Urinary trypsin inhibitor efficiently inhibits urokinase production in tumor necrosis factor-stimulated cells

We demonstrated that urinary trypsin inhibitor (UTI) efficiently inhibits soluble and tumor cell-associated plasmin activity and subsequently inhibits tumor cell invasion and metastasis. The effect of UTI on tumor necrosis factor-alpha (TNF)-induced stimulation of urokinase-type plasminogen activator (uPA) in cultured human umbilical vein endothelial cells (HUVEC) and in the promyeloid leukemia U937 cells was studied. uPA antigen was evaluated in the cell lysate and in the conditioned media by enzyme-linked immunosorbent assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot. TNF can promote the production of uPA in HUVEC and in U937 cells. The PKC inhibitors (H7, calphostin C, and staurosporine) inhibited TNF-induced uPA expression and secretion in a dose-dependent manner. Analysis of the expression of cell surface receptor-bound uPA by flow cytometry using uPA-specific MAb indicates that induction of uPA expression by TNF was inhibited when these cells were incubated with UTI. On the other hand, treatment of the cells with UTI alone failed to alter uPA production. UTI also reduced the secretion of uPA in TNF-treated cells. UTI was as effective as PKC inhibitors in inhibiting uPA expression by TNF. Incubation of the cells with UTI, however, had no effect on the ability of PMA to stimulate cell-associated uPA expression. These data suggest that UTI may influence the PKC-dependent protein kinase pathway in uPA expression. The study on intracellular pathways involved in UTI modulation of uPA will enhance our understanding of the role that UTI plays in uPA-mediated cellular invasion.     

Endothelial and macrophage upregulation of urokinase receptor expression in human renal cell carcinoma

The binding of urokinase-type plasminogen activator (u-PA) to a specific cell surface receptor (uPA-R) has been shown to enhance plasminogen activation, a process involved in extracellular matrix degradation and cell migration during angiogenesis and tumor growth. We investigated the expression of u-PA and uPA-R in renal cell carcinomas (n = 11). By immunohistochemistry using monoclonal and polyclonal anti-uPA-R antibodies, we found that tumoral capillary endothelial cells (von Willebrand factor and CD31 positive cells) overexpressed uPA-R, whereas vascular endothelial cells of the normal human kidney do not. In addition, tumor-associated macrophages (CD68-positive cells) strongly expressed uPA-R. In contrast, few tumoral cells and stromal fibroblasts expressed uPA-R. By in situ hybridization using a cDNA S35-labeled probe specific for uPA-R, we confirmed the local expression of uPA-R messenger RNA. We also detected the induction of u-PA in tumoral capillary endothelial cells and in tumor-associated macrophages. In two cases, tumoral cells themselves were also stained by anti-u-PA antibodies in focal areas. Finally tissue-type plasminogen activator (t-PA) was also overexpressed by tumoral capillary endothelial cells as compared with endothelial cells of normal human kidney vessels. These findings indicate an active invasive phenotype of endothelial cells in renal cell carcinoma and suggest a role for the plasminogen activation system in tumoral angiogenesis and invasion.     

Regulation and role of urokinase plasminogen activator in vascular remodelling

1. Urokinase plasminogen activator (uPA) is produced and secreted by multiple vascular cell types, thus influencing the processes and the extent to which the vasculature is remodelled during the development of the intima or a neointima and during hypertrophy and angiogenesis. 2. Urokinase plasminogen activator mRNA expression is up- and down-regulated by growth factors, cytokines and steroids. Urokinase plasminogen activator is secreted as a single chain inactive form that may be proteolytically converted to active or inactive forms. Targeting of proteolytic activity may occur via focalized expression of uPA and its cell surface receptors (uPAR). Proteolytic activity is also controlled through the often co-ordinated expression of specific inhibitors. 3. A proteolytic cascade involving uPA provides its major role in tissue remodelling through the primary degradation of extracellular matrix and secondarily through the activation of transforming growth factor-beta or release from the matrix of basic fibroblast growth factor. In addition, uPA secreted by growth factor-stimulated vascular cells may contribute to the chemotactic and mitogenic responses ascribed to the growth factor and recent evidence strongly suggests that uPA has direct biological actions on vascular cells. 4. The cell surface binding of uPA via its growth factor-like domain to uPAR localizes and activates the protease, but may also initiate transmembrane signalling of biological responses, including migration/invasion and proliferation. As the uPAR lacks intracellular signalling domains, the signals may be transduced via interactions between uPA/uPAR and more classical signalling receptors. The mechanism by which uPA may be involved in cell signalling is yet to be elucidated.     

