Figure 1.
Rapid AJ dissociation by LPA induces accumulation of β-catenin in a perinuclear compartment.
A431 cells were serum-starved for 18 h, incubated with PBS or 1 µM LPA for indicated times, and β-catenin expression visualized by immunofluorescence staining as described in Materials and Methods, using anti-β-catenin monoclonal IgG antibody followed by Alexa-Fluor 488-tagged anti-rabbit IgG antibody. Samples (N = 3 per group) were examined with a Zeiss LSM-510 confocal microscope (40× Plan-NEOFLUAR objective; NA 1.3; scale bar = 50 µm). When cells were LPA-treated, pockets of increased levels of ß-catenin accumulation (arrowheads) were visualized over time compared to PBS-treated cells; the apparent accumulation lasted up to approximately 6 h and declined as cells migrated out of colonies and rebuilt AJ formations.
Figure 2.
E-cadherin colocalizes with β-catenin at the perinuclear region.
A431 cells were serum-starved for 18 h, pretreated with PBS or 1 µM LPA for 1.5 h, immunostained with anti-β-catenin polyclonal rabbit IgG (red) and anti-E-cadherin monoclonal mouse IgG (green), and imaged with a Zeiss LSM-510 confocal microscope (40× Plan-NEOFLUAR objective; NA 1.3; scale bar = 50 µm). When cells were LPA-treated, β-catenin expression colocalized (yellow) with E-cadherin expression. In contrast, when cells were PBS-treated for control, no colocalization was seen (N = 3 per group).
Figure 3.
β-catenin is targeted to the perinuclear endocytic recycling compartment.
(A) A431 cells were serum-starved, pretreated with 1 µM LPA for 1 h, and incubated for 30 min in 10 µg/ml Alexa-Fluor™ 647-transferrin (colored as green). Cells were fixed, stained with anti-β-catenin monoclonal IgG antibody (colored as red), and imaged using a Zeiss LSM-510 confocal microscope (40× Plan-NEOFLUAR objective; NA 1.3; scale bar = 50 µm). The perinuclear region stained positively for transferrin under receptor recycling conditions and overlapped well, colocalizing with ß-catenin staining. (B) The distribution of β-catenin at the perinuclear region was also performed with anti-Rab11 polyclonal rabbit antibody. Cells were incubated in PBS or 1 µM LPA for 1.5 h, fixed, stained with anti-β-catenin monoclonal antibody (red) and Rab11 antibody (green), and imaged with a Zeiss LSM-510 confocal microscope. Rab11 staining was also found to highly colocalized with β-catenin staining (yellow; arrows) (N = 3 per group).
Figure 4.
ERC targeted β-catenin translocates to the nucleus in the presence of Wnt activator or GSK-3β inhibitors.
(A) A431 cells were serum starved for 18 h, incubated in PBS or 1 µM LPA for indicated time, then treated with either 40 mM KCl or LiCl for 5 min, immunostained for β-catenin using a monoclonal IgG antibody, and imaged with a Zeiss LSM-510 confocal microscope (40× Plan-NEOFLUAR objective; NA 1.3; scale bar = 50 µm). Cells treated with both LPA and LiCl displayed concentrated β-catenin accumulation in their nuclei (especially at 90 min when AJ dissociation occurs); whereas, those cells pretreated with PBS and KCl/LiCl together or LPA and KCl together (control salt) displayed no nuclear accumulation. (B) Cells were serum starved, treated with PBS or 1 µM LPA for 1.5 h, subsequently incubated in the presence or absence of 40 mM LiCl for indicated time, immunostained for β-catenin, and imaged. LPA-treated cells accumulated β-catenin at the ERC and showed higher catenin nuclear translocation than control upon treatment with LiCl, particularly at the 30 min time point. (C) Cells were serum-starved, stimulated with LPA or PBS for 1 h, pretreated with LiCl as indicated, and further incubated with an antibody against β-catenin and Alexafluor 647-transferrin for 30 min. The lateral images of cells were reconstituted from confocal z-sectioned images (β-catenin, red; transferrin, green). This view captures β-catenin co-distribution with transferrin in the perinuclear ERC when cells were stimulated with LPA alone (arrow head). Cells treated with both LPA and LiCl displayed accumulation in the central nuclear region (open arrow). In both cases, AJ β-catenin levels (arrows) were reduced. (D) Serum-starved cells were stimulated with PBS or LPA±LiCl as indicated, and the nuclear and cytoplasmic fractions were isolated. Western blot using anti-β-catenin (top panel) and anti-PCNA monoclonal antibodies (lower panel) was performed according to the Odyssey Infrared Imaging System. LiCl-LPA costimulation increased β-catenin level normalized by PCNA band intensity, compared to LPA or LiCl alone (N = 3). (E) Cells were stimulated by PBS or LPA for 1.5 h, incubated in the presence or absence of 1 µM GSK-3β inhibitor VIII or XIII for 30 min, and immunostained for β-catenin. Addition of specific GSK inhibitors resulted in β-catenin translocation to the nucleus in LPA-treated, but not control cells (N = 3).
