title: Redox processes inform multivariate transdifferentiation trajectories associated with TGFβ-induced epithelial-mesenchymal transition.
abstract: Phenotype reprogramming during transforming growth factor β (TGFβ)-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, TGFβ-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 TGFβ 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. TGFβ 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 TGFβ 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 TGFβ signaling.
authors.affiliation: The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332-0363, USA.
authors.forename: Adam F
authors.initials: AF
authors.lastname: Prasanphanich
authors.affiliation: The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332-0363, USA.
authors.forename: C Andrew
authors.initials: CA
authors.lastname: Arencibia
authors.affiliation: The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332-0363, USA. Electronic address: melissa.kemp@bme.gatech.edu.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Kemp
mesh_terms: D015153:Blotting, Western; D054338:Cell Transdifferentiation / Q000187:drug effects*; D058750:Epithelial-Mesenchymal Transition; D005453:Fluorescence; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008165:Luminol / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D051899:Smad2 Protein / Q000378:metabolism; D051901:Smad4 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta; D008165:Luminol; D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D005978:Glutathione
keywords: Epithelial–mesenchymal transition; In-cell Western assay; Multivariate analysis; Multivariate phenotype; Nucleophilic tone; Principal component analysis; Transdifferentiation; Transforming growth factor β
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