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and Represses Its Transcriptional Activity
Institut de Génétique et de Biologie Moléculaire et Cellulaire (C.B., K.S., O.A.B., J.A.), Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, BP 1042, 67404 Illkirch, France; and Department of Biosciences at Novum (E.T.), Karolinska Institute, S-14157 Huddinge/Stockholm, Sweden
Address all correspondence and requests for reprints to: Johan Auwerx, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch, France. E-mail: auwerx{at}igbmc.u-strasbg.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(NR1H3) and LXRß (NR1H2), as demonstrated by glutathione-S-transferase pull-down assays, mammalian two-hybrid, and coimmunoprecipitation experiments. In transfection assays, SHP inhibits the expression of an artificial reporter driven by an LXR-response element and represses the transcriptional activation by LXR of the human ATP-binding cassette transporter 1 (ABCA1) promoter. Treatment of Caco-2 cells with bile acids, which activate farnesoid X receptor and subsequently induce SHP, leads to the repression of the human ABCG1 gene, an established LXR target gene. These results demonstrate that SHP is able to interact with LXR and to modulate its transcriptional activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Liver X receptors (LXRs) are nuclear receptors that are activated by oxysterols (2, 3, 4, 5, 6). LXR (NR1H3, RLD-1) (2, 7) and LXRß (NR1H2, UR, NER1, OR-1, RIP15) (8, 9, 10, 11) are encoded by different genes and bind both to DNA as a heterodimer with retinoid X receptor (RXR). Whereas LXRß is ubiquitously expressed, LXR is mainly localized in the liver, intestine, kidney, and adipose tissue. LXR modulates the expression of multiple genes involved in cholesterol homeostasis such as the cholesterol-7--hydroxylase (CYP7A1) gene that encodes the rate limiting enzyme of the bile acid synthesis pathway (4, 12), the ATP-binding cassette transporters ABCA1, ABCG1, ABCG5, and ABCG8 (13, 14, 15, 16, 17, 18, 19), the cholesteryl ester transfer protein (CETP) (20), the apolipoprotein E (21), and the sterol regulatory element-binding protein-1c (6, 22, 23). Due to the coordinate regulation of several of these target genes, LXR has been suggested to play an important role in reverse cholesterol transport. For some genes, optimal activity of the LXR-RXR heterodimer is achieved when the liver receptor homolog-1 (LRH-1), an orphan nuclear receptor, is also bound to an adjacent site on the promoter of target genes, such as reported for CYP7A1 (24) or CETP (25). Therefore, LRH-1 is considered as a competence factor for the LXR-RXR heterodimer.
The small heterodimer partner (SHP) is an atypical nuclear receptor that lacks a DNA binding domain (26). It has been shown to repress the transcriptional activity of several nuclear receptors, such as the constitutive androstane receptor (26), thyroid receptor (26), RXR, retinoic acid receptor (RAR) (26), estrogen receptors (ERs) (27, 28), hepatocyte nuclear factor 4 (HNF-4) (29), peroxisome proliferator-activated receptors (30), and LRH-1 (24, 31). SHP is closely related to dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1), a corepressor for the steroidogenic factor-1 (32, 33), the paralog of LRH-1. An illustration for the functional relevance of the interaction of SHP with nuclear receptors has been provided by the existence of an autoregulatory loop controlling bile acid homeostasis involving several nuclear receptors. In fact, bile acids activate the farnesoid X receptor (FXR)/bile acid receptor, which on its turn induces the expression of SHP (24, 31, 34, 35). SHP will repress on the one hand LRH-1 activity, ultimately decreasing CYP7A1 expression (24, 31), and on the other hand RXR/RAR activity, decreasing the expression of the principal hepatic bile acid transporter sodium/taurocholate cotransporting polypeptide (36). This will coordinately down-regulate bile acid import and synthesis, thereby protecting the liver from bile acid-mediated cellular damage.
