Interaction of urokinase a chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator☆
Abstract
On the basis of a fibrinolytic assay with 125I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, we have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration (5 ng/ml) which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, we hypothesize that this mechanism may be important in vivo during the process of wound repair.
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Fibrinolytic system in the process of wound healing in rat
2000, PathophysiologyWe investigated the involvement of fibrinolytic system in the process of wound healing as well as angiogenesis in rats using a model system, in which polystyrene capsules with 100 perforated pores filled without (empty capsule) or with fibrin gel (fibrin capsule) were subcutaneously implanted into the dorsal of rats. The intracapsular tissue was accompanied by neovascularization and the tissue weight in the fibrin capsule was three times that of the empty capsule on day 5 after implantation. Both plasminogen activation and amidolytic activities of urokinase-type plasminogen activator (u-PA) in the tissue extracts and intracapsular fluid obtained from the fibrin capsule were higher than those in the empty capsule on day 5 after implantation. Both u-PA and its specific receptor (u-PAR) mRNAs were detected in the intracapsular tissues formed in both capsules. On day 5 after implantation, though the levels of u-PA mRNA in the tissue of the empty capsule and those of the fibrin capsule were not significantly different, more u-PAR mRNA was expressed in the tissue of the fibrin capsule than in the empty capsule. Furthermore, when the cultured endothelial cells were incubated with intracapsular fluids, the proliferation of the cells was significantly enhanced. These results indicate that fibrinolytic activities increase in the presence of fibrin gel, possibly by up-regulation of the u-PA/u-PAR system in the early stage of rat wound healing.
Cultivation and transplantation of epidermal keratinocytes
1999, International Review of CytologyTransplantation of autologous cultured keratinocytes is the most advanced area of tissue engineering which has clinical application in restoration of skin lesions.In vitro, disaggregated keratinocytes undergo activation and after adhesion and histogenic aggregation form three-dimensional epithelial sheets suitable for grafting on prepared wounds that provide a reparative environment. Epidermal stem cells survive and proliferate in culture, retaining their potential to differentiate and to produce neoepidermis. Reconstructed skin is physiologically compatible to split-thickness autografts. Autotransplantation of cultured keratinocytes is a promising technique for gene therapy.In many cases allografting of cultured keratinocytes promotes wound healing by stimulation of epithelialization. Banking of cryopreserved keratinocytes is a significant improvement in usage of cultured keratinocytes for wound healing. Skin substitutes reconstructed in vitro that have morphological, biochemical, and functional features of the native tissue are of interest as model systems that enable extrapolation to situations in vivo.
Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/ uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for α2-macroglobulin receptor/LDL receptor-related protein (α2MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI-1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell-bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the disappearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.
Differential expression of urokinase-type plasminogen activator (uPA), its receptor (uPA-R), and inhibitor type-2 (PAI-2) during differentiation of keratinocytes in an organotypic coculture system
1995, Experimental Cell ResearchCultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-I stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.
Vascular endothelial growth factor increases urokinase receptor expression in vascular endothelial cells
1995, Journal of Biological ChemistryVascular endothelial growth factor (VEGF) is a potent angiogenic factor and endothelial cell-specific mitogen that stimulates urokinase-type plasminogen activator (uPA) activity in vascular endothelial cells. Here, we report that VEGF increases the high affinity binding of uPA to the same cells and that this binding is prevented by a peptide corresponding to the uPA receptor (uPAR) binding growth factor-like domain of uPA. Ligand cross-linking, ligand blotting, and uPA-Sepharose affinity chromatography revealed an increase in a cell surface uPA binding protein that corresponds to the uPAR on the basis of its affinity for uPA, Mrof 50,000-55,000, and phosphatidylinositol-specific phospholipase C sensitivity. By Scatchard analysis, VEGF increased the number of uPAR molecules by 2.8-3.5-fold and concomitantly decreased their affinity for uPA. By northern blotting uPAR mRNA was increased in a dose- and time-dependent manner in response to VEGF. Taken together, these findings demonstrate that VEGF-induced angiogenesis is accompanied by increased uPAR expression and uPA activity on the endothelial cell surface. These observations are consistent with the notion that the uPA-uPAR interaction facilitates cellular invasion.
Production of second messengers following chemotactic and mitogenic urokinase-receptor interaction in human fibroblasts and mouse fibroblasts transfected with human urokinase receptor
1994, Experimental Cell ResearchWe studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transferred with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG treshold level required to trigger DNA synthesis.
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This work was supported by a grant from Ministero Pubblica Istruzione (DPR 382/80, 40% and 60%) and A.I.R.C.