Role of mitochondrial ROS in TGF-ß-induced mitochondrial translocation of SMAD4 and its interaction with COXII

Abstract

Purpose : Epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells lose their differentiated phenotype and attain mesenchymal features. Our previous work showed important role of TGF-ß₂ in the EMT of retinal pigment epithelium (RPE) cells and the involvement of nuclear SMAD4 signaling pathway. The present study describes the relationship between SMAD4 translocation to mitochondria, generation of mitochondrial ROS and the interaction of SMAD4 with Cytochrome C Oxidase II (COX II).

Methods : Human primary subconfluent RPE cells were starved overnight with serum free media and switched to DMEM containing 3% FBS. The cells were stimulated with 10 ng/ml TGF-β₂ for up to 48 h. Mitochondrial ROS production in RPE cells was studied after incubation with 10 ng/ml TGF-β₂ for 24 h. After the experiment, cells were incubated with 5 µM MitoSOX Red (mitochondrial superoxide marker) for 10 min at 37 ^ο C, washed and imaged with LSM 710. Apoptosis was monitored by TUNEL assay. SMAD4 translocation to mitochondria was studied by immunofluorescence and western blot analysis of isolated mitochondria. The effect of TGF-β treatment on the co-localization of SMAD4 and COX II was also investigated.

Results : TGF-β₂ treatment for 48 h significantly reduced E-cadherin and increased α-SMA. 24 h treatment with TGF-β increased ROS levels in the cytoplasm and mitochondria in RPE cells. To confirm that TGF-β increased mitochondrial ROS generation, we used MitoSOX Red. Confocal microscopy showed a significant increase in mitochondrial fluorescence intensity of MitoSOX. Mitochondrial ROS modulated the TGF-β-mediated activation and translocation of SMAD4 into mitochondria in immunofluorescence and western blot analysis. Further, TGF-β₂ treatment induced COX II expression in RPE cells and co-localization with SMAD4 in mitochondria during transdifferentiation to an EMT phenotype. Interestingly, no appreciable cell death in RPE cells treated with varying doses of TGF-β (5-20 ng/ml ) for 48 h was observed.

Conclusions : Our data provide evidence for mitochondrial translocation of SMAD4 in TGF-ß induced EMT in RPE which is accompanied by increased ROS generation with no apparent cell death. The interaction of SMAD4 with COX II in mitochondria may be an important mechanism in SMAD regulation of TGF-ß- mediated EMT.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.