Retinoic acid upregulates the plasminogen activator system in human epidermal keratinocytes

The activation of the proteolytic plasminogen activator system is important for the re-epithelialization of skin wounds. Keratinocytes synthesize and secrete the urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. Receptor-bound urokinase-type plasminogen activator efficiently activates cell surface bound plasminogen. This results in pericellular proteolysis, which facilitates keratinocyte migration. Urokinase-type plasminogen activator activity is specifically controlled by plasminogen activator inhibitor-1 and -2. As retinoids have been reported to accelerate epithelialization of skin wounds in animal studies and clinical settings, we investigated the effects of all-trans retinoic acid on the plasminogen activator system in human epidermal keratinocytes. As tested in a chromogenic plasminogen activation assay, incubation with 10 microM all-trans retinoic acid caused a marked induction of cell-associated plasminogen activity after 24 h, and this induction was blocked by neutralizing anti-urokinase-type plasminogen activator antibodies, but not anti-tissue-type plasminogen activator antibodies. All-trans retinoic acid lead to a strong increase in urokinase-type plasminogen activator (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) after 24 h. At this time-point, tissue-type plasminogen activator and plasminogen activator inhibitor-1 and -2 proteins were not or only slightly increased. Northern blot analyses revealed that all-trans retinoic acid caused an early and short-lived increase of plasminogen activator inhibitor-1, but a prolonged induction of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA levels. Collectively, these data suggest that all-trans retinoic acid activates the plasminogen activator system in human epidermal keratinocytes by differentially regulating activating and inhibiting components. The activation of the plasminogen activator system may be one mechanism by which all-trans retinoic acid exerts beneficial effects in cutaneous wound healing.     

Plasminogen activator system in pemphigus vulgaris

Summary Pemphigus vulgaris (PV) is caused by autoantibodies against desmosomes and is characterized by intra-epidermal blisters. The pathology of PV has been linked with plasminogen activation in lesional epidermis. The plasminogen activator system (PA system) consists of urokinase-type plasminogen activator (uPA). tissue-type PA (tPA). as well as the two types of plasminogen activator inhibitors (PAI-1 and PAI-2). In keratinocytes. uPA binds to a specific cell surface receptor for uPA (uPA-R = CD87) in an autocrine manner. Cell-bound uPA is regulated by PAIs. The central PA system component plasminogen, which is present in plasma and interstitial fluids, is bound to the keratinocyte surface via plasmin(ogen) binding sites, where it can be activated by uPA-R-bound uPA. Cell surface-associated plasmin then mediates pericellular proteolysis. As the topographical organization of the distinct PA system components in lesional epidermis of PV remained elusive, we have performed the present immunohistological analysis of lesional and non-lesional epidermis of PV. In keratinocytes directly involved in the epidermal split formation, plasmin(ogen) was stained in nine of 10 cases, uPA-R and uPA in four of 10 cases and PAI-2 in seven of 10 cases. Together, acantholytic plasmin(ogen)⁺ keratinocytes appeared in three different phenotypes: uPA-R⁺/uPA⁺ and PAI-2⁺, uPA-R^-/uPA^- and PAI-2⁺. as well as uPA-R^-/uPA^- and PAI-2^-. Our findings demonstrate that, in acantholytic keratinocytes of PV. PAs and PAIs appear as differentially regulated components of the PA system.     

Keratinocyte urokinase-type plasminogen activator is secreted as a single chain precursor

Urokinase-type plasminogen activator (uPA) is produced and secreted by cultured human keratinocytes as a single chain precursor. UPA in keratinocyte conditioned medium is not susceptible to inhibition with diisopropylfluorophosphate (DFP), and it has an apparent molecular weight of 55 kD under both reducing and nonreducing conditions. Cleavage of keratinocyte uPA by plasmin results in the formation of a 96 kD complex comprised of activated uPA and PA inhibitor 2. PA extracted from normal human epidermis is only partially inhibited by DFP, suggesting that precursor uPA is also present in vivo. The synthesis of uPA as a precursor with reduced enzymatic activity as well as decreased affinity for inhibitors is likely to be a mechanism by which normal epidermis regulates plasminogen activation in vivo.     