Although the expression patterns of LXR and LRH-1 overlap and these receptors coregulate several target genes, it has not been clearly demonstrated whether SHP is also able to interact with LXR. Therefore, we compared the interaction of SHP with LXR on the one hand and with LRH-1 on the other hand. We demonstrate here that SHP is able to bind to LXR. Helix 12 of LXR is indispensable for the interaction with SHP, whereas both the central and carboxy terminus of SHP are required for its interaction with LXR. SHP strongly represses LXR activation of an artificial reporter gene controlled by multiple LXR response elements and represses also, although to a lesser extent, the promoter of the LXR target gene ABCA1. In Caco-2 cells, the LXR-dependent induction of ABCG1 mRNA expression is repressed after induction of SHP by chenodeoxycholic acid. The finding that SHP represses the activity of both LRH-1 and LXR suggests that it is an important integrator of bile acid metabolism.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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to activate its target genes, such as CYP7A1 or CETP, is usually obtained when LRH-1, a competence factor for LXR, is also bound to an adjacent site on the promoter (20, 24, 25). Recently it has been shown that SHP inhibits LRH-1 activity, thereby attenuating the CYP7A1 promoter and establishing an inhibitory feed-back loop controlling bile acid synthesis by its end products. We investigated here the possibility that LXR might also be a molecular target for SHP. To this end, we analyzed whether LXR and SHP interact in glutathione-S-transferase (GST)-pull-down assays. Full-length LXR- and LRH-1-GST fusion proteins were incubated with 35S-radiolabeled SHP. SHP interacted to a similar extent with the LXR- and LRH-1-GST fusion proteins but was unable to bind to the GST protein itself, which served as a negative control (Fig. 1A
). Full-length GST-SHP fusion protein also interacted with 35S-radiolabeled full-length LXRß (Fig. 1B). SHP interacted with LXRs in the absence of ligand. The efficiency of the interaction of SHP with LXR was, however, significantly increased by the addition of T0901317, a synthetic ligand for LXR (6) (Fig. 1C). The binding between LXR and SHP, in this particular experiment, could not be detected anymore in the absence of ligand because the vehicle to dissolve T0901317, i.e. dimethylsulfoxide (DMSO), strongly inhibited the interaction between LXR and SHP (compare Fig. 1, A and C). Likewise and consistent with previous reports, the interaction between SHP and RXR was also enhanced in the presence of the synthetic rexinoid, LG100268 (26) (Fig. 1D).
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Coimmunoprecipitates with Endogenous SHP in vivo, coimmunoprecipitation assays were carried out. An LXR-flag fusion protein expression vector or an empty expression vector were transfected in RK-13 cells containing endogenous SHP. Cells were treated for 20 h with 10-5 M 22(R)-hydroxycholesterol, an LXR ligand, before harvesting and lysis. The lysate was then incubated with an irrelevant antibody (Fig. 2A, lanes 3 and 5) or an anti-SHP antibody (Fig. 2A, lanes 4 and 6) and the precipitated proteins were analyzed by immunoblotting with an anti-flag antibody. No band was detected in the immunoprecipitate when an irrelevant antibody was used (Fig. 2A, lane 5). In contrast, the LXR-flag protein could be detected when an anti-SHP antibody was used for immunoprecipitation (Fig. 2A, lane 6). The amount of endogenous SHP present in the cells transfected with the empty pCMX expression vector or pCMX-LXR-flag and detected by anti-SHP immunoblot was similar (Fig. 2B).
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and LRH-1 Are Necessary and Sufficient for in Vitro Interaction. The interaction domain with LRH-1 has been recently localized to the amino-terminal part of SHP (37). Various deletion mutants of SHP fused to the GST protein were generated as outlined in Fig. 3A. The fusion proteins were subsequently incubated with 35S-radiolabeled full-length RXR or LXR. Consistent with previous reports (38), the SHP mutant containing region 1 + 2 still interacted with RXR in the presence of the RXR ligand, LG100268 (Fig. 3B). In contrast to the situation for RXR, region 1 + 2 of SHP seemed less critical for the interaction with LXR (Fig. 3B). Interestingly, the carboxy-terminal region 3 of SHP by itself interacted with LXR, although the interaction was weaker than when domains 2 and 3 of SHP were combined.
