Gangliosides inhibit urokinase-type plasminogen activator (uPA)-dependent squamous carcinoma cell migration by preventing uPA receptor/alphabeta integrin/epidermal growth factor receptor interactions

The interaction of the urokinase-type plasminogen activator (uPA) receptor (uPAR) with integrins plays a critical role in the regulation of cell adhesion and migration. However, the molecular events underlying the modulation of the interaction of uPAR and integrin are poorly understood. Gangliosides are thought to regulate epithelial cell adhesion and migration by inhibiting alpha(5)beta(1) integrin and epidermal growth factor receptor (EGFR) signaling. We report here that increases in the expression of ganglioside NeuAcalpha2-->3Galbeta1-->3GalNAcbeta1-->4(NeuAcalpha2-->8NeuAcalpha2-->3)Galbeta1-->4Glcbeta1-Cer (GT1b) or NeuAcalpha2-->3Galbeta1-->4Glcbeta1-Cer (GM3) inhibit uPA-dependent cell migration by preventing the association of uPAR with alpha(5)beta(1) integrin or uPAR/alpha(5)beta(1) integrin with the EGFR, respectively. As a result, uPA-dependent focal adhesion kinase (FAK) and integrin-mediated EGFR signaling are suppressed. Both gangliosides inhibit uPAR signaling-stimulated migration; however, GM3 inhibits uPA-induced EGFR phosphorylation by blocking the crosstalk between integrin and EGFR, whereas GT1b suppresses both uPA-induced FAK and EGFR activation by preventing the activation of integrin alpha(5)beta(1).     

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.     

Cancer cell expression of urokinase-type plasminogen activator receptor mRNA in squamous cell carcinomas of the skin

In this study we have used in situ hybridization with radiolabeled antisense RNA probes to examine the expression of mRNA for urokinase-type plasminogen activator and its receptor in histologic samples of squamous cell (n = 7) and basal cell (n = 7) carcinomas of the skin. Messenger RNA for both urokinase-type plasminogen activator and its receptor were expressed in all of the squamous cell carcinomas, but could not be detected in the basal cell carcinomas. In all of the seven squamous cell carcinomas a signal for urokinase-type plasminogen activator receptor mRNA was detected focally in well-differentiated cancer cells surrounding keratinized pearls, and in four specimens urokinase-type plasminogen activator receptor mRNA was in addition expressed by cancer cells at the edge of invasively growing strands of tumor. Urokinase-type plasminogen activator mRNA expression was found in virtually all the cancer cells of the squamous cell carcinomas, and importantly we found, by hybridizations for urokinase-type plasminogen activator and its receptor mRNA on adjacent sections of squamous cell carcinomas, that it was exactly the invading cancer cells that simultaneously expressed both these components required for plasmin-mediated proteolysis at the cell surface. We have previously shown that both urokinase-type plasminogen activator and its receptor mRNA are expressed by the leading-edge keratinocytes in regenerating epidermis during mouse skin wound healing, and that wound healing is impaired in mice made deficient in plasminogen by targeted gene disruption. We propose that there are similarities between the mechanisms of generation and regulation of extracellular proteolysis during skin re-epithelialization and squamous cell carcinoma invasion. The ability of the squamous carcinoma cells to mimic the "invasive" phenotype of re-epithelializing keratinocytes may be one of the factors that make squamous cell carcinomas more aggressive tumors than basal cell carcinomas.     

Involvement of urokinase-type plasminogen activator in acantholysis induced by pemphigus IgG

Pemphigus IgG induces acantholysis in skin organ culture without the involvement of complement. Urokinase-type plasminogen activator, a proteolytic enzyme, has been implicated in the development of acantholysis. To test this hypothesis, we prepared a rabbit anti-urokinase antibody, which inhibited the plasminogen activator activity in normal human epidermis and in cultured keratinocytes. When added to skin organ cultures along with pemphigus IgG, anti-urokinase IgG completely prevented the development of acantholysis. Normal or preimmune rabbit IgG had no effect on pemphigus IgG-induced acantholysis. Plasminogen activator converts the zymogen plasminogen to its active form plasmin, a broad specificity serine proteinase. When high concentrations of plasminogen alone were added to skin organ culture, acantholysis of the pemphigus foliaceous type was induced. Anti-urokinase antibody also inhibited plasminogen-induced acantholysis. These results strongly support a pivotal role for plasminogen activator in the development of acantholysis.     

