Annelise Isabelle Wohlwend

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.

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.

Mutations in connexin genes and disease.

Eur J Clin Invest 2010; 41 (1): 103–116

Facultative polypeptide translocation allows a single mRNA to encode the secreted and cytosolic forms of plasminogen activators inhibitor 2

Two forms of plasminogen activators inhibitor 2 (PAI‐2) are synthesized by human and murine monocytes/macrophages: one accumulates in the cytosol, while the other is translocated into the endoplasmic reticulum, glycosylated and secreted. We show here that a single mRNA encodes both forms of PAI‐2. Firstly, a single PIA‐2 mRNA was detected by Northern blot hybridization and by RNase protection. Secondly, transfection of a PAI‐2 cDNA led to the synthesis of both forms of PAI‐2. Finally, in vitro translation of an mRNA transcript of the PAI‐2 cDNA in the presence of microsomal membranes generated two topologically distinct forms of PAI‐2. The cytosolic and secreted forms of PAI‐2 do not result from the use of two translation start sites, since their synthesis initiates at the same AUG, in a sequence context that is conserved between the human and murine genes. Thus, the accumulation of one polypeptide into two topologically distinct cellular compartments can be achieved by facultative translocation.

Urokinase-catalyzed plasminogen activation at the monocyte/macrophage cell surface: a localized and regulated proteolytic system

In the adult organism, monocytes and macrophages are among the few cell types that can migrate within and between body compartments. To do so, they must have the capacity to clear for themselves a path through the macromolecular barriers of basement membranes and other extracellular matrices. This requires the controlled and localized degradation of matrix proteins by extracellular proteases. Mononuclear phagocytes can produce a number of such enzymes, including collagenolytic, elastinolytic, and gelatinolytic hydrolases (Takemura and Werb 1984). Because they can, directly or indirectly, catalyze the degradation of most components of extracellular matrices, plasminogen activators (PAs) are thought to play a key role in the proteolytic events that accompany the migration of a wide variety of cell types, during ontogeny as well as in pathologic circumstances. Monocytes and macrophages can produce PAs, and the regulation of their PA-dependent proteolytic activity has been a focus of attention in recent years. The findings of a number of investigators converge to suggest that the expression of PA activity is a tightly controlled phenotypic property of human and murine mononuclear phagocytes, and that multiple mechanisms act concurrently to achieve the exquisitely focused and regulated generation of plasmin precisely where and when it is needed to allow cell migration in the context of inflammatory reactions.

Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells.

Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor.

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.

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.

Plasminogen Mediates the Pathological Effects of Urokinase-Type Plasminogen Activator Overexpression

Increased expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is associated with different pathological conditions. Both uPAR-mediated signaling and plasmin-catalyzed extracellular proteolysis may contribute to pathogenesis. To evaluate the involvement of plasminogen in such circumstances, we have taken advantage of transgenic mouse models in which overexpression of uPA and/or uPAR in enamel epithelium, basal epidermis, and hair follicles leads to a pathological phenotype; uPA transgenic mice have chalky-white incisors and, when uPAR is co-expressed, develop extensive alopecia, epidermal thickening, and subepidermal blisters. We report here that when these transgenic mice were backcrossed into a plasminogen-deficient (Plg-/-) background, the dental and skin phenotypes appeared completely normal. Heterozygous Plg+/- transgenic mice exhibited a haplo-insufficiency, with an intermediate or normal phenotype. These results do not argue in favor of a role for uPAR-mediated signaling in our experimental model; rather, they demonstrate an essential, dose-dependent, requirement for plasminogen in uPA-mediated tissue alterations. They also support the hypothesis that plasminogen could play a part in certain skin diseases.

Medial coracoclavicular ligament revisited: an anatomic study and review of the literature

The medial coracoclavicular ligament (MCCL), up to now rarely reported in the literature, was studied in a formol-fixed cadaver by means of dissection, morphometry, and light microscopy. This entity represents a true ligament within the coracoclavicular fascia. Although longer and narrower than its lateral counterpart, the medial coracoclavicular ligament follows the same morphological pattern, including the cartilage at the level of the coracoidal attachment. Its clinical significance and implications together with a review of the literature is presented.