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and a series of in vitro translated 35S-radiolabeled SHP mutants (Fig. 3C
). In this experiment, SHP domain 1+2 (SHP1 + 2) still interacted with LXR, albeit more weakly than the wild-type (full length) protein (SHPFL) (Fig. 3, D and E). Mutation of the two LXXLL motifs in domains 1 and 2 of the wild-type protein (SHPmut1+2) did not abolish the interaction but weakened it. This result confirms that domain 3 also contributes to the interaction between SHP and LXR and that region 1 + 2 is dispensable to some extent. To determine the exact domains of LXR and LRH-1 that interact with SHP, deletion mutants of LXR and LRH-1 fused to GST (outlined in Fig. 4, A and C) were incubated with 35S-radiolabeled full-length SHP. For both receptors, the LBD (or DE domain) was necessary and sufficient for the interaction with SHP (Fig. 4, B and D). This result is consistent with a previous report delimitating the LRH-1 interaction domain to the AF-2 region (37).
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in an AF-2-Dependent Manner and Interacts in Vitro with RNA Polymerase II in vivo, we used the mammalian two-hybrid system in which the LXR LBD was fused to the DNA binding domain (DBD) of the Gal4 protein and in which the full-length SHP protein was fused to the activation domain of VP16. As the consequence of a direct interaction between LXR and SHP, increased transcriptional activity of a reconstituted Gal4-VP16 transcription factor was monitored by transfecting the luciferase reporter vector under the control of a multimerized Gal4 response element (upstream activating sequence, UAS).
The ectopic expression of VP16-SHP in the presence of a DBD-Gal4-LXRLBD resulted in a ±3-fold induction in luciferase activity compared with the empty vector VP16 (Fig. 5, A and B). As for the interaction observed in GST pull-down assays between LXR and SHP, the interaction was increased in the presence of the LXR ligand, 22(R)-hydroxycholesterol. In a parallel experiment, the interaction between DBD-Gal4-LRH-1LBD and VP16-SHP was shown to lead to a more moderate (±2-fold) induction of luciferase activity, which is consistent with previously reported results (24) (Fig. 5, C and D). We also repeated the mammalian two-hybrid experiments using more detailed deletion mutants of the LBD of LXR and LRH-1 fused to the DBD of Gal4. Deletion of helices 11 and 12 completely abrogated the interaction of VP16-SHP with LXR in the presence of 22(R)-hydroxycholesterol (Fig. 5E). The deletion of helix 12 alone had the same effect as deletion of both helices 11 and 12, which confirms that helix 11, that is thought to be part of the corepressor anchoring site in some nuclear receptors (39), is not crucial for the interaction with SHP. Likewise, deletion of helix 12 of LRH-1 also abolished completely the capacity of LRH-1 to interact with VP16-SHP (Fig. 5F).
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LBD was cotransfected with either the empty expression vector pCDM8 or the pCDM8-SHP vector that codes for the full-length SHP protein without the activation domain of VP16. In the absence of the VP16 activation domain, we would hence expect SHP to decrease the capacity of LXR to activate the reporter gene. As expected, a reduction of the UAS-driven reporter activity could be observed when pCDM8-SHP was cotransfected instead of pCDM8 (Fig. 6A). This reduction in UAS-driven reporter activity was somewhat more pronounced in the presence of an LXR ligand. In general, these data are consistent with the reported moderate reduction of ER activity by SHP cotransfection (28). As the basal transcriptional activity of DBD-Gal4-LXRLBD was only slightly activated by the addition of 22(R)-hydroxycholesterol (Fig. 5A), we sought to increase the activation of the reporter gene by cotransfecting an expression vector encoding the coactivator TIF2. Cotransfection of TIF2 increased the amplitude of the ligand-induced activation of the UAS-driven reporter by the DBD-Gal4-LXRLBD fusion protein. Also, under these conditions, SHP was able to repress TIF2-mediated coactivation (Fig. 6B).
