Modulation of β-Catenin Signaling by Glucagon Receptor Activation

The glucagon receptor (GCGR) is a member of the class B G protein–coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin–mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor–related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter–mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations

Wnt/β-catenin Signaling in Normal and Cancer Stem Cells

The ability of Wnt ligands to initiate a signaling cascade that results in cytoplasmic stabilization of, and nuclear localization of, β-catenin underlies their ability to regulate progenitor cell differentiation. In this review, we will summarize the current knowledge of the mechanisms underlying Wnt/β-catenin signaling and how the pathway regulates normal differentiation of stem cells in the intestine, mammary gland, and prostate. We will also discuss how dysregulation of the pathway is associated with putative cancer stem cells and the potential therapeutic implications of regulating Wnt signaling

Blocking Lrp5/6 inhibited glucagon-induced β-catenin signaling.

<p>A). 293STF cells cultured in 24-well plate were transfected with a combination of GCGR (100 ng), Lrp5 (100 ng), Lrp5ECD (200 ng) and TKRlu (5 ng) plasmids as indicated on day 1, and then were treated with or without 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the TCF-mediated luciferase activity. Triplicate samples were used for each treatment. *p<0.005 compared with the non-treated (NT) group. ^#p<0.005 compared with the group without the Lrp5ECD transfection. B). DKK1 protein inhibits glucagon-induced β-catenin signaling. 293STF cells cultured in 24-well plate were transfected with pcDNA3.1 and GCGR plasmids (100 ng each) or GCGR and Lrp5 plasmids (100 ng each) along with 5 ng TKRlu plasmid on day 1, and then were treated with 50 nM GCG1-29±2 µg/ml DKK1 on day 2. Cells were harvested on day 3 to measure the luciferase activity. Duplicate samples were used for each treatment. *p<0.02 compared with the GCG1-29-treated group.</p

Lrp5 coexpression enhances glucagon and GLP1-induced β-catenin signaling.

<p>A). HEK293 cells cultured in 12-well plate were transfected with a combination of indicated plasmids (GCGR 1000 ng, Lrp5 500 ng) for 24 h and then treated with or without 50 nM GCG1-29 for 1 h. The cells were harvested and lysed, and samples were used for western blot analysis. The blot was first probed with anti-β-catenin antibody and then stripped and reprobed for anti-β-actin antibody as a loading control. B). 293STF cells cultured in 24-well plate were transfected with 100 ng of empty vector (pcDNA3.1) or GCGR plasmid along with the indicated amount of Lrp5 and 5 ng TKRlu plasmids on day 1, and then treated with or without 50 nM GCG1-29 on day 2. Cells were harvested for luciferase activity measurement on day 3. Triplicate samples were used for each treatment. *p<0.005 compared with non-treated group. 4C). 293STF cells cultured in 24-well plate were transfected with 100 ng GLP1R, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with the GLP1 agonist GLP1(7–36) (50 nM) or the antagonist Exendin(9–39) (50 nM) on day 2. Cells were harvested on day 3 to measure luciferase activity. Duplicate samples were used for each treatment. *p<0.05 compared with the non-treated (NT) group.</p

Lrp5 physically interacts with GCGR.

<p>A). HEK293 cells cultured in 6-well plate were transfected with 2000 ng of a control vector (GFP), v5-tagged Lrp5, HA-tagged GCGR, or both for 24 h and then were treated with or without 50 nM GCG1-29 for 1 h. Cells were harvested and lysed, and equal amounts of protein were used for western blot analysis. The blot was first probed with v5 antibody and then stripped and reprobed with HA antibody. The β-actin blot was used as a loading control. B). HEK293 cells were transfected and treated same as in A. The cells were harvested and lysed, and equal amounts of lysate were immunoprecipitated with the indicated antibody. For HA antibody, the antibody complex was pulled down by protein G beads. For v5 antibody, it was a single-step pull-down because the antibody was directly conjugated to the agarose beads. After pull-down, the beads were washed three times with 1× TBST and then incubated in 1× SDS sample buffer to release the bound proteins. The lysates were used for western blot analysis and probed with the indicated antibody. C–E). BRET data for Rlu-tagged Lrp5 and YFP-tagged GCGR expressed on COS-1 cells. Shown are the static BRET signals (C), saturation BRET analysis (D), and effect of natural agonist ligand binding on the BRET signals (E). Shaded area represents the background signal of 0.12 determined using Rlu-tagged Lrp5 with soluble YFP as noted. Coexpression of untagged GCGR or Lrp5 competitively reduced the BRET signals obtained between Lrp5-Rlu and GCGR-YFP (black bars). Saturation BRET analysis supported the specific interaction of Lrp5 and GCGR. The non-specific bystander type BRET signal (linear) was observed when the non-structurally-related CCK1 receptors were co-expressed with Lrp5. Incubation with the natural agonist peptide ligand, glucagon (up to 1 µM), did not significantly change the Lrp5 and GCGR BRET signal. Data are represented as the means ± S.E.M. from four to six independent experiments performed in triplicate. Data marked with ** were significantly different from background signals at the level of p<0.01.</p

