Abstract: Dry eye is a common disease that develops as a result of alteration of tear fluid, leading to osmotic stress and a perturbed epithelial barrier. Matrix metalloproteinase-9 (MMP-9) may be important in dry eye disease, as its genetic knockout conferred resistance to the epithelial disruption. We show that extracellular matrix metalloproteinase inducer (EMMPRIN; also termed CD147), an inducer of MMP expression, participates in the pathogenesis of dry eye through MMP-mediated cleavage of occludin, an important component of tight junctions. EMMPRIN expression was increased on the ocular surface of dry eye patients and correlated with those of MMP-9. High osmolarity in cell culture, mimicking dry eye conditions, increased both EMMPRIN and MMP-9 and resulted in the disruption of epithelial junctions through the cleavage of occludin. Exogenously added recombinant EMMPRIN had similar effects that were abrogated in the presence of the MMP inhibitor marimastat. Membrane occludin immunostaining was markedly increased in the apical corneal epithelium of both EMMPRIN and MMP-9 knock-out mice. Furthermore, an inverse correlation between EMMPRIN and occludin membrane staining was consistently observed both in vitro and in vivo as a function of corneal epithelial cells differentiation. These data suggest a possible role of EMMPRIN in regulating the amount of occludin at the cell surface in homeostasis beyond pathological situations such as dry eye disease, and EMMPRIN may be essential for the formation and maintenance of organized epithelial structure.
Abstract: Matrix metalloproteinases (MMPs) are thought to play an important role in skeletal muscle cell growth and differentiation. In view of the MMP inducing function of EMMPRIN/CD147, its role in myogenic cell differentiation was investigated. EMMPRIN level increased during differentiation of both rat primary myoblasts derived from satellite cells and mouse C2.7 myogenic cells and was associated with an alteration in its molecular forms. In parallel, expression of pro-MMP-9 gradually decreased and that of pro-MMP-2 and active MMP-2 increased. While small interfering RNA (siRNA) inhibition of EMMPRIN expression accelerated cell differentiation, exogenously added recombinant EMMPRIN inhibited differentiation by an MMP-mediated mechanism, as the MMP inhibitor marimastat abrogated EMMPRIN's effect. Our results further suggest that EMMPRIN regulates differentiation through an MMP activation of transforming growth factor beta (TGFβ), a known inhibitor of myoblast's differentiation, as the increased activation and signaling of TGFβ by EMMPRIN was attenuated in the presence of marimastat. EMMPRIN inhibition may thus represent a novel strategy in the treatment of muscular degenerative disorders.
Abstract: The activation of epidermal growth factor receptor (EGFR) by its ligands constitutes an important step in the metastatic process but the clinical response to its inhibition in breast cancer patients has so far been very low. In this work, we investigated the role of the EGFR ligand amphiregulin (AR) in modulating EGFR activation. For this, transformed epithelial mammary tumor cells NS2T2A1 were used in which AR or EGFR expression was down-regulated by antisense cDNA technique. This down-regulation was associated with a significant inhibition of matrix metalloproteinase-9 production as well as cell proliferation, but this inhibition was only minimally reversed by exogenously added AR or EGF. EGFR protein levels were not affected but EGFR-tyrosine phosphorylation in response to EGF was markedly reduced. Thus, the inhibition of AR expression, which impairs EGFR response to its exogenously available ligands, may represent an alternative anti-EGFR therapeutic strategy in breast cancer.
Abstract: Polysulfone (PSU) membranes, coated and uncoated hyaluronan (HA), were compared for their ability to allow dermal fibroblast express genes related to extracellular matrix synthesis and remodeling. Fibroblasts type I and type III collagens were studied on both types of membranes; only type I collagen was synthesized on control cultures in plastic Petri dishes, whereas type III collagen was also expressed on PSU membranes. Expression of metalloproteinase (MMP)1, MMP3, and MMP2 was enhanced on PSU and HA-coated PSU membranes, with a lower level of MMP2 on HA-covered membranes. These membranes promote fetal- like matrices that provide good support for skin wound healing as well as favor nonscarring tissue repair.
