The cardiotonic steroid Marinobufagenin (MBG) induces Epithelial-Mesenchymal Transition (EMT) in LLC-PK1 cells
Cardiotonic steroids such as MBG have been shown to induce cardiac fibrosis. MBG contributes to renal fibrosis as well by inducing epithelial to mesenchymal transition (EMT). The porcine kidney cell line, LLC-PK1, acquires mesenchymal features such as a fibroblast-like phenotype, scattering and invasive properties and increased expression of mesenchymal proteins fibronectin and vimentin in a time and concentration dependent fashion. To examine the mechanisms which are operant, Western blotting and immunostaining were used. LLC-PK1 cells were grown to complete confluence and treated with 100 nM of MBG for 72-96 hours. At this concentration, MBG induces profound EMT (more than 50% of cells show fibroblast-like morphology; scattered cells occupy more than 80% of the cell- grown surface). It decreases the expression of epithelial proteins-E-cadherin, occludin and claudin-1 and increases the expression of mesenchymal proteins-vimentin and fibronectin by two fold. These alterations of LLC-PK1 cells are accompanied by 1) translocation of transcription factor Snail into nuclei after 72-96 hours post treatment and 2) de novo expression of transcription factor LEF1 at 72 hours post treatment, and its accumulation in the nuclei at 96 hours. Interestingly, distribution of the LEF1 co-activator, beta-catenin, does not change with MBG treatment. ROS scavenger NAC completely prevents morphological alteration of LLC-PK1 cells and inhibits the up-regulation of fibronectin caused by MBG. The Src inhibitor, PP2, shows only partial attenuation of the morphological alterations of LLC-PK1 cells induced by MBG but it completely abolishes MBG-induced up regulation of fibronectin. This study indicates that MBG induces EMT in LLC-PK1 cells and the two transcription regulators of the EMT transcriptome, Snail and LEF1 in a process requiring reactive oxygen species.
School:University of Toledo Health Science Campus
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:cardiotonic steroids emt na pump renal failure mbg
Date of Publication:01/01/2008