Abstract
Tetracycline-inducible gene expression systems have been used successfully to study gene function in vivo and in vitro renal epithelial models but the effects of the common inducing agent, doxycycline (DOX), on gene expression are not well appreciated. Here, we evaluated the DOX effects on the transcriptome of a widely used renal epithelial cell model, mIMCD3 cells, to establish a reference. Cells were grown on permeable filter supports in the absence and presence of DOX (3 or 6 days), and genome-wide transcriptome profiles were assessed using RNA-Seq. We found DOX significantly altered the transcriptome profile, changing the abundance of 1,549 transcripts at 3 days and 2,643 transcripts at 6 days. Within 3 days of treatment, DOX significantly decreased the expression of multiple signaling pathways (ERK, cAMP, and Notch) that are associated with cell proliferation and differentiation. Genes associated with cell cycle progression were subsequently downregulated in cells treated with DOX for 6 days, as were genes involved in cellular immune response processes and several cytokines and chemokines, correlating with a remarkable repression of genes encoding cell proliferation markers. The results provide new insight into responses of renal epithelial cells to DOX and a establish a resource for DOX-mediated gene expression systems.
Highlights
Gene expression systems utilizing drug-induced trans-activation provide the means to conditionally investigate gene function in a temporal manner
Mesenchymal cell marker genes, namely Cdh2, Fn1, Sparc, and Vim, were more abundant in mIMCD3 cells grown on the solid support compared to mIMCD3 cells grown on the filter support (Figure 1E)
In mIMCD3 cells grown on the filter supports, tight junction proteins, Cldn4, Cldn7, and Epcam, were more abundant than in cells on the solid support (Figure 1F)
Summary
Gene expression systems utilizing drug-induced trans-activation provide the means to conditionally investigate gene function in a temporal manner. Effects of DOX on a global gene expression and cellular processes in renal epithelial cell models are not known. In ex vivo studies with surgically removed pterygia tissue from the eye, RNA sequencing (RNA-Seq)-based transcriptomic analysis (Larrayoz et al, 2012) revealed DOX affected the expression of mitochondrial genes, the ER stress cascade, growth factors, interleukins, cell cycle regulators, integrins, and components of the Doxycycline Effects on mIMCD3 Transcriptome extracellular matrix. More studies are required with other cell types to identify common pathways It remains unknown if DOX affects gene expression in renal epithelial cells
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