Calpain as an effector of the Gq signaling pathway for inhibition of Wnt/beta -catenin-regulated cell proliferation

Abstract

Signaling pathways interact to integrate and regulate information flow in evoking complex cellular responses. We have studied the mechanisms and consequences of interactions between the Gq and Wnt/beta-catenin pathways. In human colon carcinoma SW480 cells, activation of the Gq pathway inhibits beta-catenin signaling as determined by transcriptional reporter and cell proliferation assays. Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Galphaq does not inhibit the effects of constitutively activated DeltaN-XTCF3-VP16 chimera in SW480 cells. Similarly, in HEK293 cells the Gq pathway suppresses beta-catenin-T cell factor/lymphocyte enhancer factor-1 transcriptional activity induced by Wnt/Frizzled interaction or glycogen synthase kinase-3beta-resistant beta-catenin, but not DeltaN-XTCF3-VP16. We conclude that Gq signaling promotes nuclear export and calpain-mediated degradation of beta-catenin, which therefore contributes to the inhibition of Wnt/beta-catenin pathway.

Figure 1

Effect of the Gq signaling pathway on TCF/LEF-1-mediated transcriptional activity and proliferation. (a) Effects of various Gα subunits on β-catenin-dependent TCF/LEF-1 transcriptional activity. SW480 cells were transfected with 025 μg of empty vector as control or cDNAs encoding various constitutively activated Gα subunits, and relative TCF/LEF-1 activity was measured. The results are expressed as mean ± SD of duplicates. (b) Activation of Gq-coupled receptor inhibits β-catenin-dependent TCF/LEF-1 transcriptional activity. SW480 cells were transfected with empty vector as control or cDNAs expressing WT or mutant activated Gαq, M2R, or M3R as indicated. Carbachol (1 mM) was added 24 h after transfection, and transcriptional activity was measured 48 h after transfection. (c) Q209L–Gαq down-regulates the cyclin D1 protein level. SW480 cells were transfected with empty plasmid pIRES2-EGFP as control or pQ209L-Gαq-IRES2-EGFP expression plasmid. EGFP-positive cells were enriched by fluorescence-activated cell sorting. Cell extracts were prepared, electrophoretically resolved, and immunoblotted with cyclin D1 antibody. β-actin was used for loading control. WB, Western blot. (d) Q209L–Gαq inhibits proliferation of SW480 cells. Cells were prepared as in c. Indicated number of cells was plated on 96-well plates and assayed for proliferation by using a colorimeteric assay. Cell proliferation was normalized with corresponding control cells transfected with empty vector and shown as mean ± SD.

Figure 2

Activation of the Gαq signaling pathway promotes calpain-mediated β-catenin proteolysis in a Ca²⁺-dependent manner. (a) The inhibitory effect of the Gq signaling pathway on TCF/LEF-1-mediated transcription is not mediated by PKC. SW480 cells were transfected with empty plasmid as control, Q209L-Gαq, or M3R expression plasmids. PKC inhibitor bisindolylmaleimide I (1 μM) was added on transfection and carbachol (1 mM) was added 24 h posttransfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. (b) Thapsigargin mimics the effect of Q209L–Gαq. SW480 cells were transfected with indicated expression plasmids. Thapsigargin (50 nM) was added on transfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. (c) Q209L–Gαq induces reduction in cytosolic β-catenin levels. SW480 cells were transfected with empty plasmid as control or Q209L–Gαq expression plasmid. Cycloheximide (25 μg/ml) was added 24 h after transfection. Cells were harvested at the indicated times after cycloheximide addition. Cytosolic fraction was prepared, resolved by SDS/PAGE, and blotted with antibody specific against the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. (d) Q209L–Gαq induces cleavage of cytosolic β-catenin. Cytosolic fractions of SW480 cells were prepared at the indicated time points after cycloheximide treatment, electrophoretically resolved, and blotted with β-catenin antibody specific for the C terminus. Proteolytic products of β-catenin are indicated by arrowheads. (e) Thapsigargin induces calpain-mediated proteolysis of β-catenin in a calcium-dependent manner. SW480 cells were pretreated with DMSO or inhibitors for 10 min, and then treated with thapsigargin (50 nM) for 30 min. The untreated cells were used as control. The concentrations of the various inhibitors were BAPTA/AM (40 μM), NH₄Cl (1 mM), calpeptin (50 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Whole-cell extract was prepared, resolved by SDS/PAGE, and blotted with the C-terminal β-catenin antibody. Proteolytic products of β-catenin are indicated by arrowheads. (f) Activation of Gq-coupled M3R induces calpain-mediated proteolysis of β-catenin. SW480 cells stably expressing M3R were treated with carbachol (1 mM), lactacystin (10 μM), or calpeptin (10 μM) as indicated. Whole-cell lysates were prepared at the indicated time points, and β-catenin fragments were detected by antibody specific for the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. (g and h) Calpain-mediated cleavage occurs at the N-terminal region of β-catenin. As described in Materials and Methods, HEK293 cell extracts were incubated at 37°C for 30 min alone as control or with CaCl₂ (0.1 mM) and μ-calpain in the presence of the indicated reagents: EGTA (1 mM), ALLN (10 μM), ALLM (10 μM), E-64 (25 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Cleavage of β-catenin was assessed with antibodies specific for the N-terminal or C-terminal regions of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. WB, Western blot; NT, N terminus; CT, C terminus.