Abstract: OBJECTIVE: Glycosaminoglycans (GAG) are major components of bone marrow extracellular matrix because they have the property to interact with cells and growth factors in hematopoietic niches. In this study, we investigated the effect of two different chemically defined GAG mimetics on mobilization of hematopoietic stem and progenitor cells (HSPCs) in mice peripheral blood. MATERIALS AND METHODS: Mobilization was achieved by intraperitoneal injection of GAG mimetics. Mobilized cells were characterized phenotypically by reverse transcription polymerase chain reaction and fluorescence-activated cell sorting analysis and functionally by colony-forming cell, cobblestone area-forming cell and long-term culture-initiating cell assays in vitro. Radioprotection assays were performed to confirm the functionality of primitive hematopoietic cells in vivo. Involvement of stromal-derived factor-1 (SDF-1) and matrix metalloproteinase-9 (MMP-9) were investigated. RESULTS: GAG mimetics treatment induces hyperleukocytosis and mobilization of HSPC. They synergize with the effects of granulocyte colony-stimulating factor or AMD3100 on hematopoietic progenitors mobilization. Reconstitution of lethally irradiated recipient mice with peripheral blood mononuclear cells from GAG mimetic-treated donor mice improves engraftment and survival. BiAcore studies indicate that the mimetics interact directly with SDF-1. In addition, GAG mimetics-induced mobilization is associated with increased levels of pro- and active MMP-9 from bone marrow cells and increased level of SDF-1 in peripheral blood. Finally, mobilization is partially inhibited by co-injection with anti-SDF-1 antibody. CONCLUSION: This study demonstrates that GAG mimetics induce efficient mobilization of HSPCs, associated with an activation of pro-MMP-9 and a modification in the SDF-1 concentration gradient between bone marrow and peripheral blood. We suggest that structural features of GAGs can modify the nature of mobilized cells.
Abstract: Dentin Matrix Protein 1 (DMP1) plays a regulatory role in dentin mineralization and can also function as a signaling molecule. MMP-2 (matrix metalloproteinase-2) is a predominant protease in the dentin matrix that plays a prominent role in tooth formation and a potential role during the carious process. The possibility that MMP-2 can cleave DMP1 to release biologically active peptides was investigated in this study. DMP1, both in the recombinant form and in its native state within the dentin matrix, was shown to be a substrate for MMP-2. Proteolytic processing of DMP1 by MMP-2 produced two major peptides, one that contains the C-terminal region of the protein known to carry both the ASARM (aspartic acid and serine rich domain) domain involved in biomineralization and the DNA binding site of DMP1. In vitro experiments with recombinant N- and C-terminal polypeptides mimicking the MMP-2 cleavage products of DMP1 demonstrated an effect of the C-polypeptide on the differentiation of dental pulp stem/progenitor cells to a putative odontoblast phenotype. In vivo implantation of this peptide in a rat injured pulp model induced a rapid formation of a homogeneous dentin bridge covered by a palisade of orientated cells expressing dentin sialoprotein (DSP) and DMP1, attesting an efficient repair process. These data suggest that a peptide generated through the proteolytic processing of DMP1 by MMP-2 can regulate the differentiation of mesenchymal cells during dentinogenesis and thus sustain reparative dentin formation in pathological situations such as carious decay. In addition, these data open a new therapeutic possibility of using this peptide to regenerate dentin after an injury.
Abstract: In the cornea, the epithelium and the underlying stroma are separated by the basement membrane and Bowman's layer. The disruption of these anatomical barriers during wound healing represents a key step which initiates tissue remodeling through the modification of the epithelial-stromal interactions (ESI). Diffusible cytokines are generally viewed as central modulators in the bidirectional communication between these epithelial and stromal compartments and their implication in all stages of the wound healing process has been an active area of research for many years. Our studies which aimed to explore mechanisms of matrix degradation in pathological corneal wound healing have shown that EMMPRIN, a glycoprotein expressed on corneal epithelial cell surface, can induce matrix metalloproteinase (MMP) production and myofibroblasts differentiation after direct interaction with corneal fibroblasts. EMMPRIN appears therefore as a potential mediator of ESI by direct cell-cell contact which represents a new mechanism for dysregulated MMPs' induction observed in corneal ulcerations. These direct epithelial-stromal interactions (direct-ESI) can occur when delayed epithelial healing prevents regeneration of the basement membrane and allows the two cell types to come into close proximity. We propose that prevention of these interactions through inhibition of EMMPRIN may represent a promising therapeutic strategy in the inhibition of MMP induction in ulceration.
Abstract: Emmprin/CD147 is a cell membrane glycoprotein that belongs to the Ig superfamily and is involved in numerous physiological and pathological systems. Through its ability to interact with multiple partners within the cell surface and its potential to regulate the expression of several targets within the cell, emmprin may have different functions depending on the cell or tissue type. However, its role in tissue remodeling remains the most clearly demonstrated. Emmprin is able to induce, in the same cellular model, both the matrix metalloproteinases and the serine protease urokinase plasminogen activator, whose concerted action in the breakdown of the extracellular matrix (ECM) during various physiopathological situations has been reported. In addition, emmprin also promotes myofibroblasts' differentiation and tissue contraction through the induction of alpha smooth muscle actin, thus expanding on the mechanism by which emmprin remodels ECM.
