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1.
J Biol Chem ; 291(30): 15687-99, 2016 07 22.
Article in English | MEDLINE | ID: mdl-27231342

ABSTRACT

α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the ß-catenin·cadherin complex, whereas the homodimer does not bind ß-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The ß-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the ß-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks ß-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion.


Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Cadherins/metabolism , Hypertrophy, Right Ventricular/metabolism , Multiprotein Complexes/metabolism , Myocardium/metabolism , Myocytes, Cardiac/metabolism , alpha Catenin/metabolism , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/genetics , Actins/chemistry , Actins/genetics , Animals , Cadherins/chemistry , Cadherins/genetics , Hypertrophy, Right Ventricular/genetics , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Myocardium/pathology , Myocytes, Cardiac/pathology , Protein Binding , alpha Catenin/chemistry , alpha Catenin/genetics
2.
Cancer Res ; 73(15): 4781-90, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23749643

ABSTRACT

Hypoxia promotes angiogenesis, proliferation, invasion, and metastasis of pancreatic cancer. Essentially, all studies of the hypoxia pathway in pancreatic cancer research to date have focused on fully malignant tumors or cancer cell lines, but the potential role of hypoxia inducible factors (HIF) in the progression of premalignant lesions has not been critically examined. Here, we show that HIF2α is expressed early in pancreatic lesions both in human and in a mouse model of pancreatic cancer. HIF2α is a potent oncogenic stimulus, but its role in Kras-induced pancreatic neoplasia has not been discerned. We used the Ptf1aCre transgene to activate Kras(G12D) and delete Hif2α solely within the pancreas. Surprisingly, loss of Hif2α in this model led to markedly higher, rather than reduced, number of low-grade pancreatic intraepithelial neoplasia (mPanIN) lesions. These lesions, however, failed to progress to high-grade mPanINs, and displayed exclusive loss of ß-catenin and SMAD4. The relationship among HIF2α, ß-catenin, and Smad4 was further confirmed in vitro, where silencing of Hif2α resulted in reduced ß-catenin and Smad4 transcript levels. Thus, with oncogenic Ras expressed in the pancreas, HIF2α modulates Wnt-signaling during mPanIN progression by maintaining appropriate levels of both Smad4 and ß-catenin.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Carcinoma in Situ/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Pancreatic Neoplasms/metabolism , Wnt Signaling Pathway/physiology , Animals , Blotting, Western , Carcinoma, Pancreatic Ductal/pathology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Humans , Immunohistochemistry , Mice , Mice, Transgenic , Pancreatic Neoplasms/pathology , Real-Time Polymerase Chain Reaction , Smad Proteins/metabolism , beta Catenin/metabolism
3.
Neoplasia ; 15(4): 421-34, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23555187

ABSTRACT

ß-Catenin is important in liver homeostasis as a part of Wnt signaling and adherens junctions (AJs), while its aberrant activation is observed in hepatocellular carcinoma (HCC). We have reported hepatocyte-specific ß-catenin knockout (KO) mice to lack adhesive defects as γ-catenin compensated at AJ. Because γ-catenin is a desmosomal protein, we asked if its increase in KO might deregulate desmosomes. No changes in desmosomal proteins or ultrastructure other than increased plakophilin-3 were observed. To further elucidate the role and regulation of γ-catenin, we contemplate an in vitro model and show γ-catenin increase in HCC cells upon ß-catenin knockdown (KD). Here, γ-catenin is unable to rescue ß-catenin/T cell factor (TCF) reporter activity; however, it sufficiently compensates at AJs as assessed by scratch wound assay, centrifugal assay for cell adhesion (CAFCA), and hanging drop assays. γ-Catenin increase is observed only after ß-catenin protein decrease and not after blockade of its transactivation. γ-Catenin increase is associated with enhanced serine/threonine phosphorylation and abrogated by protein kinase A (PKA) inhibition. In fact, several PKA-binding sites were detected in γ-catenin by in silico analysis. Intriguingly γ-catenin KD led to increased ß-catenin levels and transactivation. Thus, γ-catenin compensates for ß-catenin loss at AJ without affecting desmosomes but is unable to fulfill functions in Wnt signaling. γ-Catenin stabilization after ß-catenin loss is brought about by PKA. Catenin-sensing mechanism may depend on absolute ß-catenin levels and not its activity. Anti-ß-catenin therapies for HCC affecting total ß-catenin may target aberrant Wnt signaling without negatively impacting intercellular adhesion, provided mechanisms leading to γ-catenin stabilization are spared.


