Biomedical subjects

K Brix

Publications and source records attributed to K Brix.

16 recordsLinked to original sources

PubMed2001

Cysteine proteinases mediate extracellular prohormone processing in the thyroid.

Thyroglobulin, the precursor of thyroid hormones, is extracellularly stored in a highly condensed and covalently cross-linked form. Solublization of thyroglobulin is facilitated by cysteine proteinases like cathepsins B and K which are proteolytically active at the surface of thyroid epithelial cells. The cysteine proteinases mediate the processing of thyroglobulin by limited extracellular proteolysis at the apical plasma membrane, thereby rapidly liberating thyroxine. The trafficking of cysteine proteinases in thyroid epithelial cells includes their targeting to lysosomes where they become maturated before being transported to the apical plasma membrane and, thus, into the extracellular follicle lumen. We propose that thyroid stimulating hormone regulates extracellular proteolysis of thyroglobulin in that it enhances the rate of exocytosis of lysosomal proteins at the apical plasma membrane. Later, thyroid stimulating hormone upregulates thyroglobulin synthesis and its secretion into the follicle lumen for subsequent compaction by covalent cross-linking. Hence, cycles of thyroglobulin proteolysis and thyroglobulin deposition might result in the regulation of the size of the luminal content of thyroid follicles. We conclude that the biological significance of extracellularly acting cysteine proteinases of the thyroid is the rapid utilization of thyroglobulin for the maintenance of constant thyroid hormone levels in vertebrate organisms.

Animals↗

PubMed2000

Thyroglobulin type-I-like domains in invariant chain fusion proteins mediate resistance to cathepsin L digestion.

The MHCII associated invariant chain isoform Ii41 shows homology to a repeat in thyroglobulin (TgR). We show that the Ii31 isoform, which lacks the TgR-like domain, is sensitive to cathepsin L treatment whereas Ii41 displays substantial resistance. The TgR-like sequence of Ii41 was exchanged for thyroglobulin type-IA and -IB repeats, that contain six or four cysteine residues. Resistance to cathepsin L digestion was maintained upon substitution of the Ii41 TgR for homologous sequences from TgR type-IA. Mutation of a conserved cysteine in the TgR domain of an Ii fusion protein strongly reduced resistance to cathepsin L digestion.

Animals↗

PubMed2000

Hyperproliferative hepatocellular alterations after intraportal transplantation of thyroid follicles.

The thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is a strong direct hepatocyte mitogen in vivo. The effects of T3 resemble those of peroxisome proliferators, which are known to induce hepatocellular tumors in rats. With the aim of studying long-term local effects of thyroid hormones on liver parenchyma, small pieces of thyroid tissue were transplanted via the portal veins into the livers of thyroidectomized male Lewis rats. At 1 week, 3 weeks, 3 months, and 18 months after transplantation, the transplants were found to proliferate, to synthesize thyroglobulin, and to release thyroxine and T3. At 3 and 18 months after transplantation, the hepatocytes of the liver acini downstream of the transplanted follicles showed an increase in cytoplasmic basophilia, a loss of glycogen, an enlargement and hyperchromasia of their nuclei, and a strong increase in cell turnover compared with unaltered liver acini. The altered hepatocytes exhibited an increase in the activities of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, malic enzyme, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome-c-oxidase, and acid phosphatase; the activities of glycogen synthase and glycogen phosphorylase were strongly decreased. The hepatocytic alterations downstream of the transplanted follicles could be explained by effects of T3. On the other hand, they resembled alterations characteristic of amphophilic preneoplastic liver foci observed in different models of hepatocarcinogenesis.

Animals↗

PubMed2000

Covalent cross-linking of secreted bovine thyroglobulin by transglutaminase.