Glucagon agonist activates the CRE-Luc activity in GCGR-expressing cells.

<p>A). Protein expression for GCGR in HEK293 cells and primary hepatocytes (isolated from BL/6 mice). HEK293 cells cultured in 6-well plate were transfected with 2000 ng of pcDNA3.1 (empty vector), GCGR or HA tagged GCGR for 24 h and cells were lysed for western blot analysis as described. B). HEK293 cells cultured in 24-well plate were transfected with pcDNA3.1 and CRE-Luc (50 ng each) or CRE-Luc and GCGR plasmids (50 ng each) along with 5 ng TKRlu (an internal control) on day 1; cells were treated with or without the glucagon agonist GCG1-29 (50 nM), the antagonist GCG9-29 (50 nM) or forskolin (FSK, 10 µM) on day 2, and were harvested for luciferase activity measurements after about 17 h on day 3. Triplicate samples were used for each treatment. In all experiments, the CRE promoter–driven firefly luciferase activity was normalized to <i>Renilla</i> luciferase activity driven by the thymidine kinase promoter (for transfection control). Luc activity for non-treated group was set to 1 and Luc activities for treated groups were adjusted accordingly. *p<0.005 compared with the non-treated group. C). Primary hepatocytes cultured in 12-well plates were transfected with CRE-Luc (400 ng) and TKRlu (10 ng) plasmids on day 1, then treated with or without 50 nM GCG1-29 on day 2 and harvested on day 3 for luciferase activity measurement. Duplicate samples were used for each treatment. *p<0.001 compared with the non-treated group.</p

Glucagon agonist activates the β-catenin signaling in GCGR-expressing cells.

<p>A). 293STF cells cultured in 24-well plate were transfected with 100 ng GCGR and 10 ng TKRlu plasmids on day 1 and then treated with the indicated amounts of GCG1-29, GCG9-29, or LiCl, a positive control on day 2. Cells were harvested on day 3 for luciferase activity measurements. Triplicate samples were used for each group. *p<0.005 compared with the non-treated (NT) group. B). Primary liver cells cultured in 12-well plates were transfected with 400 ng TCF-Luc and 10 ng TKRlu plasmids on day 1 and then treated with the indicated amounts of GCG1-29, GCG9-29, or PTH1-34 on day 2. Cells were harvested for luciferase activity measurement on day 3. Duplicate samples were used for each treatment. ^#p<0.02, *p<0.005 compared with the non-treated group.</p

Glucagon agonist induces β-catenin stabilization in GCGR-expressing cells.

<p>A). Lanes 1–3, HEK293 cells cultured in 6-well plate were transfected with 2000 ng GCGR for 24 h and then either left non-treated(NT) or treated with 50 nM of glucagon agonist (GCG1-29) or antagonist (GCG9-29) for 1 h. Lanes 4–5, HEK293 cells cultured in 6-well plate were transfected with 2000 ng pcDNA3.1 empty vector for 24 h and then treated without or with 20 mM LiCl for 1 h. The cells were harvested and lysed, and equal amounts of protein for each sample were used for western blot analysis. The blot was first probed with anti-β-catenin antibody, then stripped and reprobed for anti-β-actin antibody as a loading control. B). Hep3B hepatocarcinoma cells were serum starved for 4 h and then treated with 50 nM GCG1-29 for the indicated time in serum-free medium. The cells were harvested and lysed. Cell lysates were used for western blot analysis. C). Mouse primary hepatocytes were treated without or with 50 nM GCG1-29 for 1 h and cells were harvested and lysed for western blot analysis.</p