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. 2013 Aug 22;154(11):4352–4364. doi: 10.1210/en.2013-1358

Figure 3.

Figure 3.

GSK3β and its targets are regulated by ERβ ligands to prevent cardiac hypertrophy. A, Left panels, AngII causes the inhibitory phosphorylation of GSK3β at serine 9 in cultured cardiomyocytes, inhibited by E2 or β-LGND2. Total GSK3β serves as loading control. Data are from three experiments combined. *, P < .05 for control vs AngII; +, P < .05 for AngII vs AngII + E2 or β-LGND2. Right panels, In vivo stimulation of GSK3β phosphorylation at serine 9 by AngII and inhibition by E2 or β-LGND2. The latter was seen only in WT mouse ventricles, and bar graph is from four to six mice per condition. *, P < .05 for control vs AngII in both mouse models or AngII + β-LGND2 in ERβ KO mice; +, P < .05 for AngII vs AngII + β-LGND2 in WT mice. B, Left panels, Serine 473 Akt phosphorylation in response to AngII ± E2 or β-LGND2 in vitro. Right panels, In vivo regulation of Akt phosphorylation. All data are analyzed as in panel A. C, Ser9 phosphorylation of GSK3β induced by AngII in cardiomyocytes is prevented by an Akt inhibitor (LY294002). Data are from three experiments combined, and GAPDH serves as loading control. *, P < .05 for control vs AngII; +, P < .05 for AngII vs AngII + LY294002. D, GATA4 mRNA (upper panel) and protein (lower panel) expression in cardiomyocytes are stimulated by AngII but inhibited by E2 or β-LGND2. *, P < .05 for control vs AngII; +, P < .05 for AngII vs AngII plus E2 or β-LGND2. E, AngII activates GATA4 transcriptional activity, inhibited by estrogenic compounds. Cardiomyocytes were transfected with a GATA4-luciferase reporter construct and recovered, and luciferase activity was determined after 24 hours of incubation. The study was done three times and data were combined. *, P < .05 for control vs AngII; +, P < .05 for AngII vs AngII + E2 or β-LGND2. F, AngII-stimulated β-MHC mRNA in cardiomyocytes is dependent on GATA4. GAPDH is the loading control. siRNA validation is shown. Data are from three experiments; +, P = .05 for control vs AngII + control siRNA; +, P < .05 for AngII + control siRNA vs AngII + GATA4 siRNA. G, Inpp5f protein (left panel) and mRNA (right panel) expression are suppressed by AngII but prevented by E2 or β-LGND2 in cardiomyocytes. Data are from three experiments. *, P < .05 for control vs AngII; +, P < .05 for AngII vs AngII + E2 or β-LGND2. H, E2 or β-LGND2 derepresses the effects of AngII on Inpp5f protein (left panels) and mRNA (right panels) expression only in WT mouse ventricles. AngII also stimulates β-MHC mRNA expression in vivo, prevented by E2 or β-LGND2. *, P < .05 for control vs AngII or AngII + E2 or β-LGND2 in ERβ KO mice; +, P < .05 for AngII vs AngII plus E2 or β-LGND2 in WT mice. I, Inpp5f knockdown prevents β-LGND2 inhibition of AngII activation of Akt and the resulting inhibitory phosphorylation of GSK3β. Data are from three experiments. *, P < .05 for control vs AngII under either siRNA condition or AngII + β-LGND2 + control siRNA vs AngII + β-LGND2 + Inpp5f siRNA; +, P < .05 for AngII vs AngII + E2 or β-LGND2.