Extracellular storage of thyroglobulin (TG) is a prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of TG within the follicle lumen is achieved by compactation and by the formation of covalent cross-links between TG molecules. In bovine thyroids, approximately 75% of the cross-links are other than disulfide bonds (J. Cell Biol. 180, 1071-1081). We have now shown that polymeric TG contains a large number of N(epsilon)(gamma-glutamyl)lysine cross-links and that only traces of these can be found in the soluble form of TG. Because such isopeptide bridges are generated usually by the action of a transglutaminase, it is reasonable to propose that the covalent polymerization of TG in the globules is under the control of this enzyme. Soluble TG was shown to be a substrate for transglutaminase in vitro; moreover, the presence of transglutaminase was demonstrated by immunofluorescence and by immunoblotting in freshly isolated bovine thyroid globules. With immunoelectron microscopy, transglutaminase was detected in the cytoplasm of thyrocytes, but not in compartments of the secretory pathway. Only one messenger RNA for transglutaminase was found by Northern blotting. Sequencing of the cloned gene failed to reveal a secretory signal, which supports the notion that the thyroid transglutaminase is the cytosolic type. Apparently, the enzyme reaches the lumen of the follicle by an as yet unknown pathway to catalyze the covalent cross-linking of thyroid globules in this extracellular compartment.

Animals↗

PubMed2000

Cathepsin K in thyroid epithelial cells: sequence, localization and possible function in extracellular proteolysis of thyroglobulin.

Extracellular proteolysis of thyroglobulin at the apical surface of thyroid epithelial cells results in liberation of thyroxine, and is mediated by lysosomal cysteine proteases such as cathepsins B and L. Here, we report on the expression of the cysteine protease cathepsin K in thyroid epithelial cells. The cDNA for porcine thyroid cathepsin K showed homologies ranging from 71% to 94% to the cDNA of cathepsin K from various species and cell types. The deduced amino acid sequence of porcine thyroid cathepsin K predicted a 37 kDa preproenzyme, with the active site residues Cys-140, His-277 and Asn-297, and one potential N-glycosylation site. The localization of cathepsin K was not restricted to lysosomes. Rather, secreted cathepsin K was predominantly found within the follicular lumen and in association with the apical plasma membrane of thyroid epithelial cells. Enzyme cytochemistry showed that cell-surface associated cathepsin K was proteolytically active at neutral pH. In vitro, recombinant cathepsin K liberated thyroxine from thyroglobulin by limited proteolysis at neutral pH. We postulate that its localization enables cathepsin K to contribute to the extracellular proteolysis of thyroglobulin, i.e. thyroid hormone liberation, at the apical surface of thyroid epithelial cells in situ.

Amino Acid Sequence↗

PubMed1999

Subcellular distribution, secretion, and posttranslational modifications of clusterin in thyrocytes.

A prominent secretory glycoprotein was detected in the culture medium of porcine thyrocytes which was identified as clusterin by microsequencing. Treatment of thyrocytes with thyroid stimulating hormone revealed a tight regulation of both synthesis and secretion of clusterin, with a distinct fraction of clusterin being always associated with the cells. At least three N-bound glycans were found on each subunit of clusterin, receiving most of the incorporated [(32)P]phosphate-label. Binding of clusterin to the immobilized cation-independent mannose 6-phosphate (M6P) receptor indicated that part of the phosphate label was contained in M6P moieties. Immunolabeling of cultured thyrocytes and of thyrocytes in situ showed clusterin on the apical cell surfaces where it colocalized with gp330/megalin, which is known to serve as a binding site for clusterin. The association with the apical plasma membrane, which, in thyrocytes, carries the iodinating system, was confirmed by biosynthetic iodination, an as yet unknown posttranslational modification of clusterin. On the basolateral plasma membranes clusterin was found within distinct, bipartite patches, suggesting that it is a constituent of cell-adhesion complexes and that it participates in cell-cell and cell-matrix interactions.

Amidohydrolases↗

PubMed1998

Extracellularly occurring histone H1 mediates the binding of thyroglobulin to the cell surface of mouse macrophages.

