Abstract
The human constitutive androstane receptor (CAR, CAR1) regulates the expression of genes involved in xenobiotic metabolism in the liver. The CAR gene uses multiple alternative splicing events during pre-mRNA processing, thereby enhancing the CAR transcriptome. Previous reports have identified two prominent human CAR variants, CAR2 and CAR3, that possess four- and five-amino acid insertions in their ligand binding domains, respectively. Unlike the constitutively active reference form of the receptor, we now demonstrate that CAR2 is a ligand-activated receptor and comprises approximately 30% of the reference transcript level in human liver tissues in human hepatocytes. Furthermore, we identify the common plasticizer, di(2-ethylhexyl) phthalate (DEHP), as a highly potent and uniquely selective agonist of CAR2. Results from reporter transactivation and mammalian two-hybrid assays reveal that DEHP activates CAR2 at low nanomolar concentrations, results further supported by analysis of CAR target gene expression in primary human hepatocytes. In addition, comparative genomic analyses show that the typical mouse, rat, and marmoset models of DEHP toxicity cannot accurately profile potential human toxicity because of these species' inability to generate a CAR2-like transcript. The discovery that CAR2 is an ultimate human DEHP receptor identifies a novel pathway modulating human DEHP toxicity with potential clinical implications for a subset of patients undergoing critical care medical interventions.
Footnotes
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This work was supported, in part, by the National Institutes of Health National Institute of General Medical Sciences [Grant GM66411]; by the Intramural Research program of the National Institutes of Health National Institute of Environmental Health Sciences; and by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Contract N01-DK-7-0004/HHSN267200700004c]. The Quattro Premier XE mass spectrometer was purchased with funds from the National Science Foundation [Grant DBI-0619489].
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ABBREVIATIONS: CAR, constitutive androstane receptor; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime; DEHP, di(2-ethylhexyl) phthalate; DMSO, dimethyl sulfoxide; RXR, retinoid X receptor; CMV, cytomegalovirus; FBS, fetal bovine serum; SRC1, steroid receptor coactivator 1; MEHP, mono(ethylhexyl) phthalate.
- Received November 25, 2008.
- Accepted February 5, 2009.
- U.S. Government work not protected by U.S. copyright
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