Retinoic Acid Receptor-Dependent, Cell-Autonomous, Endogenous Retinoic Acid Signaling and Its Target Genes in Mouse Collecting Duct Cells

Yuen Fei Wong,Matthew Arno,Patricia D Wilson,Qihe Xu,Robert J Unwin,Bruce M Hendry,Jill T Norman,Jean-Claude Dussaule

doi:10.1371/journal.pone.0045725

Yuen Fei Wong, Matthew Arno + Show 6 more

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https://doi.org/10.1371/journal.pone.0045725

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Abstract

BackgroundVitamin A is necessary for kidney development and has also been linked to regulation of solute and water homeostasis and to protection against kidney stone disease, infection, inflammation, and scarring. Most functions of vitamin A are mediated by its main active form, all-trans retinoic acid (tRA), which binds retinoic acid receptors (RARs) to modulate gene expression. We and others have recently reported that renal tRA/RAR activity is confined to the ureteric bud (UB) and collecting duct (CD) cell lineage, suggesting that endogenous tRA/RARs primarily act through regulating gene expression in these cells in embryonic and adult kidney, respectively.Methodology/Principal FindingsTo explore target genes of endogenous tRA/RARs, we employed the mIMCD-3 mouse inner medullary CD cell line, which is a model of CD principal cells and exhibits constitutive tRA/RAR activity as CD principal cells do in vivo. Combining antagonism of RARs, inhibition of tRA synthesis, exposure to exogenous tRA, and gene expression profiling techniques, we have identified 125 genes as candidate targets and validated 20 genes that were highly regulated (Dhrs3, Sprr1a, and Ppbp were the top three). Endogenous tRA/RARs were more important in maintaining, rather than suppressing, constitutive gene expression. Although many identified genes were expressed in UBs and/or CDs, their exact functions in this cell lineage are still poorly defined. Nevertheless, gene ontology analysis suggests that these genes are involved in kidney development, renal functioning, and regulation of tRA signaling.Conclusions/SignificanceA rigorous approach to defining target genes for endogenous tRA/RARs has been established. At the pan-genomic level, genes regulated by endogenous tRA/RARs in a CD cell line have been catalogued for the first time. Such a catalogue will guide further studies on molecular mediators of endogenous tRA/RARs during kidney development and in relation to renal defects associated with vitamin A deficiency.

Highlights

All-trans retinoic acid is the primary bioactive form of endogenous retinoids derived from dietary vitamin A and plays important roles in regulating a myriad of physiological events [1]
By treating mIMCD-3 cells with AGN193109, basal retinoic acid response elements (RAREs)-luciferase activity was reduced to about 50% of that of the vehicle control group; when exogenous trans retinoic acid (tRA) was added with AGN193109, the reduction of RARE-luciferase activity was abolished in a dose-dependent manner (Figure 2Bi)
Under culture conditions, when RARE-luciferase transfected mIMCD-3 cells were treated with exogenous tRA alone, the induction of RARE-luciferase activity was weak and did not reach statistical significance (Figure 2Biii), suggesting that the constitutive RARE activity induced by endogenous tRA/retinoic acid receptors (RARs) was close to saturation for activating the reporter construct

Summary

Introduction

All-trans retinoic acid (tRA) is the primary bioactive form of endogenous retinoids derived from dietary vitamin A and plays important roles in regulating a myriad of physiological events [1]. One of the major mechanisms through which tRA exerts its biological activity is by binding and activating its cognate nuclear receptors, the retinoic acid receptors (RARs) a, b, c, and retinoid X receptors (RXRs) a, b, c, which heterodimerize to act as transcription factors, thereby modulating gene transcription [2]. Most functions of vitamin A are mediated by its main active form, all-trans retinoic acid (tRA), which binds retinoic acid receptors (RARs) to modulate gene expression. We and others have recently reported that renal tRA/RAR activity is confined to the ureteric bud (UB) and collecting duct (CD) cell lineage, suggesting that endogenous tRA/RARs primarily act through regulating gene expression in these cells in embryonic and adult kidney, respectively

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