Preferential Physical and Functional Interaction of Pregnane X Receptor with the SMRT Isoform

The silencing mediator for retinoid and thyroid hormone receptors (SMRT) serves as a platform for transcriptional repression elicited by several steroid/nuclear receptors and transcription factors. SMRT exists in two major splicing isoforms, and , with SMRT containing only an extra 46-amino acid sequence inserted immediately downstream from the C-terminal corepressor motif. Little is known about potential functional differences between these two isoforms. Here we show that the pregnane X receptor (PXR) interacts more strongly with SMRT than with SMRT both in vitro and in vivo. It is interesting that the PXR-SMRT interaction is also resistant to PXR ligandinduced dissociation, in contrast to the PXR-SMRT interaction. SMRT consistently inhibits PXR activity more efficiently than does SMRT in transfection assays, although they possess comparable intrinsic repression activity and association with histone deacetylase. We further show that the mechanism for the enhanced PXR-SMRT interaction involves both the 46-amino acid insert and the C-terminal corepressor motif. In particular, the first five amino acids of the SMRT insert are essential and sufficient for the enhanced binding of SMRT to PXR. Furthermore, we demonstrate that Tyr2354 and Asp2355 residues of the SMRT insert are most critical for the enhanced interaction. In addition, expression data show that SMRT is more abundantly expressed in most human tissues and cancer cell lines, and together these data suggest that SMRT may play a more important role than SMRT in the negative regulation of PXR. Transcriptional regulation is a dynamic process involving both association and dissociation of the transcription factor with various coactivators and corepressors. One well investigated system is the prominent effects of coactivators and corepressors on the transcriptional activity of steroid/nuclear hormone receptors (NRs) (Westin et al., 2000). The silencing mediator of retinoid and thyroid hormone receptors (SMRT) (Chen and Evans, 1995; Ordentlich et al., 1999; Park et al., 1999) and the nuclear receptor corepressor (N-CoR) (Horlein et al., 1995) are two related corepressors known to mediate repression by several unliganded NRs through the recruitment of histone deacetylases (HDACs) (Nagy et al., 1997; Guenther et al., 2000). These corepressors are believed to act as protein platforms for the assembly of corepressor complexes necessary for transcriptional repression. Transcription repression is an important genomic event involved in many physiological processes such as development, homeostasis, cell growth, and differentiation (Privalsky, 2004). In the absence of ligand, SMRT and N-CoR bind to the unliganded receptor through their NR-interacting domains (IDs) (Hu and Lazar, 1999; Ghosh et al., 2002). Upon ligand binding, the receptors undergo a conformational change, leading to the alteration of the corepressor-binding pocket that causes the release of corepressors and recruitment of coactivators. The human pregnane X receptor (PXR, also known as SXR and PAR) is a promiscuous sensor for several xenobiotic compounds (Watkins et al., 2001), and it binds to a diverse group of endogenous and exogenous ligands (Synold et al., 2001; Moore et al., 2003). PXR directly activates a subset of genes involved in drug metabolism (Xu et al., 2002). Therefore, drugs that activate PXR are likely to cause a This work was supported by the National Institutes of Health [Grant DK52542]. 1 Current affiliation: MD Anderson Cancer Center, University of Texas, Houston, Texas. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.047845. ABBREVIATIONS: NR, nuclear receptor; PXR, pregnane X receptor; SMRT, silencing mediator for retinoid and thyroid hormone receptors; N-CoR, nuclear receptor corepressor; HDAC, histone deacetylase; ID, interacting domain; RXR, retinoid X receptor; CAR, constitutive androstane receptor; TR, thyroid hormone receptor; PCN, pregnenolone-16 -carbonitrile; GST, glutathione transferase; Rif, rifampicin; CTZ, clotrimazole; HA, hemagglutinin; aa, amino acid(s); PCR, polymerase chain reaction; AD, activation domain; DMSO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; HEK, human embryonic kidney; RAR, retinoic acid receptor; VDR, vitamin D receptor; ROR, retinoid-related orphan receptor; EGFP, enhanced green fluorescent protein. 