Overexpression of plasminogen activator inhibitor type 2 in basal keratinocytes enhances papilloma formation in transgenic mice.

The serpin plasminogen activator inhibitor (PAI) type 2 is expressed in differentiated epidermal keratinocytes. To explore its role in this tissue, we studied the impact of PAI-2 overexpression on epidermal differentiation and skin carcinogenesis. A mouse PAI-2-encoding transgene was targeted to basal epidermis and hair follicles under the control of the bovine keratin type 5 gene promoter. Two mouse lines were established, one of which strongly expressed the transgene and produced elevated levels of PAI-2 in the epidermis. Although it had no manifest impact on cellularity or differentiation of skin or hair follicles, PAI-2 overexpression rendered the mice highly susceptible to skin carcinogenesis induced by a single application of 7,12-dimethylbenz(a)anthracene (initiation) followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate [TPA (promotion)]. In transgenic mice, papillomas could be observed after 3 weeks of promotion; after 8 weeks, 94% (31 of 33) of transgenic mice had developed readily visible papillomas, whereas only 35% (7 of 20) of control mice (transgene-negative littermates) had barely detectable lesions. After 11 weeks, all but 1 (32 of 33) of the transgenic mice had papillomas as compared with only 65% (13 of 20) of control mice. After 11 weeks of promotion, application of TPA was terminated. In control mice, papillomas regressed and eventually disappeared; in transgenic mice, there was continued growth of papillomas, some of which further progressed to carcinomas. In contrast to massive apoptosis in regressing papillomas of control mice, only a few apoptotic cells were detected in transgenic papillomas after the cessation of TPA application. The effect of PAI-2 on papilloma formation did not appear to involve inhibition of the secreted protease urokinase-type plasminogen activator (uPA): PAI-2 accumulated predominantly in cells, and PAI-2 overexpression failed to alleviate a phenotype induced by uPA secretion, as demonstrated by a double transgenic strategy. In addition, in situ hybridization revealed that uPA mRNA is not expressed concomitantly with PAI-2 in developing papillomas. We conclude that overexpression of PAI-2 promotes the development and progression of epidermal papillomas in a manner that does not involve inhibition of its extracellular target protease, uPA, but appears to be related to an inhibition of apoptosis.

Plasminogen activation in human acute leukaemias.

Plasminogen activation is implicated in solid tumour growth, invasion and metatastic spread. However, little is known about its role in leukaemia. We investigated the production by leukaemic cells of plasminogen activators [urokinase (uPA) and tissue-type PA (tPA)], cell surface receptor for uPA (uPAR) and PA inhibitors (PAI-1 and PAI-2). Leukaemic cells from 37 patients [26 with acute myeloid leukaemia (AML) and 11 with acute lymphoid leukaemia (ALL)] were analysed for mRNA content and enzymatic activities. High levels of uPA mRNA were found in M1, M2, M3 and M4-M5 AMLs, whereas tPA mRNA was not detected in any of the analysed cases. uPAR mRNA was confined to subtypes M4-M5. PAI-1 mRNA was detected in M3 and M4-M5. PAI-2 mRNA was found predominantly in M2 and M4-M5. SDS-PAGE/zymography analyses of cell extracts and supernatants after 24 and 48 h of culture confirmed the production of active uPA by AML cells (mainly M4-M5). but not by ALL. The finding of uPA, uPAR, PAI-1 and PAI-2 synthesized by leukaemic cells suggests that plasminogen activation may contribute to the invasive behaviour of these cells, the fibrinolytic imbalance observed in leukaemic patients and the differentiation and proliferation of M4-M5 by interaction of uPA with uPAR.

Extracellular proteolysis alters tooth development in transgenic mice expressing urokinase-type plasminogen activator in the enamel organ.

By catalyzing plasmin formation, the urokinase-type plasminogen activator (uPA) can generate widespread extracellular proteolysis and thereby play an important role in physiological and pathological processes. Dysregulated expression of uPA during organogenesis may be a cause of developmental defects. Targeted epithelial expression of a uPA-encoding transgene under the control of the keratin type-5 promoter resulted in enzyme production by the enamel epithelium, which does not normally express uPA, and altered tooth development. The incisors of transgenic mice were fragile, chalky-white and, by scanning electron microscopy, their labial surface appeared granular. This phenotype was attributed to a defect in enamel formation during incisor development, resulting from structural and functional alterations of the ameloblasts that differentiate from the labial enamel epithelium. Immunofluorescence revealed that disorganization of the ameloblast layer was associated with a loss of laminin-5, an extracellular matrix molecule mediating epithelial anchorage. Amelogenin, a key protein in enamel formation, was markedly decreased at the enamel-dentin junction in transgenics, presumably because of an apparent alteration in the polarity of its secretion. In addition, increased levels of active transforming growth factor-beta could be demonstrated in mandibles of transgenic mice. Since the alterations detected could be attributed to uPA catalytic activity, this model provides evidence as to how dysregulated proteolysis, involving uPA or other extracellular proteases, may have developmental consequences such as those leading to enamel defects.