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.     

Immunohistochemical characterization of the plasminogen activator system in psoriatic epidermis

The relative topographical distribution of urokinase-type plasminogen activator (uPA), tissue-type PA (tPA). PA-inhibitor-1 (PAI-1). PA-inhibitor-2 (PAI-2), plasmin(ogen), α₂-antiplasmin, and α₂-macroglobulin was studied in lesional epidermis of psoriasis vulgaris, and in normal epidermis, by immunohistochemistry. In psoriatic epidermis, tPA predominated, although uPA was found in some biopsies. PAs were not detected in normal epidermis. PAI-1 was not detected in normal epidermis and was only present in a proportion of biopsies of psoriatic lesions. PAI-2 was found in normal and psoriatic epidermis. Plasmin(ogen) was confined to the basal cell layer of normal epidermis, whereas in lesional psoriatic skin it was scattered throughout the epidermis, α₂-antiplasmin and α₂-macroglobulin were not found in the epidermis of normal skin. In psoriatic epidermis α₂-antiplasmin was confined to the subcorneal layer, whereas staining for α₂-macroglobulin was found only in a proportion of biopsies, in the upper epidermis. Our immunohistological findings indicate that colocalization of tPA and its substrate plasminogen may allow efficient generation of plasmin, and that the focal absence of plasmin inhibitors may then favour the persistence of plasmin activity.     

Plasminogen activation in lesional skin of Pemphigus vulgaris type Neumann

Zymographic and immunological studies revealed that primarily tissue-type plasminogen activator and to a lesser extent urokinase-type plasminogen activator were present in fluids of pemphigus vulgaris (type Neumann) skin blisters. Furthermore, plasmin activity was detected in pemphigus blister fluids using chromogenic peptide substrate assays. In pemphigus, but not in control, suction blister fluids plasmin/₂-antiplasmin and plasmin/ ₂-macroglobulin complexes were found by immunoprecipitation or by testing in immunoassays after fractionation by molecular-sieve chromatography. Plasmin activity, detected by a low molecular weight chromogenic peptide assay, was ascribed to plasmin/₂-macroglobulin complexes. Since formation of plasmin/inhibitor complexes requires active plasmin, the finding indicates previous activation of plasminogen in pemphigus lesions.     

Mite and cockroach allergens activate protease-activated receptor 2 and delay epidermal permeability barrier recovery

Protease-activated receptor-2 (PAR-2) is known to be involved in epidermal permeability barrier function homeostasis. PAR-2 activation occurs after barrier disruption and further activation of PAR-2 by activating peptide significantly delays barrier recovery rate. Cockroach and house dust mite allergens, both known to be associated with the development of asthma, allergic rhinitis, and atopic dermatitis, have protease activity, which can activate PAR-2. In this study, we investigated the effects of both allergens on the epidermal barrier function as well as on the epidermal calcium gradient. Both allergens, when topically applied on the barrier-disrupted site, increased protease activities in the epidermis and delayed barrier recovery and lamellar body secretion in murine skin. The topical application of PAR-2-specific antagonist or protease inhibitors normalized the barrier recovery. Cockroach allergens induced intracellular calcium oscillations in cultured human keratinocytes through PAR-2-involved pathway, which was confirmed by desensitization protocol. Abnormal calcium ion distribution after barrier disruption was also observed in allergens-applied skin. These results suggest that allergens with protease activity can influence the epidermal permeability barrier homeostasis through PAR-2 activation and consequent modulation of the calcium ions in skin.     

