Identification of compounds that modulate retinol signaling using a cell-based qHTS assay
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.
Section snippets
Reagents
Chemicals purchased from Cayman Chemical (Ann Arbor, MI) include 5-azacytidine, Bay 11-7085, camptothecin, d-ribofuranosylbenzimidazole, H-8 HCl, kenpaullone, 3,4-Methylenedioxy-β-nitrostyrene (MNS), niclosamide, PD 98059, and topotecan hydrochloride. Citral, DMSO, LY-294002, retinol, and SU 4312 were purchased from Sigma-Aldrich (St. Louis, MO). CD437, ER50891, HX531, and SR11237 were purchased from Torcris (Minneapolis, MN). AM580 was purchased from Enzo Life Sciences (Farmingdale, NY).
Establishment of the C3RL4 reporter gene cell line
The parental C3H10T1/2 [Clone8] cell line is an embryonic multipotent mesenchymal cell line capable of differentiating into the muscle, adipose, bone and cartilage cells (Reznikoff et al., 1973, Hisada et al., 2013). This cell is inducible for alkaline phosphatase enzyme expression by atRA (Reese et al., 1992) and the results from our preliminary tests show that this cell contains a functional RSP to metabolize retinol to atRA (unpublished data). To create stable clones, a reporter construct
Discussion
Birth defects, which occur to approximately 120,000 (~ 3%) newborns in the U.S. every year (U.S. CDC (Rynn et al., 2008)), are thought to be caused by a mix of both genetic inheritance and environmental factors, which include exposure to chemicals during pregnancy. Studies have shown that maternal exposure to chemicals via certain behaviors (e.g., smoking (Hackshaw et al., 2011)), medications (Goldsmith et al., 2004), and contaminated consumer products (e.g., heavy metal (Needham et al., 2005))
Conflict of interest
The authors declare that there is no conflict of interest.
Transparency Document
Acknowledgment
This work was supported in part through the interagency agreement IAG #NTR 12003 from the National Institute of Environmental Health Sciences/Division of the National Toxicology Program to the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH). This project was also supported in part by the appointment of Dr. Yanling Chen to the Research Participation Program at the Center for Food Safety and Applied Nutrition administrated by the Oak Ridge
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