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

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.

Keywords: Developmental toxicant; High-throughput screening; LOPAC; Retinol signaling; Stem cell; Tox21.

Figure 1. Major steps in the RSP and the RARE-Luc reporter construct in C3RL4 cells

Retinol is converted to retinal and then to atRA via two enzymatic reactions in the RSP. atRA is an activating ligand for the RAR/RXR nuclear receptors that bind to the RARE to regulate downstream gene expression.

Figure 2. RARE assay optimization in 96-well format

(A) Retinol dose-dependent expression of Luc (open diamond) in C3RL4 cells. Closed diamonds indicate relative cell viability. (B) Citral inhibits the Luc expression that was induced by retinol at different concentrations. Closed diamonds indicate relative cell viability. (C) Compound dose-dependent induction of Luc expression by agonists of the RAR/RXR receptors. Open shapes indicate relative Luc expression; closed shapes indicate cell viability. (D) Inhibition of retinol-induced Luc expression by antagonists of the RAR/RXR receptors. (E) EC₅₀/IC₅₀ values of the tested compounds. RLU, Relative Luminescence Units. Values are expressed as mean ± s.e.m.; n=8.

Figure 3. Compound effect on the Hoxa1 gene expression in P19 cells

(A) The relative Hoxa1 gene expression levels in cells that were treated with indicated compounds for 6 hr. *, p < .01, compared to the DMSO-treated control. (B) Inhibition of retinol-induced Hoxa1 gene expression by the indicated compounds. *, p < .01, compared to the retinol-treated control. The floating bars show relative P19 cell viability determined in the MTT assays. Values are expressed as mean ± s.e.m.; n=4.

Figure 4. Possible interactions between compounds and signaling pathways/biological processes

Diagram is drawn based on published literatures. Pathway crosstalk and the regulatory effects on each other are shown as arrows to indicate activation or inhibition. Dashed lines indicate that further mechanistic studies are required to prove direct compound action on the RSP.