Shintaro Hiraoka

PPARγ agonists inhibit cell growth and suppress the expression of cyclin D1 and EGF‐like growth factors in ras‐transformed rat intestinal epithelial cells

Peroxisome proliferator‐activated receptor γ (PPARγ) inhibits the growth of several types of cancer cells. However, the mechanisms by which this occurs are poorly understood. The goal of the present study was to investigate the effects of PPARγ on mutated ras‐induced cell growth, activation of transcription factors and expression of genes associated with cellular transformation in rat intestinal epithelial cells. A human PPARγ cDNA was introduced to the activated H‐ras‐transfected IEC‐6 cells (IECras) and 1 clone (IECrasPR82) that stably expresses both activated ras and PPARγ was obtained. Thiazolidinedione derivatives such as troglitazone and rosiglitazone, selective ligands for PPARγ, inhibited the cellular growth of IECrasPR82 cells in a time‐dependent manner and induced G1 cell cycle arrest. Treatment with troglitazone (20 μM) decreased the expression of cyclin D1, heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and amphiregulin and suppressed the promoter activities of cyclin D1 and HB‐EGF. Furthermore, a luciferase assay and an electrophoretic mobility shift assay showed that thiazolidinedione derivatives suppressed the transcriptional activities of AP‐1 and Ets, both of which play crucial roles in the expression of cyclin D1 and HB‐EGF. These findings suggest that reduction of EGF‐like growth factors and cyclin D1 through the suppression of AP‐1 and Ets may be 1 mechanism whereby PPARγ inhibits their growth.

Calphostin C induces expression of amphiregulin mRNA via reactive oxygen species in IEC-6 cells.

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, is generally used as a specific inhibitor of protein kinase C. It is known that 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, induces expression of mRNA for amphiregulin (AR), a member of EGF-related polypeptides, in mammalian epithelial cells. In this work, we determined the effect of calphostin C on AR mRNA expression in IEC-6 cells, a rat intestinal epithelial cell line, and unexpectedly found that this compound enhanced the TPA-induced expression of AR mRNA. Moreover, calphostin C alone induced expression of AR mRNA in a light-dependent manner, and this effect was abrogated by pretreatment with N-acetylcysteine. These results suggest that calphostin C can upregulate expression of AR mRNA via reactive oxygen species.