Watching proteases in action.
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Binding Sites↗
Biomedical subjects
Klaudia Brix
Publications and source records attributed to Klaudia Brix.
HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration.
Cathepsin B, a lysosomal cysteine proteinase, was detected within vesicles of cellular protrusions forming cell-cell contact sites between keratinocytes of the stratum spinosum of human skin. This observation suggested the possibility that secretion of the protease into the pericellular spaces could be involved in the dissociation of cell-cell contacts to enable intraepidermal keratinocyte migration. To determine whether cathepsin B is indeed secreted from migrating keratinocytes, we first used subconfluent HaCaT cells as a culture model to study spontaneous keratinocyte migration. A cathepsin B-specific fluorescent affinity label proved the association of mature cathepsin B with the surfaces of HaCaT cells at the leading edges of growing cells. Second, we used scratch-wounds of confluent HaCaT monolayers as a model of induced keratinocyte migration. Cathepsin B was detected within lysosomes, i.e. vesicles within the perinuclear region of non-wounded cells. Expression of cathepsin B was up-regulated and cathepsin B-positive vesicles showed a redistribution from perinuclear to peripheral regions of keratinocytes at the wound margins within 4 h after wounding. Enzyme cytochemistry further showed that cell surface-associated cathepsin B was proteolytically active at the leading fronts of migrating keratinocytes. In addition, increased amounts of mature forms of cathepsin B were detected within the conditioned media of HaCaT cells during the first 4 h after scratch-wounding. In contrast, and as a control, the activity of the cytosolic enzyme lactate dehydrogenase was not significantly higher in media of wounded cells as compared with non-wounded controls, arguing for a specific induction of cathepsin B secretion upon wounding and migration of the cells. This was further substantiated by applying various cathepsin B-specific inhibitors after wounding. These experiments showed that the migration ability of keratinocytes was reduced due to the blockage of functional cathepsin B. Thus, our results strongly suggest that cell surface-associated cathepsin B is a protease that contributes to the remodelling of the extracellular matrix and thereby promotes keratinocyte migration during wound healing.
Cathepsin B↗
Evidence for a differential expression of the FcepsilonRIgamma chain in dendritic cells of atopic and nonatopic donors.
While mast cells and basophils constitutively express the high-affinity IgE receptor (Fc epsilon RI), it is absent or weakly expressed on APCs from normal donors. Fc epsilon RI is strongly upregulated on APCs from atopic donors and involved in the pathophysiology of atopic diseases. Despite its clinical relevance, data about Fc epsilon RI regulation on APCs are scarce. We show that in all donors intracellular alpha chain of the Fc epsilon RI (Fc epsilon RI alpha) accumulates during DC differentiation from monocytes. However, expression of gamma chains of the Fc epsilon RI (Fc epsilon RI gamma), mandatory for surface expression, is downregulated. It is low or negative in DCs from normal donors lacking surface Fc epsilon RI (Fc epsilon RI(neg) DCs). In contrast, DCs from atopics express surface Fc epsilon RI (Fc epsilon RI(pos) DCs) and show significant Fc epsilon RI gamma expression, which can be coprecipitated with Fc epsilon RI alpha. In Fc epsilon RI(neg) DCs lacking Fc epsilon RI gamma, immature and core glycosylated Fc epsilon RI alpha accumulates in the endoplasmic reticulum. In Fc epsilon RI(pos) DCs expressing Fc epsilon RI gamma, an additional mature form of Fc epsilon RI alpha exhibiting complex glycosylation colocalizes with Fc epsilon RI gamma in the Golgi compartment. IgE binding sustains surface-expressed Fc epsilon RI on DCs from atopic donors dependent on baseline protein synthesis and transport and enhances their IgE-dependent APC function. We propose that enhanced Fc epsilon RI on DCs from atopic donors is driven by enhanced expression of otherwise limiting amounts of Fc epsilon RI gamma and is preserved by increased IgE levels.
Antigens↗
Thyroid functions of mouse cathepsins B, K, and L.
Thyroid function depends on processing of the prohormone thyroglobulin by sequential proteolytic events. From in vitro analysis it is known that cysteine proteinases mediate proteolytic processing of thyroglobulin. Here, we have analyzed mice with deficiencies in cathepsins B, K, L, B and K, or K and L in order to investigate which of the cysteine proteinases is most important for proteolytic processing of thyroglobulin in vivo. Immunolabeling demonstrated a rearrangement of the endocytic system and a redistribution of extracellularly located enzymes in thyroids of cathepsin-deficient mice. Cathepsin L was upregulated in thyroids of cathepsin K(-/-) or B(-/-)/K(-/-) mice, suggesting a compensation of cathepsin L for cathepsin K deficiency. Impaired proteolysis resulted in the persistence of thyroglobulin in the thyroids of mice with deficiencies in cathepsin B or L. The typical multilayered appearance of extracellularly stored thyroglobulin was retained in cathepsin K(-/-) mice only. These results suggest that cathepsins B and L are involved in the solubilization of thyroglobulin from its covalently cross-linked storage form. Cathepsin K(-/-)/L(-/-) mice had significantly reduced levels of free thyroxine, indicating that utilization of luminal thyroglobulin for thyroxine liberation is mediated by a combinatory action of cathepsins K and L.
