Otmar Huber

Nuclear localization of β-catenin by interaction with transcription factor LEF-1

Vertebrate beta-catenin and Drosophila Armadillo share structural similarities suggesting that beta-catenin, like Armadillo, has a developmental signaling function. Both proteins are present as components of cell adherens junctions, but accumulate in the cytoplasm upon Wingless/Wnt signaling. beta-Catenin has axis-inducing properties like Wnt when injected into Xenopus blastomeres, providing evidence for participation of beta-catenin in the Wnt-pathway, but until now no downstream targets for beta-catenin have been identified. Here we demonstrate that beta-catenin binds to the HMG-type transcription factor lymphoid enhancer factor-1 (LEF-1), resulting in a nuclear translocation of beta-catenin both in cultured mouse cells and after ectopic expression of LEF-1 in two-cell mouse embryos. LEF-1/beta-catenin complexes bind to the promoter region of the E-cadherin gene in vitro, suggesting that this interaction could regulate E-cadherin transcription. As shown for beta-catenin, ectopic expression of LEF-1 in Xenopus embryos caused duplication of the body axis, indicating a regulatory role for a LEF-1-like molecule in dorsal mesoderm formation.

CADHERINS AND CATENINS IN DEVELOPMENT

Cadherins and catenins represent key molecules during development. Recent findings demonstrate the involvement of cadherins and catenins in signaling pathways. In a working hypothesis, signaling via beta-catenin regulates the epithelial-mesenchymal transition in vertebrate development.

Pontin52 and Reptin52 function as antagonistic regulators of β‐catenin signalling activity

In Wnt‐stimulated cells, β‐catenin becomes stabilized in the cytoplasm, enters the nucleus and interacts with HMG box transcription factors of the lymphoid‐enhancing factor‐1 (LEF‐1)/T‐cell factor (TCF) family, thereby stimulating the transcription of specific target genes. We recently identified Pontin52 as a nuclear protein interacting with β‐catenin and the TATA‐box binding protein (TBP), suggesting its involvement in regulating β‐catenin‐mediated transactivation. Here, we report the identification of Reptin52 as an interacting partner of Pontin52. Highly homologous to Pontin52, Reptin52 likewise binds β‐catenin and TBP. Using reporter gene assays, we show that the two proteins antagonistically influence the transactivation potential of the β‐catenin–TCF complex. Furthermore, we demonstrate the evolutionary conservation of this mechanism in Drosophila. dpontin and dreptin are essential genes that act antagonistically in the control of Wingless signalling in vivo. These results indicate that the opposite action of Pontin52 and Reptin52 on β‐catenin‐mediated transactivation constitutes an additional mechanism for the control of the canonical Wingless/Wnt pathway.

FUNCTIONAL CHARACTERIZATION OF MULTIPLE TRANSACTIVATING ELEMENTS IN BETA -CATENIN, SOME OF WHICH INTERACT WITH THE TATA-BINDING PROTEIN IN VITRO

β-Catenin, a member of the family of Armadillo repeat proteins, plays a dual role in cadherin-mediated cell adhesion and in signaling by Wnt growth factors. Upon Wnt stimulation β-catenin undergoes nuclear translocation and serves as transcriptional coactivator of T cell factor DNA-binding proteins. Previously the transactivation potential of different portions of β-catenin has been demonstrated, but the precise location of transactivating elements has not been established. Also, the mechanism of transactivation by β-catenin and the molecular basis for functional differences between β-catenin and the closely related proteins Armadillo and Plakoglobin are poorly understood. Here we have used a yeast system for the detailed characterization of the transactivation properties of β-catenin. We show that its transactivation domains possess a modular structure, consist of multiple subelements that cover broad regions at its N and C termini, and extend considerably into the Armadillo repeat region. Compared with β-catenin the N termini of Plakoglobin and Armadillo have different transactivation capacities that may explain their distinct signaling properties. Furthermore, transactivating elements of β-catenin interact specifically and directly with the TATA-binding proteinin vitro providing further evidence that a major function of β-catenin during Wnt signaling is to recruit the basal transcription machinery to promoter regions of Wnt target genes.

The Histidine Triad Protein Hint1 Triggers Apoptosis Independent of Its Enzymatic Activity

Hint1 is a member of the evolutionarily conserved family of histidine triad proteins that acts as a haplo-insufficient tumor suppressor inducing spontaneous tumor formation in Hint+/- and Hint-/- mouse models. However, the molecular mechanisms for the tumor-suppressing activity are poorly defined. In this respect, we have recently shown that Hint1, by interaction with Pontin and Reptin, inhibits T-cell factor/β-catenin-mediated transcription of Wnt target genes. In this study, we have found that, after transient transfection with Hint1, SW480 and MCF-7 cells undergo apoptosis as analyzed by pro-caspase-3 and poly(ADP-ribose) polymerase cleavage, M30 CytoDEATH staining, cytochrome c release, and DNA fragmentation enzyme-linked immunosorbent assay. Hint1 is involved in the regulation of apoptotic pathways by inducing an up-regulation of p53 expression coinciding with an up-regulation of the proapoptotic factor Bax and a concomitant down-regulation of the apoptosis inhibitor Bcl-2. Bad and Puma levels remained unchanged. Further analyses revealed that Hint1 is associated with the Bax promoter and is a component of the Tip60 histone acetyltransferase complex and, in this context, appears to be involved in the regulation of Bax expression. Knockdown of Hint1 by short hairpin RNA resulted in down-regulation of p53 and Bax but had no effect on Bcl-2 expression. A mutant Hint1 (H112N) protein defective in enzymatic activity as an AMP-NH2 hydrolase was not impaired in induction of apoptosis, suggesting that the Hint1 pro-apoptotic activity is independent of the Hint1 enzymatic activity.