Differential expression of urokinase-type plasminogen activator and its type-1 inhibitor during healing of mouse skin wounds

The expression of urokinase-type plasminogen activator (u-PA) and its type-1 inhibitor (PAI-1) was examined in vivo in mouse wounds by in situ hybridization and immunohistochemistry. u-PA mRNA was present in both basal and suprabasal keratinocytes in the regenerative epithelial outgrowths at the edge of the wounds. In the same area, PAI-1 mRNA was only present in the basal keratinocytes. u-PA protein was detected in keratinocytes in several layers of the epithelial outgrowth, whereas PAI-1 protein was confined to the basal keratinocytes and to the area of the basal membrane. The two proteins and their mRNA were not detected in normal epidermis or in normal-looking epidermis adjacent to the wounds. Fibroblast-like cells and fairly large stellate cells (possibly macrophages) in the granulation tissue underneath the wound contained both the two proteins and their mRNA. The large stellate cells, showing a strong hybridization signal for PAI-1 mRNA, were especially abundant at the border between the necrotic wound and the newly formed granulation tissue. The specificity of these results was supported by the use of two different non-overlapping antisense probes, sense mRNA probes, antibody preparations preabsorbed with purified proteins, and Northern analysis of tissue extracts. The localized and regulated expression of u-PA and PAI-1 seen in this study may reflect that plasminogen activation plays a role in the migration of keratinocytes and connective tissue cells during reepithelialization and tissue remodeling in wound healing.     

Relationship between urokinase plasminogen activator receptor (uPAR) and the invasion of human prenatal hair follicle

During the morphogenesis of hair follicles, the invasive migration of basal keratinocytes resembles cell’s dissemination of tissue remodeling. The urokinase-type plasminogen activator receptor (uPAR) appears to be a key molecule in the metastasis. In order to elucidate the relationship between uPAR and the invasion of the human hair follicle, immunohistochemistry, RT-PCR, plasmids transfection, and western blot were used. The results showed that uPAR was expressed in the outermost epithelial cells of the hair follicle and the basal keratinocytes of epidermis, and that the expression decreased with the development of the hair follicle. The cells of the outer root sheath (ORS) and interfollicle epidermis, which overexpressed uPAR, acquired increased invasiveness; however, they showed decreased invasion with overexpression of the urokinase-type plasminogen activator amino terminal fragment (uPA ATF), which inhibited the combination of uPAR and uPA competitively, and the cell invasive migration with overexpressed uPAR was required activated extracellular signal-regulated kinases (ERK). These results implied that overexpression of uPAR promote the invasive migration of hair follicle into the dermis in uPA-dependent and independent manner during human prenatal development.     

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Studies on two siblings with recessive dystrophic epidermolysis bullosa (Hallopeau-Siemens) and the plasminogen activator and its inhibitor in the lesion

Activities of plasminogen activators (PA) and their inhibitors were studied in the bullous lesions of 2 siblings with recessive dystrophic epidermolysis bullosa (RDEB). The hematological findings of the patients revealed hyperfibrinogenemia, hyper-gamma-globulinemia and marked thrombocytosis. The immunofluorescent studies showed strong deposits of plasminogen, fibrin-degenerative products and alpha 1-antitrypsin around the blister lesions. On the other hand, alpha 2-macroglobulin (alpha 2-M) was sparsely deposited. The levels of alpha 2-M in the blister fluid of the patients were also decreased in comparison with those from patients with other bullous dermatoses. The topical application of antibiotic ointment with strong PA inhibitor was clinically effective when applied to the blister and eroded lesions of the patients. These findings suggest that the increased activity of PA may play an important role in the development of blister formation in patients with RDEB.     

Modulation of urokinase-type and tissue-type plasminogen activator occurs at an early stage of progressing stages of chronic venous insufficiency

Chronic venous insufficiency (CVI) progresses through a series of clinical stages, from healthy skin to poorly healing leg ulcers. The aim of this study was to analyse the distribution pattern and activity level of urokinase-type (uPA) and tissue-type plasminogen activators (tPA) in normal skin and in tissue biopsies of progressing stages of CVI, prior to and including venous ulceration. Biopsies 6 mm thick were taken from 14 healthy volunteers and 37 patients with 5 different stages of CVI: telangiectases; stasis dermatitis; hyperpigmentation; lipodermatosclerosis; and leg ulcer. Changes in the enzymatic activity and spatial localization of uPA and tPA during the progression of CVI were examined using in situ histological zymography. Normal skin and skin with telangiectases showed a punctate PA activity, consisting of both uPA and tPA activity. As CVI progressed, an increase in the distribution of uPA and a decrease in tPA activity was observed. The spatial localization of uPA was widespread within the dermis of biopsies from stasis dermatitis and lipodermatosclerosis and was associated in particular with the dermoepidermal junction. Hyperpigmented skin revealed a pattern of PA expression similar to that of healthy skin. However, leg ulcer specimens exhibited peak levels of uPA with little tPA. Furthermore, a plasminogen-independent protease activity that was not present in any of the earlier stages of CVI appeared. Our results indicate that there are profound changes in PA activity during the progression of CVI and that these changes begin early in CVI, for example, in stasis dermatitis. We hypothesize that the balance or imbalance of the PA activity in the later stages of CVI is an important pathogenic factor for the development of venous leg ulcer.     