Animals↗
Trafficking of lysosomal cathepsin B-green fluorescent protein to the surface of thyroid epithelial cells involves the endosomal/lysosomal compartment.
Cathepsin B, a lysosomal cysteine proteinase, is involved in limited proteolysis of thyroglobulin with thyroxine liberation at the apical surface of thyroid epithelial cells. To analyze the trafficking of lysosomal enzymes to extracellular locations of thyroid epithelial cells, we have expressed a chimeric protein consisting of rat cathepsin B and green fluorescent protein. Heterologous expression in CHO cells validated the integrity of the structural motifs of the chimeric protein for targeting to endocytic compartments. Homologous expression, colocalization and transport experiments with rat thyroid epithelial cell lines FRT or FRTL-5 demonstrated the correct sorting of the chimeric protein into the lumen of the endoplasmic reticulum, and its subsequent transport via the Golgi apparatus and the trans-Golgi network to endosomes and lysosomes. In addition, the chimeras were secreted as active enzymes from FRTL-5 cells in a thyroid-stimulating-hormone-dependent manner. Immunoprecipitation experiments after pulse-chase radiolabeling showed that secreted chimeras lacked the propeptide of cathepsin B. Thus, the results suggest that cathepsin B is first transported to endosomes/lysosomes from where its matured form is retrieved before being secreted, supporting the view that endosome/lysosome-derived cathepsin B contributes to the potential of extracellular proteolysis in the thyroid.
Animals↗
Thyroid stimulating hormone upregulates secretion of cathepsin B from thyroid epithelial cells.
Constant levels of thyroid hormones in the blood are principal requirements for normal vertebrate development. Their release depends on the regulated proteolysis of thyroglobulin which is extracellularly stored in the follicle lumen under resting conditions. Thyroglobulin is proteolytically degraded to a major part in lysosomes, but in part also extracellularly leading to the release of thyroxine. Extracellularly occurring lysosomal enzymes are most probably involved in the proteolytic release of thyroxine. In this study we have analyzed the secretion of cathepsin B by thyroid follicle cells (primary cells as well as FRTL-5 cells) and its regulation by thyroid stimulating hormone, which stimulated the secretory release of the proenzyme as well as of mature cathepsin B. Within one to two hours of stimulation with thyroid stimulating hormone, the cathepsin B activity associated with the plasma membrane increased significantly. This increase correlated closely with the localization of lysosomes in close proximity to the plasma membrane of cultured thyrocytes as well as with the thyroxine liberating activity of thyrocyte secretion media. These observations indicate that thyroid stimulating hormone induces the secretion of cathepsin B, which contributes to the extracellular release of thyroxine by thyrocytes.
Animals↗
Altered dye diffusion and upregulation of connexin37 in mouse aortic endothelium deficient in connexin40.
Connexin40 (Cx40), connexin37 (Cx37) and connexin43 (Cx43) are subunit proteins of gap junction channels in the vascular wall which are presumably involved in the propagation of vasomotor signals. In this study we have investigated in Cx40-deficient versus wild-type aortic endothelium to which extent loss of Cx40 impairs intercellular communication. We show in Cx40-deficient mice that expression of both Cx37 and Cx43 protein was increased approximately 3- and 2-fold over the level in wild-type endothelium, respectively. Furthermore, Cx37 immunosignals were distributed more homogeneously on contacting plasma membranes in Cx40-deficient versus with wild-type endothelium. Cx43 was not detected in endothelium but only in smooth muscle cells of the vessel wall. Iontophoretic injection of Lucifer Yellow or neurobiotin into aortic endothelium of Cx40-deficient mice showed extensive intercellular transfer of neurobiotin but not of Lucifer Yellow. In contrast, intercellular spreading of Lucifer Yellow was observed in endothelium of wild-type aorta. As shown by electron microscopy, gap junctions in Cx40-deficient endothelium were morphologically different from those of wild-type vessels. These results demonstrate that dye diffusibility of endothelial gap junctions is different in Cx40-deficient and wild-type mice, although Cx40-deficient mice retain the capability of intercellular communication. Apparently, Cx40-deficient endothelial cells upregulate and redistribute Cx37 as a molecular adaptation to the lack of Cx40.
Animals↗