Pericellular Proteolytic Activity Research Articles

Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment-Peculiar Effect of Epigallocatechin-3-Gallate.

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.

Pirfenidone inhibits motility of NSCLC cells by interfering with the urokinase system

Pirfenidone (PFD) is an orally available synthetic drug which has been approved for the treatment of idiopathic pulmonary fibrosis. In addition to its anti-fibrotic properties, PFD also exerts anti-tumor effects in cancer models by inducing alterations in the tumor microenvironment. Here, we demonstrate that PFD reduces proliferation, 2D- and 3D-migration as well as colony formation of the non-small-cell lung carcinoma (NSCLC) cells. On a molecular level, we show that PFD on the one hand interacts with plasminogen activator inhibitor-1 (PAI-1; Kd of 46.2±11.3nM) and affects its inhibitory potency, but on the other hand it also increases PAI-1 expression; in both cases consequently leading to the reduction of urokinase (uPA) activity. Finally, we report that the effect of PFD on 2D-migration of NSCLC cells depends on PAI-1 expression and thus on the activity of the uPA system whereas the PFD-induced changes in cancer cell proliferation, 3D-migration and colony formation are PAI-1 independent. To conclude, a direct interference of PFD with the uPA-PAI-1 system may deregulate pericellular proteolytic activity and thereby influence the stability of the tumor blood vessels and the matrix architecture within tumor stroma.

Invadosomes are coming: new insights into function and disease relevance.

Invadopodia and podosomes are discrete, actin-based molecular protrusions that form in cancer cells and normal cells, respectively, in response to diverse signaling pathways and extracellular matrix cues. Although they participate in a host of different cellular processes, they share a common functional theme of controlling pericellular proteolytic activity, which sets them apart from other structures that function in migration and adhesion, including focal adhesions, lamellipodia, and filopodia. In this review, we highlight research that explores the function of these complex structures, including roles for podosomes in embryonic and postnatal development, in angiogenesis and remodeling of the vasculature, in maturation of the postsynaptic membrane, in antigen sampling and recognition, and in cell-cell fusion mechanisms, as well as the involvement of invadopodia at multiple steps of the metastatic cascade, and how all of this may apply in the treatment of human disease states. Finally, we explore recent research that implicates a novel role for exosomes and microvesicles in invadopodia-dependent and invadopodia-independent mechanisms of invasion, respectively.

Abstract 363: Thrombin Activatable Fibrinolysis Inhibitor is a Regulator of Angiogenic Potential in Endothelial Cells

Angiogenesis, the sprouting of new blood vessels from existing vessels, is a process that is fundamental to both normal physiology, as well as disease processes such as atherosclerosis and cancer metastasis. A key regulator of the angiogenesis is the elaboration of pericellular proteolytic activity including plasmin and the matrix metalloproteases (MMPs). Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that can be cleaved by thrombin, plasmin, or thrombin in complex with thrombomodulin (TM) to form activated TAFI (TAFIa). TAFIa cleaves carboxyl terminal lysine and arginine residues from substrates, including plasminogen-binding sites on cell surface receptors. TAFIa is thus a regulator of pericellular plasminogen activation. Plasmin regulates angiogenesis through MMP activation and degradation of the extracellular matrix (ECM). Previous studies have shown that TAFIa downregulates endothelial tube formation in a fibrin matrix, likely through inhibition of fibrin degradation. The current study was undertaken to evaluate if TAFIa is capable of modulating angiogenic responses of endothelial cells in vitro in the context of reconstituted basement membrane. We found that treatment of primary human umbilical vein endothelial cells (HUVECs) with a specific inhibitor of TAFIa, potato tuber carboxypeptidase inhibitor, resulted in an increase in proliferation, invasion, tube formation, and collagen degradation. On the other hand, treatment with a stable variant of TAFIa (TAFIa-CIIYQ) with a 150-fold increased half-life at 37°C resulted in a decrease in HUVEC tube formation and collagen degradation of HUVECs. Furthermore, a mutant soluble form of TM that supports TAFI, but not protein C, activation also had an inhibitory effect on HUVEC tube formation and collagen degradation. Finally, we assessed the effect of TAFIa on plasminogen activation. Treatment of HUVECs with various concentrations of TAFIa resulted in a decrease in uPA-mediated plasminogen activation on the surface of HUVECs. Together these experiments provide direct evidence of an anti-angiogenic effect of TAFIa. Our results suggest that promotion of TAFI activation or TAFIa stability may be viable therapeutic approaches for inhibition of angiogenesis.