0026-895X/09/7502-363–373$20.00 MOLECULAR PHARMACOLOGY Vol. 75, No. 2 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 47845/3426573 Mol Pharmacol 75:363–373, 2009 Printed in U.S.A. 363 at A PE T Jornals on Sptem er 8, 2017 m oharm .aspeurnals.org D ow nladed from higher risk of drug-drug interactions (Harmsen et al., 2007; Urquhart et al., 2007; Wipf et al., 2007). The ability of PXR to regulate gene expression depends on its ability to form heterodimers with the retinoid X receptor (RXR) and bind to several PXR response elements. Several PXR response elements are found within the CYP3A promoters configured as direct repeats separated by three nucleotides (Kliewer et al., 1998), everted repeats separated by six nucleotides (Lehmann et al., 1998), or inverted repeats separated by eight nucleotides (Kast et al., 2002). In addition, the PXR-RXR heterodimers also bind tightly to several natural DR4 (direct repeats separated by four nucleotides)-type response elements. These include a DR4 motif in the intestinal multidrug resistance gene promoter responsible for its induction by rifampin (Geick et al., 2001) and a similar motif in the nitric-oxide synthase promoter responsible for its induction by clotrimazole (Toell et al., 2002), both through the PXR-RXR heterodimers. PXR-RXR heterodimers also consistently bind well to synthetic AG(G/T)TCA repeats of different spacing with a preferred affinity toward a DR4 element (Blumberg et al., 1998). Once activated, the PXR-RXR heterodimer recruits transcriptional coactivators such as the p160 proteins (Leo and Chen, 2000) to form a multiprotein complex to activate transcription (Kliewer et al., 1998). In addition, PXR can also cross-talk with other NR response elements, including those recognized by the constitutive androstane receptor (CAR) (Muangmoonchai et al., 2001; Kodama et al., 2004) and the antioxidant response element on the rat glutathione transferase A2 gene (Falkner et al., 2001). SMRT is known to exist in cells as at least two major splicing isoforms: and (Goodson et al., 2005). Compared with SMRT , SMRT contains an extra small exon encoding a 46-amino acid sequence inserted after residue Gly2352, immediately downstream to the distal corepressor motif (ID2, residues 2342– 2350). SMRT and SMRT reportedly interact with thyroid hormone receptors (TRs) with different affinities (Goodson et al., 2005); however, the molecular mechanism of such a differential affinity remains unknown. SMRT has also been shown to interact directly with and regulate the transcriptional activity of PXR in a PXR ligand-sensitive manner (Johnson et al., 2006; Wang et al., 2006); however, it was unknown whether and how PXR might interact with SMRT . In this study, we compared the binding affinities of SMRT and SMRT toward several NRs, with a focus on PXR. We found that, in contrast to other NRs, PXR uniquely displayed a preferential binding toward SMRT . It is interesting that this SMRT interaction is resistant to PXR ligand-induced dissociation, and SMRT elicited a greater inhibition on PXR activity than SMRT . It is noteworthy that we also uncovered critical residues in SMRT that are responsible for its higher affinity toward PXR and showed that SMRT is the dominant form expressed in most surveyed human tissues and cancer cells. Materials and Methods Chemicals. Rifampicin (Rif), clotrimazole (CTZ), and pregnenolone-16 -carbonitrile (PCN) were purchased from Sigma (St. Louis, MO). The rabbit anti-HA and mouse anti-FLAG antibodies were purchased from MBL International (Woburn, MA) and Stratagene (La Jolla, CA), respectively. All other reagents, including culture media for bacteria, yeast, and mammalian cells, were purchased from standard sources. Plasmids. The expression vectors pGEX-SMRT S1/2 (aa 2077– 2471) and pGEX-SMRT S1/2 (aa 2077–2517) were as described previously (Goodson et al., 2005) and were kindly provided by Dr. Martin Privalsky. The pCMX-FLAG-cSMRT (2095–2471) and pCMX-FLAG-cSMRT (2095–2517) were constructed by subcloning the Hind III to Nhe1 fragments of pGEX-SMRT S1/2 and pGEX-SMRT S1/2 into the pCMX-FLAG vector, respectively. The SMRT ID1 (aa 2107–2187), SMRT ID2 (aa 2284–2379), SMRT ID2 (aa 2284–2425), SMRT ID1–2 (aa 2107–2379), and SMRT ID1–2 (aa 2107–2425) fragments were generated by PCR reactions with pfu polymerase (New England Biolabs, Ipswich, MA) and subcloned into various plasmid vectors. The full-length pCMX-F-SMRT and pCMX-F-SMRT were constructed by assembling the Asp718 to Hind III fragment of pCMX-hSMRTe (Park et al., 1999) into the pCMX-FLAG-cSMRT and pCMX-FLAG-cSMRT plasmids and then subcloned into pEGFP-C1 and pCMX-GAL4 plasmids at Asp718 and Nhe1 sites. The full-length human PXR (hPXR) and its AF2 (aa 1–422) mutant in pGBT9, pCMXHA, and pCMX-GAL4 vectors were as described previously (Johnson et al., 2006). The point mutations mID1 (V2142A/I2143A), mID2 (I2345A/I2346A), mID1–2 (V2142A/I2143A, I2345A/I2346A), m3 (S2285E/K2286E/K2287E), and m4 (L2467A/ I2468A, based on SMRT sequence) were as described previously (Ghosh et al., 2002). These point mutants were regenerated in the SMRT template by QuikChange site-directed mutagenesis (Stratagene). All constructs were double-confirmed by restriction enzyme digestion and DNA sequencing, and further information is available