Thyroglobulin is the major secretory protein of thyroid epithelial cells. Part of thyroglobulin reaches the circulation of vertebrates by transcytosis across the epithelial wall of thyroid follicles. Clearance of thyroglobulin from the circulation occurs within the liver via internalization of thyroglobulin by macrophages. Here we have analyzed the interaction of thyroglobulin with the cell surface of J774 macrophages with the aim to identify the possible thyroglobulin-binding sites on macrophages. Binding of thyroglobulin to J774 cells was saturated at approximately 100 nM thyroglobulin with a Kd of 50 nM, and it was competed by the ligand itself. Preincubation of J774 cells with thyroglobulin resulted in downregulation of thyroglobulin-binding sites, indicating internalization of thyroglobulin and its binding proteins. By affinity chromatography, two proteins from J774 cells were identified as thyroglobulin-binding proteins with an apparent molecular mass of approximately 33 kD. Unexpectedly, both proteins were identified as histone H1 by protein sequencing. The occurrence of histone H1 at the plasma membrane was further proven by biotinylation or immunolabeling of J774 cells. The in vitro interaction between histone H1 and thyroglobulin was analyzed by surface plasmon resonance that revealed a Kd at 46 nM. In situ, histone H1 was colocalized to FITC-Tg-containing endocytic compartments of Kupffer cells, i.e., liver macrophages. We conclude that histone H1 is detectable at the cell surface of macrophages where it serves as a thyroglobulin-binding protein and mediates thyroglobulin endocytosis.

Amino Acid Sequence↗

PubMed1998

Iodination of mature cathepsin D in thyrocytes as an indicator for its transport to the cell surface.

Thyrocytes are known for their ability to iodinate thyroglobulin from which the thyroid hormones are generated. In the intact thyroid gland the iodination process is almost exclusively executed at the apical plasma membrane of thyroid epithelial cells. Here, we show that freshly isolated thyrocytes iodinated polypeptides other than thyroglobulin and that one of the major iodinated polypeptides was the mature form of the lysosomal protease cathepsin D (CD). The detection of mature CD as an iodinated polypeptide suggested that a fraction of the lysosomally maturated enzyme was delivered to the apical plasma membrane where it became available for iodination. After labeling of thyrocytes with [35S]methionine/cysteine overnight part of the mature CD was released into the culture medium. This was abolished by inhibiting maturation of CD with NH4Cl, indicating that mature CD appeared in the medium after its proteolytic maturation in an acidic compartment. Besides CD other soluble lysosomal polypeptides like the beta-N-acetylhexosaminidase and the sphingolipid-activating protein D (Sap D) were iodinated and partially secreted as mature polypeptides. In contrast, the membrane-associated lysosomal ceramidase was iodinated and partially secreted as immature single-chain enzyme and not as fully maturated two-chain enzyme. These data indicate that a portion of mature CD and other soluble lysosomal enzymes is delivered from lysosomes to the cell surface whereas some membrane-associated enzymes from the terminal lysosomal compartment are efficiently excluded from this process.

Amidohydrolases↗

PubMed1997

Epithelial folding in vitro: studies on the cellular mechanism underlying evagination of thyrocyte monolayers.

Epithelial monolayers in suspension culture fold in a way which closely resembles epithelial evagination. We have used freshly isolated segments of porcine thyroid follicles to study the mechanism underlying this evagination process. Epithelial folding was accompanied by dramatic changes in cell shape: the cells elongated and apical cell surfaces widened, whereas the basal cell portions were narrowed to about 20% of their original width. Apparently, enzymatic separation of thyroid epithelial cells from their underlying extracellular matrix resulted in an extension of the lateral cell-cell interactions on the expense of the basal cell surface area. Epithelial folding in vitro was Ca2+ dependent and reversibly blocked by cytochalasin D, by which the reorganization of the F-actin network was disturbed. This inhibitory effect was also observed by the action of cAMP analogues known to cause rounding of cells by their effect on cortical F-actin. Moreover, evagination in vitro was reversibly blocked at intracellular pH values of 5.8 and below. Under these conditions, protein phosphorylation was entirely inhibited. Inhibitors of protein kinases, specifically of myosin light chain kinase, were able to disrupt the evagination process, suggesting that protein phosphorylation, presumably of the myosin light chain, was essential for folding. We conclude that enzymatic separation of epithelial monolayers from their extracellular matrix initiated a cascade consisting of extended cell-cell interactions of the lateral plasma membranes and of reorganization of the apical actin-myosin network, finally resulting in profound changes in cell shape characteristic of epithelial evagination.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Paracrine interaction between hepatocytes and macrophages after extrathyroidal proteolysis of thyroglobulin.