Dynamic Reorganization and Enzymatic Remodeling of Type IV Collagen at Cell-Biomaterial Interface.

Vascular basement membrane remodeling involves assembly and degradation of its main constituents, type IV collagen (Col IV) and laminin, which is critical during development, angiogenesis, and tissue repair. Remodeling can also occur at cell-biomaterials interface altering significantly the biocompatibility of implants. Here we describe the fate of adsorbed Col IV in contact with endothelial cells adhering on positively charged NH2 or hydrophobic CH3 substrata, both based on self-assembly monolayers (SAMs) and studied alone or mixed in different proportions. AFM studies revealed distinct pattern of adsorbed Col IV, varying from single molecular deposition on pure NH2 to network-like assembly on mixed SAMs, turning to big globular aggregates on bare CH3. Human umbilical endothelial cells (HUVECs) interact better with Col IV adsorbed as single molecules on NH2 surface and readily rearrange it in fibril-like pattern that coincide with secreted fibronectin fibrils. The cells show flattened morphology and well-developed focal adhesion complexes that are rich on phosphorylated FAK while expressing markedly low pericellular proteolytic activity. Conversely, on hydrophobic CH3 substrata HUVECs showed abrogated spreading and FAK phosphorylation, combined with less reorganization of the aggregated Col IV and significantly increased proteolytic activity. The later involves both MMP-2 and MMP-9, as measured by zymography and FITC-Col IV release. The mixed SAMs support intermediate remodeling activity. Taken together these results show that chemical functionalization combined with Col IV preadsorption provides a tool for guiding the endothelial cells behavior and pericellular proteolytic activity, events that strongly affect the fate of cardiovascular implants.

Regulation of plasminogen activator activity and expression by cyclic mechanical stress in rat mandibular condylar chondrocytes

To investigate the mechanism of cartilage degradation induced by overloading in the temporomandibular joint (TMJ), the effect of cyclic mechanical compressive stress on the activity of plasminogen activator (PA) and the expression of the predominant components of the PA system were analyzed in cultured mandibular condylar chondrocytes (MCCs) in rats. MCCs were exposed to cyclic mechanical compressive stress (2000, 4000 and 6000 µ strain) at 0.5 Hz by a four‑point bending system. The activity of PA was determined by hydrolysis of the chromogenic substrate H‑D-Val-Leu-Lys‑pNA (S‑2251). The mRNA and protein expression levels of urokinase‑type PA (uPA), tissue‑type PA (tPA), uPA receptor (uPAR) and PA inhibitor 1 (PAI‑1) were detected by qPCR and western blot analysis, respectively. Cyclic mechanical stress at 4000 and 6000 µ strain induced the expression of uPA, tPA and uPAR, and increased the activity of PA. Furthermore, cyclic mechanical stress at 6000 µ strain also inhibited the expression of PAI‑1. Analysis of pericellular proteolytic activity demonstrated that PA functioned as the active enzyme in excessive mechanical stress responsiveness (e.g., 4000 and 6000 µ strain) largely via uPAR, not PAI‑1. Cyclic mechanical stress at 2000 µ strain induced the expression of tPA and PAI‑1; however, it did not change the activity of PA. These results suggested that the mechanical induction of uPA, tPA and uPAR upregulated PA activity, which may provide a proteolytic environment of extracellular matrix components and subsequently contribute to the cartilage degradation in TMJ osteoarthritis.

MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Abstract 3536Poster Board III-473Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34+ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34+ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34+ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34+ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34+ cells is necessary for proMMP-2 activation as media conditioned by CD34+ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation. Disclosures:No relevant conflicts of interest to declare.

Regulation of cell surface protease matriptase by HAI2 is essential for placental development, neural tube closure and embryonic survival in mice

