Annette S. Flozak

Activity of the β-catenin phosphodestruction complex at cell-cell contacts is enhanced by cadherin-based adhesion

It is well established that cadherin protein levels impact canonical Wnt signaling through binding and sequestering β-catenin (β-cat) from T-cell factor family transcription factors. Whether changes in intercellular adhesion can affect β-cat signaling and the mechanism through which this occurs has remained unresolved. We show that axin, APC2, GSK-3β and N-terminally phosphorylated forms of β-cat can localize to cell–cell contacts in a complex that is molecularly distinct from the cadherin–catenin adhesive complex. Nonetheless, cadherins can promote the N-terminal phosphorylation of β-cat, and cell–cell adhesion increases the turnover of cytosolic β-cat. Together, these data suggest that cadherin-based cell–cell adhesion limits Wnt signals by promoting the activity of a junction-localized β-cat phosphodestruction complex, which may be relevant to tissue morphogenesis and cell fate decisions during development.

Nuclear β-Catenin Is Increased in Systemic Sclerosis Pulmonary Fibrosis and Promotes Lung Fibroblast Migration and Proliferation

Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis-associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.

β-Catenin/T-cell Factor Signaling Is Activated during Lung Injury and Promotes the Survival and Migration of Alveolar Epithelial Cells

The Wnt/β-catenin signaling cascade activates genes that allow cells to adopt particular identities throughout development. In adult self-renewing tissues like intestine and blood, activation of the Wnt pathway maintains a progenitor phenotype, whereas forced inhibition of this pathway promotes differentiation. In the lung alveolus, type 2 epithelial cells (AT2) have been described as progenitors for the type 1 cell (AT1), but whether AT2 progenitors use the same signaling mechanisms to control differentiation as rapidly renewing tissues is not known. We show that adult AT2 cells do not exhibit constitutive β-catenin signaling in vivo, using the AXIN2+/LacZ reporter mouse, or after fresh isolation of an enriched population of AT2 cells. Rather, this pathway is activated in lungs subjected to bleomycin-induced injury, as well as upon placement of AT2 cells in culture. Forced inhibition of β-catenin/T-cell factor signaling in AT2 cultures leads to increased cell death. Cells that survive show reduced migration after wounding and reduced expression of AT1 cell markers (T1α and RAGE). These results suggest that AT2 cells may function as facultative progenitors, where activation of Wnt/β-catenin signaling during lung injury promotes alveolar epithelial survival, migration, and differentiation toward an AT1-like phenotype.

β-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from β-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling

C. elegans and Drosophila generate distinct signaling and adhesive forms of β-catenin at the level of gene expression. Whether vertebrates, which rely on a single β-catenin gene, generate unique adhesive and signaling forms at the level of protein modification remains unresolved. We show that β-catenin unphosphorylated at serine 37 (S37) and threonine 41 (T41), commonly referred to as transcriptionally Active β-Catenin (ABC), is a minor nuclear-enriched monomeric form of β-catenin in SW480 cells, which express low levels of E-cadherin. Despite earlier indications, the superior signaling activity of ABC is not due to reduced cadherin binding, as ABC is readily incorporated into cadherin contacts in E-cadherin-restored cells. β-catenin phosphorylated at serine 45 (S45) or threonine 41 (T41) (T41/S45) or along the GSK3 regulatory cassette S33, S37 or T41 (S33/37/T41), however, is largely unable to associate with cadherins. β-catenin phosphorylated at T41/S45 and unphosphorylated at S37 and T41 is predominantly nuclear, while β-catenin phosphorylated at S33/37/T41 is mostly cytoplasmic, suggesting that β-catenin hypophosphorylated at S37 and T41 may be more active in transcription due to its enhanced nuclear accumulation. Evidence that phosphorylation at T41/S45 can be spatially separated from phosphorylations at S33/37/T41 suggests that these phosphorylations may not always be coupled, raising the possibility that phosphorylation at S45 serves a distinct nuclear function.