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SHP Represses LXR Transcriptional Activity on an Artificial and a Natural Promoter
Because SHP interacts with LXR both in vitro and in vivo and because this interaction is associated with moderate repressive effect on transcriptional activity in our mammalian two-hybrid assays, we investigated next the possibility that LXR transactivation might be attenuated by SHP. We therefore transfected CV-1 cells with a luciferase reporter gene under the control of a multimerized response element for LXR. When the heterodimer LXR/RXR was cotransfected with increasing amounts of SHP, transcriptional activity was strongly decreased (Fig. 7A). This repression was abrogated when a SHP construct containing mutations in the LXXLL motifs present in domain 1 and 2 was used (Fig. 7B). These data support the concept that these motifs, although dispensable for the interaction between SHP and LXR, are crucial for the repressive activity of SHP on LXR.
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but not to human LRH-1 (25). CV-1 cells were therefore cotransfected with an ABCA1 reporter luciferase vector containing the region between -400 and +181 harboring the LXR/RXR response element. In the presence of increasing amounts of cotransfected SHP, a repression of ABCA1 promoter was observed (Fig. 7C). Under similar conditions, we demonstrated that increasing amounts of SHP blocked the capacity of LRH-1 to activate the SHP promoter, as previously published (24) (Fig. 7D).
SHP Represses LXR Transcriptional Activity in Vivo
To validate our in vitro data in vivo, we performed an experiment in differentiated Caco-2 cells derived from a human colon adenocarcinoma. Caco-2 cells express LXR, FXR, and SHP (Fig. 8A and data not shown), and are an adequate model to study certain steps of cholesterol absorption and bile acid metabolism. Caco-2 cells were therefore treated with T0901317 (10-5 M), a synthetic LXR agonist, and/or chenodeoxycholic acid (CDCA; 250 µM), an FXR ligand that induces SHP. RNA was isolated from these cells and analyzed by Northern blot hybridization (Fig. 8A). Interestingly, T0901317 addition induced the expression of SHP (12-fold induction) (Fig. 8A, lane 2). When CDCA and T0901317 were added together to the Caco-2 cells, SHP mRNA was, however, induced to a much higher level (40-fold induction) (Fig. 8A, lane 4). Unexpectedly, ABCA1 mRNA was induced by CDCA (30-fold induction), and no clear conclusions could be made regarding its expression (Fig. 8A, lane 3). In absence of the bile acids, treatment with T0901317 induced ABCG1 mRNA (Fig. 8A, lane 2), another LXR target gene (16, 21). The up-regulation of ABCG1 mRNA levels by T0901317 was completely abrogated when the Caco-2 cells were treated with CDCA, which coincided with stronger SHP expression (Fig. 8A, lane 4). The fact that ABCG1 expression was independent of LRH-1 was established by the demonstration that ABCG1 expression was not up-regulated in mouse hepatic (BNL-CL.2) cells that were infected with a retrovirus that expressed LRH-1 (Fig. 8C, lane 2). In this case, a RT-PCR was used because of the low expression of the different genes. SHP, a well-established LRH-1 target gene, was induced in these cells (Fig. 8C, lane 2). We have also treated noninfected BNL-CL.2 cells with T0901317 to demonstrate that ABCG1 mRNA can be induced upon treatment with an LXR agonist (Fig. 8B, lane 2). These reults hence confirm our in vitro data and claim a role of SHP in the direct down-regulation of specific LXR target genes.
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DISCUSSION |
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has never been fully explored. The evaluation of an eventual interaction of SHP and LXR is confounded by the fact that most of the currently known promoters that are activated by LXR also require the presence of LRH-1 as a competence factor [CYP7A1, CETP (24, 25)].