Subject(s)
Adherens Junctions/metabolism , Desmoplakins/metabolism , beta Catenin/genetics , Animals , Cell Adhesion , Cell Membrane/metabolism , Cell Movement , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Desmogleins/metabolism , Desmoplakins/genetics , Desmosomes/metabolism , Gene Expression , Gene Knockdown Techniques , Hep G2 Cells , Humans , Liver Neoplasms , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Plakophilins/metabolism , Protein Processing, Post-Translational , Protein Stability , RNA, Small Interfering/genetics , Wnt Signaling Pathway , beta Catenin/metabolism , gamma Catenin
4.
Hepatology ; 54(4): 1333-43, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21721031

ABSTRACT

UNLABELLED: Prolonged exposure of mice to diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in hepatobiliary injury, atypical ductular proliferation, oval cell appearance, and limited fibrosis. Previously, we reported that short-term ingestion of DDC diet by hepatocyte-specific ß-catenin conditional knockout (KO) mice led to fewer A6-positive oval cells than wildtype (WT) littermates. To examine the role of ß-catenin in chronic hepatic injury and repair, we exposed WT and KO mice to DDC for 80 and 150 days. Paradoxically, long-term DDC exposure led to significantly more A6-positive cells, indicating greater atypical ductular proliferation in KO, which coincided with increased fibrosis and cholestasis. Surprisingly, at 80 and 150 days in KO we observed a significant amelioration of hepatocyte injury. This coincided with extensive repopulation of ß-catenin null livers with ß-catenin-positive hepatocytes at 150 days, which was preceded by appearance of ß-catenin-positive hepatocyte clusters at 80 days and a few ß-catenin-positive hepatocytes at earlier times. Intriguingly, occasional ß-catenin-positive hepatocytes that were negative for progenitor markers were also observed at baseline in the KO livers, suggesting spontaneous escape from cre-mediated recombination. These cells with hepatocyte morphology expressed mature hepatocyte markers but lacked markers of hepatic progenitors. The gradual repopulation of KO livers with ß-catenin-positive hepatocytes occurred only following DDC injury and coincided with a progressive loss of hepatic cre-recombinase expression. A few ß-catenin-positive cholangiocytes were observed albeit only after long-term DDC exposure and trailed the appearance of ß-catenin-positive hepatocytes. CONCLUSION: In a chronic liver injury model, ß-catenin-positive hepatocytes exhibit growth and survival advantages and repopulate KO livers, eventually limiting hepatic injury and dysfunction despite increased fibrosis and intrahepatic cholestasis.


Subject(s)
Chemical and Drug Induced Liver Injury, Chronic/pathology , Hepatocytes/drug effects , Pyridines/pharmacology , beta Catenin/metabolism , Animals , Blotting, Western , Cells, Cultured , Chronic Disease , Disease Models, Animal , Hepatocytes/pathology , Immunohistochemistry , Liver Function Tests , Liver Regeneration/physiology , Mice , Mice, Knockout , Random Allocation , Real-Time Polymerase Chain Reaction
5.
J Hepatol ; 55(6): 1256-62, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21703193

ABSTRACT

BACKGROUND & AIMS: Wnt/ß-catenin signaling is important in liver physiology. Moreover, ß-catenin is also pivotal in adherens junctions (AJ). Here, we investigate hepatocyte-specific ß-catenin conditional null mice (KO) for any alterations in AJ and related tight junctions (TJ). METHODS: Using gene array, PCR, Western blot, immunohistochemistry, immunofluorescence, and co-immunoprecipitation, we compare and contrast the composition of AJ and TJ in KO and littermate wild-type (WT) control livers. RESULTS: We show association of E-cadherin with ß-catenin in epithelial cells of WT livers, which is lost in the KOs. While total levels of α-catenin, E-cadherin, and F-actin were modestly decreased, KO livers show increased γ-catenin/plakoglobin. By co-immunoprecipitation, E-cadherin/ß-catenin/F-actin association was observed in WT livers, while the association of E-cadherin/γ-catenin/F-actin was evident in KO livers. γ-Catenin was localized at the hepatocyte membrane at baseline in the KO liver. While γ-catenin gene expression remained unaltered, an increase in serine- and threonine-phosphorylated, but not tyrosine-phosphorylated γ-catenin was observed in KO livers. A continued presence of γ-catenin at the hepatocyte membrane, without any nuclear localization, was observed in liver regeneration after partial hepatectomy at 40 and 72 h, in both KO and WT. Analysis of TJ revealed lack of claudin-2 and increased levels of JAM-A and claudin-1 in KO livers. CONCLUSIONS: ß-Catenin adequately maintains AJ in the absence of ß-catenin in hepatocytes; however, it lacks nuclear localization. Moreover, ß-catenin/claudin-2 may be an important mechanism of crosstalk between the AJ and TJ.


Subject(s)
Adherens Junctions/metabolism , Hepatocytes/metabolism , beta Catenin/metabolism , gamma Catenin/metabolism , Actins/metabolism , Animals , Base Sequence , Cadherins/metabolism , Cell Nucleus/metabolism , Cell Proliferation , Hepatocytes/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Models, Biological , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Tight Junctions/metabolism , beta Catenin/deficiency , beta Catenin/genetics
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