Functional involvement of urokinase-type plasminogen activator receptor in pemphigus acantholysis

Urokinase-type plasminogen activator (uPA) has been well documented in the development of pemphigus acantholysis. The function of its receptor (uPA-R) in pemphigus acantholysis has only recently attracted attention. Increased expression of uPA-R has been demonstrated in pemphigus vulgaris. In this study, we have further explored the functional involvement of uPA-R in pemphigus acantholysis. Our results show that uPA-R expression is significantly increased in acantholytic foci of pemphigus vulgaris and pemphigus foliaceus but not in bullous pemphigoid or normal skin specimens; the expression of uPA-R in cultured human keratinocytes is subjected to regulation by pemphigus vulgaris IgG but not by pemphigoid IgG or normal human IgG; furthermore, anti-uPA-R monoclonal antibody effectively inhibits pemphigus vulgaris IgG induced acantholysis in skin organ cultures. These data suggest that uPA-R may play an important role in the pathogenesis of pemphigus acantholysis.     

PKC regulation of microfilament network organization in keratinocytes defined by a pharmacological study with PKC activators and inhibitors

The modulation by PKC activators and inhibitors of adhesion, spreading, migration, actin cytoskeleton organization, and focal complex formation in keratinocytes attaching to type I collagen was studied. Two actin microfilament networks, stress fibers and cortical actin, could be distinguished on the basis of cellular distribution and opposite regulation by growth factors, tyrosine kinase inhibitors, and PKC activators. Stress fiber formation was stimulated by growth factors and by PMA (100 ng/ml) and these stimulations were blocked by tyrosine kinase inhibitors (0.3 mM genistein and 1 microM herbimycin A). By contrast, the cortical network occurred in quiescent cells, was unaffected by tyrosine kinase inhibitors, and was broken down after PKC activation by PMA. Spreading, migration, and actin polymerization were completely blocked while adhesion efficacy was significantly decreased by three specific PKC inhibitors. Half-inhibition of migration was obtained with 0.025, 1, and 3 microM concentrations of calphostin C, chelerytrine chloride, and D-erythrosphingosine, respectively, which are concentrations close to those known to inhibit the PKC kinase function in vitro. Paxillin clustering, which was observed even in the presence of tyrosine kinase inhibitors, disappeared only when actin polymerization was completely impaired, i.e., in cells treated with PKC inhibitors or with both tyrosine kinase inhibitors and PMA, which indicated that focal complex formation was highly dependent on microfilament reorganization. The analysis of these data underscores a major regulation function of PKC in the molecular events involved in growth factor and adhesion-dependent regulation of microfilament dynamics.     

Migration of human keratinocytes in electric fields requires growth factors and extracellular calcium

Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human keratinocytes in vitro and may promote wound healing in vivo. We have examined the effects of growth factors and calcium, normally present in culture medium and the wound fluid, on the directional migration of human keratinocytes in culture. In electric fields of physiologic strength (100 mV per mm), keratinocytes migrated directionally towards the cathode at a rate of about 1 microm per min. This directional migration requires several growth factors. In the absence of these growth factors, the cell migration rate decreased but directionality was maintained. Epidermal growth factor alone restored cell migration rates at concentrations as low as 0.2 ng per ml. Insulin at 5-100 microg per ml or bovine pituitary extract at 0.2%-2% vol/vol also stimulated keratinocyte motility but was not sufficient to fully restore the migration rate. Keratinocyte migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 microM to 3.3 mM did not significantly change keratinocyte migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to migration medium reduced, and eventually abolished, keratinocyte motility. Our results show that (i) growth factors and extracellular calcium are required for electric field-induced directional migration of human keratinocytes, and (ii) keratinocytes migrate equally well in low and high calcium media.     