In the present study, we provide evidence for a direct interaction of LXR and SHP in the absence and presence of ligand. A solid binding between both subtypes of LXR and SHP was demonstrated by GST pull-down assays in vitro in the absence of ligand. In addition, the efficiency of interaction between LXR and SHP was significantly increased in the presence of a synthetic LXR ligand. Consistent with these in vitro interaction data, a direct in vivo interaction between LXR and endogenous SHP was also demonstrated by immunoprecipitation in cells. The carboxy-terminal domain of SHP, in combination with the central domain, is implicated in the interaction with LXR. It was shown previously that the carboxy-terminal domain of SHP was necessary for the repressive activity of SHP but most likely dispensable for the interaction with nuclear receptors, which required mainly its central domain (38). Consistent with these reports, we also demonstrated that the central domain of SHP, and not the carboxy-terminal region, is necessary for the interaction with RXR. The fact that LXR interacts with two different domains in SHP, including the C-terminal domain, which is not implicated in the binding of other nuclear receptors such as RXR, ER and HNF-4 (27, 29, 38, 41), hence suggests that the interaction domains between SHP and its partners slightly differs according to the nature of the nuclear receptor. It is likely that the carboxy-terminal region of SHP is more involved in the ligand-independent interaction with LXR, whereas the LXXLL motifs, present in the N-terminal and central part of SHP, could be responsible for the ligand-dependent interaction. In support of this hypothesis, SHP inhibition of LXR on a synthetic promoter is more effective when the two LXXLL motifs of SHP, found in domains 1 and 2, are intact.
On the receptor side, the LBDs of both LXR and LRH-1 are necessary for the interaction with SHP in both GST pull-down and mammalian two-hybrid assays. The interaction of SHP with the LBD of LXR was significantly stronger than the interaction of SHP with the LBD of LRH-1. This binding was reinforced by the presence of a ligand. Consistent with a role of ligand binding in the SHP/LXR interaction is the fact that the interaction between SHP and LXR or between SHP and LRH-1 is completely abrogated when the minimal AF-2 domain (contained within helix 12) of both nuclear receptors is deleted. This is in line with previous reports where it has been hypothesized that SHP acts as a corepressor by competing with the binding of coactivators to helix 12, which constitutes an interaction surface for coactivators in ligand-bound nuclear receptors (28).
SHP acts as a corepressor of LXR transactivation both on a heterologous reporter construct responsive to LXR and on a reporter construct controlled by the human ABCA1 promoter. We have chosen to work with the ABCA1 promoter because, unlike the promoter of other LXR target genes, such as CYP7A1 and CETP (24, 25), the expression of the human ABCA1 promoter activity is unaffected by LRH-1. Therefore, this promoter is a bona fide system to characterize an eventual effect of SHP on LXR activity (25). Although the inhibition of the LXR-mediated activation of the ABCA1 promoter by SHP is weaker compared with its capacity to inhibit the LRH-1-activated SHP promoter, the molar ratio of SHP necessary to observe a repression is in accordance with what was reported in the literature for other nuclear receptors, such as ER (27, 28, 41) and HNF-4 (29).
We have tried to validate the relevance of the LXR-SHP interaction in differentiated human colon adenocarcinoma (Caco-2) cells treated with CDCA, a bile acid that activates FXR and hence induces SHP. In these cells, the expression of two reported LXR target genes, ABCA1 and ABCG1 was analyzed. ABCA1 mRNA was weakly induced after treatment with a synthetic LXR ligand, T0901317, whereas a more robust induction was seen with the FXR ligand CDCA. This therefore suggests that FXR might also directly regulate the expression of ABCA1. Hence, no clear conclusions concerning the effect of SHP on the in vivo regulation of ABCA1 by LXR could be made. This direct induction of ABCA1 expression by FXR agonists is, however, not very surprising on a physiological point of view because it has been reported that FXR modulates the expression of other transporters such as the bile salt export pump (BSEP) (42, 43) and ABCC2 (44). Interestingly, ABCG1 mRNA levels were robustly induced upon treatment with T0901317 in Caco-2 and mouse hepatic (BNL-CL.2) cells. This induction was abrogated when Caco-2 cells were treated with CDCA, which induces SHP expression. Importantly, we established that the expression of the ABCG1 gene is not affected by LRH-1 because its expression does not change in BNL-CL.2 cells that retrovirally overexpress LRH-1. In contrast, classical LRH-1 target genes, such as SHP, are induced in the LRH-1 infected cells. This effect of FXR activation on an LXR (but not LRH-1) target gene is consistent with our in vitro data and supports the notion that SHP can directly down-regulate specific LXR target genes.