Wnt Coreceptor Lrp5 Is a Driver of Idiopathic Pulmonary Fibrosis

RATIONALE Wnt/β-catenin signaling has been implicated in lung fibrosis, but how this occurs and whether expression changes in Wnt pathway components predict disease progression is unknown. OBJECTIVES To determine whether the Wnt coreceptor Lrp5 drives pulmonary fibrosis in mice and is predictive of disease severity in humans. METHODS We examined mice with impaired Wnt signaling caused by loss of the Wnt coreceptor Lrp5 in models of lung fibrosis induced by bleomycin or an adenovirus encoding an active form of transforming growth factor (TGF)-β. We also analyzed gene expression in peripheral blood mononuclear cells (PBMC) from patients with idiopathic pulmonary fibrosis (IPF). MEASUREMENTS AND MAIN RESULTS In patients with IPF, analysis of peripheral blood mononuclear cells revealed that elevation of positive regulators, Lrp5 and 6, was independently associated with disease progression. LRP5 was also associated with disease severity at presentation in an additional cohort of patients with IPF. Lrp5 null mice were protected against bleomycin-induced pulmonary fibrosis, an effect that was phenocopied by direct inhibition of β-catenin signaling by the small molecular inhibitor of β-catenin responsive transcription. Transplantation of Lrp5 null bone marrow cells into wild-type mice did not limit fibrosis. Instead, Lrp5 loss was associated with reduced TGF-β production by alveolar type 2 cells and leukocytes. Consistent with a role of Lrp5 in the activation of TGF-β, Lrp5 null mice were not protected against lung fibrosis induced by TGF-β. CONCLUSIONS We show that the Wnt coreceptor, Lrp5, is a genetic driver of lung fibrosis in mice and a marker of disease progression and severity in humans with IPF. Evidence that TGF-β signaling can override a loss in Lrp5 has implications for patient selection and timing of Wnt pathway inhibitors in lung fibrosis.

α-Catenin is an inhibitor of transcription.

Significance Recent studies have shown that actin-binding and -regulating proteins, originally characterized in the context of cytoskeletal events, can also modify gene expression through directly impacting actin-dependent transcription. This study shows α-catenin (α-cat), an actin-binding protein that is essential for cell–cell adhesion and contact-dependent growth inhibition, can antagonize Wnt/β-catenin–mediated transcription and impact nuclear actin properties, suggesting that these events may be related. These findings establish α-cat as one of a growing list of actin-binding proteins that can modulate transcription, possibly by controlling actin dynamics in the nucleus. α-Catenin (α-cat) is an actin-binding protein required for cell–cell cohesion. Although this adhesive function for α-cat is well appreciated, cells contain a substantial amount of nonjunctional α-cat that may be used for other functions. We show that α-cat is a nuclear protein that can interact with β-catenin (β-cat) and T-cell factor (TCF) and that the nuclear accumulation of α-cat depends on β-cat. Using overexpression, knockdown, and chromatin immunoprecipitation approaches, we show that α-cat attenuates Wnt/β-cat–responsive genes in a manner that is downstream of β-cat/TCF loading on promoters. Both β-cat– and actin-binding domains of α-cat are required to inhibit Wnt signaling. A nuclear-targeted form of α-cat induces the formation of nuclear filamentous actin, whereas cells lacking α-cat show altered nuclear actin properties. Formation of nuclear actin filaments correlates with reduced RNA synthesis and altered chromatin organization. Conversely, nuclear extracts made from cells lacking α-cat show enhanced general transcription in vitro, an activity that can be partially rescued by restoring the C-terminal actin-binding region of α-cat. These data demonstrate that α-cat may limit gene expression by affecting nuclear actin organization.

Issues associated with assessing nuclear localization of N-terminally unphosphorylated β-catenin with monoclonal antibody 8E7

Backgroundβ-catenin is a dual function adhesion/transcriptional co-activator protein, and both functions are critical for normal tissue homeostasis. Since the transcriptional functions of β-catenin are more often implicated in various disease processes, there is much interest in the development and use of reagents to interrogate spatial and temporal evidence of β-catenin nuclear signaling in cells and tissues. An important study demonstrated that the signaling form of β-catenin is specifically unphosphorylated at residues S37 and T41, and suggested that this form exhibits a propensity for cytosolic/nuclear accumulation relative to the total pool of β-catenin.ResultsWe show that monoclonal antibody, 8E7, which recognizes the signaling form of β-catenin specifically unphosphorylated at S37 and T41 (Active B-Catenin, ABC), also cross-reacts with a widely expressed, variably accessible nuclear antigen that is not β-catenin. In cell types commonly used to study Wnt activation, this non-specific nuclear staining can be robust, obscuring the ABC signal. Definitive detection of nuclear localized ABC can be confirmed through an ability of classical cadherins to sequester ABC to cell junctions. In tissues, milder antigen retrieval methods can reduce the accessibility of mAb 8E7 to this cross-reacting nuclear antigen.ConclusionThese findings reveal that interpretation of nuclear, signaling active β-catenin using monoclonal antibody 8E7 should be considered judiciously, and in conjunction with independent methods.ReviewersThis article was reviewed by Frank J. T. Staal (nominated by Rachel Gerstein), Jyoti M. Sen (nominated by Avinash Bhandoola) and Manabu Sugai.

