c-erbB-3: a nuclear protein in mammary epithelial cells

c-erbB receptors are usually located in cell membranes and are activated by extracellular binding of EGF-like growth factors. Unexpectedly, using immunofluorescence we found high levels of c-erbB-3 within the nuclei of MTSV1-7 immortalized nonmalignant human mammary epithelial cells. Nuclear localization was mediated by the COOH terminus of c-erbB-3, and a nuclear localization signal was identified by site-directed mutagenesis and by transfer of the signal to chicken pyruvate kinase. A nuclear export inhibitor caused accumulation of c-erbB-3 in the nuclei of other mammary epithelial cell lines as demonstrated by immunofluorescence and biochemical cell fractionation, suggesting that c-erbB-3 shuttles between nuclear and nonnuclear compartments in these cells. Growth of MTSV1-7 on permeable filters induced epithelial polarity and concentration of c-erbB-3 within the nucleoli. However, the c-erbB-3 ligand heregulin β1 shifted c-erbB-3 from the nucleolus into the nucleoplasm and then into the cytoplasm. The subcellular localization of c-erbB-3 obviously depends on exogenous stimuli and on the stage of epithelial polarity and challenges the specific function of c-erbB-3 as a transmembrane receptor protein arguing for additional, as yet unidentified, roles of c-erbB-3 within the nucle(ol)us of mammary epithelial cells

Hepatocyte specific expression of an oncogenic variant of β-catenin results in cholestatic liver disease

[Background] The Wnt/β-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous β-catenin signaling in hepatocytes remain elusive. [Results] Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated β-catenin. [Materials and Methods] Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of β-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. [Conclusion] Expression of a dominant stable form of β-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis

Expression patterns of protein C inhibitor in mouse development

Proteolysis of extracellular matrix is an important requirement for embryonic development and is instrumental in processes such as morphogenesis, angiogenesis, and cell migration. Efficient remodeling requires controlled spatio-temporal expression of both the proteases and their inhibitors. Protein C inhibitor (PCI) effectively blocks a range of serine proteases, and recently has been suggested to play a role in cell differentiation and angiogenesis. In this study, we mapped the expression pattern of PCI throughout mouse development using in situ hybridization and immunohistochemistry. We detected a wide-spread, yet distinct expression pattern with prominent PCI levels in skin including vibrissae, and in fore- and hindgut. Further sites of PCI expression were choroid plexus of brain ventricles, heart, skeletal muscles, urogenital tract, and cartilages. A strong and stage-dependent PCI expression was observed in the developing lung. In the pseudoglandular stage, PCI expression was present in distal branching tubules whereas proximal tubules did not express PCI. Later in development, in the saccular stage, PCI expression was restricted to distal bronchioli whereas sacculi did not express PCI. PCI expression declined in postnatal stages and was not detected in adult lungs. In general, embryonic PCI expression indicates multifunctional roles of PCI during mouse development. The expression pattern of PCI during lung development suggests its possible involvement in lung morphogenesis and angiogenesis

Morphology of nuclear transcription

Gene expression control is a fundamental determinant of cellular life with transcription being the most important step. The spatial nuclear arrangement of the transcription process driven by RNA polymerases II and III is nonrandomly organized in foci, which is believed to add another regulatory layer on gene expression control. RNA polymerase I transcription takes place within a specialized organelle, the nucleolus. Transcription of ribosomal RNA directly responds to metabolic requirements, which in turn is reflected in the architecture of nucleoli. It differs from that of the other polymerases with respect to the gene template organization, transcription rate, and epigenetic expression control, whereas other features are shared like the formation of DNA loops bringing genes and components of the transcription machinery in close proximity. In recent years, significant advances have been made in the understanding of the structural prerequisites of nuclear transcription, of the arrangement in the nuclear volume, and of the dynamics of these entities. Here, we compare ribosomal RNA and mRNA transcription side by side and review the current understanding focusing on structural aspects of transcription foci, of their constituents, and of the dynamical behavior of these components with respect to foci formation, disassembly, and cell cycle.(VLID)340415

Histochemistry and Cell Biology / Nucleolus and chromatin

The nucleolus as site of ribosome biogenesis holds a pivotal role in cell metabolism. It is composed of ribosomal DNA (rDNA), which is present as tandem arrays located in nucleolus organizer regions (NORs). In interphase cells, rDNA can be found inside and adjacent to nucleoli and the location is indicative for transcriptional activity of ribosomal genesinactive rDNA (outside) versus active one (inside). Moreover, the nucleolus itself acts as a spatial organizer of non-nucleolar chromatin. Microscopy-based approaches offer the possibility to explore the spatially distinct localization of the different DNA populations in relation to the nucleolar structure. Recent technical developments in microscopy and preparatory methods may further our understanding of the functional architecture of nucleoli. This review will attempt to summarize the current understanding of mammalian nucleolar chromatin organization as seen from a microscopists perspective.(VLID)359162

Histochemistry and Cell Biology / Histone deacetylase 1 expression is inversely correlated with age in the short-lived fish Nothobranchius furzeri

Aging is associated with profound changes in the epigenome, resulting in alterations of gene expression, epigenetic landscape, and genome architecture. Class I Histone deacetylases (HDACs), consisting of HDAC1, HDAC2, HDAC3, and HDAC8, play a major role in epigenetic regulation of chromatin structure and transcriptional control, and have been implicated as key players in the pathogenesis of age-dependent diseases and disorders affecting health and longevity. Here, we report the identification of class I Hdac orthologs and their detailed spatio-temporal expression profile in the short-lived fish Nothobranchius furzeri from the onset of embryogenesis until old age covering the entire lifespan of the organism. Database search of the recently annotated N. furzeri genomes retrieved four distinct genes: two copies of hdac1 and one copy of each hdac3 and hdac8. However, no hdac2 ortholog could be identified. Phylogenetic analysis grouped the individual killifish class I Hdacs within the well-defined terminal clades. We find that upon aging, Hdac1 is significantly down-regulated in muscle, liver, and brain, and this age-dependent down-regulation in brain clearly correlates with increased mRNA levels of the cyclin-dependent kinase inhibitor cdkn1a (p21). Furthermore, this apparent reduction of class I HDACs in transcript and protein levels is mirrored in the mouse brain, highlighting an evolutionarily conserved role of class I HDACs during normal development and in the aging process.(VLID)484087

Phylogenetic analysis and expression profiling of the Klotho gene family in the short-lived African killifish Nothobranchius furzeri

Members of the Klotho gene family have been identified as modulators of the aging process. Deletion of klotho in the mouse results in a syndrome resembling rapid human aging. Conversely, overexpression of klotho extends mammalian lifespan. Here, we identify klotho orthologs in the vertebrate aging model Nothobranchius furzeri and provide a detailed spatio-temporal expression profile of both paralogs, and klotho, from embryogenesis until old age spanning the entire life cycle of the organism. Specifically, we observe low levels of expression of both paralogs during embryogenesis followed by a significant transcriptional induction as development proceeds. In adult killifish, klotho is predominantly expressed in the liver, the kidney, and the developing pharyngeal teeth. Particularly high levels of Klotho protein were identified in the kidney tubules, closely resembling mammalian expression patterns. Prominent klotho expression was detected in the killifish intestine and liver. Overall, qRT-PCR analysis of Klotho members as a function of age revealed steady transcript levels, except for klotho expression in the liver which was significantly downregulated with age. This spatio-temporal expression profiling may serve as a useful starting point to further investigate the distinct physiological roles of Klotho members during the aging process.(VLID)473639