Hypomorphic mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis, including organ and digit duplications, atresia, fistulas, hypertelorism, cleft palate and hamartoma. SPINT2 encodes the transmembrane serine protease inhibitor HAI2 (placental bikunin), and the severe developmental effects of decreased HAI2 activity can be hypothesized to be a consequence of excess pericellular proteolytic activity. Indeed, we show here that HAI2 is a potent regulator of protease-guided cellular responses, including motogenic activity and transepithelial resistance of epithelial monolayers. Furthermore, we show that inhibition of the transmembrane serine protease matriptase (encoded by St14) is an essential function of HAI2 during tissue morphogenesis. Genetic inactivation of the mouse Spint2 gene led to defects in neural tube closure, abnormal placental labyrinth development associated with loss of epithelial cell polarity, and embryonic demise. Developmental defects observed in HAI2-deficient mice were caused by unregulated matriptase activity, as both placental development and embryonic survival in HAI2-deficient embryos were completely restored by the simultaneous genetic inactivation of matriptase. However, neural tube defects were detected in HAI2-deficient mice even in the absence of matriptase, although at lower frequency, indicating that the inhibition of additional serine protease(s) by HAI2 is required to complete neural development. Finally, by genetic complementation analysis, we uncovered a unique and complex functional interaction between HAI2 and the related HAI1 in the regulation of matriptase activity during development. This study indicates that unregulated matriptase-dependent cell surface proteolysis can cause a diverse array of abnormalities in mammalian development.

Selective Inhibition of Matrix Metalloproteinase-14 Blocks Tumor Growth, Invasion, and Angiogenesis

Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.

Transforming Growth Factor-β1 Promotes Matrix Metalloproteinase-9–Mediated Oral Cancer Invasion through Snail Expression

Oral squamous cell carcinoma (OSCC), which is the most common malignancy of the oral cavity, is often associated with local and regional invasion. Increased expression of matrix metalloproteinase-9 (MMP-9) is correlated with invasive behavior of OSCC. Because transforming growth factor beta1 (TGF-beta1) is up-regulated in OSCC tumors, we examined the relationship between TGF-beta1 signaling and MMP-9 in human OSCC specimens. Evaluation of human specimens showed that tumors with enhanced TGF-beta1 signaling also showed increased MMP-9 expression. Because the transcription factor Snail has been determined to be a key mediator of TGF-beta1 signaling, we evaluated the role of Snail in TGF-beta1-mediated MMP-9 expression. Initially, we examined the extent to which TGF-beta1 regulated Snail expression in oral keratinocytes and in OSCC cell lines. TGF-beta1 enhanced Snail expression in a majority of the cell lines examined, with the largest induction of Snail detected in UMSCC1 cells. Interestingly, overexpression of Snail in UMSCC1 cells enhanced MMP-9 and tissue inhibitor of metalloproteinase-1 protein levels. Despite the increase in the tissue inhibitor of metalloproteinase-1 protein, there was a net increase in the pericellular proteolytic activity as shown by enhanced MMP-9-dependent Matrigel invasion. Moreover, Snail-specific siRNA blocked TGF-beta1-induced MMP-9 expression and Matrigel invasion. In addition, Snail increased Ets-1 levels and Ets-1-specific siRNA blocked both Snail- and TGF-beta1-mediated MMP-9 expression and Matrigel invasion. Thus, these data show that Snail functions as a molecular mediator of TGF-beta1-regulated MMP-9 expression by increasing Ets-1 and thereby contributing to oral cancer progression.

Probing cellular microenvironments and tissue remodeling by atomic force microscopy

The function of cells is strongly determined by the properties of their extracellular microenvironment. Biophysical parameters like environmental stiffness and fiber orientation in the surrounding matrix are important determinants of cell adhesion and migration. Processes like tissue maintenance, wound repair, cancer cell invasion, and morphogenesis depend critically on the ability of cells to actively sense and remodel their surroundings. Pericellular proteolytic activity and adaptation of migration tactics to the environment are strategies to achieve this aim. Little is known about the distinct regulatory mechanisms that are involved in these processes. The system's critical biophysical and biochemical determinants are well accessible by atomic force microscopy (AFM), a unique tool for functional, nanoscale probing and morphometric, high-resolution imaging of processes in live cells. This review highlights common principles of tissue remodeling and focuses on application examples of different AFM techniques, for example elasticity mapping, the combination of AFM and fluorescence microscopy, the morphometric imaging of proteolytic activity, and force spectroscopy applications of single molecules or individual cells. To achieve a more complete understanding of the processes underlying the interaction of cells with their environments, the combination of AFM force spectroscopy experiments will be essential.

