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TFF3 is a member of the TFF-domain peptide family which is constitutively expressed in mucous epithelial tissues where it acts as a motogenic factor and plays an important role during epithelial restitution after wounding and during inflammation. In contrast to these beneficial functions, TFFs were also reported to be involved in cell scattering and tumor invasion. These changes in epithelial cell morphology and motility are associated with a modulation of cell contacts. In this respect, we here investigated the E-cadherin/catenin cell adhesion complex in FLAG-hTFF3-transfected HT29/B6 and MDCK cells. In hTFF3-transfected cells the amount of E-cadherin is reduced with a concomitant reduction of alpha- and beta-catenin levels. On one hand, E-cadherin expression is lowered at the transcriptional level as shown by multiplex RT-PCR analysis. This decrease does not depend on differences in the promoter methylation status as shown by methylation-specific PCR. On the other hand, pulse-chase experiments showed a reduction in the E-cadherin half-life in hTFF3-transfected cells reflecting increased E-cadherin degradation. In summary, hTFF3 induces transcriptional and posttranslational processes resulting in a modulation of E-cadherin-mediated cell-cell contacts that may play an important role in the paradoxical benefical and pathogenic function of TFF peptides.

Increasing evidence suggests that morphogenesis of the distinct developmental structures derived from the same organ-committed epithelium is controlled by differential mechanisms. As was recently shown in mice with mutations in the downless (dL) gene, induction of primary or tylotrich hair follicles is strikingly dependent of signaling through the Tnf receptor homologue, Edar. Here, we show that dorsal skin of murine embryos with constitutive deletion of the BMP2/4 antagonist noggin, after transplantation into SCID mice, is characterized by the lack of induction of secondary hair follicles, and by the arrest of primary hair follicle development prior to hair shaft formation. The loss of noggin activity was associated with failure to express genes that specify hair follicle cell fates in the epidermis (Lef-1, beta-catenin, Shh) and dermal papilla (p75 kDa neurotrophin receptor, alkaline phosphatase). This suggests that regulation of BMP2/4 signaling by noggin is essential for the induction of secondary hair follicles, as well as for advanced stages of development in primary hair follicles.

In epithelial and endothelial cell layers tight junctions form selective apicolateral paracellular barriers separating luminal and extracellular spaces from the underlying tissues. Within the tight junctions the tetraspan transmembrane proteins occludin, claudins, and tricellulin form anastomosing strands of protein complexes, which interconnect opposing membranes of neighboring cells. Phosphorylation of tight junction components is critically involved in the regulation of tight junction assembly, maintenance, and function. This chapter compares occludin and tricellulin phosphorylation by the serine/threonine kinases CK2 and CK1. [ABSTRACT FROM AUTHOR]

Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I. Nucleolar localization of Pontin was confirmed by its detection in nucleolar extracts and by electron microscopy, which revealed Pontin accumulation specifically in the nucleolar fibrillar centers. Pontin localization in the nucleolus was dynamic and Pontin accumulated in large nucleolar dots mainly during S-phase. Pontin concentration in the large nucleolar dots correlated with reduced transcriptional activity of nucleoli. In addition, Pontin was found to associate with RNA polymerase I and to interact in a complex with c-Myc with rDNA sequences indicating that Pontin is involved in the c-Myc-dependent regulation of rRNA synthesis. [ABSTRACT FROM AUTHOR]

Pontin and Reptin previously were identified as nuclear β-catenin interaction partners that antagonistically modulate β-catenin transcriptional activity. In this study, Hint1/PKCI, a member of the evolutionary conserved family of histidine triad proteins, was characterised as a new interaction partner of Pontin and Reptin. Pull-down assays and co-immunoprecipitation experiments show that Hint1/PKCI directly binds to Pontin and Reptin. The Hint1/PKCI-binding site was mapped to amino acids 214–295 and 218–289 in Pontin and Reptin, respectively. Conversely, Pontin and Reptin bind to the N-terminus of Hint1/PKCI. Moreover, by its interaction with Pontin and Reptin, Hint1/PKCI is associated with the LEF-1/TCF-β-catenin transcription complex. In this context, Hint1/PKCI acts as a negative regulator of TCF-β-catenin transcriptional activity in Wnt-transfected cells and in SW480 colon carcinoma cells as shown in reporter gene assays. Consistent with these observations, Hint1/PKCI represses expression of the endogenous target genes cyclin D1 and axin2 whereas knockdown of Hint1/PKCI by RNA interference increases their expression. Disruption of the Pontin/Reptin complex appears to mediate this modulatory effect of Hint1/PKCI on TCF-β-catenin-mediated transcription. These data now provide a molecular mechanism to explain the tumor suppressor function of Hint1/PKCI recently suggested from the analysis of Hint1/PKCI knockout mice. [ABSTRACT FROM AUTHOR]