Abstract: Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein enriched on tumor cells and normal epithelia. It is mainly known for its ability to induce matrix metalloproteinase production in fibroblasts following epithelial-stromal interaction. We sought to examine whether EMMPRIN has a broader role promoting fibroblast-to-myofibroblast differentiation. Because alpha-smooth muscle actin (alphaSMA) is considered a marker of this differentiation process, we analyzed the effect of EMMPRIN on its expression in corneal and skin fibroblasts by Western blots, immunocytochemistry, and a functional assay of collagen lattice contraction. Increasing EMMPRIN expression by cDNA transfection or by treatment with exogenously added recombinant EMMPRIN resulted in an up-regulation of alphaSMA expression. EMMPRIN also increased the contractile properties of the treated fibroblasts as demonstrated by the immunohistochemical appearance of stress fibers and by the accelerated contraction of fibroblast-embedded collagen lattices. Blocking EMMPRIN expression by small interfering RNA inhibited alphaSMA and collagen gel contraction induced not only by EMMPRIN but also by transforming growth factor-beta, a major mediator of myofibroblast differentiation that also regulated EMMPRIN expression. These findings, combined with the fact that EMMPRIN and alphaSMA colocalized to the same cells in the stroma of pathological corneas, expand on the mechanism by which EMMPRIN remodels extracellular matrix during wound healing and cancer.
Abstract: Burn-related skin fibrosis leads to loss of tissue function and hypertrophic scar formation with damaging consequences for the patient. There is therefore a great need for an efficient agent to treat burned skin. We report that ReGeneraTing Agent (RGTA) reduces burn-induced skin alteration. The tissue-regenerating effect of RGTA OTR4120 was evaluated after 1-6 days and after 10 months in a rat skin burn model. This effect was also examined in vitro using fibroblasts isolated from control and 6-day-old burned skins. We measured production of dermal collagen I, III, and V and activities of metalloproteinases 2 and 9 (MMP-2 and MMP-9). Ratio of collagen III over collagen I production increased 6 days after the burn, because of a decrease in collagen I production. After 10 months, ratio of collagen III over collagen I in burn sites was still increased compared with control skin, because of an increase in collagen III production. Both abnormalities were corrected by OTR4120. OTR4120 increased pro- and active MMP-2 and MMP-9, compared with healthy and burned controls and therefore accelerated remodeling. Similar data were obtained with cultured fibroblasts from healthy and burned skins. OTR4120 enhanced healing in short- and long-term after burns, reducing the formation of fibrotic tissue, and then represents a potential agent to improve burned skin healing.
Abstract: Elastin-derived peptides display a wide range of biological activities in a number of normal and transformed cells but their involvement in angiogenesis has not been reported. In the present study, we show that kappa-elastin and VGVAPG hexapeptide elastin motif accelerated angiogenesis in the chick chorio-allantoic membrane in an in vivo model. They also stimulated pseudotube formation from human vascular and microvascular endothelial cells in the matrigel and collagen models as well as cell migration in an in vitro wound healing assay. Confocal and scanning electron microscopy analyses revealed the main reorganization of actin filaments mediated by elastin-derived peptides and changes in cell shape that correlated with a decrease of the cell form factor determined by computerized image analysis. Such elastin-derived peptide effects were attributed to upregulation of proMT1-MMP and proMMP-2 expression and activation at both the mRNA and protein levels. Batimastat, an inhibitor of furin convertase and TIMP-2, but not TIMP-1, totally abolished the influence of elastin-derived peptides (EDPs) on cell migration and tubulogenesis, thus favoring the involvement of MT1-MMP in such processes. To assess its contribution to EDP-mediated angiogenesis further, we used a small interfering RNA (siRNA) approach for specifically silencing MT1-MMP in human microvascular endothelial cells. Four sets of 21 bp siRNA duplexes targeting MT1-MMP mRNA were synthesized by in vitro transcription. Two of them proved to inhibit MT1-MMP expression efficiently but did not affect MT2-, MT3- and MT5-MMP expression. Seventy-two hours after transfection with 25 nM siRNAs EDP-induced MT1-MMP expression at the mRNA and protein levels was decreased fourfold. In parallel, proMMP-2 activation was inhibited. A scrambled siRNA, used as a negative control, had no effect. Finally, the effect of elastin peptides on pseudotube formation in MT1-MMP-siRNA transfected cells was totally abolished. These data emphasise the crucial role of MT1-MMP in the elastin-induced angiogenic phenotype of endothelial cells.