Figure 5.
β-catenin from the cadherin-bound AJ pool translocates to the nucleus.
(A) β-catenin-ΔGSK-PA-GFP construct (green) was co-expressed in A431 cells with mRFP-actin (red). The AJ area was photo-activated selectively by 405 nm light using a Zeiss LSM510 confocal microscope (pre, pre-activation; 4 s, 4 sec after activation). The fluorescence of PA-GFP (green) was monitored at the indicated times, together with of mRFP-actin (red). One µM LPA stimulation images are shown in panel (PBS-treated cells not shown). The fluorescence levels of the nuclear (Nuc) and AJ areas (representative areas shown in bottom image) were measured over time using Zeiss LSM510 software. (B) LPA-stimulated cells exhibited a steady decrease of AJ expression, coupled with a steady increase of nuclear β-catenin expression, over time. In contrast, PBS-treated cells showed a steady level of nuclear β-catenin. Since cells displayed a wide variety of morphologies and initial fluorescence intensities per region, all measurements were normalized by plotting data as the fold-change of the fluorescence ratio (against corresponding measure for 0 h) of nuclear fluorescence/AJ fluorescence over time, for both PBS- and LPA-stimulated cells (N = 3). LPA-treated cells displayed significantly (P<0.05) increased levels of nuclear β-catenin over time, compared to PBS-treated control cells.
Figure 6.
TCF/LEF-dependent transcriptional activity TOPflash.
In order to measure TCF/LEF-dependent transcriptional activity of A431 and HEK293 cell lines, cells were transfected with a TOPflash reporter plasmid. After overnight starvation, cells were stimulated by PBS or LPA, in the presence or absence of LiCl for 18 h, and were assessed for transcriptional activity using the Steady-Glo Luciferase assay system. (A) A431 cells treated with either PBS or LPA alone showed negligible levels of transcriptional activity (N = 3). In contrast, cells treated with LiCl alone showed significant increase in signal (N = 3; P<0.0001), which was significantly diminished by co-treatment with LPA (N = 3; P = 0.001). (B) Similarly, HEK293 cells treated with either PBS or LPA alone showed negligible levels of transcriptional activity (N = 3). Cells treated with LiCl alone showed significant increase in signal (N = 3; P = 0.024), which was significantly diminished by co-treatment with LPA (N = 3; P = 0.017).
Figure 7.
A schematic model for the intersection of cadherin-bound β-catenin pool with Wnt-signaling upon AJ dissociation by LPA stimulation.
ß-catenin (ß) is recruited to sites of cell-cell interactions and binds E-cadherin (E-cad) establishing adhesion via formation of adherens junctions (AJ). Upon LPA stimulation, AJ dissociate and cadherin-bound ß-catenin travels to the early endosome (EE), endocytic recycling compartment (ERC) [and potentially the recycling endosome (RE) before its exit back to AJ area], before being translocated into the nucleus (Nuc) of the cell, where it engages transcription factors such as TCF and LEF (altering gene expression). Translocation is dependent upon LiCl treatment or Wnt pathway activation (via binding to receptors encoded by frizzled genes), as these steps prevent glycogen synthase kinase 3ß (GSK3ß) from phosphorylating the (APC) complex and ß-catenin substrate (ß). Unphosphorylated ß either degrades or escapes to the nucleus where it engages transcription factors such as TCF and LEF.