Key Words
15d-PGJ2 ? EGFP-Smad2 ? Translocation ? IN Cell
Analyzer ? Fluorescence ? Rosiglitazone
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
J2 (15d-PGJ2), 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-PGJ2 could regulate Smad2
activity by depending on PPARγ through PPARγ/TGFβ/
Smad2 pathway. In this work, by analyzing the EGFPSmad2
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-PGJ2 was quantitatively
investigated. The results showed that TGFβ could
induce EGFP-Smad2 translocation from cytoplasm to
nucleus by EC50 of 8.83 pM, and 15d-PGJ2 could
impede the TGFβ-stimulated Smad2 translocation by
IC50 of 0.68 μM. Moreover, GW9662, a PPARγ
antagonist, could attenuate such a 15d-PGJ2
inhibitory activity by almost one order of magnitude.
This result thereby implies that 15d-PGJ2 might inhibit
Smad2 translocation through PPARγ/TGFβ/Smad2
pathway. Further investigation discovered that
different from the case for 15d-PGJ2, rosiglitazone,
another PPARγ agonist, could enhance Smad2
translocation to nucleus, suggesting that rosiglitazone
and 15d-PGJ2 might take different modes in the
activation of PPARγ within the signaling pathway.