α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism

ABSTRACT The cadherin–catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian &agr;E-catenin (also known as catenin &agr;-1) and Drosophila &agr;-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of &agr;-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal &agr;-catenin function during development and collective cell migration, although no obvious defects were found in cadherin–catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of &agr;-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of &agr;-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the &agr;-catenin P-linker are required for normal cadherin–catenin complex function in Drosophila and mammalian cells.

Lamellipodial motility in wounded endothelial cells exposed to physiologic flow is associated with different patterns of β1‐integrin and vinculin localization

Integrins‐ and cytoskeletal‐associated focal adhesion proteins may participate in the process of endothelial wound closure, but their relationship in these wounds and in the presence of shear forces has not been defined. The goal in this study was to test the hypotheses that (1) modulation of β1‐integrin in human coronary artery endothelial cells (HCAEC) would alter endothelial wound closure under shear stress, and (2) β1‐integrin association with vinculin would be necessary for mediating this closure. HCAEC monolayers were pre‐conditioned to attain alignment by shearing at 12 dynes/cm2 for 18 h in a parallel‐plate flow chamber. Subsequently, they were divided into three groups: (a) control, (b) treated with anti‐β1‐integrin adhesion blocking antibody, or (c) treated with anti‐β1‐integrin adhesion promoting antibody. Next, the monolayers were wounded with a metal spatula, and re‐sheared at 20 dynes/cm2 or left static. Time‐lapse imaging and deconvolution microscopy were then performed for 3 h. Immunocytochemistry for β1‐integrin expression and vinculin was performed on all wounded monolayers. Under shear stress, vinculin localized to the ends of stress fibers, while β1‐integrin took on an intracellular macroaggregate appearance. Treatment with anti‐β1‐integrin adhesion blocking antibody enhanced wound closure, left the vinculin staining at the lamellipodial tips unchanged, but was associated with β1‐integrin staining at the lateral cell edges. Treatment with the anti‐β1‐integrin adhesion promoting antibody retarded wound closure, increased vinculin staining at cell‐cell junctions, and was associated with a fibrillar pattern of β1‐integrin staining. Modulation of β1‐integrin and changes in β1‐integrin and vinculin localization may further our understanding of laminar shear stress‐induced endothelial repair in the coronary circulation.

Wound Closure in Sheared Endothelial Cells is Enhanced by Modulation of Vascular Endothelial-Cadherin Expression and Localization:

We previously demonstrated that laminar shear stress enhances human coronary artery endothelial cell (HCAEC) wound closure via the mechanisms of cell spreading and migration. Because cell–cell junctional proteins such as vascular endothelial cell cadherin (VE–cadherin) are critical to cell–cell adhesion and motility, we tested the hypothesis that modulation of VE–cadherin expression under shear stress may be linked to this enhancement in wound closure. HCAEC monolayers were preconditioned to attain cellular alignment by shearing at 12 dynes/cm2 for 18 hr in a parallel-plate flow chamber. Subsequently, they were divided into the following three groups: (i) control; (ii) treated with anti-cadherin-5 antibody; or (iii) treated with the calcium chelating agent EGTA. Next, the monolayers were wounded with a metal spatula and resheared at 20 dynes/cm2 or left static. Time-lapse imaging was performed during the first 3 hr after imposition of these conditions, immunocytochemistry or Western blot analyses for VE–cadherin expression were performed on all wounded monolayers. Deconvolution microscopy, three-dimensional cell–cell junctional reconstruction images, and histogram analyses of interendothelial junction signal intensities were performed on cells at the wound edge of a monolayer. Under shear, HCAEC demonstrated increased VE–cadherin immunofluorescence and protein expression despite an enhancement in wound closure compared with static conditions. In separate experiments, application with anti-cadherin-5 antibody or treatment with EGTA attenuated VE–cadherin expression and further enhanced wound closure compared with control shear and all static conditions. In addition, the pattern of VE–cadherin localization with these treatments became more intracellular and nuclear in appearance. These findings of changes in this junctional adhesion protein expression and localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.