Thyroglobulin (Tg), the precursor of the thyroid hormones triiodothyronine (T3) and thyroxine (T4), is known to derive from thyroid epithelial cells. Part of Tg reaches the circulation as an intact molecule by transcytosis across the epithelial wall of thyroid follicles. Circulating Tg is a potential ligand for the asialoglycoprotein receptor of hepatocytes. In this report we show, however, that clearance of circulating Tg occurred exclusively by endocytosis in liver macrophages, whereas hepatocytes did not participate in this process. The biological significance of this Tg uptake by the macrophages might consist in an increase of thyroid hormones in close proximity to the macrophages, thereby affecting the hepatocyte metabolism. To test this hypothesis, co-cultures of hepatocytes and macrophages were incubated with Tg, which resulted in the release of thyroid hormones and in a significant increase in the activity of lipogenesis and of hepatocellular key enzymes of the hexose monophosphate shunt. This effect of Tg could be mimicked by equivalent amounts of T3 or T4 exclusively in the co-cultures. When hepatocytes were incubated with thyroid hormones in the absence of macrophages, no or only little effect was observed, indicating that the interaction of macrophages and hepatocytes was a prerequisite for the stimulation of the hepatocellular metabolism. We conclude that the paracrine effect on HepG2 cells results from the degradation of Tg in J774 cells. Apparently, this process is not confined to the release of thyroid hormones, but it requires the interaction of both cell types, possibly mediated by an additional, as yet unknown stimulus.

Animals↗

PubMed1996

Evidence for extracellularly acting cathepsins mediating thyroid hormone liberation in thyroid epithelial cells.

Thyroglobulin (Tg) is the major secretory product of thyroid epithelial cells and is stored in the lumen of thyroid follicles at high concentrations. Thyroid hormone liberation is assumed to occur separately from this storage compartment within lysosomes. However, for the transfer of Tg to lysosomes, mechanisms to solubilize the luminal content must precede its endocytosis, because part of the luminal Tg occurs in a covalently cross-linked form. Here, by immunoprecipitation and immunoblotting we show that the majority of procathepsin B or L and a fraction of mature cathepsin B are released from porcine thyrocytes in vitro. Released cathepsins were detectable on the cell surface of the thyrocytes by immunocytochemistry and amounted to 27% of the total cathepsin B. Cytochemical studies revealed the proteolytic activity of cathepsin B at neutral pH on the cell surface of thyrocytes. Therefore, the possibility of extracellular proteolysis by cathepsins was investigated by incubating plasma membrane preparations, conditioned media, or lysosomes with Tg. The liberation of thyroid hormones was quantitated by RIA, and the degradation of Tg was determined by SDS-PAGE. Extracellular and plasma membrane-associated proteases rapidly mediated up to 54% of the total T4 liberation by limited proteolysis of Tg at neutral pH under conditions where cysteine proteases were reactivated. We propose that released and proteolytically active cysteine protease i.e. cathepsins B and L, provide thyrocytes with a pathway of limited extracellular proteolysis of Tg before endocytosis.

Animals↗

PubMed1994

Extrathyroidal release of thyroid hormones from thyroglobulin by J774 mouse macrophages.

