Figure 2. Glucagon agonist induces b-catenin stabilization in GCGR-expressing cells. 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-b-catenin antibody, then stripped and reprobed for anti-b- 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. doi:10.1371/journal.pone.0033676.g002 Glucagon-induced TCF reporter activity was PKA- dependent To understand the mechanism of glucagon-induced b-catenin signaling, we first asked whether glucagon-induced cAMP/PKA activity is required for activating the b-catenin pathway. HEK293 cells were transfected with GCGR and treated with GCG1-29 and H89, a PKA inhibitor. Inhibition of PKA activity completely blocked the activation of the b-catenin pathway induced by GCG1-29 (Fig. 5A). In another experiment, HEK293 cells were cotransfected with GCGR and Lrp5 and then treated with GCG1- 29 in the presence or absence of H89 inhibitor. Treatment with H89 also completely abolished glucagon-induced b-catenin transcription activity (Fig. 5B). In these two experiments, we demonstrated that the glucagon-induced b-catenin signaling required PKA activity, consistent with previous reports on GLP- 1R [13] or PTH1R [10,11]. Inhibition of Lrp5/6 blocked glucagon-induced TCF reporter activity We next asked whether inhibition of Lrp5/6 would reduce glucagon-induced b-catenin signaling, and we used two approach- es. First, we used a dominant negative inhibitor of Lrp5/6, the Lrp5 extracellular domain (ECD) [21,22]. Lrp5ECD inhibited glucagon-induced TCF luciferase activity when HEK293 cells were transfected with GCGR alone or GCGR+Lrp5 (Fig. 6A). In the other approach, we used Dickkopf-1 (DKK1), a known Glucagon Induced b-Catenin Signaling Pathway Figure 3. Glucagon agonist activates the b-catenin signaling in GCGR-expressing cells. 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. doi:10.1371/journal.pone.0033676.g003 inhibitor of Lrp5/6, to block Lrp5/6 activity. In this experiment, DKK1 completely blocked glucagon-induced TCF luciferase activity when HEK293 cells were transfected with either GCGR alone or GCGR+Lrp5 (Fig. 6B). These two experiments demonstrated that inhibiting Lrp5/6 activity blocked the gluca- gon-induced b-catenin signaling, suggesting that Lrp5/6 is required for glucagon-induced b-catenin dependent transcription. Lrp5 physically interacts with GCGR Because cotransfection with GCGR and Lrp5 increases glucagon-induced b-catenin stabilization and TCF luciferase activity, we examined whether GCGR and Lrp5 physically interact with each other by immunoprecipitation. HEK293 cells PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33676