EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 42, No. 11, 731-738, November 2010 Estrogen modulates transactivations of SXR-mediated liver X receptor response element and CAR-mediated phenobarbital response element in HepG2 cells Gyesik Min1 by moxestrol in the presence of ER. Thus, ER may play both stimulatory and inhibitory roles in modulating Department of Pharmaceutical Engineering CAR-mediated transactivation of PBRU depending on Jinju National University the presence of their ligands. In summary, this study Jinju 660-758, Korea demonstrates that estrogen modulates transcriptional 1Correspondence: Tel, 82-55-751-3396; activity of SXR and CAR in mediating transactivation Fax, 82-55-751-3399; E-mail, [email protected] of LXRE and PBRU, respectively, of the nuclear re- DOI 10.3858/emm.2010.42.11.074 ceptor target genes through functional cross-talk be- tween ER and the corresponding nuclear receptors. Accepted 14 September 2010 Available Online 27 September 2010 Keywords: constitutive androstane receptor; estro- gen; liver X receptor; phenobarbital; pregnane X re- Abbreviations: CAR, constitutive androstane receptor; CYP, cyto- ceptor; transcriptional activation chrome P450 gene; E2, 17-β estradiol; ER, estrogen receptor; ERE, estrogen response element; GRIP, glucocorticoid receptor interacting protein; LRH, liver receptor homolog; LXR, liver X receptor; LXREs, LXR response elements; MoxE2, moxestrol; PB, Introduction phenobarbital; PBRU, phenobarbital-responsive enhancer; PPAR, Estrogen plays important biological functions not peroxisome proliferator activated receptor; RXR, retinoid X receptor; only in the development of female reproduction SRC, steroid hormone receptor coactivator; SXR, steroid and and cellular proliferation but also in lipid meta- xenobiotic receptor; TCPOBOP, 1,4-bis-(2-(3,5-dichloropyridoxyl)) bolism and biological homeostasis in different tis- benzene sues of body (Archer et al., 1986; Croston et al., 1997; Blum and Cannon, 2001; Deroo and Korach, 2006; Glass, 2006). Estrogen action is mediated by Abstract the nuclear estrogen receptor (ER), which is a ligand-dependent transcription factor and consists The nuclear receptors, steroid and xenobiotic receptor of different regulatory domains with distinct bio- (SXR) and constitutive androstane receptor (CAR) play logical functions (Evans, 1988; Beato, 1989). Li- important functions in mediating lipid and drug metab- gand-bound ER either binds to estrogen response olism in the liver. The present study demonstrates element (ERE) directly or interacts indirectly to the modulatory actions of estrogen in transactivations DNA through protein-protein interactions with other of SXR-mediated liver X receptor response element transcriptional factors in estrogen-responsive tar- (LXRE) and CAR-mediated phenobarbital response el- get genes (Paech et al., 1997). Ligand binding ement (PBRU). When human estrogen receptor (hERα) induces a conformational change of the ER and and SXR were exogenously expressed, treatment with recruits different sets of coactivators or corepre- either rifampicin or corticosterone promoted signi- ssors that determine biological activity by changing ficantly the SXR-mediated transactivation of LXRE re- the transcriptional responses according to the porter gene in HepG2. However, combined treatment physiological needs (Katzenellenbogen and Kat- zenellenbogen, 2000). with estrogen plus either rifampicin or corticosterone Recent studies (Min et al., 2002b) demonstrated resulted in less than 50% of the mean values of the that the action of ER in transcriptional activity can transactivation by rifampicin or corticosterone alone. be modulated by functional cross-talk between ER Thus, it is suggested that estrogen may repress the and other nuclear receptors. Specifically, the xeno- SXR-mediated transactivation of LXRE via functional biotic nuclear receptors, constitutive androstane cross-talk between ER and SXR. The CAR-mediated receptor (CAR) and steroid and xenobiotic receptor transactivation of PBRU was stimulated by hERα in the (SXR), inhibit ER-mediated transactivation. These absence of estrogen. However, the potentiation by xenobiotic receptors play important roles in mo- CAR agonist, TCPOBOP, was significantly repressed dulating steroid hormone homeostasis and drug 732 Exp. Mol. Med. Vol. 42(11), 731-738, 2010 metabolism by mediating cellular physiological re- compounds can also interact with classic steroid sponses not only to the endogenous compounds hormone receptors suggest that multiple signal such as hormones but also to the exogenous cross-talk mechanisms might be involved. chemical compounds such as drugs and environ- The liver X receptor (LXR) is an orphan nuclear mental pollutants. receptor that plays important