We have recently documented that troglitazone, a ligand for PPARγ, inhibited proliferation of human hepatoma cells, involving an increased intracellular protein stability of p27Kip1 (Hepatology 2001, Hepatology 2003). Although this accumulation of p27Kip1 was responsible for, at least in part, transcriptional down-regulation of Skp2, it remained unclear whether or not the accumulation was entirely dependent on ligand-activated PPARγ. In addition, intracellular localization of the accumulated p27Kip1 should be determined, because some types of highly-malignant neoplasms are known to have systems to mislocalize nuclear p27Kip1 to the cytoplasm (Nature Rev Cancer 2004). Thus, the AIM of this study was to assess alteration in expression level and localization of p27Kip1 in both the PPARγ2-overexpressing hepatoma cells and the cells treated by highly-specific ligands for PPARγ, such as rosiglitazone. Methods: HLF hepatoma cells were used. Myc-PPARγ2-overexpressing HLF clones were established using G418. Expression levels in PPARγ, phosphorylated extracellular signal-related kinases (Erks) 1/2, Skp2, and its known substrates, such as p27Kip1 and p21WAF1/Cip1 were examined by Western blot. Subcellular localization of immunoreactive p27Kip1 was observed by confocal laser microscopy. Results: A significant inhibition in cell proliferation was found in the PPARγ2-overexpressing HLF cells, showing 58.1% reduction in cell number at day 5, in comparison with the mock-transfected cells. The PPARγ2-overexpressing cells disclosed a clear nuclear accumulation of p27Kip1. In these cells, p21WAF1/Cip1, another substrate of Skp2, was also accumulated to the nucleus. In rosiglitazone-treated HLF cells, an immediate accumulation of p27Kip1 was evident, accompanied by the phosphorylation of Erk1/2. Since Erk1/2 are phosphorylated through the activation of PPARγ (J Biol Chem 2001), we investigated involvement of the PPARγ-Erk1/2 axis in the increased expression of p27Kip1. U0126, a MEK inhibitor, completely inactivated Erk1/2 and prevented the accumulation of p27Kip1, suggesting involvement of the phosphorylated Erk1/2 in the rosiglitazone-induced intracellular retention of p27Kip1. This phenomenon was reproduced using GW9662, a selective inhibitor for PPARγ, suggesting that PPARγ activation was required for the PPARγ/Erk1/2-mediated accumulation of p27Kip1. Nuclear localization of the accumulated p27Kip1 was confirmed by confocal laser scanning microscopy. Conclusions: We have first demonstrated that both a profound expression of PPARγ2 and a specific ligand-activated PPARγ had potential to restore mislocalization of p27Kip1 in human hepatoma cells. It was suggested that the PPARγ-dependent phosphorylation of Erk1/2 played a critical role in PPARγ-mediated intracellular retention of p27Kip1.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]