Figure 1.
TCR signalling increases the level of β-catenin.
T cells were stimulated with anti-CD3 antibodies for the indicated times. (A) Cell aliquots were analysed for expression of α-catenin or β-catenin protein levels by Western blotting. Western blotting with anti-ERK was used to control for protein loading. (B) Antibody specificity was confirmed by transfecting T cells with control RNAi (Luciferase) or RNAi directed against β-catenin (Ctnnb1) prior to T cell stimulation for 18 hours. (C) mRNA levels for α-catenin (Ctnna1) and β-catenin (Ctnnb1) were measured by qPCR relative to expression of 18srRNA (endogenous control). Two independent experiments are shown for comparison. Experiments were performed in triplicate with similar results obtained when compared against alternative endogenous controls actin, GAPDH and HPRT (not shown). The cells used in Expt1 were used for Western blotting in (A).
Figure 2.
GSK3 regulates the expression of β-catenin downstream of TCR signalling.
(A) Primary human T cells were left unstimulated (lane 1) or stimulated for 18 hours with plate bound anti-CD3 and anti-CD28 antibodies (lane 2), or with increasing concentrations of LiCl (0, 0.2, 2 or 20 mM, indicated by black triangles, lanes 3–6), or with anti-CD3/CD28 and increasing LiCl concentrations together (lanes 7–10). Total cell lysates were immunoblotted with antibodies specific for β-catenin, phospho-β-catenin (Ser33/37/Thr41, herein referred to as p-β-catenin), phospho-GSK3α/β (Ser21/9, herein referred to as p-GSK3α/β) and GSK3. (B) T cells were stimulated for 18 hours with plate-bound anti-CD3 as indicated, either in the absence or presence of the GSK3 inhibitor Bio-ac at 1 µM. Total cell lysates were analysed by Western blot with anti-β-catenin. An anti-tubulin blot is shown as a control for protein loading. This experiment was repeated twice with similar results.
Figure 3.
Induction of β-catenin correlates with a modulation of TCF1 isoform usage.
(A) T cells were stimulated with plate bound anti-CD3 and anti-CD28 antibodies for the indicated times and immediately frozen as dry pellets on dry ice. Total cell lysates were prepared and run on SDS-PAGE, immunblotted for β-catenin, phospho-β-catenin and TCF-1. Total ERK expression served as a loading control. (B) T cells were fractionated based on the expression of CD4 and CD8 as well as CD45 isoform expression (CD45RA: naïve T cells or CD45RO: memory T cells) as outlined in materials and methods. The cells were either left unstimulated or were stimulated overnight with anti-CD3/CD28 antibodies. Total cell lysates were immunoblotted for β-catenin and TCF1.
Figure 4.
TCR stimulation leads to nuclear translocation of β-catenin.
(A) T cells were stimulated with plate bound anti-CD3/CD28 in the presence or absence of 20 mM LiCl for 18 hours, as indicated, following which cytoplasmic and nuclear fractions were separated. Fractions were immunoblotted for β-catenin and phospho-β-catenin expression. Cytoplasmic and nuclear fractionation was confirmed by tubulin (cytosol) and lamin-B1 (nuclear) expression. (B) T cells were stimulated with anti-CD3/CD28 magnetic beads for the indicated times. CD25 (IL-2Rα) mRNA levels were measured by qRT-PCR, using Abl1 as an internal control, to verify successful T cell activation. (C) The expression of the TCF/β-catenin target gene Axin2 was measured by qRT-PCR following stimulation of T cells with anti-CD3/CD28 (same samples as in B). (D) In parallel to the samples in B and C, T cells and HEK293T cells were left untreated or incubated with 20 mM lithium for 7 hours and measured for Axin2 mRNA levels relative to those of Abl1. (E) Left: T cells were stimulated with anti-CD3/CD28 for the indicated times and Axin2 and DKK-1 mRNA levels were determined by qRT-PCR relative to Abl and PPIA. The expression relative to Abl1 is shown, with calculations relative to PPIA yielding comparable results. Right: HEK293T cells were left untreated or incubated with 20 mM lithium for 7 hours and measured for DKK-1 mRNA levels relative to those of Abl1. All qRT-PCR reactions were performed in triplicate.
Figure 5.
PI3K regulates the expression of β-catenin downstream of TCR signalling.
(A) T cells were left untreated or were incubated for 18 hours with the proteasome inhibitor MG132, 20 mM LiCl, plate bound anti-CD3 (10 µg/ml), plate bound anti-CD28 (10 µg/ml), plate bound anti-CD3 plus anti-CD28 (10 µg/ml each). Total cell lysates were used for Western blotting with anti-β-catenin, anti-phospho-β-catenin and anti-GSK3αβ. (B) T cells were stimulated for 18 hours with two different concentrations of plate-bound anti-CD3 in the absence or presence of anti-CD28. Total cell lysates were analysed by Western blot with anti-β-catenin, anti-p-GSK3αβ and anti-tubulin. This experiment was performed twice with similar results. (C) T cells were pre-treated with the PI3K inhibitor Ly294002 (50 µM) or the MEK inhibitor PD98059 (50 µM) for 1 hour prior to 18 hours stimulation with plate bound anti-CD3 in the presence of these inhibitors. Total cell lysates were immunoblotted with indicated antibodies.
Figure 6.
PKC can also regulate the expression of β-catenin.
(A) T cells were incubated for 18 hours with either Ionomycin (I, 100 nM), PDBu (P, 10 ng/ml), or a combination of PDBu and Ionomycin (P+I). Total cell lysates were used for Western blotting with anti-β-catenin, anti-phospho-β-catenin and anti-GSK3. (B) T cells were stimulated for 18 hours with either anti-CD3 or PDBu in the absence or presence of the PI3K inhibitor Ly294002. Total cell lysates were analysed by Western blot with anti-β-catenin and anti-phospho-GSK3. (C) T cells were stimulated for 18 hours with either plate-bound CD3 (CD3), lithium (Li+) or PDBu (P), as shown, and analysed by Western blot with the antibodies indicated. (D) T cells were stimulated with anti-CD3 for 18 hours either in the absence or presence of the PI3K inhibitor Ly294002 (20 and 50 µM) or the PLC inhibitor U73122 (10 µM). Total cell lysates were analysed by Western blot with anti-β-catenin, anti-p-GSK3α/β and anti-tubulin. A representative figure of two experiments is shown.