An immunohistochemical study of the distribution of plasminogen and plasminogen activators in bullous pemphigoid

Abnormalities of the cutaneous plasminogen/plasminogen activator system have been associated with acantholytic disorders, psoriasis, keratinocytes in culture, and epidermis in healing wounds. The present study was undertaken to investigate the possible role of the plasmin/plasminogen protease system in lesion development in bullous pemphigoid (BP). Using polyclonal antibodies and a fluorescent technique, the immunohistochemical distribution of plasmin/plasminogen, fibrinogen and the plasminogen activators, urokinase (uPA) and tissue plasminogen activator (tPA), were studied in lesional and non-fesional skin from nine BP patients, one with linear IgA disease (LAD) and one with pemphigoid gestationis (PG). The distribution of the proteases was compared with that in normal skin (n=4) and in suction blisters (n=2). In normal skin, fibrinogen, tPA and uPA were absent from the epidermis and plasminogen was confined to the basal layer. Uninvolved BP skin was identical to controls. Focal areas of suprabasal plasminogen expression in the region of a blister was seen in 3/9 BP lesions and in 1/2 suction blisters. In 6/9 BP lesions and both uninvolved and lesional LAD and PG skin were identical to controls, and no suprabasal expression of plasminogen was present. These findings suggest that suprabasal plasminogen expression is unlikely to play a fundamental role in the pathogenesis of blister formation in BP as enhanced expression was not present in every case and the finding was not specific to BP, also occurring in a suction blister. Enhanced plasminogen expression rather may be a reflection of the processes of tissue repair.     

Do plasminogen activators play a role in lichen sclerosus?

The histological changes of lichen sclerosus suggest that significant remodelling of the extracellular matrix is occurring. As the proteases of the plasminogen activator system have been implicated in tissue remodelling, cell migration and tumour invasion, we performed an immunohistochemical study to look for evidence of alteration in the expression of plasminogen/plasmin, urokinase-type plasminogen activator, tissue-type plasminogen activator and alpha2-antiplasmin in biopsies of clinically typical vulval lichen sclerosus obtained from 11 untreated adult women. Normal vulva obtained from gynaecological procedures and samples of the patients' uninvolved thigh tissue were used as controls. No significant difference was seen in the staining pattern between the lichen sclerosus tissue and control tissue. However, although we found no immunohistochemical evidence that the plasminogen activator system is involved in the pathogenesis of vulval lichen sclerosus, it may be that other proteases are involved.     

A monoclonal antibody recognizing plasminogen and plasmin—altered reactivity in psoriatic lesions

To study the organization of the plasminogen activator/plasminogen-plasmin (PA/PG-P) system in human epidermis we raised monoclonal antibodies with specificity for human plasminogen and plasmin (PG-P). Monoclonal antibody P2, which appeared most suitable for immunohistology, is a mouse monoclonal antibody of IgG1 subtype, specific for the precursor enzyme plasminogen and for the high molecular weight chain of the active enzyme, plasmin. Immunofluorescence analysis of normal human epidermis revealed that reactivity with P2 was confined to the basal cell layer. In psoriatic lesions, however, this regional organization was not found. Immunoreactivity in this case was scattered throughout all the hyperproliferating cell layers, which is taken as evidence for an altered distribution of PG-P in psoriatic lesions. In psoriasis other components of the PA/PG-P system have previously been found to be altered. In this context our findings add to the hypothesis that this system may be involved in the pathology or the pathogenesis of psoriatic lesions.     

Topical peroxisome proliferator activated receptor activators accelerate postnatal stratum corneum acidification

Previous studies have shown that pH declines from between 6 and 7 at birth to adult levels (pH 5.0-5.5) over 5-6 days in neonatal rat stratum corneum (SC). As a result, at birth, neonatal epidermis displays decreased permeability barrier homeostasis and SC integrity, improving days 5-6. We determined here whether peroxisome proliferator-activated receptor (PPAR) activators accelerate postnatal SC acidification. Topical treatment with two different PPARalpha activators, clofibrate and WY14643, accelerated the postnatal decline in SC surface pH, whereas treatment with PPARgamma activators did not and a PPARbeta/delta activator had only a modest effect. Treatment with clofibrate significantly accelerated normalization of barrier function. The morphological basis for the improvement in barrier function in PPARalpha-treated animals includes accelerated secretion of lamellar bodies and enhanced, postsecretory processing of secreted lamellar body contents into mature lamellar membranes. Activity of beta-glucocerebrosidase increased after PPARalpha-activator treatment. PPARalpha activator also improved SC integrity, which correlated with an increase in corneodesmosome density and increased desmoglein-1 content, with a decline in serine protease activity. Topical treatment of newborn animals with a PPARalpha activator increased secretory phospholipase A2 activity, which likely accounts for accelerated SC acidification. Thus, PPARalpha activators accelerate neonatal SC acidification, in parallel with improved permeability homeostasis and SC integrity/cohesion. Hence, PPARalpha activators might be useful to prevent or treat certain common neonatal dermatoses.     