Thyroglobulin appears in the circulation of vertebrates at species-specific concentrations. We have observed that the clearance of thyroglobulin from the circulation occurs in the liver by macrophages. Here we show that the thyroid hormones T3 and T4 were released by incubation of mouse macrophages (J774) with thyroglobulin. Thyroid hormone release was a fast process, with an initial rate of approximately 20 pmol T4/mg per min and approximately 0.6 pmol T3/mg per min, indicating that macrophages preferentially release T4. The bulk of released thyroid hormones appeared after 5 min of incubation of macrophages with thyroglobulin, whereas degradation of the protein was detectable only after several hours. During internalization of thyroglobulin, endocytic vesicles and endosomes were reached at 5 min and lysosomes at 60 min. T4 release started extracellularly by secreted proteases and continued along the endocytic pathway of thyroglobulin, whereas T3 release occurred mainly intracellularly when thyroglobulin had reached the lysosomes. This shows that the release of both hormones occurred at distinct cellular sites. Our in vitro observations suggest that macrophages in situ represent an extrathyroidal source for thyroid hormones from circulating thyroglobulin.

Animals↗

PubMed1990

Dynamics of the cytoskeleton in Amoeba proteus. III. Influence of microinjected antibodies on the organization and function of the microfilament system.

Affinity-purified antibodies against actin, myosin, alpha-actinin and vinculin cross-reacted with corresponding proteins from Amoeba proteus in immunoblotting experiments. Antibody staining of cells fixed during locomotion revealed different distribution patterns with a local concentration of anti-actin in the intermediate and of anti-myosin in the uroid region. Anti-alpha-actinin labeled a thin layer at the internal face of the plasma membrane, whereas anti-vinculin was distinctly concentrated at the base of advancing pseudopodia. Microinjection of different control solutions or antibodies against actin, myosin and alpha-actinin neither influenced the normal morphology and motile activity of amoebae nor changed the cellular distribution pattern of complementary antigens. However, antibodies against vinculin disorganized controlled locomotion and altered the spatial morphology of the microfilament system as well as the localization of the vinculin antigen thus pointing to a function of this protein in adhesion and locomotion of A. proteus. The results of the present paper show similarities to observations on mammalian tissue culture cells.

Actin Cytoskeleton↗

PubMed1987

Studies on microplasmodia of Physarum polycephalum. VII. Adhesion-dependent changes in the organization of the fibrillar actin system.

Axenically-grown microplasmodia of the acellular slime mold Physarum polycephalum were used to study adhesion-dependent changes in the spatial organization of the cytoplasmic microfilament system. Results obtained by light- and electron microscopical techniques demonstrate the presence of a membrane-bound filament cortex in all microplasmodia, and the expression of additional cytoplasmic fibrils in specimens with tight contact to a substratum. The fibrils partly terminate in focal adhesion-sites and rather seem to serve a cytoskeletal than a contractile function.

Actin Cytoskeleton↗

PubMed1987

Chemically induced changes in the morphology, dynamic activity and cytoskeletal organization of Physarum cell fragments.

Spherical cell fragments derived from Physarum polycephalum by caffeine-treatment were used as an experimental system to investigate the influence of 15 externally applied substances on the general morphology, motile behavior and cytoskeletal organization of the acellular slime mold. In comparison to controls, the most obvious changes observed after chemical stimulation proved to be cytokinetic activities, ameboid-like movement phenomena, intense cell surface dynamics and formation of cytoplasmic actin fibrils. The results demonstrate the high adaptability of the microfilament system in Physarum even when subjected to extreme conditions in the external environment.

Caffeine↗

PubMed1987

Occupation and industry data obtained from death certificates: the effect and influence of case selection.

This study first examined the accuracy of death certificate diagnoses of 4,954 cases of cancer of the lung, liver, nasopharynx, and pleura/peritoneum, then compared usual occupation and industry based on case selection from the Metropolitan Detroit Cancer Surveillance System (MDCSS), a population-based cancer registry, with cases selected from death certificates for the above types of tumors to examine the effect of misclassification. Accuracy of death certificate cancer diagnoses ranged from 93.4% for lung cancer to 28.6% for malignancies of the pleura/peritoneum. The mix of usual occupation/industry titles obtained from death certificate cases and MDCSS cases was similar for lung cancer but not for malignancies of the pleura/peritoneum (35.7% of cases from the registry v 11.1% from death certificates for the automobile industry, P = .05). The effect of misclassification and utility of usual occupation/industry statements on death certificates is discussed.

Death Certificates↗