The mechanism by which SHP represses the activity of nuclear receptors is still not completely elucidated. SHP was first thought to inhibit the DNA binding of receptors via dimerization, such as was suggested for the RAR/RXR heterodimer (26), but this hypothesis was discarded for ERs as SHP interacts with their LBD (27). SHP was then shown to form a ternary complex with dimeric ERß on DNA (41). It has thus been hypothesized that SHP recruits corepressor complexes, or even directly represses transcription. SHP does not seem to be able to interact with the nuclear receptor corepressor N-CoR and probably does not recruit histone deacetylases (29, 38). In contrast, SHP can recruit the mouse E1A-like inhibitor of differentiation 1, which could antagonize the CBP/p300-dependent coactivator functions (45). DAX-1, a nuclear receptor that resembles SHP both structurally and in its function as a corepressor, is thought to recruit corepressor complexes such as N-CoR and/or Alien (46, 47). In addition, it has been suggested that DAX-1 acts by binding to hairpin-loop structures of DNA (48). In another study, DAX-1 was shown to bind to RNA and exert regulatory functions at a posttranscriptional level (49). More recently, it has been also suggested that DAX-1 inhibits the androgen receptor function by cytoplasmic tethering (50).
Our data, which show that SHP decreases TIF2 stimulation of LXR activity in the presence of a ligand, would be rather consistent with the hypothesis that the repression function of SHP is at least in part linked to competition with cofactor binding, as previously proposed for ER and HNF-4 (29, 41). Nevertheless, the fold induction (with and without ligand) is affected to a relatively low extent; therefore, it cannot be excluded that the results shown on Fig. 6B are due to a more general inhibition of the basal transcription rate by SHP. Indeed, in an attempt to explore other possible molecular mechanisms underlying SHP activity we have found that SHP can interact in vitro with RNA polymerase II. This interaction could be an additional way how SHP could inhibit both basal and induced transactivation. Further experiments are actually ongoing to explore how this interaction could mediate repression by SHP.
In conclusion, we report the interaction of LXR with SHP and mapped the interaction domain to the AF-2 domain of LXR. SHP can inhibit the expression of an artificial reporter gene, whose expression is under the control of LXR, as well as the human ABCA1 promoter, a natural LXR target gene. Furthermore, the induction of SHP by CDCA in differentiated Caco-2 cells leads to a dramatic down-regulation of the expression of the LXR target gene ABCG1. In combination, our data suggest that the interaction between SHP and LXR is relevant in vivo and that this interaction leads to the repression of LXR transcriptional activity on specific LXR target genes.
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MATERIALS AND METHODS |
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Plasmids
pCMX-SHP was obtained by insertion of a PCR product corresponding to the mouse SHP cDNA into the pCMX vector using EcoRI and XmaI restriction sites. pCMX-LRH-1 was produced by insertion of a PCR product, corresponding to the human LRH-1 cDNA, into the pCMX vector. For protein expression, cDNA fragments encoding the deletion mutant proteins were inserted in the pGex-4T-1 bacterial expression vector (Amersham Pharmacia Biotech, Orsay, France). The various full-length cDNA or deletion mutants of SHP, LXR, and LRH-1 cloned into pGex-4T-1 were obtained by restriction digestion or PCR amplification. The SHP mutants obtained by PCR were cloned using EcoRI and XmaI sites. SHP "1+2" mutant was obtained by digestion of pGex-4T-1-SHP by Eco47III and SmaI. The LXR mutants obtained by PCR were cloned into pGex-4T-1 using BamHI and XmaI sites. The LRH-1 mutants obtained by PCR were cloned into pGex-4T-1 using EcoRI and XmaI sites. LRH-1 "AB" mutant was obtained by digestion of pGex-4T-1-LRH-1 with BglII and XmaI.