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.     

Establishment of a mouse skin model of the lichenification in human chronic eczematous dermatitis

BACKGROUND: Repeated mechanical stresses, such as scratching and rubbing, on a lesional skin area induce a rough skin condition known as lichenification in patients with chronic eczematous dermatitis. For ethical reasons, the pathomechanisms involved are difficult to study, so an animal model is required. OBJECTIVES: To study the pathomechanisms of the unique rough skin changes seen in chronic eczematous dermatitis, we established a mouse skin model by repeated tape stripping to inflict stratum corneum (SC) barrier disruption. The skin characteristics of the model were investigated biologically, histologically and pharmacologically. METHODS: Tape stripping was done on mouse back skin three times a week for 4 weeks. The skin changes were studied by obtaining negative replicas, haematoxylin and eosin staining, immunostaining for CD31 and BrdU, and measuring epidermal and cutaneous thickness and skin capacitance. Activities of matrix metalloproteinase (MMP)-2, 9 and urokinase-type plasminogen activator (uPA) in the skin tissues were analysed by zymography. The effects of MMP inhibitor and glycine were assessed. RESULTS: The repeated tape stripping produced crusting and desquamation at 48 h, followed 1 week later by the formation of shallow furrows, which became much deeper after 4 weeks, appearing as fine and regular wrinkles. The resultant wrinkled skin resembled lichenified skin seen in patients with chronic eczematous dermatitis. Histopathologically, we found acanthosis, hypergranulosis and hyperkeratosis even at 48 h, and the skin was 2.5 times thicker than untreated control skin at 4 weeks. We observed angiogenesis in the upper dermis at 1 and 4 weeks. Skin capacitance, a parameter of SC hydration, displayed consistently low levels throughout the experimental period. Although the dermis was also thickened, the activity of MMP-9 was sharply increased only at 24 and 48 h after tape stripping, declining thereafter to the control level. Topical applications of CGS-27023A (CGS), an MMP inhibitor, failed to suppress this tape-stripping-induced wrinkle formation. In contrast, topical applications of a barrier recovery accelerator, glycine, effectively inhibited the wrinkle formation induced by repeated tape stripping. CONCLUSIONS: The induction of fine and regular wrinkles by inflicting chronic SC barrier disruption in this model involves mainly epidermal changes, which is in sharp contrast to the mainly dermal changes induced by chronic ultraviolet B irradiation.     

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.     

Functional consequences of a neutral pH in neonatal rat stratum corneum

At birth, neonatal stratum corneum (SC) pH is close to neutral but acidifies with maturation, which can be ascribed, in part, to secretory phospholipase A(2) and sodium/hydrogen antiporter 1 (NHE1) activities. Here we assessed the functional consequences of a neutral SC pH in a newborn rat model. While basal transepidermal water loss rates are near normal, barrier recovery (BR) rates after acute barrier disruption were delayed in newborn animals. The abnormality in barrier homeostasis could be improved by topical applications of an acidic buffer, indicating that barrier abnormality is primarily due to high SC pH. The delay in BR correlated with incompletely processed lamellar membranes and decreased activity of beta-glucocerebrosidase. Inhibition of NHE1 delayed BR after acute barrier perturbation. SC integrity was abnormal in newborn animals. Electron microscopy demonstrated decreased corneodesmosomes (CD) in newborn animals with decreased expression of desmoglein 1 and corneodesmosin. Serine protease activation appears to be responsible for CD degradation in newborn animals, because serine protease activity is increased in the SC and it can be reduced by acidification of the SC. The delay in acidification of neonatal SC results in abnormalities in permeability barrier homeostasis and SC integrity and are likely due to pH-induced modulations in enzyme activity.     

