Key Words
15d-PGJ2 ? EGFP-Smad2 ? Translocation ? IN Cell
Analyzer ? Fluorescence ? Rosiglitazone
Abstract
Smad2 is an important factor in TGF/Smad2 signal
transduction pathway with ability for signal
propagation, it could translocate from cytoplasm to
nucleus after the TGF receptor-mediated
phosphorylation. 15-deoxy-delta(12,14)-prostaglandin
J2 (15d-PGJ2), a natural agonist of the
peroxisome proliferator-activated receptor (PPAR),
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 PPAR through PPAR/TGF/
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, TGF stimulated EGFP-Smad2
translocation regulated by 15d-PGJ2 was quantitatively
investigated. The results showed that TGF could
induce EGFP-Smad2 translocation from cytoplasm to
nucleus by EC50 of 8.83 pM, and 15d-PGJ2 could
impede the TGF-stimulated Smad2 translocation by
IC50 of 0.68 M. Moreover, GW9662, a PPAR
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 PPAR/TGF/Smad2
pathway. Further investigation discovered that
different from the case for 15d-PGJ2, rosiglitazone,
another PPAR agonist, could enhance Smad2
translocation to nucleus, suggesting that rosiglitazone
and 15d-PGJ2 might take different modes in the
activation of PPAR within the signaling pathway.