For the mammalian two-hybrid assays, a full-length mouse SHP cDNA PCR product was inserted downstream the pCMX-VP16 activation domain with BamHI and NheI to generate the pCMX-VP16-SHP expression vector. The LBDs of human LXR and human LRH-1 (or their deletion mutants) were cloned in pCMX-BDGal4 vector, downstream of the Gal4 DNA binding domain, using KpnI and XmaI for LXR, and EcoRI and XmaI for LRH-1.
The pGL3-(LXRE)5TK-Luc reporter construct contains five tandem repeats of the DR-4 LXR response element (5'-gcggttcccagGGTTTAAATAAGTTCAtctagat) cloned upstream of the herpes simplex virus thymidine kinase (TK) promoter and the luciferase (Luc) reporter gene. pGL3-hABCA1-Luc was obtained by amplifying the -400/+181 part of the human ABCA1 promoter by PCR using human genomic DNA as a template. The luciferase reporter construct pGL3-(UAS)5TK-Luc comprises five tandem repeats of the Gal4 UAS cloned in front of the TK promoter. pCMV-ßGal was used as an internal control for transfection efficiency. All constructs were verified by DNA sequencing. pCDM8-mSHP, pCMX-hLXR, pBluescript-mLXRß, pCMX-LXR-flag, pSG5-mRXR, pGex-2T-mRXR, pSG5-hTIF2, and the human LRH-1 PCR product were gifts. pSG5-SHP, pSG5-SHP mut1+2, pSG5-SHP1+2 (also called pSG5-SHP159) and pGL3-hSHP (569)-Luc were described elsewhere (24, 41).
Cell Culture, Transient Transfection Assays, and Retroviral Infection
CV-1, RK-13, BNL-CL.2, and Caco-2 cells (ATCC, Manassas, VA) were maintained at 37 C, 5% CO2 and respectively grown in DMEM supplemented with 5 or 10% fetal calf serum (FCS), MEM supplemented with 10% FCS, or MEM supplemented with 20% FCS, L-glutamine, and antibiotics (penicillin-streptomycin-seromed A2213). Caco-2 cells were differentiated as described (51). Unless specified, cells were transfected by the calcium phosphate-DNA coprecipitation technique as described (52). In general, cells were transfected in six-well plates. Empty expression vectors were used to maintain equivalent amounts of expression vector for each transfection condition. Luciferase data were normalized to an internal ß-galactosidase control and represent the mean (±SD) of triplicate assays. When indicated, lipoprotein-deficient and charcoal-stripped FCS (LPDS-DCC) was used as described (53). For retroviral infection, 293 cells stably expressing Moloney gag and pol (293 gp) were maintained in DMEM supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 at 37 C. Virus production for infection was performed by cotransfection with lipofectamine (Life Technologies, Inc., Gaithersburg, MD) of the 293 gp packaging cell line with 15 µg of pLPCX retroviral vector containing the cDNA of mouse LRH-1, or the empty vector, and 5 µg of the ecotropic vector SV-E-MLV-env containing the Moloney virus envelope cDNA downstream of the Simian virus 40 promoter enhancer. After 48 h, the medium was removed and filtered, polybrene (Sigma) was then added (8 µg/ml). Infection of BNL-CL.2 cells was performed by adding 3 ml of supernatant containing the viruses to approximately 105 cells for 8 h. The cells were then allowed to expand for 48 h and the infected cells were selected using 2.5 µg/ml of puromycin (Sigma).
Protein Production and GST Pull-Down Assays
GST fusion proteins were expressed in Escherichia coli and purified on a glutathione affinity matrix (Amersham Pharmacia Biotech). Purified proteins (TFIIE, TFIID, and RNA polymerase II) or in vitro35S-radiolabeled translated proteins (TNT T7 Quick Rabbit Reticulocyte, Promega Corp., Madison, WI) were incubated 1 h at 25 C in pull-down buffer (PBS 1x, glycerol 10%, Triton 0.1%, protease inhibitor cocktail) with GST or the GST fusion proteins linked to glutathione-Q sepharose beads in the presence of vehicle or T0901317 (10-4 M final concentration) or LG100268 (10-6 M final concentration). Beads were then washed five times in pull-down buffer and boiled in SDS-PAGE sample buffer. Samples were separated on a 7.5–15% sodium dodecyl sulfate (SDS) polyacrylamide gel and protein interaction was visualized by Western blot or autoradiography.
