Quantitative characterization of 15-deoxy-delta(12,14)-prostaglandin J 2 in regulating EGFP-Smad2 translocation in CHO cells through PPARγ/TGFβ/Smad2 pathway

Abstract

Smad2 is an important factor in TGFβ/Smad2 signal transduction pathway with ability for signal propagation, it could translocate from cytoplasm to nucleus after the TGFβ receptor-mediated phosphorylation. 15-deoxy-delta(12,14)-prostaglandin J 2 (15d-PGJ 2 ), a natural agonist of the peroxisome proliferator-activated receptor γ (PPARγ), is found recently to be able to function in the regulation of Smad2 activity. However, no quantification data have been yet reported, and it still keeps suspenseful whether or not 15d-PGJ 2 could regulate Smad2 activity by depending on PPARγ through PPARγ/TGFβ/ Smad2 pathway. In this work, by analyzing the EGFP-Smad2 location in CHO cells according to the Nucleus Trafficking Analysis Module based on IN Cell Analyzer 1000 platform, TGFβ stimulated EGFP-Smad2 translocation regulated by 15d-PGJ 2 was quantitatively investigated. The results showed that TGFβ could induce EGFP-Smad2 translocation from cytoplasm to nucleus by EC 50 of 8.83 pM, and 15d-PGJ 2 could impede the TGFβ-stimulated Smad2 translocation by IC 50 of 0.68 μM. Moreover, GW9662, a PPARγ antagonist, could attenuate such a 15d-PGJ 2 inhibitory activity by almost one order of magnitude. This result thereby implies that 15d-PGJ 2 might inhibit Smad2 translocation through PPARγ/TGFβ/Smad2 pathway. Further investigation discovered that different from the case for 15d-PGJ 2 , rosiglitazone, another PPARγ agonist, could enhance Smad2 translocation to nucleus, suggesting that rosiglitazone and 15d-PGJ 2 might take different modes in the activation of PPARy within the signaling pathway. Copyright © 2006 S. Karger AG.