Caroline E. Chwieralski

Cathepsin-regulated apoptosis.

Apoptosis can be mediated by different mechanisms. There is growing evidence that different proteolytic enzymes are involved in the regulation of apoptosis. Cathepsins are proteases which, under physiologic conditions, are localized intralysosomally. In response to certain signals they are released from the lysosomes into the cytoplasm where they trigger apoptotic cell death via various pathways, including the activation of caspases or the release of proapoptotic factors from the mitochondria. Here, we review different mechanisms that induce the release of lysosomal enzymes, and the functional relevance of defined cathepsins in defined models of apoptosis.

Trefoil factor family (TFF) expression in the mouse brain and pituitary: changes in the developing cerebellum

Trefoil factor family (TFF) peptides, besides their prominent expression in mucous epithelia, are also synthesized in the central nervous system. Previously TFF1 expression was observed in mouse brain astrocytes, while oxytocinergic neurons of the hypothalamo-pituitary axis are recognized sites of TFF3 synthesis. Here, the expression of TFF1, TFF2, and TFF3 was systematically studied using reverse transcription-polymerase chain reaction (RT-PCR) analysis of dissected adult mouse brain regions including the pituitary. Additionally, the developmental profile of TFF expression in murine cerebral cortex and cerebellum was monitored. Overall, the expression patterns of the three TFF genes differed. The TFF1 and TFF2 profiles shared some similarities, whereas the TFF3 expression pattern was completely different. TFF1 was nearly uniformly, but weakly expressed in all brain regions tested. The TFF1 and TFF2 expression patterns differed characteristically in the pituitary where abundant TFF2 transcription was detected in the anterior and not the posterior lobe and the expression level in males was higher than in females. In contrast, TFF3 expression was limited to the hippocampus, the temporal cortex, and the cerebellum, the latter being surprisingly the major site of expression. Here, TFF3 mRNA appeared to be restricted mainly to neurons and not glial cells. Cerebellar TFF3 expression is clearly developmentally regulated (maximum at P15), indicating a role for TFF3 during postnatal cerebellar development.

Gene Targeting of the Cysteine Peptidase Cathepsin H Impairs Lung Surfactant in Mice

Background The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed. Methodology/Principal Findings Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh −/− mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh −/− mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh+/+ and Ctsh −/− mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh −/− mice. Conclusions/Significance We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.

Modulation of cytokine production and silica-induced lung fibrosis by inhibitors of aminopeptidase N and of dipeptidyl peptidase-IV-related proteases

AIMS Dipeptidyl peptidase IV (DP IV)-related proteases and aminopeptidase N (APN) are drug targets in various diseases. Here we investigated for the first time the effects of DP-IV-related protease inhibitors and APN inhibitors on chronic inflammatory lung diseases. MAIN METHODS A murine model of silica (SiO2)-induced lung fibrosis and in vitro cultures of human lung epithelial cells and monocytes have been used and the influence of silica-treatment and inhibitors on inflammation and fibrosis has been measured. KEY FINDINGS We found increased inflammation and secretion of the chemokines IL-6, MCP-1 and MIP-alpha 2 weeks after SiO2 application, and increased lung fibrosis after 3 months. Treatment with the APN inhibitor actinonin reduced chemokine secretion in the lung and bronchoalveolar lavage fluid, and in cell culture, and decreased the level of fibrosis after 3 months. Treatment with inhibitors of DP-IV-related proteases, or a combination of DP IV inhibitors and APN inhibitors, had no significant effect. We found no obvious side effects of long-term treatment with inhibitors of APN and DP IV. SIGNIFICANCE Overall, our findings show that actinonin, an inhibitor of aminopeptidase N, might modulate chemokine secretion in the lung and thus attenuate the development of lung fibrosis. Additional targeting of DP-IV-related proteases had no significant effect on these processes.

Radiation-induced matrix production of lung fibroblasts is regulated by interleukin-8

Purpose: Lung fibrosis can be caused by radiation therapy during cancer treatment and therefore can be the limiting factor of the treatment. The factors that cause the actual fibrosis and the interaction between different cell types were investigated. Materials and methods: Epithelial lung cells and fibroblasts were irradiated and different cytokines were measured in the supernatant. Also effects of radiation on the matrix production of fibroblasts were investigated. Results: Irradiation of isolated lung fibroblasts did not cause increased extracellular matrix production; however, the co-culturing of fibroblasts and irradiated lung epithelial cells or the treatment of fibroblasts with supernatants of irradiated epithelial cells did result in an increase. We were able to show that increased interleukin-8 (IL-8) levels led to increased matrix production. Conclusions: IL-8 is not only a proinflammatory cytokine but it also stimulates collagen synthesis and matrix production and therefore could be a possible drug target in preventing radiation damage during cancer therapy.

Allergic Airway Inflammation in Mice Deficient for the Antigen-Processing Protease Cathepsin E

Background: Allergic asthma is a Th2-type chronic inflammatory disease of the lung. It is characterized by infiltration of eosinophils, neutrophils, mast cells and T lymphocytes into the airways. Th2 cytokines like interleukin (IL)-4, IL-5 and chemokines like eotaxin are increased in the asthmatic response. The processing and presentation of exogenous antigens is important in the sensitization to an allergen. Cathepsin E (Ctse) is an intracellular aspartic endoprotease which is expressed in immune cells like dendritic cells (DCs). It was found to play an essential role in the processing and presentation of ovalbumin (OVA). The aim of the present study was to investigate the inhibition of Ctse in two different experimental models of allergic airway inflammation. Methods: Ctse wild-type (Ctse+/+) and Ctse-deficient (Ctse–/–) bone marrow-derived DCs (BMDCs) were pulsed with OVA/OVA peptide and cocultured with OVA transgenic T II (OT II) cells whose proliferation was subsequently analyzed. Two different in vivo asthma models with Ctse+/+ and Ctse–/– mice were performed: an acute OVA-induced and a subchronic Phleum pratense-induced airway inflammation. Results: Proliferation of OT II cells was decreased when cocultured with BMDCs of Ctse–/– mice as compared to cells cocultured with BMDCs of Ctse+/+ mice. In vivo, Ctse deficiency led to reduced lymphocyte influx after allergen sensitization and challenge in both investigated airway inflammation models, compared to their control groups. Conclusion: Ctse deficiency leads to a reduced antigen presentation in vitro. This is followed by a distinct effect on lymphocyte influx in states of allergic airway inflammation in vivo.