Novel mutations in the gene encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) and description of five ancestral haplotypes in patients with Mal de Meleda

Mal de Meleda is a recessive, transgressive palmoplantar keratoderma for which we previously identified mutations in the gene encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1). In this report we describe two new mutations: (i) a founder mutation, which changes a conserved cysteine residue to tyrosine (C99Y) in a large inbred Tunisian pedigree, and (ii) a signal sequence mutation (W15R), which was homozygous in a German family and heterozygous in a Scottish patient. Four ancestral haplotypes were observed in 69 patients from countries around the Mediterranean basin, and an additional haplotype was found in the German and Scottish patients.     

失血性休克血清对内皮细胞t-PA和PAI-1mRNA表达的影响

目的探讨失血性休克大鼠血清对大鼠主动脉内皮细胞（SVAREC）表达组织型纤溶酶原激活剂（t—PA）和纤溶酶原激活剂抑制物-1（PAI-1）的影响。方法建立失血性休克大鼠模型,取假休克组、休克组血清,并将胎牛血清作为正常对照组,无菌处理后加入到培养基,分别与内皮细胞分别培养6h、12h和18h,用逆转录-聚合酶链反应（RT—PCR）检测t—PA和PAI—1mRNA的表达。结果与正常对照组相比,休克组血清刺激SVAREC6h、18h后t—PAmRNA均升高。与正常对照组及假休克组相比,休克组血清刺激SVAREC12h后,PAI-1mRNA升高-P〈0．05）;培养18h后,与正常对照组比,PAI-1mRNA升高（P〈0．01）。结论失血性休克血清上调内皮细胞t—PA和PAI-1mRNA表达,提示休克血清对内皮细胞有损伤作用,休克中应注意保护内皮细胞。     

Interleukin-1β upregulates tissue-type plasminogen activator in a keratinocyte cell line (HaCaT)

Human keratinocytes synthesize and secrete tissue-type plasminogen activator (tPA). tPA converts the inactive precursor enzyme plasminogen into the trypsin-like proteinase plasmin. tPA is not found in normal epidermis, but in lesional epidermis from patients with a variety of cutaneous diseases, including psoriasis, pemphigus and pemphigoid. The presence of tPA is probably a reaction to the disease process rather than the initiating event in these etiologically and histopathologically diverse lesions. However, the factor(s) that upregulate tPA expression and secretion in keratinocytes have remained largely elusive. We sought to determine whether the inflammatory cytokine interleukin-1β (IL-1β), which is commonly present in diverse epidermal lesions, influences tPA production. Accordingly, we studied the influence of IL-1β on secretion of tPA by cells of the human keratinocyte cell line HaCaT. We found that IL-1β increased tPA secretion in these cells. Given the observation that IL-1β is a common proinflammatory mediator in cutaneous diseases, our findings may explain the increase in tPA in clinically and etiologically diverse inflammatory epidermal lesions. Received: 9 October 1995     

Characterization of keratinocyte plasminogen activator inhibitors and demonstration of the prevention of pemphigus IgG-induced acantholysis by a purified plasminogen activator inhibitor

To investigate the mechanisms by which cutaneous plasminogen activator (PA) may be regulated, we have tested cultured keratinocytes for the presence of PA inhibitors. Using biosynthetic labeling experiments with 35S-methionine in conjunction with specific antibody precipitation, we have shown that human keratinocytes in culture synthesized and secreted both PA inhibitor 1 and PA inhibitor 2. PA inhibitor 1 was present in conditioned media in the inactive form, but it could be detected with reverse phase autography. PA inhibitor 2 was detected by its ability to form complexes with 125I-uPA. Potential therapeutic relevance for cutaneous PA inhibitor 2 was suggested in skin organ culture experiments which demonstrated that purified PA inhibitor 2 from human placenta was able to prevent the acantholytic changes induced by pemphigus IgG.