Abstract: C18 unsaturated fatty acids were here found to inhibit proMMP (matrix metalloproteinase)-3 activation by plasmin. This effect was suppressed by lysine ligand competitors, indicating that it was mediated by binding to kringle domains. Surface plasmon resonance analysis demonstrated that oleic acid interacted to a similar extent with plasmin and kringle 5 (KD values of 3.4 x 10(-8) and 5.9 x 10(-8)M) while interaction with kringles 1-2-3 was 10-fold lower. Furthermore, oleic acid stimulated the amidolytic activity of plasmin and mini-plasmin, but not micro-plasmin. Oleic acid also enhanced u-PA (urokinase-type plasminogen activator)-mediated plasminogen activation over 50-fold. Taken together, these data indicate that inhibition of plasmin-induced proMMP-3 activation by unsaturated fatty acids was mediated through their preferential binding to kringle 5. The influence of elaidic acid on the plasmin/MMP-3/MMP-1 proteolytic cascade was assessed ex vivo. Exogenous addition of plasmin to dermal fibroblasts or supplementation of gingival fibroblast culture medium with plasminogen triggered this cascade. In both instances, elaidic acid totally abolished proMMP-3 and proMMP-1 activation. Additionally, a significant decrease in lattice retraction and collagen degradation in a range similar to that obtained with Batimastat was observed when human gingival fibroblasts were cultured in plasminogen-containing type I collagen gels, indicative of the dual influence of unsaturated fatty acids on MMP activation and activity. In conclusion, unsaturated fatty acids or molecules with similar structures could be attractive target for the development of natural pharmacological inhibitors directed against plasmin and/or MMPs in different pathological contexts such, skin UV irradiation, vascular diseases and tumour growth and invasion.
Abstract: HT-1080 fibrosarcoma cells express at their plasma membrane the elastin-binding protein (EBP). Occupancy of EBP by elastin fragments, tropoelastin or XGVAPG peptides was found to trigger procollagenase-1 (proMMP-1) overproduction by HT-1080 cells at the protein and enzyme levels. RT-PCR analysis indicated that elastin peptides did not modify the MMP-1 mRNA steady state levels, suggesting the involvement of a post-transcriptional mechanism. We previously reported that binding of elastin peptides to EBP induced other matrix metalloproteinases (MMP-2 and MT1-MMP) expression. Since those peptides were here found to also accelerate the secretion of urokinase from HT-1080 cells, culture medium was supplemented with plasminogen together with elastin peptides at aims to induce or potentiate MMPs activation cascades. In such conditions, plasmin activity was generated and exacerbate proMMP-1 and proMMP-2 activation. As a consequence, elastin peptides and plasminogen-treated HT-1080 cells displayed a significant type I collagen matrix invasive capacity.
Abstract: Soluble elastin-derived peptides from alkaline or elastase hydrolysis of insoluble elastin, as well as tropoelastin, increase matrix metalloproteinase-2 (MMP-2) production by human skin fibroblasts in culture as determined by gelatin zymography and ELISA. Such an effect is time and concentration dependent; it can be reproduced by synthetic elastin: VGVAPG, PGAIPG, and laminin: LGTIPG, hexapeptides and inhibited by lactose and is therefore elastin receptor-mediated. The steady state levels of MMP-2 mRNAs are invariant following elastin-fibroblasts interaction. Inhibition of phospholipase C (D-609), ADP-ribosylation factor (brefeldin), protein kinase C (RO-318220) and phospholipase D (1-propanol) totally abolished the elastin-mediated increase of MMP-2 production. It suggested that the post-transcriptional mechanism controlling the elastin-mediated overproduction of MMP-2 involved a cascade leading to phospholipase D activation.
Abstract: TSH-treated pig thyroid cells reorganize into follicle-like structures and exhibit differentiated functions. TSH also induces a phosphotyrosine phosphatase (PTPase) activity evaluated by phosphorylated substrate hydrolysis. Incubation of thyrocytes with various concentrations of 8-bromo-cyclic AMP or forskolin induces an increase of PTPase activity in a dose-dependent manner. During the culture period, adenylyl cyclase sensitivity, protein binding iodine and PTPase activity progressively increase from the first to the fourth day of the culture. Chronic treatment with phorbol 12-myristate 13-acetate (PMA) significantly inhibits PTPase activity during the first 24 h following PMA addition. GF 109203X, a specific inhibitor of protein kinase C, abolishes the inhibitory effect of PMA. Electrophoresis of membrane extracts allowed us to demonstrate a phosphatase activity at 111 kDa (p111). Vanadate inhibits this activity, indicating that p111 is a PTPase. This p111 is significantly reduced in PMA-treated cells. These data suggest that PTPase activity evidenced at 111 kDa is correlated with a differentiated state of primary cultured pig thyroid cells induced by TSH.