Roles and regulation of membrane-associated serine proteases

Pericellular proteolytic activity affects many aspects of cellular behaviour, via mechanisms involving processing of the extracellular matrix, growth factors and receptors. The serine proteases have exquisitely sensitive regulatory mechanisms in this setting, involving both receptor-bound and transmembrane proteases. Receptor-bound proteases are exemplified by the uPA (urokinase plasminogen activator)/uPAR (uPAR receptor) plasminogen activation system. The mechanisms initiating the activity of this proteolytic system on the cell surface, a critical regulatory point, are poorly understood. We have found that the expression of the TTSP (type II transmembrane serine protease) matriptase is highly regulated in leucocytes, and correlates with the presence of active uPA on their surface. Using siRNA (small interfering RNA), we have demonstrated that matriptase specifically activates uPAR-associated pro-uPA. The uPA/uPAR system has been implicated in the activation of the plasminogen-related growth factor HGF (hepatocyte growth factor). However, we find no evidence for this, but instead that HGF can be activated by both matriptase and the related TTSP hepsin in purified systems. Hepsin is of particular interest, as the proteolytic cleavage sequence of HGF is an 'ideal substrate' for hepsin and membrane-associated hepsin activates HGF with high efficiency. Both of these TTSPs can be activated autocatalytically at the cell surface, an unusual mechanism among the serine proteases. Therefore these TTSPs have the capacity to be true upstream initiators of proteolytic activity with subsequent downstream effects on cell behaviour.

MT1-MMP Controls Tumor-induced Angiogenesis through the Release of Semaphorin 4D

The semaphorins are a family of proteins originally identified as regulators of axon growth that recently have been implicated in blood vessel development. The plexins are high affinity receptors for the semaphorins and are responsible for initiation of signaling upon ligation. Emerging evidence indicates that many human cancers overexpress Semaphorin 4D, which promotes neovascularization upon stimulating its receptor, Plexin-B1, on endothelial cells. However, to exert its pro-angiogenic functions, Semaphorin 4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. Here we show that Semaphorin 4D is a novel target for the membrane-tethered collagenase membrane type 1-matrix metalloproteinase. We demonstrate that this metalloproteinase, which is not expressed in normal or immortal but non-tumorigenic epithelial cell lines, was present in several head and neck squamous cell carcinoma cell lines and was required for processing and release of Semaphorin 4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and blood vessel growth in vivo. These results suggest that the proteolytic cleavage of Semaphorin 4D may provide a novel molecular mechanism by which membrane type 1-matrix metalloproteinase controls tumor-induced angiogenesis.

Double-Layered Collagen Gel Hemisphere for Cell Invasion Assay: Successful Visualization and Quantification of Cell Invasion Activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].

A Novel Role for Thrombin in Hematopoietic Stem/Progenitor Cell Homing.

Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (Blood 2005; 105:40). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.

Tetraspanin CD151 is expressed in osteoarthritic cartilage and is involved in pericellular activation of pro–matrix metalloproteinase 7 in osteoarthritic chondrocytes

The proenzyme of matrix metalloproteinase 7 (proMMP-7), which can degrade various extracellular matrix (ECM) and non-ECM molecules after being activated, is overexpressed in osteoarthritic (OA) articular cartilage, but the process of its activation in the cartilage remains unknown. The present study was undertaken to investigate the expression of tetraspanin CD151 in OA cartilage and its involvement in proMMP-7 activation. The expression of CD151 in articular cartilage was examined by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunohistochemistry, in situ hybridization, and immunoblotting. Chondrocytes were used to study the interaction between CD151 and proMMP-7, and activation of proMMP-7. RT-PCR revealed expression of CD151 messenger RNA in all OA cartilage samples, but in only 30% of normal control cartilage samples. Immunohistochemistry and in situ hybridization findings indicated that CD151 was coexpressed with proMMP-7 in chondrocytes, mainly in the superficial and transitional zones of OA cartilage. CD151 immunoreactivity directly correlated with the Mankin score (r = 0.757, P < 0.0001 [n = 30]) and the degree of chondrocyte cloning (r = 0.83, P < 0.0001 [n = 30]) in the cartilage samples. Complexes CD151 and proMMP-7 and their colocalization on the cell membranes were demonstrated by immunoprecipitation and double fluorescence immunostaining of the OA chondrocytes. In situ zymography indicated that chondrocytes exhibit pericellular proteolytic activity, which was abolished by treatment with MMP inhibitors, anti-MMP-7 antibody, or anti-CD151 antibody. These data demonstrate that CD151 is overexpressed in OA cartilage and suggest that CD151 plays a role in the pericellular activation of proMMP-7, leading to cartilage destruction and/or chondrocyte cloning.

Regulation of Urokinase Receptor Proteolytic Function by the Tetraspanin CD82

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolytic activity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by approximately 50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin alpha(5)beta(1), which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with alpha(5)beta(1), but not with a variety of other integrins, including alpha(3)beta(1). These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and alpha(5)beta(1) to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolytic activity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1.