Wnt Signaling Inhibits Forkhead Box O3a-induced Transcription and Apoptosis through Up-regulation of Serum- and Glucocorticoid-inducible Kinase 1

In human cancers, mutations in components of the Wnt signaling pathway lead to β-catenin stabilization and result in augmented gene transcription. HCT116 colon cancer cells carry stabilizing mutations in β-catenin and exhibit an elevated activation of Wnt signaling. To clarify the role of an overactive Wnt signaling, we used DNA microarray analysis to search for genes whose expression is up-regulated after knockdown of the wild type adenomatous polyposis coli (APC) tumor suppressor in HCT116 cells, which further enhances Wnt signaling activation. Serum and glucocorticoid-inducible kinase 1 (SGK1) was among the most up-regulated genes following APC knockdown through small interfering RNA. Up-regulation of SGK1 in response to small interfering RNA against APC was inhibited by concomitant knockdown of β-catenin. Quantitative real time reverse transcription-PCR, Western blot, and chromatin immunoprecipitation analyses confirmed that SGK1 is a direct β-catenin target gene. SGK1 negatively regulates the pro-apoptotic transcription factor Forkhead box O3a (FoxO3a) via phosphorylation and exclusion from the nucleus. We show that Wnt signaling activation results in FoxO3a exclusion from the nucleus and inhibits expression of FoxO3a target genes. Importantly, FoxO3a mutants that fail to be phosphorylated and therefore are regulated by SGK1 are not influenced by activation of Wnt signaling. In line, knockdown of SGK1 relieves the effects of Wnt signaling on FoxO3a localization and FoxO3a-dependent transcription. Finally, we show that induction of Wnt signaling inhibits FoxO3a-induced apoptosis. Collectively our results indicate that evasion of apoptosis is another feature employed by an overactive Wnt signaling.

The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF–β-catenin-mediated transcription

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.

In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization.

Summary Tight junctions seal the paracellular cleft of epithelia and endothelia, form vital barriers between tissue compartments and consist of tight-junction-associated marvel proteins (TAMPs) and claudins. The function of TAMPs and the interaction with claudins are not understood. We therefore investigated the binding between the TAMPs occludin, tricellulin, and marvelD3 and their interaction with claudins in living tight-junction-free human embryonic kidney-293 cells. In contrast to claudins and occludin, tricellulin and marvelD3 showed no enrichment at cell–cell contacts indicating lack of homophilic trans-interaction between two opposing cell membranes. However, occludin, marvelD3 and tricellulin exhibited homophilic cis-interactions, along one plasma membrane, as measured by fluorescence resonance energy transfer. MarvelD3 also cis-interacted with occludin and tricellulin heterophilically. Classic claudins, such as claudin-1 to -5 may show cis-oligomerization with TAMPs, whereas the non-classic claudin-11 did not. Claudin-1 and -5 improved enrichment of occludin and tricellulin at cell–cell contacts. The low mobile claudin-1 reduced the membrane mobility of the highly mobile occludin and tricellulin, as studied by fluorescence recovery after photobleaching. Co-transfection of claudin-1 with TAMPs led to changes of the tight junction strand network of this claudin to a more physiological morphology, depicted by freeze-fracture electron microscopy. The results demonstrate multilateral interactions between the tight junction proteins, in which claudins determine the function of TAMPs and vice versa, and provide deeper insights into the tight junction assembly.

Amyloid Precursor Protein (APP) Metabolites APP Intracellular Fragment (AICD), Aβ42, and Tau in Nuclear Roles

Amyloid precursor protein (APP) metabolites (amyloid-β (Aβ) peptides) and Tau are the main components of senile plaques and neurofibrillary tangles, the two histopathological hallmarks of Alzheimer disease. Consequently, intense research has focused upon deciphering their physiological roles to understand their altered state in Alzheimer disease pathophysiology. Recently, the impact of APP metabolites (APP intracellular fragment (AICD) and Aβ) and Tau on the nucleus has emerged as an important, new topic. Here we discuss (i) how AICD, Aβ, and Tau reach the nucleus and how AICD and Aβ control protein expression at the transcriptional level, (ii) post-translational modifications of AICD, Aβ, and Tau, and (iii) what these three molecules have in common.

ADP-ribosylation Factor-like GTPase ARFRP1 Is Required for Trans-Golgi to Plasma Membrane Trafficking of E-cadherin *□

ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1-/- embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1vil-/-), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1vil-/- enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk-Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, α-catenin, β-catenin, γ-catenin, and p120ctn from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function.