A new all-ceramic post and core system: clinical, technical, and in vitro results.

Root-filled teeth with fractured or discolored coronal aspects invariably need to be restored by crowns. The prepared abutment tooth is usually reinforced by a metallic post and core system. The grayish discoloration of the root, and consequently of the gingiva, caused by the metal color may be an enormous esthetic disadvantage in the anterior teeth. In 1993 ceramic posts made of zirconia were introduced by the authors, allowing a new all-ceramic concept for nonvital abutment teeth. A new ceramic post and core system has now been developed with the idea of further improving esthetic appearance. In this system the core material is heat pressed directly onto the zirconia post. This article describes the material and the fabrication procedures (chairside and in the laboratory) of the system. Clinical results are presented. The retention of the core material is evaluated by in vitro tests.

Protease nexin 1 in the murine kidney: glomerular localization and up-regulation in glomerulopathies.

Protease nexin 1 (PN-1), a potent serpin-class antiprotease, is thought to be synthesized in the murine kidney. However, neither the cellular localization of PN-1 synthesis nor its role has yet been defined. To address these questions, we determined by in situ hybridizations RNase protection assay and immunoblotting, the sites of PN-1 mRNA accumulation in normal mouse kidneys and the modulation of PN-1 expression in several pathological conditions. In normal kidneys, PN-1 mRNA was detected primarily in glomeruli, most likely in mesangial cells. The glomerular expression of PN-1 was substantially enhanced not only in lupus-like glomerulonephritis (induced by IgG3 monoclonal rheumatoid factors or occurring spontaneously in lupus-prone mice), but also in mild glomerular lesions associated with intracapillary thrombi induced by IgG3 anti-trinitrophenyl monoclonal antibodies. In contrast, no modulation of PN-1 mRNA levels was observed during the course of lipopolysaccharide-induced acute tubular necrosis. A constitutive PN-1 gene expression and its up-regulation during glomerular injury suggest a possible role for PN-1 in glomerular biology. In view of its high inhibitory activity towards thrombin, mesangial PN-1 may be involved in the control of glomerular coagulation following initial glomerular injuries.

TGF-beta 1 overexpression in murine pancreas induces chronic pancreatitis and, together with TNF-alpha, triggers insulin-dependent diabetes.

We have generated transgenic mice overexpressing TGF-beta 1 in pancreatic beta cells. This resulted in massive fibrosis of the pancreas; in adult mice, most of the acini were replaced by fibrotic and adipose tissues. A conspicuous disorganization of the islets of Langerhans was also observed; however, the number of beta cells was not decreased and the mice were normoglycemic. Backcrossing to transgenic mice overexpressing TNF-alpha in their islet beta cells (which also remain normoglycemic, (1)) yielded double transgenics, most of which became diabetic by the age of 4 months; histological analysis revealed a dramatic decrease in insulin-containing beta cells.

Regulation of parathyroid hormone-related protein production in a human lung squamous cell carcinoma line.

The synthesis and release of parathyroid hormone-related protein (PTHrP) could be influenced in a paracrine or autocrine manner by substances present around or inside tumours, such as bone or stromal cell-derived cytokines, factors produced by the tumour itself or by peritumoural inflammatory cells. We investigated the effects of various cytokines known to be synthesized by osteoblasts, stromal cells, leucocytes or cancer cells, on PTHrP production by the human lung squamous cell carcinoma line BEN. The influence of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) was studied, and compared with those of insulin-like growth factors-I and -II (IGF-I, IGF-II), or macrophage- or granulocyte-macrophage colony-stimulating factors (M-CSF, GM-CSF). TNF-alpha caused a 1.9 +/- 0.1-fold increase in immunoreactive PTHrP production, which was maximal by 24 h of incubation. IL-6 caused a 2.3 +/- 0.2-fold increase, which was maximal by 16 h. These effects, which were time- and concentration-dependent, were blocked by monoclonal antibodies raised against the corresponding cytokine. An increase of PTHrP mRNA was found in IL-6-treated cells. IGF-I and IGF-II increased PTHrP production by 2.0 +/- 0.3- and 2.3 +/- 0.1-fold respectively. Neither M-CSF nor GM-CSF altered PTHrP production up to 64 h of incubation. PTHrP production was not affected by varying extracellular calcium concentrations, but was decreased by incubation with 100 nmol/l dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

Human clusterin gene expression is confined to surviving cells during in vitro programmed cell death.

Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death (PCD). However, no study has so far directly shown that cells expressing clusterin in these tissues are actually apoptotic as defined by morphological and biochemical criteria. We have studied cellular clusterin gene expression in vitro using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and (c) dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7. In all three models, the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by Northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. Our results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD.

Maxillary anterior single-tooth replacement: comparison of three treatment modalities.

The correct treatment of a single missing maxillary anterior tooth in the aesthetically prominent area has become more challenging. The missing tooth can today be replaced by one of three prosthodontic treatment modalities--conventional fixed bridge, resin-bonded bridge, and single-tooth implant. The most important factors to be considered are the predictability of aesthetics, the preservation of the enamel shield and the dentinal and pulpal tissue, and the preservation of the periodontium and the alveolar bone. The learning objective of this article is a critical discussion of these three modalities as a replacement of a missing anterior single tooth to aid in selection of the most appropriate treatment in each individual clinical case.

Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis.

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

Extracellular proteolysis in the adult murine brain.

Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

Protease-nexin I as an androgen-dependent secretory product of the murine seminal vesicle.

A search for inhibitors of urokinase-type plasminogen activator (uPA) in the male and female murine genital tracts revealed high levels of a uPA ligand in the seminal vesicle. This ligand is functionally, biochemically and immunologically indistinguishable from protease-nexin I (PN-I), a serpin ligand of thrombin and uPA previously detected only in mesenchymal cells and astrocytes. A survey of murine tissues indicates that PN-I mRNA is most abundant in seminal vesicles, where it represents 0.2-0.4% of the mRNAs. PN-I is synthesized in the epithelium of the seminal vesicle, as determined by in situ hybridization, and is secreted in the lumen of the gland. PN-I levels are much lower in immature animals, and strongly decreased upon castration. Testosterone treatment of castrated males rapidly restores PN-I mRNA levels, indicating that PN-I gene expression is under androgen control.

Effects of veneering and glazing on the strength of heat-pressed ceramics.

A newly developed press-type all-ceramic crown system, the IPS-Empress system (Ivoclar), has recently been introduced. Two methods may be used to obtain the desired shade: surface staining and glazing; veneer technique. The purpose of this study was to determine whether these two methods affected flexure strength of Empress glass ceramic. Eight groups of test bars were pressed. In groups 1 and 2, one surface was stained and glazed. The bars were placed face down (1) or up (2) for testing. For comparison, group 3 was heat-treated only (simulating stain and glaze firing). In groups 4-7, one surface of the bars was either veneered with porcelain on the bottom (4) or top surface (5) and then subsequently glazed (6 and 7). Group 8 was just heat-treated (simulating veneer and glaze firings). The results showed that there were no significant differences in strength between groups 2, 5 and 7 compared to the reference groups 3 and 8 (159 +/- 28 and 175 +/- 32 MPa, respectively), indicating that, from the mechanical point of view, the two surface techniques can be equally used on Empress ceramic. If the porcelain veneer supported the ceramic (4), the strength was significantly decreased. The highest mean value was obtained in group 1 (220 +/- 34 MPa).

Polarized secretion of urokinase-type plasminogen activator by epithelial cells.

Numerous epithelial cell types produce and secrete plasminogen activators (PAs) and/or PA inhibitors (PAIs). When epithelial cells were grown on polycarbonate filters and their apical and basolateral secretion products analyzed, PA activity accumulated in a highly polarized fashion; depending upon the cell line, the compartment of PA accumulation was either apical (MDCK I cells and HBL-100 cells) or basolateral (LLC-PK1, CaCo-2, and HeLa cells). By contrast, PAI-1 was recovered in roughly equal amounts in both compartments. Basolateral accumulation of urokinase-type plasminogen activator (uPA), but not its apical targeting, required an acidic compartment and the integrity of the cytoskeleton. Polarity of uPA accumulation did not result from removal of the free enzyme from the opposite compartment through its binding to the cell surface. Transfection with wild-type or mutated murine uPA demonstrated that neither the "growth factor" domain nor the kringle domain is required for the appropriate sorting of the protein. We propose that polarized secretion of PAs is one mechanism whereby cells spatially control extracellular proteolysis.

Heat-pressed ceramics: technology and strength.

The flexural strength of a new heat-pressed ceramic material (IPS-Empress) was measured before and after pressing and/or simulated firing treatments (eg, veneering, surface coloring, glazing). Heat pressing the material significantly improved its flexure strength whereas heat treating the material alone did not. Additional firings (heat treatments) after heat pressing further increased material strength. The final strength values ranged between 160 and 180 MPa and were comparable to some other all-ceramic systems. No clinical implications were drawn from these data.