Coimmunoprecipitation and Western Blot Analysis
RK-13 cells were grown in 100-mm dishes and were transfected with 10 µg of either the pCMX or the pCMX LXR-flag expression vectors, using lipofectamine (Life Technologies, Inc.). After 4 h, medium was changed and cells were treated for another 20 h with 22(R)-hydroxycholesterol (10-5 M). Cells from were lysed in 400 µl of IP buffer (NaCl 150 mM; Nonidet P-40 1%; Tris, pH 8; protease inhibitor cocktail) for 30 min at 4 C. Supernatant was incubated 1 h at 25 C with rabbit preimmune serum and 2.5 mg of protein A sepharose (Amersham Pharmacia Biotech) to preclear the samples. Half of the supernatant was first incubated with the anti-SHP antibody or an irrelevant antibody, and consecutively incubated with 2.5 mg of protein A sepharose overnight at 4 C. Beads were washed three times in lysis buffer and three times in wash buffer (KCl 250 mM, dithiothreitol 1 mM, PBS 1x). Beads were then boiled in SDS-PAGE sample buffer. Proteins were separated on a 7.5% (12% for the SHP input) SDS acrylamide gel and electroblotted to a nitrocellulose membrane. Membranes were incubated in PBS Tween (0.05%) containing 5% nonfat dry milk 1 h at 25 C and subsequently with the anti-flag primary antibody (1/500 dilution) or the anti-SHP antibody (1/2000 dilution) in PBS Tween (0.05%) containing 5% nonfat dry milk overnight at 4 C. Membranes were washed in PBS Tween and incubated with a secondary goat antimouse or antirabbit antibody coupled to horseradish peroxidase (1/5000 dilution) for 1 h at 25 C. Membranes were washed in PBS Tween and proteins were visualized with the enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech).
Cell Treatment, Northern Blot Analysis, and RT-PCR
Caco-2 cells were first treated with 10-5 M T0901317 or DMSO during 4 h and then with 250 µM chenodeoxycholic acid (Sigma) or ethanol during 24 h in normal medium supplemented with 20% LPDS-DCC. RNA extraction and Northern blot analysis of RNA were performed as described (52). A human acidic ribosomal phosphoprotein 36B4 cDNA clone was used as control (54). All probes were labeled by random priming (Roche Molecular Biochemicals, Mannheim, Germany). BNL-CL.2 cells were treated with 10-5 M T0901317 or DMSO during 24 h in normal medium supplemented with 10% LPDS-DCC. RNA extraction was performed as described (52). RT-PCR was performed using random hexanucleotides and Superscript reverse transcriptase (Invitrogen, Cergy Pontoise, France).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ABCA1, ABCG1, ABCG5, and ABCG8, ATP-binding cassette transporters; CDCA, chenodeoxycholic acid; CETP, cholesteryl ester transfer protein; CYP7A1, cholesterol-7--hydroxylase; DAX-1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1; DBD, DNA binding domain; DMSO, dimethylsulfoxide; ER, estrogen receptor; FCS, fetal calf serum; FXR, farnesoid X receptor; GST, glutathione-S-transferase; HNF, hepatocyte nuclear factor; LBD, ligand binding domain; LPDS-DCC, lipoprotein-deficient and charcoal-stripped FCS; LRH, liver receptor homolog; Luc, luciferase; LXR, liver X receptor; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SHP, small heterodimer partner (NR0B2); TFIID, general transcription initiation factor II D; TFIIE, general transcription initiation factor II E; TK, thymidine kinase; UAS, upstream activating sequence.
Received for publication August 17, 2001. Accepted for publication May 30, 2002.
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