ve to GCG1-29, but not to GCG9-29 . As a GW 501516 site 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 9720791 until after transfection with a GCGR expression plasmid. 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. 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. catenin protein levels relative to a control, non-treated sample or that treated with the antagonist GCG9-29. As a positive control, treatment with lithium chloride 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. Treatment of primary hepatocytes also caused an increase in b-catenin protein. These experiments demonstrate that activation of the GCGR receptor in cell lines and primary cells leads to bcatenin stabilization. Activation of the b-catenin pathway leads to stabilization of bcatenin in the cytosol, which can translocate into the nucleus and associate with TCF transcription factors to activate TCF promotermediated 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 promotermediated luciferase activity, an indicator for an active b-catenin signaling pathway. 293STF cells 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. Treatment with LiCl also caused an increase in TCF luciferase activity. 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. These experiments demonstrate that activation of the GCGR receptor increases TCF promoter activity. Together with the western results, they demonstrated that activation of GCGR receptor leads to active b-catenin signaling. Coexpression of Lrp5 potentiated glucagon and GLP-1induced b-catenin signaling We observed an increase in b-catenin protein level and TCFmediated 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 glucagon-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. Next, we examined 15523001” the effect of coexpression of GCGR and Lrp5 on glucagon-induced TCF luciferase activity. As expected, we observed a larger increase in glucagon-induced TCF luciferase activity in HEK293 cells cotransfected with GCGR and Lrp5, relative to cells transfected with GCGR alone. As a contro
Heme Oxygenase heme-oxygenase.com
Just another WordPress site