Mouse dendritic epidermal T cells exhibit chemotactic migration toward PAM 212 keratinocyte culture supernatants

Dendritic epidermal T cells (DETCs) are Thy-1+, CD45+, CD3+, CD4-, CD8-, and T-cell receptor-V gamma 3/V delta 1+ leukocytes that reside normally in adult mouse skin. We have demonstrated previously that keratinocytes serve as adhesion substrates for DETCs, and that interleukin 7 (IL-7), which is produced by keratinocytes, serves as a growth factor for DETCs. The present study was conducted to address the mechanisms by which DETCs migrate into the epidermis, reasoning that keratinocytes may also be a source of chemotactic activity. Short-term DETC lines were 35S-labeled and tested for migration toward Pam 212 keratinocyte culture supernatants using a modified Boyden chamber method; cell movement from upper chambers toward test samples in lower chambers was traced by counting radioactivity. DETC displayed rapid (within 60 min) and marked (> 50%) migration toward keratinocyte supernatants. The majority of cells that had migrated into keratinocyte supernatants expressed the V gamma 3 T-cell receptor, thus verifying that the migrating cells were DETCs. Addition of keratinocyte supernatants to the upper chambers completely blocked migration, suggesting its chemotactic nature. By contrast, no DETC migration was observed toward 3T3 fibroblast supernatants. Chemotactic activities were 1) produced by Pam 212 cells even in the absence of serum; 2) greater than 12 kD in size; 3) heat and pH labile; 4) trypsin sensitive; and 5) precipitated by 60-100% ammonium sulfate. Several cytokines (e.g., IL-1 alpha and IL-8) failed to mediate DETC migration when added to the lower chambers. Likewise, the same cytokines, when added to the upper chambers, failed to inhibit DETC migration toward Pam 212 supernatants. These results support our hypothesis that keratinocytes facilitate the residence of DETC in epidermis by secreting unique chemotactic factors, by providing adhesion substrates, and by elaborating specific growth factors.     

EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.     

A case of congenital complete heart block in a mother with anti-52 kD SS-A/Ro antibodies

Amphiregulin and transforming growth factor-alpha, agonists for the epidermal growth factor receptor, are the major autocrine growth factors for cultured keratinocytes, and their substantial overexpression in psoriatic lesions suggests that they are crucial to the basal hyperplasia that characterizes psoriasis. Amphiregulin binds to heparin and related highly sulfated polysaccharides, and exogenous heparin blocks its growth factor activity, rationalizing previous reports that psoriasis responds to heparin therapy. Differentiating keratinocytes produce increased amounts of protein-bound as well as free-chain heparan sulfates, which may function physiologically as amphiregulin antagonists. By promoting keratinocyte synthesis of these heparan sulfates, glucosamine administration may inhibit amphiregulin function and thus provide therapeutic benefit in psoriasis. Concurrent ingestion of fish oil, by impeding the excessive activation of protein kinase C, may decrease keratinocyte production of amphiregulin and other autocrine growth factors, thus complementing the postulated benefits of glucosamine.     