The polarized morphology of epithelial cells depends on the establishment and maintenance of characteristic intercellular junctions. The dramatic morphological changes observed in apoptotic epithelial cells were ascribed at least in part to the specific fragmentation of components of adherens junctions and desmosomes. Little, however, is known about tight junctions during apoptosis. We have found that after induction of apoptosis in epithelial cells, tight junction proteins undergo proteolytic cleavage in a distinctive manner correlated with a disruption of tight junctions. The transmembrane protein occludin and, likewise, the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage. In addition, occludin is cleaved at an extracellular site by a metalloproteinase. The caspase cleavage site in occludin was mapped Cterminally to Asp320 within the C-terminal cytoplasmic domain. Mutagenesis of this site efficiently blocked fragmentation. In the presence of caspase and/or metalloproteinase inhibitors, fragmentation of occludin, ZO-1 and ZO-2 was blocked and cellular morphology was almost fully preserved. Interestingly, two members of the claudin family of transmembrane tight junction proteins exhibited a different behavior. While the amount of claudin-2 protein was reduced similarly to occludin, ZO-1 and ZO-2, claudin-1 was either fully preserved or was even increased in apoptotic cells. [ABSTRACT FROM AUTHOR]

Presents information on a study on the identification of an interaction partner of beta-catenin, a nuclear protein designated Pontin52. Materials and methods; Results; Discussion.

The exact sites, structures, and molecular mechanisms of interaction between junction organizing zona occludence protein 1 (ZO-1) and the tight junction protein occludin or the adherens junction protein α-catenin are unknown. Binding studies by surface plasmon resonance spectroscopy and peptide mapping combined with comparative modeling utilizing crystal structures led for the first time to a molecular model revealing the binding of both occludin and α-catenin to the same binding site in ZO-1. Our data support a concept that ZO-1 successively associates with α-catenin at the adherens junction and occludin at the tight junction. Strong spatial evidence indicates that the occludin C-terminal coiled-coil domain dimerizes and interacts finally as a four-helix bundle with the identified structural motifs in ZO-1. The helix bundle of occludin406-521 and α-catenin509-906 interacts with the hinge region (ZO-1591-632 and ZO-1591-622, respectively) and with (ZO-1726-754 and ZO-1756-781) in the GuK domain of ZO-1 containing coiled-coil and α-helical structures, respectively. The selectivity of both protein-protein interactions is defined by complementary shapes and charges between the participating epitopes. In conclusion, a common molecular mechanism of forming an intermolecular helical bundle between the hinge region/GuK domain of ZO-1 and α-catenin and occludin is identified as a general molecular principle organizing the association of ZO-1 at adherens and tight junctions. [ABSTRACT FROM AUTHOR]

Assembly of tight junctions at the most apical part of the lateral cell membrane is a key event in the differentiation of polarized epithelial cells. Claudin-2, a transmembrane protein involved in tight junction strand formation, has turned out to play a crucial role for the paracellular barrier function by opening pores for small cations. Physiological and pathological variations of epithelial barrier function are accompanied by differential expression of tight junction proteins. Therefore, we characterized molecular mechanisms regulating claudin-2 gene expression. Genomic DNA containing the transcription start point of human claudin-2 was isolated and functionally characterized by reporter gene assays. Activity of the claudin-2 promoter was elevated in mouse mammary epithelial C57 cells expressing Wnt-1. LEF-1, a nuclear effector of the Wnt signaling pathway which is involved in the regulation of cell differentiation and polarization, was found to bind directly to the claudin-2 promoter as revealed by electrophoretic mobility shift assays. Expression of LEF-1 and β-catenin both enhanced claudin-2 promoter activity. This increase was reduced after mutation of LEF-1 binding sites within the claudin-2 promoter. Furthermore, claudin-2 promoter activity was found to be enhanced by the TCF-4/β-catenin transcription complex. Therefore, we conclude that gene expression mediated by the promoter of the human tight junction protein claudin-2 is regulated by factors involved in Wnt signaling. Moreover, a functional crosstalk between Wnt signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins could be demonstrated in the regulation of claudin-2 promoter-mediated gene expression. [Copyright &y& Elsevier]