Identification of compounds that modulate retinol signaling using a cell-based qHTS assay

Highlights

• We developed a reporter gene assay to identify modulators of retinol signaling.

• The assay was validated in a qHTS format using LOPAC.

• Potential agonists and antagonists were identified.

• Active compounds were verified in mouse pluripotent cells.

• This assay can be used to screen large compound libraries.

Abstract

In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.

Introduction

Retinol (vitamin A) and its chemical analogs (retinoids (Sporn et al., 1976)) are involved in the regulation of diverse biological processes including cell growth, vision, reproduction, immune response and embryonic development (Ross et al., 2000, Collins and Mao, 1999, Duester, 2008, Stephensen, 2001, Cunningham and Duester, 2015, Janesick et al., 2015). In vertebrates, dietary retinol is metabolized to various retinoids. Among these, atRA is the predominant natural metabolite and also the major biologically active form of retinol (Napoli, 2012). atRA is an activating ligand for the retinoic acid receptors (RARs) (Chambon, 1996, Mark et al., 2009) that form heterodimers with the retinoid X receptors (RXRs) (Evans and Mangelsdorf, 2014) on the retinoic acid response element (RARE) (Mader et al., 1993, Kurokawa et al., 1994), an enhancer for transcription. Binding of atRA to the RAR partner of the heterodimers activates the RAR/RXR nuclear receptor to initiate transcription of the RARE-regulated genes (Chambon, 1996, Rochette-Egly, 2015). Through activating the RAR/RXR receptors, atRA modulates the expression of over 500 protein-coding genes (Balmer and Blomhoff, 2002) and possibly a large number of regulatory RNAs (Cawley et al., 2004) that are necessary for embryonic development and cellular functions in adults. RAR ligands have been found to be potent teratogens, whereas RXR ligands have not (Collins and Mao, 1999), suggesting that retinol signaling for the regulation of embryonic development depends on atRA and the RAR receptors (Collins and Mao, 1999, Mark et al., 2009).

The intracellular levels of atRA are regulated by the retinol signaling pathway (RSP) that controls the biosynthesis and catabolism of atRA. In the RSP, retinol is oxidized to retinaldehyde (retinal) by alcohol dehydrogenases, which is subsequently oxidized to atRA by retinaldehyde dehydrogenases (Fig. 1). atRA is further oxidized to polar metabolites by the Cyp26 cytochrome p450 enzymes for removal (Sonneveld et al., 1999). The RSP maintains the physiological homeostasis of atRA that is required for normal cellular functions.

Deviation of atRA levels from cell-defined limits, which can be a result of improper administration of retinoids or dysregulation of the RSP, may be teratogenic. For example, the offspring of pregnant animals fed on vitamin A-deficient diets or diets containing excess vitamin A (presumably retinyl esters) were shown to have congenital malformations in different organs (Collins and Mao, 1999, Mark et al., 2009); the use of isotretinoin (13-cis retinoic acid) for the treatment of severe acne and skin cancers may also lead to teratogenic outcomes (Goldsmith et al., 2004). In addition, disruption of the regulatory function of the RSP can also result in abnormal levels of atRA and consequently aberrant expression of developmental genes. Therefore, chemicals that interfere with the RSP have the potential to be developmental toxicants.

A large number of chemicals found in commercial products lack toxicity data (Judson et al., 2009) and to what extent these chemicals could adversely affect retinol signaling to influence embryonic development remains largely unknown. The use of animal bioassays for large-scale chemical assessment is limited due to factors such as high costs, long study periods, low-throughput, and ethical concerns over animal use. To overcome these limitations, we have developed a reporter gene cell line, designated C3RL4, to identify chemicals that disrupt the RSP using high-throughput screening. The C3RL4 clone contains a functional RSP and the firefly luciferase gene (Luc) under the control of the RARE. The expression of the RARE-Luc reporter construct is determined by the intracellular concentrations of atRA, which in turn is determined by the RSP-mediated biosynthesis/degradation of atRA (Fig. 1). A RARE reporter gene assay based on this clone has been developed and validated for use in a qHTS format by screening the 1280-compound LOPAC plus a set of control chemicals in the Tox21 robotic system (Sakamuru et al., 2012, Attene-Ramos et al., 2013). A group of compounds that have not been shown previously to affect the RSP were identified and these compound activities were subsequently confirmed in a series of follow-up tests. The results suggest that the RARE assay is a reliable and effective approach for screening large chemical libraries to identify and prioritize potential developmental toxicants that act by interfering with retinol signaling.