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2014 ; 76
(ä): 1-13
Nephropedia Template TP
Free Radic Biol Med
2014[Nov]; 76
(ä): 1-13
PMID25088330
show ga
Phenotype reprogramming during transforming growth factor ? (TGF?)-induced
epithelial-mesenchymal transition (EMT) is an extensive and dynamic process,
orchestrated by the integration of biological signaling across multiple time
scales. As part of the numerous transcriptional changes necessary for EMT,
TGF?-initiated Smad3 signaling results in remodeling of the redox environment and
decreased nucleophilic tone. Because Smad3 itself is susceptible to attenuated
activity through antioxidants, the possibility of a positive feedback loop
exists, albeit the time scales on which these mechanisms operate are quite
different. We hypothesized that the decreased nucleophilic tone acquired during
EMT promotes Smad3 signaling, enhancing acquisition and stabilization of the
mesenchymal phenotype. Previous findings supporting such a mechanism were
characterized independent of each other; we sought to investigate these
relationships within a singular experimental context. In this study, we
characterized multivariate representations of phenotype as they evolved over
time, specifically measuring expression of epithelial/mesenchymal
differentiation, redox regulators, and Smad transcription factors. In-cell
Western (ICW) assays were developed to evaluate multivariate phenotype states as
they developed during EMT. Principal component analysis (PCA) extracted
anticorrelations between phospho-Smad3 (pSmad3) and Smad2/Smad4, which reflected
a compensatory up-regulation of Smad2 and Smad4 following cessation of TGF?
signaling. Measuring transcript expression following EMT, we identified
down-regulation of numerous antioxidant genes concomitant with up-regulation of
NADPH oxidase 4 (NOX4) and multiple mesenchymal phenotype markers. TGF? treatment
increased CM-H2DCF-DA oxidation, decreased H2O2 degradation rates, and increased
glutathione redox potential. Our findings suggest that the decreased nucleophilic
tone during EMT coincides with the acquisition of a mesenchymal phenotype over
too long a time scale to enable enhanced Smad3 phosphorylation during initiation
of EMT. We further challenged the mesenchymal phenotype following EMT through
antioxidant and TGF? inhibitor treatments, which failed to induce a
mesenchymal-epithelial transition (MET). Our characterization of multivariate
phenotype dynamics during EMT indicates that the decrease in nucleophilic tone
occurs alongside EMT; however, maintenance of the mesenchymal phenotype following
EMT is independent of both the nascent redox state and the continuous TGF?
signaling.
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