physiological roles in The orphan nuclear receptor CAR mediates the regulation of lipid metabolism. LXR serves as a induction of cytochrome P450 (CYP)2B genes, sensor of cellular cholesterol and regulate the which are steroid hydroxylases, by the classical expression of genes such as CYP7A1 involved in inducer of drug metabolism, phenobarbital (PB), in the maintenance of cholesterol homeostasis and the liver (Wei et al., 2000; Min et al., 2002a, fatty acid biosynthesis (Glass, 2006). LXR recog- 2002b). CAR is sequestered in the cytoplasm and nizes DR-4 LXR response elements (LXREs) as a upon exposure to agonists, such as PB and heterodimer with RXR, and 9-cis retinoic acid was 1,4-bis-(2-(3,5-dichloropyridoxyl))benzene reported to be a potent activator of LXR tran- (TCPOBOP) (Tzameli et al., 2000), translocated scriptional activity (Willy et al., 1995; Glass, 2006: into the nucleus (Kawamoto et al., 1999; Min et al., Matsukuma et al., 2007). A previous report also 2002a), where it binds to its cognate recognition demonstrated that LXR heterodimerizes with pe- sites called nuclear receptor (NR)-1 and NR-2 as a roxisome proliferator activated receptor (PPAR) to CAR/retinoid X receptor (RXR) heterodimer in the regulate gene expression suggesting possible in- phenobarbital-responsive enhancer (PBRU) of the teractions with other related nuclear receptors CYP2B genes (Trottier et al., 1995; Honkakoski et (Miyata et al., 1996). al., 1998; Kawamoto et al., 1999; Kim et al., 2001; Nuclear receptors can positively or negatively Min et al., 2002a). Although transcriptional activity regulate gene expression by several mechanisms. of CAR is ligand-independent, some of CAR Many nuclear receptors can inhibit signal-depen- inducers such as TCPOBOP can bind CAR directly dent activation of promoters by other classes of and enhance its transcriptional activity by pro- transcription factors in a ligand-dependent manner. moting the interaction of CAR with the coactivators, An example of this is the ability of the gluco- steroid hormone receptor coactivator (SRC)-1 corticoid receptor to inhibit activation of inflam- (Forman et al., 1998; Moore et al., 2000; Tzameli matory response genes by signal-dependent tran- et al., 2000) and glucocorticoid receptor interacting scription factors such as activator protein-1 and protein (GRIP)-1 (Min et al., 2002a). NF-κB (De Bosscher et al., 2003; Glass, 2006). The steroid and xenobiotic nuclear receptor SXR The nuclear receptors are tethered to target pro- can modulate transcription in response to diverse moters through protein-protein interactions (Glass, natural and synthetic compounds with a broad 2006). This trans-repression accounts for a ma- spectrum of ligand specificity and low- affinity. It is jority of the anti-inflammatory actions of synthetic highly expressed in liver, the major expression site glucocorticoids such as dexamethasone (Glass, of steroid and xenobiotic-metabolizing enzymes, 2006) and may be an important mechanism by and intestine known to play important role in the which other nuclear receptor ligands such as first stage of metabolism of dietary compounds estrogens exert modulatory effects. (Kolars et al., 1991; Blumberg et al., 1998). SXR The liver is the major organ that metabolizes forms a heterodimer with RXR that can bind to and steroids and xenobiotic chemical compounds, and induce transcription from the response elements of one of the target organs for estrogen action in the cytochrome P450 genes including CYP3A4 and body (Min et al., 2002b). ER and the orphan CYP2A that are involved in steroid and xenobiotic nuclear receptors, SXR and CAR, and their he- metabolism (Gonzalez, 1992; Blumberg et al., terodimeric partner RXR are all expressed in the 1998). SXR is activated by a variety of xenobiotic liver (Kliewer et al., 1999). Although several compounds, including drugs such as rifampicin, studies have been reported on the modulation of steroid receptor agonists and antagonists such as ER transcriptional activity by other nuclear re- estrogen and tamoxifen respectively, and bioactive ceptors (Glass et al., 1988; Kraus et al., 1995; dietary compounds such as phytoestrogens Nuñez et al., 1997; Duan et al., 1999; Ricci et al., (Blumberg et al., 1998). Thus, it was hypothesized 1999; Min et al., 2002b), it is not known whether that SXR acting as a broad sensor nuclear ER can modulate transcriptional activities of the receptor may monitor aggregate levels of diverse xenobiotic nuclear receptors, SXR or CAR. Ac- inducers in order to modulate expression of xe- cordingly, this study examined modulatory actions nobiotic-metabolizing enzymes (Blumberg et al., of estrogen in transactivations of both SXR- 1998). The structural diversity of compounds that mediated LXRE and CAR-mediated PBRU