Retinoid X Receptor Modulators Transcriptionally Regulate Human Aldehyde Oxidase Expression

Abstract

Aldehyde oxidase‐1 (AOX1) is an oxidative drug‐metabolizing enzyme. Retinoid X receptor (RXR) is a nuclear receptor that elicits various physiological functions and is an obligate partner in specific cell signaling pathways. It regulates gene expression by forming a homodimer or heterodimer with another nuclear receptor, such as retinoic acid receptor (RAR) or peroxisome proliferator‐activated receptor (PPAR). Given the central role of RXR in controlling gene expression, we hypothesized that RXR modulators regulate AOX1 expression in cultured MCF‐7 human breast adenocarcinoma cells. 9‐Cis‐retinoic acid (10 μM), a pan‐RXR and RAR agonist, increased AOX1 mRNA expression at 24, 48, 72, 96, and 120 h post‐treatment, and a plateau was achieved at 96 h. To determine the effect of RXR activation on AOX1 expression, we compared the effect of several RXR‐selective modulators and 9‐cis‐retinoic acid. Non‐cytotoxic concentrations (as assessed by lactate dehydrogenase assay) of LG100268, CD3254, LG100754, SR11237, bexarotene, and 9‐cis‐retinoic acid increased AOX1 mRNA level in a concentration‐dependent manner with EC50 values of 4, 6, 12, 39, 42, and 334 nM, and Emax values of 5, 4, 9, 5, 15, and 9‐fold, respectively. The EC50 values of bexarotene and 9‐cis‐retinoic acid were less than the plasma concentrations of bexarotene (3 μM) and 9‐cis‐retinoic acid (0.5 μM) achieved in cancer patients administered either of these drugs. In contrast to other RXR activators, LG101506, which has no effect on RXR‐RXR homodimer or RXR‐RAR heterodimer and is a selective RXR‐PPAR agonist, decreased AOX1 expression with an EC50 of 1 μM and Emax of 94% decrease. Pretreatment of cells with actinomycin D (a RNA synthesis inhibitor) abolished AOX1 induction by 9‐cis‐retinoic acid, bexarotene, CD3254, SR11237, LG100268, and LG100754, indicating that the induction occurred by a transcriptional mechanism. RXRα mRNA was 6‐fold greater than RXRβ mRNA in MCF‐7 cells. To evaluate the role of RXR isoforms in AOX1 induction, genetic knock‐down approach was used. Transfection of cells with RXRα or RXRβ siRNA decreased the specific RXR mRNA level by 70–80%. RXRα siRNA attenuated AOX1 induction by 9‐cis‐retinoic acid, bexarotene, CD3254, SR11237, LG100268, and LG100754 by 55–70%, whereas RXRβ siRNA attenuated AOX1 induction by only 15–30%. To determine whether RXR transactivates AOX1 promoter, a reporter gene assay was conducted. 9‐Cis‐retinoic acid and selective RXR activators increased luciferase activity in cells transfected with an AOX1 (−1467 to +10) promoter plasmid, indicating that RXR transactivated this region of the promoter. In conclusion, RXR is the first nuclear receptor that transcriptionally regulates AOX1 expression. RXR activators differentially regulate AOX1 expression and this occurs primarily by activating the RXRα isoform. Given that LG100268 is an agonist of RXR homodimer and has no effect on RXR‐RAR heterodimer, whereas LG100754 is a RXR homodimer antagonist and a RXR‐RAR and RXR‐PPAR heterodimer agonist, the results suggest a role of both RXR homodimer and heterodimers in AOX1 induction. Our novel findings suggest interaction between RXR modulators and AOX1‐mediated drug metabolism and toxicity.Support or Funding InformationSingapore Ministry of Education Academic Research Fund Tier 1 and Ministry of Health National Medical Research Council Grants