Epithelial tissue-type plasminogen activator expression, unlike that of urokinase, its receptor, and plasminogen activator inhibitor-1, is increased in chronic venous ulcers

The plasminogen activation system represents a potent mechanism of extracellular proteolysis and is an essential component of normal wound healing. It has also been implicated in the pathogenesis of chronic, nonhealing ulcers. Traditionally, urokinase-type plasminogen activator (uPA) has been associated with pericellular proteolytic activity involved in tissue remodelling processes, and tissue-type plasminogen activator (tPA) mainly with intravascular fibrinolysis. The present study was conducted to characterize the spatial distribution of the various plasminogen activation system components in chronic ulcers and acute, well-granulating wounds. The expression of uPA, tPA, urokinase receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and vitronectin was investigated by immunohistochemical staining, in addition to uPA, tPA and PAI-1 expression by in-situ hybridization, in samples from eight chronic venous ulcers, five decubitus ulcers, five well-granulating acute wounds and five normal skin samples. In chronic venous leg ulcers tPA mRNA was detected in basal and suprabasal keratinocytes at the leading wound edge, while in well-granulating wounds and in decubitus ulcers tPA mRNA was expressed only in a few keratinocytes. However, tPA was widely expressed in fibroblast- and macrophage-like cells in the stroma of well-granulating wounds, while less tPA was detected in the granulation tissue of chronic ulcers. tPA mRNA and protein were localized in the superficial granular layers in normal skin. Although no qualitative differences in expression of uPA, PAI-1 or uPAR in the wound edge keratinocytes in chronic ulcers vs. normally granulating wounds were found, their expressions were more pronounced in the granulation tissue of well-granulating wounds. These results suggest that in poorly healing venous leg ulcers, the pattern of tPA expression is altered in keratinocytes at the leading edge of the wound, and the patterns of tPA, uPA and PAI-1 expression are altered in the granulation tissue.

Upregulation of MT1-MMP Expression by Hyaluronic Acid Enhances Homing-Related Responses of Hematopoietic CD34+ Cells to an SDF-1 Gradient.

Hyaluronic acid (HA), the bone marrow (BM) extracellular matrix microenvironment (ECM) component, not only supports cell adhesion but also promotes migration and homing of hematopoietic stem/progenitor cells (HSPC) by interacting with its cell surface receptor CD44. CD44 has been shown to co-localize with matrix metalloproteinases (MMPs), particularly membrane-type (MT)-1 MMP and MMP-9, at the leading edge of migrating tumor cells, and the cleavage of CD44 by MT1-MMP is critical for tumor cell migration and invasion. MT1-MMP has strong pericellular proteolytic activity and also activates latent forms of MMP-2 and MMP-9. In this study we examined the effect of HA on MT1-MMP expression and migration of BM, peripheral blood and cord blood CD34+ cells. We found that HA upregulates mRNA for MT1-MMP and MMP-9, increases MT1-MMP protein (as evaluated by Western blotting) and stimulates MMP-9 and MMP-2 activity (as determined by zymography) in CD34+ cells. In chemotaxis assays HA alone did not show any chemotactic activity but primed the chemotaxis of CD34+ cells to a low SDF-1 gradient (10 ng/mL) and their trans-Matrigel chemoinvasion to a low SDF-1 gradient. Similarly, SDF-1 besides stimulating MMP-2 and MMP-9 (as we previously described in Exp Hematol 2000; 28:1274), also upregulated MT1-MMP in CD34+ cells. In addition, we found that the HA enhanced activation of latent MMP-2 in co-cultures of CD34+ cells with HUVEC. In conclusion, we demonstrate for the first time that (i) HA primes CD34+ cell chemotaxis and chemoinvasion to a low SDF-1 gradient; (ii) that both HA and SDF-1 stimulate MT1-MMP, MMP-2 and MMP-9 in CD34+ cells, and (iii) HA enhances activation of proMMP-2 in the ECM. Hence the interaction of HA and SDF-1 with MT1-MMP could play an important role in HSPC migration and homing and we postulate that pretreatment of HSPC with HA before transplantation could enhance their homing by inducing expression of MMPs that is optimal for stem cell engraftment.

Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator.

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA-/-) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1). developed a novel methodology to detect pericellular proteolytic function, (2). demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3). demonstrated its usefulness in real-time studies of cell migration, and (4). showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.