M. Järvinen

A protein reminiscent of the epidermal SH-protease inhibitor occurs in squamous epithelia of man and rat

The occurence of the human and rat epidermal SH-protease inhibitors in various human and rat tissues was studied by double radial immunodiffusion against specific antisera to the inhibitors. An immunoreactive protein was found in the extracts prepared from human and rat epidermis and from eosophageal and vaginal squamous epithelia, and from rat pro-ventricular squamous epithelium. No immunoreactive protein was found in man or rat in any other of their tissues, studied by us. The results strongly suggest that a protein reminiscent of the human or rat epidermal SH-protease inhibitor is present in squamous epithelia but not in other tissues. The identity of the epidermal inhibitor and the immunoreactive protein in the other squamous epithelia was confirmed by immunodiffusion, immunoelectrophoresis and gel chromatography, and by immunoinhibition of the papain inhibiting activity of the human epidermal and oesophageal inhibitors by gammaglobulins separated from antiserum to the human epidermal inhibitor.

Dexamethasone-induced plasminogen activator inhibitor: characterization, purification, and preparation of monoclonal antibodies.

SummaryThe effects of dexamethasone on protein synthesis were studied in human fibrosarcoma (HT-1080) cells. Dexamethasone induced a new protein of 46 kD which was rapidly secreted into the medium, while neither progesterone nor estradiol would induce the synthesis of this protein and only a small increase in its amount could be seen in the presence of testosterone. The 46 kD protein was partially purified by ammonium sulfate precipitation and gel filtration and mouse monoclonal antibodies to it were produced in mouse hybrid cells. Altogether 13 positive clones were found, of which six reacted only with native and seven reacted with the unreduced 46 kD protein in Western blotting. It was possible by using polyclonal antibodies to plasminogen activator inhibitor type I (PAI-1) and purified plasminogen activator inhibitor type I to confirm that the 46 kD protein purified and characterized here was PAI-1. In addition, the 46 kD protein clearly inhibited plasminogen activation, thus further confirming that protein isolated was an inhibitor of plasminogen activator. Since the induction of PAI-1 by dexamethasone was very extensive, it is possible that glucocorticoids regulate proteolysis and fibrinolysis in vivo by increasing the amount of the inhibitor of plasminogen activator and thus preventing the activation of plasminogen to plasmin. The reduction of activation of plasminogen to plasmin by glucocorticoid-induced inhibitor could be of great importance, e.g., in various blistering diseases, in metastases from malignant cells, and in the migration of inflammatory cells.

Acid and neutral cysteine proteinase inhibitor in normal uterine portio and in squamo-epithelial metaplasia, dysplasias and infiltrative carcinoma of the uterine portio.

Acid cysteine proteinase inhibitor (ACPI) and neutral cysteine proteinase inhibitor (NCPI) were localized in formalin-fixed normal human uterine portio as well as in the squamous cell carcinoma and dysplasias of the uterine portio. The peroxidase-antiperoxidase technique was used. In the squamous epithelium of normal uterine portio, ACPI and NCPI were localized in the cells of the upper and middle layers, mainly in the cytoplasm. In the precursors of cancer, immunoreactivity for ACPI and NCPI declined, and neither of the inhibitors was demonstrable immunohistochemically in the anaplastic squamo-epithelial carcinoma of the uterine portio. Our results suggest that ACPI and NCPI are associated with squamo-epithelial differentiation and that they may also be of significance for the regulation of cysteine proteinase activity in normal tissue and malignant growth.

Localization of cathepsin H and its inhibitor in the skin and other stratified epithelia.

SummaryThe rat-skin-derived cysteine proteinase, so-called BANA-hydrolase, which is capable of hydrolysing benzoylarginine naphthylamide and leucine naphthylamide was shown to be immunologically identical to cathepsin H purified from rat liver. The enzyme was immunocytochemically localized in the basal cell layer of rat epidermis. A natural inhibitor of cathepsin H with a molecular weight of about 13,000 was mainly localized in the keratinizing cell layers and showed only a weak reaction in the basal cells. Thus, cathepsin H appears to be a characteristic feature of the proliferating cell layer, whereas the cysteine-proteinase inhibitor is a characteristic feature of keratinizing cells.

A 43-kDa papain inhibitor produced by a cultured human epidermal cell line

SummaryEpidermis-derived cells (NCTC 2544) were cultured and the proteins of the culture medium, as well as of the cells, were fractionated by gel-chromatography. The fractions were analyzed for their papain-inhibitory capacity and for the presence of socalled 43-kDa papain inhibitor. A papain inhibitor was identified with molecular weight and immunological characteristics similar to the original 43-kDa inhibitor that was isolated from psoriatic scales. The result proves that NCTC-2544 cells can produce the so-called psoriasis inhibitor under culture conditions.

