Di(2-ethylhexyl) phthalate Is a Highly Potent Agonist for the Human Constitutive Androstane Receptor Splice Variant CAR2

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 fourand 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 ligandactivated 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.

Di-(2-ethylhexyl)-phthalate (DEHP) activates the constitutive androstane receptor (CAR): A novel signalling pathway sensitive to phthalates

“…Similar to their observations using in vitro reporter assays [56], we show here that MEHP, the main DEHP metabolite does not induce Cyp2b10 in our JWZ-CAR cell line. It remains to be established whether DEHP or one if its metabolite other than MEHP directly binds to mouse and/or human CAR.…”

“…Interestingly, another group has very recently reported that the CAR2 splice variant of human CAR is activated by DEHP [56]. Similar to their observations using in vitro reporter assays [56], we show here that MEHP, the main DEHP metabolite does not induce Cyp2b10 in our JWZ-CAR cell line.…”

A Single Amino Acid Controls the Functional Switch of Human Constitutive Androstane Receptor (CAR) 1 to the Xenobiotic-Sensitive Splicing Variant CAR3

“…Recently, DeKeyser et al reported the common plasticizer, di(2-ethylhexyl) phthalate, known to be a ligand for PXR but not an activator of reference hCAR, is a highly potent and selective activator of hCAR2, another prominent hCAR splicing variant (DeKeyser et al, 2009). Molecular modeling analysis suggested that hCAR3 is the only differentially spliced hCAR variant with an unaltered ligand-binding pocket (Auerbach et al, 2003).…”

The Orphan Nuclear Receptor DAX-1 Functions as a Potent Corepressor of the Constitutive Androstane Receptor (NR1I3)

“…This constitutive activity is thought to result from a shortened AF-2 domain and hydrogen bonding interactions that stabilize the AF-2 domain in the active conformation (Xu et al, 2004). Alternative mRNA splicing results in CAR variants, including CAR2 and CAR3 (Auerbach et al, 2003), and unlike the constitutively active CAR1, CAR2 and CAR3 require ligand for activation and exhibit differences in ligand selectivity (Auerbach et al, 2005;Dekeyser et al, 2009;Dekeyser et al, 2011). CAR and CAR variant transcripts are detectable in many tissues but are expressed primarily in the liver, where they act as xenosensors, controlling the expression of phase I and phase II metabolic enzymes and phase III transporter (Timsit and Negishi, 2007).…”

“…Culture conditions for the maintenance of COS-1 cells (American Type Culture Collection, Manassas, VA) were published previously (Dekeyser et al, 2009). COS-1 cells were used because they are devoid of endogenous CAR expression/activity as demonstrated in previous reports (Auerbach et al, 2005(Auerbach et al, , 2007.…”