Figure 3

Ca²⁺ release promotes nuclear export of β-catenin. (a) Ca²⁺-dependent nuclear export of β-catenin in SW480 cells. Subcellular localization of endogenous β-catenin was detected by immunofluorescence staining with anti-β-catenin (I–III). (IV–VI) Light images corresponding to I–III. (I and IV) Untreated SW480 cells as a control. (II and V) Cells that were treated with thapsigargin (50 nM) for 30 min. (III and VI) Cells that were pretreated with BAPTA/AM (50 μM) for 10 min and then incubated with thapsigargin (50 nM) for 30 min. (b) Ca²⁺-dependent proteolysis of β-catenin occurs in the cytoplasm. Total lysate, cytosolic, and nuclear fractions were prepared as described in Materials and Methods and incubated with the indicated amount of Ca²⁺ at 37°C for 30 min. β-Catenin cleavage products were detected by antibody specific for the C-terminal region after being resolved by SDS/PAGE. Proteolytic products of β-catenin are indicated by arrowheads. WB, Western blot; CT, C terminus. (c) Nuclear β-catenin is down-regulated by Q209L–Gαq. Q209L–Gαq-transfected cells were labeled by EGFP expression (I). Q209L–Gαq-expressing cells are shown by arrowheads (I–III). (Scale bars: 5 μm.)

Figure 4

Constitutively active TCF3 rescues effect of Gq signaling pathway on TCF/LEF-1-mediated transcriptional activity and cell proliferation in SW480 cells. (a) A constitutively activated form of TCF3, ΔN-XTCF3-VP16, rescues the effect of Gq signaling on TCF/LEF-1-mediated transcriptional activity in SW480 cells. SW480 cells were transfected with empty plasmid as a control or indicated expression plasmids. Relative TCF/LEF-1 transcriptional activity was assayed as described. (b) ΔN-XTCF3-VP16 rescues the effect of Gq signaling on cyclin D1 expression in SW480 cells. SW480 cells stably expressing M3R alone or together with ΔN-XTCF3-VP16 were treated without or with carbachol (1 mM) for 12 h. Total lysates were prepared and resolved by SDS/PAGE. Immunoblotting was performed with antibody against cyclin D1. The untreated M3R-expressing cells were used as control and β-actin was the loading control. WB, Western blot. (c) ΔN-XTCF3-VP16 rescues the effect of Gq signaling on SW480 cell proliferation. A total of 2.5 × 10³ cells stably expressing M3R alone or together with ΔN-XTCF3-VP16 were plated on 96-well plates. Cells were treated with carbachol (1 mM) for 12 h as indicated. Cell proliferation was assessed colorimeterically. The untreated M3R-expressing cells were used as control. Data shown are mean ± SD.

Figure 5

Schematic representation of the interactions between the Gq and Wnt/β-catenin signaling pathways. As shown in the model, APC promotes β-catenin nuclear export and facilitates proteasome-mediated degradation of β-catenin after sequential phosphorylation by CKIα and GSK-3β on the Axin-based complex. Wnt binding to coreceptors LRP and Frizzled blocks proteasome-mediated degradation, which further leads to nuclear localization of β-catenin, gene expression, and cell proliferation. As an alternative degradation pathway, Ca²⁺ activation by Gq signaling pathway promotes nuclear export of β-catenin into cytoplasm, where it is further degraded by calpain in a Ca²⁺-dependent manner. Consequently, β-catenin–TCF/LEF-dependent cell proliferation is inhibited. LRP, low-density-lipoprotein receptor-related protein; UPS, ubiquitin-proteasome system; PLCβ, phospholipase C β; PIP₂, phosphatidylinositol 4,5-bisphosphate; IP₃R, IP₃ receptor.