Polymorphism of the HLA-C promotor region

We have studied how keratinocytes cultured under hyperthermal conditions modulate skin fibroblast growth potential and their biosynthetic phenotypes in vitro. When keratinocytes were cultured at 30, 34, 37 or 39 degrees C, the conditioned medium of the keratinocytes cultured at 39 degrees C showed a greater inhibitory activity for fibroblast proliferation and greater synthetic activities of collagen and glycosaminoglycans than those incubated at 30, 34, or 37 degrees C. Transforming growth factor (TGF) beta 1 production in skin fibroblasts was also stimulated by the keratinocyte conditioned medium cultured at 39 degrees C. The stimulating activity of collagen and glycosaminoglycan syntheses of keratinocyte conditioned medium may be explained at least partly by enhanced TGF beta 1 production. The results indicate that keratinocytes cultured at a higher temperature (39 degrees C) may secrete factor(s) which modulate both fibroblast growth and matrix synthesis. This may provide evidence that under hyperthermal conditions epidermis can influence the functions of skin fibroblasts.     

A competitive chromogenic assay to study the functional interaction of urokinase-type plasminogen activator with its receptor

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.     

End-stage renal disease and its management in older adults

Receptor tyrosine kinases are key regulators of cellular function including cell growth, differentiation, migration, and morphogenesis. Disruptions of receptor tyrosine kinase signaling pathways are often associated with changes in cellular proliferative capacity and tumorigenesis. Both receptor-specific and cell type-specific factors may contribute to the ultimate cellular responses observed after receptor activation. In this regard, we find that both normal keratinocytes and their tumorigenic counterparts display differential responses to activation of receptor tyrosine kinases. Multiple ligands were mitogenic for keratinocytes, but only epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), and scatter factor/hepatocyte growth factor (SF/HGF) promoted cell motility as assessed by colony dispersion (scattering) and in vitro reepithelialization. Interestingly, growth factor specificity for motility coincided with ligand-mediated cell invasion through a reconstituted basement membrane and induction of the 92-kDa metalloproteinase (MMP-9) activity as determined by gelatin zymogram analysis. Inhibitors of MMP activity or addition of an MMP-9 neutralizing antibody resulted in the loss of growth factor-induced colony dispersion, suggesting a functional role for MMP-9 induction during this response. Coordinate regulation of MMP-9 induction and the migratory response are likely to contribute to the enhanced invasive potential observed in response to EGF and SF/HGF. Our findings suggest that alternate receptor-mediated signaling pathways leading to differences in gene expression may be involved in complex cellular responses such as colony dispersion or invasion.     

Involvement of urokinase and its receptor in the remodeling of normal and pathological tissue

Urokinase is the main component of the fibrinolytic system and comprises two functions. Namely, the catalytic domain enables to trigger the proteolytic cascade in blood and in the vessel wall, stimulating the activation of growth factors, matrix remodelling and smooth muscle cell proliferation. On the other hand, the amino terminal domain of urokinase that is homologous to epidermal growth factor promotes urokinase to bind to the specific receptor. The cell surface binding of uPA may initiate the activation of the second messengers, protein phosphorylation and cell migration. This dual function of urokinase affects vascular growth, as well as the intimal thickening of the vessel wall after intravascular injury. Also uPA and its receptor are implicated in many physiological and pathological processes, including ovulation, embryogenesis, cancer metastasis, inflammation, and wound healing.     

Requirement of receptor-bound urokinase-type plasminogen activator for integrin alphavbeta5-directed cell migration

The urokinase plasminogen activator (uPA) interacts with its cell surface receptor (uPAR), providing an inducible, localized cell surface proteolytic activity, thereby promoting cellular invasion. Evidence is provided for a novel function of cell surface-associated uPA.uPAR. Specifically, induction of cell surface expression of uPA. uPAR by growth factors or phorbol ester was necessary for vitronectin-dependent carcinoma cell migration, an event mediated by integrin alphavbeta5. Cell migration on vitronectin was blocked with either a soluble form of uPAR, an antibody that disrupts uPA binding to uPAR, or a monoclonal antibody to alphavbeta5. Moreover, plasminogen activator inhibitor type 2 blocked this migration event but did not affect adhesion, suggesting a direct role for uPA enzyme activity in this process and that migration but not adhesion of these cells is regulated by uPA.uPAR. Growth factor-mediated induction of uPA.uPAR on the carcinoma cell surface promotes a specific motility event mediated by integrin alphavbeta5, since cells transfected with the beta3 integrin subunit expressed alphavbeta3 and migrated on vitronectin independently of growth factors or uPA.uPAR expression. This relationship between alphavbeta5 and the uPA.uPAR system has significant implications for regulation of motility events associated with development, angiogenesis, and tumor metastasis.     

