu are distinctive hallmarks of numerous diseases such as various cancers, inflammatory disorders, rheumatoid arthritis, bone disorders and heart diseases. In this review, we discuss cysteine cathepsins with a major focus on their extracellular roles and extracellular proteolytic targets beyond degradation of the extracellular matrix. We further highlight the perspectives of cathepsin research and novel avenues in cathepsin-based diagnostic and therapeutic applications.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.