dependent pathways in MC3T3-E1 cells [12]. Activation of glucagon-like peptide 1 receptor (GLP-1R) by glucagon-like peptide-1 (GLP-1) and exendin-4 (Exd4) peptides induces b- catenin signaling through the activation of cAMP/PKA and AKT pathways [13]. It was reported that binding of parathyroid hormone (PTH) to its receptor PTH1R induces activation of the b-catenin pathway through Lrp6 [14], yet another report suggested that PTH activates b-catenin signaling in a LRP5/6- and Wnt-independent manner [15]. So, whether b-catenin signaling activated through PTH1R requires Lrp6 remains a topic of debate. Mutations in Lrp5/6 are associated with bone disorders, abnormal ocular vascularization, early onset cardiovascular disease and metabolic syndrome [5,16,17]. These phenotypes may not all be attributable to altered responses to Wnt proteins. For instance, in eye vascularization, Lrp5 functions in a Norrin-mediated b-catenin signaling pathway [18]. Moreover, it was recently discovered that Lrp6 is not only a coreceptor for the Wnt/b-catein signaling pathway, it is also required for cAMP production for Gas-coupled GPCRs including PTH1R and GCGR [19]. Because PTH1R, GLP-1R, and GCGR are well-known targets involved in regulating bone development and glucose metabolism and the genetic link between Lrp5/6 mutations and bone and metabolic disorders, we hypothesize that Lrp5/6 plays a role in mediating the b-catenin pathway induced by activation of these receptors. Consistent with previous studies on PTH1R [10,14] and GLP-1R [13], in this study we found that activation of GCGR by glucagon also induced the b-catenin signaling pathway. Importantly, we found that Lrp5/6 is required for glucagon-induced b-catenin signaling. These results may help to explain the pleiotropic phenotypes of Lrp5 and 6 mutations and have important implications in understanding the role of Lrp5/6 in metabolic syndrome. Results Glucagon agonist induced the cAMP/PKA pathway in GCGR-expressing cells As a classical GPCR, activation of the glucagon receptor causes an increase of intracellular cAMP level, which in turn activates the PKA signaling pathway to activate cAMP-response element (CRE)-mediated gene expression. Using the CRE-Luc reporter construct, we found that HEK293 without GCGR transfection did not respond to GCG1-29 stimulation (Fig. 1B). After transfecting with GCGR, HEK293 cells became responsive to GCG1-29 (a GCGR agonist), but not to GCG9-29 (an antagonist) (Fig. 1B). As a control, forskolin, a direct PKA activator, activated CRE luciferase activity independent of GCGR expression. Using western blot, we confirmed that HEK293 cells have no detectable expression of GCGR until after transfection with a GCGR expression plasmid (Fig. 1A). These experiments suggest that HEK293 cells can be used to model GCGR signaling after ectopic expression of the receptor. We also asked if we could detect CRE luciferase activity in cells with endogenous GCGR expression. Primary liver hepatocytes are known to have endogenous GCGR expression [20]. We found that the GCG1-29 could directly activate CRE luciferase activity in primary liver cells without the need to transfect with a GCGR plasmid (Fig. 1C). Glucagon agonist induced b-catenin signaling in GCGR- expressing cells To check whether glucagon can activate the b-catenin signaling pathway, we first used HEK293 cells transfected with the GCGR receptor. Treatment with the agonist GCG1-29 increased b- Glucagon Induced b-Catenin Signaling Pathway catenin protein levels relative to a control, non-treated sample (NT) or that treated with the antagonist GCG9-29 (Fig. 2A). As a positive control, treatment with lithium chloride (LiCl) also caused an increase in b-catenin levels, an indication of activation of the Wnt/b-catenin signaling pathway. To confirm this result, we also examined cells with endogenous GCGR expression, including the hepatocarcinoma cell line Hep3B and primary liver cells. Treatment of Hep3B cells with GCG1-29 caused a rapid increase of b-catenin protein levels within 15 minutes (Fig. 2B). Treatment of primary hepatocytes also caused an increase in b-catenin protein (Fig. 2C). These experiments demonstrate that activation of the GCGR receptor in cell lines and primary cells leads to b- catenin stabilization. Activation of the b-catenin pathway leads to stabilization of b- catenin in the cytosol, which can translocate into the nucleus and associate with TCF transcription factors to activate TCF promoter–mediated gene expression. Because we observed the stabilization of b-catenin protein upon activation of GCGR receptor, we next examined whether activation of GCGR stimulated TCF promoter–mediated luciferase activity, an indica- tor for an active b-catenin signaling pathway. 293STF cells (HEK293 cells stably transfected with TCF-Luc reporter DNA) [18] were transfected with the GCGR receptor and then treated with GCG1-29 or GCG9-29 peptides. We observed a small but statistically significant increase in TCF-mediated luciferase activity upon treatment with GCG1-29, but not with GCG9-29 (Fig. 3A). Treatment with LiCl also caused an increase in TCF luciferase activity (Fig. 3A). Similarly, we observed a dose-dependent increase in TCF luciferase activity in primary hepatocytes treated with GCG1-29, but not with GCG9-29 or PTH1-34 peptides (Fig. 3B). These experiments demonstrate that activation of the GCGR receptor increases TCF promoter activity. Together with the western results (Fig. 2), they demonstrated that activation of GCGR receptor leads to active b-catenin signaling. Coexpression of Lrp5 potentiated glucagon and GLP-1- induced b-catenin signaling We observed an increase in b-catenin protein level and TCF- mediated luciferase activity upon activation of GCGR. Because Lrp5/6 is an essential coreceptor for Wnt/b-catenin signaling, we asked whether cotransfection with Lrp5/6 will potentiate gluca- gon-induced b-catenin signaling. HEK293 cells transfected with GCGR had a modest increase in b-catenin protein level upon GCG1-29 treatment, whereas cotransfection with GCGR and Lrp5 caused a larger increase (Fig. 4A). Next, we examined the effect of coexpression of GCGR and Lrp5 on glucagon-induced TCF luciferase activity. As expected, we observed a larger increase (2 to 3-fold vs 1.5-fold) in glucagon-induced TCF luciferase activity in HEK293 cells cotransfected with GCGR and Lrp5, relative to cells transfected with GCGR alone (Fig. 4B & 3A). As a control, transfection with high dose of Lrp5 alone also caused a small increase in TCF luciferase activity, which was not responsive to GCG1-29 treatment (Fig. 4B). Similar results were obtained when HEK293 cells were cotransfected with GCGR and Lrp6 plasmids (data not shown). Considering the report that GLP-1 also induced b-catenin signaling in GLP1R expressing cells [13], we examined the role of Lrp5 in GLP-1-induced b-catenin signaling. We found that cotransfection of HEK293 cells with GLP-1R and Lrp5 increased GLP1 agonist induced TCF luciferase activity to a similar level as observed for cotransfection of HEK293 cells with GCGR and Lrp5 (Fig. 4C). This is consistent with the hypothesis that there is a common mechanism for activation of the b-catenin signaling pathway through both GCGR and GLP1R receptors. PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33676