Alpha-thiolproteinase inhibitor, alpha1-antitrypsin and serum proteins in suction blister fluid; effect of local glucocorticosteroid treatment

In addition to the seven well-known proteinase inhibitots acting on serine proteinases, human serum contains at least two inhibitors with no activity against serine proteinases, These are beta~-collagenase inhibitor and alpha-thiot-proteinase inhibitor [1]. Alpha-thiolproteinase inhibitor is group-specific against thiolproteinases such as papain, ficin, and cathepsin B. The inhibitor can be fractionated by ionexchange chromatography into two compounds (possibly three [7]) having the mobilities of alpha1 and alpha2 proteins in agarose electrophoresis, The compound with alpha~ mobility has a molecular weight of about 190,000 and that with alpha2 mobility about 90,000. The alpha1 and alpha2 forms of the inhibitor are immunologically closely related and immunization of rabbits with either of them has produced antisera precipitating both the alphat and alpha2 forms of the inhibitor [2]. In this communication, we present concentrations of the alpha-thiolproteinase inhibitor (a-TPI) in suction blister fluid from human skin, using a rocket immunoelectrophoresis technique, and compare the levels of this inhibitor with the concentrations of alpha~-antitrypsin (c~-IAT) and other serum proteins. In addition, the effect of local glucocorticosteroid treatment on these values was evaluated. Four healthy male volunteers (aged 1 9 21 years) received local 0.05% clobetasol-17-propionate (Dermovate ointment, Glaxo) treatment without occlusion three times a day on the skin of one side of

Production of acid and neutral cysteine-proteinase inhibitors by a cultured human skin epithelium cell line

SummaryHuman skin epithelial-like cells (NCTC-strain 2544) were grown in RPMI-1640 medium supplemented with foetal calf serum for up to 2 weeks. The culture medium and extracts made from the cells were subjected to gel-filtration chromatography in a Sephacryl S-200 column for fractionation of the proteins. The fractions were assayed for acid and neutral cysteine-proteinase inhibitor (ACPI, NCPI) using time-resolved fluoroimmunoassay and radioimmunoassay, and the cysteine-proteinase-inhibiting activities were assayed using papain. Free NCPI, i.e. a molecule with isoelectric variants at pHs 6.0 and 6.5, which has an Mr of around 12,000 and is capable of inhibiting papain, was detected both in the culture medium and in the cells. Immunodiffusion studies revealed its immunological identity with human spleen-derived NCPI. The amount of NCPI increased during the incubation period. ACPI — characterized as a molecule having an isoelectric point of 4.9, an Mr of about 12,000, papain-inhibiting capacity and antigenic reactivity with spleen-derived ACPI — was not detected in the culture medium. It was, however, detected in the cells after 2 weeks in culture. These data prove that ACPI and NCPI are synthesized by the NCTC-2544 cells under the present culture conditions.

The 43 kDa papain inhibitor in normal and diseased skin.

The presence of 43 kDa papain inhibitor in 43 different skin diseases was immunohistochemically studied by using both poly‐and monoclonal antibodies. Psoriasis and various eczematoid reactions as well as viral infections showed the most pronounced staining in the squamous cells of the epidermis. The antigen was also present in benign tumours or precancerous lesions which showed keratinization. Cells of poorly differentiated squamous cell carcinomas, basal cell carcinomas and melanocytic tumours were negative. The antigen seems to be related to disturbed keratinization and benign proliferation in non‐neoplastic derma‐toses and it is also present in differentiating squamous neoplasms.

The 43 kDa papain-inhibiting protein in psoriatic epidermis is identical to squamous cell carcinoma antigen (SCC-antigen).

The scales of patients suffering psoriasis as a rich source of cysteine proteinase inhibitors. The major inhibitor in psoriatic epidermis is cystatin A (1), which also is highly expressed in normal epidermis (2). In addition to cystatin A (12.6 kDa), psoriatic epidermis contains a high amount of another papain-inhibiting protein (43 kDa) which can easily be separated from cystatin A by gel chromatography and then purified by ion exchange chromatography (3). By isoelectric focusing, three major activity peaks with pI’s of 7.3, 6.9 and 6.5 are separated. By immunohistochemical techniques, using antibodies against the isoelectric variant pi 6.9, the inhibitor is located in the cytoplasm of suprabasal layers of the psoriatic epidermis. In the immature psoriatic stratum corneum the staining for the 43 kDa inhibitor is very variable and uneven: sometimes a strong uniform staining is seen and sometimes the stained and unstained areas vary both vertically and horizontally in a single section (3). In addition to psoriasis, the expression of the inhibitor is elevated in several skin disorders where proliferation of the epidermal cell is accelerated (4). In normal epidermis the inhibitor is seen only in granular layer and in stratum corneum. The inhibitor seems to be a constant property of various squamous epithelia of skin, mouth, oesophagus, uterine portio and thymus and it is also expressed in squamous cell carcinomas and in the so-called basal cells of bronchial epithelium (5).