Plasmin induces acantholysis in skin organ cultures

Summary Addition of human plasminogen to three different pemphigus plasma samples showed a synergistic effect on acantholysis in the skin organ culture model. Human plasmin itself, without addition of pemphigus plasma, induced typical acantholytic changes in the skin explants, causing different types of acantholysis in a dose- and time-dependent manner: in the presence of 3 CU plasmin per ml culture medium, focal suprabasilar acantholysis of pemphigus vulgaris type could be detected after 72 h incubation, whereas 15 CU/ml caused extended acantholysis of pemphigus foliaceus type in the upper epidermal layers after 24 h, and extended acantholysis of benign chronic pemphigus (Hailey-Hailey disease) type comprising all layers of the epidermis after 48 h incubation. Plasminogen activator levels (Mr 55,000 urokinase type) in tissue extracts of skin explants and in culture media were reduced after 24 and 48 h incubation with pemphigus IgG as compared to control experiments with normal human igG; this probably resulted from urokinase inactivation by reaction with inhibitors. These results lend support to the hypothesis proposed by Hashimoto et al. in 1983 that the plasminogen activator-plasmin system could play an essential role in the protease mechanisms of pemphigus acantholysis.This work was supported by a grant from the Swiss National Science Foundation 3.075-1.84. Presented in part at the 67th Congress of the Swiss Society of Dermatology and Venereology, October 19, 1985, Zürich, Switzerland     

Increased stratum corneum serine protease activity in acute eczematous atopic skin

Background Atopic dermatitis (AD) is a chronic inflammatory disease associated with changes in stratum corneum (SC) structure and function. The breakdown of epidermal barrier function in AD is associated with changes in corneocyte size and maturation, desquamation, lipid profiles, and some protease activities. Objectives The purpose of this study was: (i) to examine physiological changes in lesional (L) skin of acute eczematous AD, compared with nonlesional (NL) AD skin and healthy (H) skin, using sequential tewametry and SC protein analysis to estimate SC thickness; and (ii) to assess which serine proteases might be involved in pathogenesis. Methods Six subjects with H skin, six AD patients with NL skin and six AD patients with mild to moderate eczema (L skin) were enrolled. Skin was assessed using several noninvasive techniques but SC thickness was estimated using tewametry and SC protein content of D‐Squame strippings. SC integrity was determined by sequential tape stripping (D‐Squame) and infrared densitometry. Kallikreins, plasmin, urokinase and leucocyte elastase protease activities together with a novel SC tryptase‐like enzyme activity were quantified. Results Transepidermal water loss (TEWL) levels after D‐Squame stripping were elevated in L compared with NL and H skin at all sampling points (P < 0·05). Conversely, the amount of SC removed by sequential tape stripping was decreased in L skin, indicating increased intracorneocyte cohesion (P < 0·05). By correlating 1/TEWL values and SC removed as an estimate of SC thickness, a significantly thinner SC was observed in L compared with NL and H skin (P < 0·05). Elevated extractable serine protease activity was measured in AD skin in the order: SC tryptase‐like enzyme (45×), plasmin (30×), urokinase (7·1×), trypsin‐like kallikreins (5·8×) and chymotrypsin‐like kallikreins (3·9×). Leucocyte elastase activity was not detected in H and NL skin but was observed in AD SC samples (L skin). All enzymes were elevated in the deeper layers of L SC compared with NL and H SC samples. All consistently elevated SC protease activities were significantly correlated with the bioinstrumental data. Conclusions We report increased serine protease activities in acute eczematous AD, especially in deeper layers of the SC, including SC tryptase‐like enzyme, plasmin, urokinase and leucocyte elastase activities. These elevations in protease activities were associated with impaired barrier function, irritation, and reduced skin capacitance. Increased SC cohesion was apparent despite elevated TEWL during tape stripping, which would indicate reduced SC thickness in acute eczematous lesions of AD. Indeed, this was observed using an estimate of SC thickness.