The relation between diabetes mellitus and IFN-gamma, IL-12 and IL-10 productions by CD4+ alpha beta T cells and monocytes in patients with pulmonary tuberculosis

Membrane glycoproteins (gps) play an important role in cell-cell interactions during epidermal maturation, and we have previously shown an up-regulation of PNA-binding gps in cultured human keratinocytes treated with interferon gamma (IFN-gamma). The protein kinase C (PKC) pathway is known to play a key role in the regulation of proliferation and differentiation of keratinocytes and is also reported to be involved in some IFN-gamma-mediated effects. In order to evaluate the cellular mechanisms and whether PNA-binding gp expression is related to the differentiative activity of the lymphokine, we studied the effects of PKC agonists and antagonists and the role of retinoic acid (RA), in the induction of these gps in cultured human keratinocytes stimulated with IFN-gamma and processed for protein analysis. The expression of PNA-binding gps was revealed by incubation of SDS-polyacrylamide gels with 125I-PNA. The PKC antagonists (H7, sphingosine) as well as RA downregulated the IFN-gamma-induced PNA-reactive gps, whereas staurosporine and TPA upregulated their expression. These results provide evidence that PNA-reactive gps are late highly IFN-gamma-sensitive markers of keratinocyte differentiation, drastically modulated through selective isoforms of PKC.     

Urokinase receptor-dependent upregulation of smooth muscle cell adhesion to vitronectin by urokinase

The plasminogen activator system has been implicated in the modulation of the response to vascular injury. Although urokinase-type plasminogen activator (uPA) and its receptor (uPAR) may enhance matrix degradation as well as migration and invasion by smooth muscle cells (SMCs), their roles in cell adhesion are uncertain. Therefore, we examined the ability of uPA and uPAR to modulate adhesion of cultured human vascular SMCs to various matrices. We demonstrated a dose-dependent stimulation of adhesion by single-chain uPA (scuPA) to vitronectin (maximum 1.55-fold [+/-0. 04-fold] increase, 10 nmol/L, P<0.002) but not to laminin, collagen I, or collagen IV. Baseline adhesion to vitronectin was completely inhibited by both EDTA and RGD peptide but was restored to >40% of control in the presence of scuPA (P=0.001 and 0.046, respectively). Adhesion to vitronectin was also significantly enhanced by the amino-terminal fragment of uPA (P=0.007) and two-chain, high-molecular-weight uPA (P<0.01) but not by the low-molecular-weight fragment of uPA, which lacks the receptor-binding domain. Aprotinin, a plasmin inhibitor, had no effect on baseline or scuPA-stimulated adhesion, suggesting a plasmin-independent process. Preincubation of scuPA with soluble uPAR inhibited scuPA stimulation of adhesion by 88+/-14% (P=0.01), as did pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes glycophosphatidylinositol-anchored proteins, including uPAR. Antibodies to both alphavbeta3 and alphavbeta5 integrin inhibited baseline adhesion but not scuPA stimulation. Finally, coating plates with scuPA alone enabled cell adhesion, which could be inhibited by both soluble uPAR and anti-uPAR antibodies. These data suggest that uPA stimulates adhesion of SMCs specifically to vitronectin and that it is mediated by an interaction with uPAR. Upregulation of both proteins after vascular injury may facilitate migration through stimulation of both matrix degradation and cell adhesion.     

EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.