FIG. 9.

Downregulation of GSK3β in young mice inhibits liver proliferation, while ectopic expression of GSK3β in old liver accelerates proliferation of the liver. (A) Downregulation of GSK3β in the liver activates the cyclin D3-C/EBPα pathway of inhibition of proliferation. Protein levels of GSK3β, cyclin D3, and C/EBPα were examined by Western blotting with corresponding antibodies. The 42- and 40-kDa isoforms of C/EBPα are shown. CRM, cross-reactive molecule observed on the C/EBPα membrane. C/EBPα-IP, C/EBPα was immunoprecipitated from nuclear extracts and probed with antibodies to ph-S193 and to cdk2. IgG, heavy and light chains of immunoglobulin G. (B) Expression of cell cycle proteins in livers with inhibited levels of GSK3β. Western blotting was performed with nuclear extracts isolated from control and siGSK3β-treated mice at different time points after PH. (C) Inhibition of GSK3β in the liver leads to delayed and reduced DNA synthesis after PH. The upper panel shows BrdU staining; the lower panel shows a summary of three independent experiments. Error bars indicate standard deviations. (D) Expression of His-GSK3β and PCNA after PH in livers of young mice, old mice, and old mice injected with GSK3β. Western blotting was performed with nuclear extracts isolated at different time points after PH (shown on the top) using antibodies to the His tag and to PCNA. The filter was reprobed with β-actin. (E) Ectopic expression of GSK3β corrects expression of PCNA in old livers. Levels of PCNA were calculated as ratios to β-actin. (F) Ectopic expression of GSK3β accelerates proliferation of old livers after PH. BrdU uptake was examined in livers of young mice (Y), old mice (O), and old mice injected with GSK3β. A summary of three independent experiments is shown.