Abstract: We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.
Abstract: The matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the physiological and pathological breakdown of several extracellular matrix proteins. In the present study, we show that long-chain fatty acids (e.g. oleic acid, elaidic acid, and cis- and trans-parinaric acids) inhibit gelatinase A as well as gelatinase B with K(i) values in the micromolar range but had only weak inhibitory effect on collagenase-1 (MMP-1), as assessed using synthetic or natural substrates. The inhibition of gelatinases depended on fatty acid chain length (with C18 > C16, C14, and C10), and the presence of unsaturations increased their inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid) protect collagen and elastin fibers against degradation by gelatinases A and B, respectively. In order to understand why gelatinases are more susceptible than collagenase-1 to inhibition by long-chain fatty acids, the possible role of the fibronectin-like domain (a domain unique to gelatinases) in binding inhibitory fatty acids was investigated. Affinity and kinetic studies with a recombinant fibronectin-like domain of gelatinase A and with a recombinant mutant of gelatinase A from which this domain had been deleted pointed to an interaction of long-chain fatty acids with the fibronectin-like domain of the protease. Surface plasmon resonance studies on the interaction of long-chain fatty acids with the three individual type II modules of the fibronectin-like domain of gelatinase A revealed that the first type II module is primarily responsible for binding these compounds.
Abstract: To examine the effects of the uPA/plasmin system on cell migration in relation to the activation of MMP-9, we used ex vivo and in vitro wound-repair models of human bronchial epithelial cells and videomicroscopy techniques that make possible cell tracking and quantification of cell migration speeds. We observed that uPA was only detected in migrating cells at the wound edges and located at crucial sites for cell/extracellular matrix interactions. The implication of uPA in human bronchial epithelial cell migration was studied by incubating cultures with a monoclonal antibody raised against uPA and these experiments led to a 70% reduction in cell velocity. To examine the effects of the plasmin system on cell migration, we incubated cultures with increasing concentrations of plasmin or activated MMP-9. We observed a significant dose-dependent increase in cell migration velocity with plasmin (P < 0.001) and MMP-9 (P < 0.001). Moreover, addition of exogenous plasmin led to a twofold increase of activated MMP-9 in migrating cells. We also demonstrated that the addition of anti-uPA IgG led to an inhibition of 43% of activated MMP-9. In conclusion, these results show that uPA is involved in human bronchial epithelial cells migration. This action is mediated by the generation of plasmin, which in turn activates MMP-9, thus making possible cell migration.
Abstract: Invasiveness and metastatic capacity of tumor cells have been related to increased expression and activation of gelatinase-A (MMP-2). 9-octadecenoic acid (oleic acid, OA), a long-chain cis-unsaturated fatty acid, has been shown to partially inhibit the formation of lung metastatic colonies in an ex vivo model of implantation of metastatic cells into nude mice. Reduction of metastasis formation was suggested to be due to a decrease of MMP-2 activity in tumor tissue extracts. Since regulation of MMP-2 activity occurs at different levels, including gene expression, pro-enzyme activation and finally active enzyme inhibition, we here investigated the precise level of the inhibitory effect of OA on MMP-2 activity by oncogene-transformed human bronchial epithelial cells (BZR cells). OA, at the dose of 5 x 10(-5) M, was shown to inhibit by 50% MMP-2 activity released from BZR cells. Northern-blot analysis of mRNA encoding MMP-2, MT1-MMP, the physiological activator of MMP-2, and TIMP-2, the natural inhibitor of MMP-2, revealed that OA did not alter the steady-state levels of MMP-2, MT1-MMP and TIMP-2 mRNA. Also, gelatin zymography demonstrated that the extent of MMP-2 activation was not modified by OA treatment. On the contrary, OA could inhibit the fluorogenic quenching substrate (7-methoxycoumarin-4-yl)acetyl-L-Pro-Leu-Gly-Leu-[N-3-(2,4-dinitrophenyl)-L-2, 3-diaminopropionyl]-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) hydrolysis by recombinant MMP-2 with Ki = 4.3 microM. These data suggest that the beneficial influence of OA on the formation of lung metastatic colonies was independent of its influence on MMP-2 expression and/or activation, but could be attributed to inhibition of MMP-2 activity.
Notes: Meeting Abstract
Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO 2005)
Ft Lauderdale, FL, MAY 01-05, 2005
Assoc Res Vision Ophthalmol
