text:
Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4α markedly transactivated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning −1191 to −700 base pairs (bp) and −140 to −79 bp were essential for the transactivation by HNF-4α. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4α, and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4α, respectively. An electrophoretic mobility shift assay showed that HNF-4α bound to DR-2 and IR-8 under the conditions of HNF-4α overexpression. Furthermore, under normal conditions, HNF-4α bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4α regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4α.
- proximal tubule
- promoter
- inverted repeat-8
the organic anion transporter (OAT) family plays an important role in the renal excretion of endogenous and exogenous organic anions, including drugs, toxins, and hormones (8, 18, 33, 41). Among the OAT family, OAT1 (SLC22A6) and OAT3 (SLC22A8) are localized to the basolateral membranes of the renal proximal tubular epithelial cells (26), and mRNA levels of both transporters were higher than those of other members of organic ion transporter (SLC22A) family in the human kidney cortex (42). So far, the molecular natures of OAT1 and OAT3 have been well characterized with regard to transport characteristics, structure-function relationships, and regulation (1, 8, 42, 47). As for regulation, various pathophysiological conditions, including hyperuricemia (14), renal failure (25, 36), bilateral ureteral obstruction (43), and acute biliary obstruction (6), affected the expression of OAT1 and/or OAT3. Although the elucidation of their regulatory mechanisms is quite important, studies that address the point are limited.
Recently, Kikuchi et al. (23) and we (28) reported the transcriptional regulation of the OAT3 gene. In contrast, as for the OAT1 promoter, a computational analysis but not a functional analysis has been conducted (2). In the present study, therefore, we attempted to characterize the transcriptional regulation of OAT1 by focusing on two kinds of transcription factors: hepatocyte nuclear factor (HNF)-1α/β and HNF-4α. The former is reported to stimulate the promoter activity of OAT3 (23), while the latter transactivates the OAT2 and OCT1 genes, which are other members of the SLC22A family mainly expressed in the liver (31, 34).
HNF-1α/β are homeodomain-containing transcription factors that are expressed in the liver, kidney, intestine, stomach, and pancreas (3, 10). In the kidney, HNF-1α is reported to control the expression of transporters such as sodium/glucose cotransporter 2 and sodium/phosphate cotransporter 1 besides OAT3 (9, 30). Furthermore, Oat1 had markedly lower renal mRNA levels in Hnf-1α-null mice than in wild-type mice (24). On the other hand, HNF-4α is an orphan member of the nuclear receptor superfamily that is expressed in the liver, kidney, intestine, stomach, and pancreas (27, 40). Although few reports are available on the physiological role of HNF-4α in the kidney, HNF-4α protein has been found only in proximal tubular epithelial cells in the kidney (22), where OAT1 protein is located (26). Accordingly, it is possible that the OAT1 gene is regulated by HNF-1α/β or HNF-4α. In the present study, we cloned the human OAT1 promoter region and examined whether the OAT1 gene is regulated by these transcription factors.
[γ-32P]ATP was obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Restriction enzymes were from New England Biolabs (Beverly, MA). An antibody against HNF-4 α (H-171) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The human HNF-1α and HNF-1β expression vectors were kindly supplied by Dr. Marco Pontoglio (Institute Pasteur, Paris, France). The human HNF-4α (transcript variant 2) expression vector was from OriGene Technologies (Rockville, MD).
5′-Rapid amplification of cDNA ends.
To identify the transcription start site of human OAT1, 5′-rapid amplification of cDNA ends (5′-RACE) was carried out using Human Kidney Marathon-Ready cDNA (Clontech, Mountain View, CA) according to the manufacturer's instructions. The primers for 5′-RACE were as follows: a gene-specific primer for OAT1 (accession number NM_004790), 5′-GGTCCCACTCAGTCACGATGGTAGATGG-3′ (683 to 656); and a nested gene-specific primer for OAT1, 5′-CGACACCCCCCACCTGCTGCAGGAGG-3′ (347 to 322). The PCR products were subcloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu, Kyoto, Japan).
Cloning of the 5′-regulatory region of the OAT1 gene.
Based on the human genomic sequence (accession number NT_033903), the 2,747-base pair (bp) flanking region upstream of the transcription start site was cloned by PCR using the primers listed in Table 1 and human genomic DNA (Promega). The PCR product was isolated by electrophoresis and subcloned into the firefly luciferase reporter vector pGL3-Basic (Promega) at the NheI and XhoI sites. This full-length reporter plasmid is hereafter referred to as −2,747/+88.
Preparation of deletion reporter constructs.
The 5′-deleted constructs (−1851/+88, −1191/+88) were generated by digestion of the −2747/+88 construct with MluI and either NdeI or PstI. The ends were blunted with T4 DNA polymerase (Takara Bio, Otsu, Japan) and then self-ligated. The other 5′-deleted constructs were generated by PCR with primers containing an NheI site and XhoI site (Table 1). The site-directed mutation in direct repeat (DR)-2 was introduced into the −2747/+88 construct and the mutation in inverted repeat (IR)-8 was introduced into the −2747/+88 construct and −140/+88 construct with a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the primers listed in Table 1. The nucleotide sequences of these deleted or mutated constructs were verified.
Cell culture, transfection, and luciferase assay.
Opossum kidney (OK) cells were cultured in medium 199 (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen) without antibiotics, in an atmosphere of 5% CO2-95% air at 37°C, and subcultured every 7 days using 0.02% EDTA and 0.05% trypsin. OK cells were plated into 24-well plates (4 × 106 cells/well) and transfected the following day with the reporter constructs and expression vector, and 25 ng of the Renilla reniformis vector pRL-TK (Promega), using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendation. The firefly and Renilla activities were determined 48 h after the transfection using a dual luciferase assay kit (Promega) and a LB940 luminometer (Berthold, Bad Wildbad, Germany). The firefly activity was normalized to Renilla activity.
Nuclear extract was prepared from OK cells transiently transfected with HNF-4α or not, according to the method of Shimakura et al. (37). The double-stranded oligonucleotides used in the EMSA are listed in Table 1. The OAT1 probes (−882/−853) and (−127/−100) were end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (Takara Bio) and purified through a Sephadex G-25 column (GE Healthcare). EMSA was performed according to Prieur et al. (32) but with some modifications. The OK nuclear extract (10 μg) was incubated in binding buffer [40 mM KCl, 10 mM Tris·HCl (pH 8.0), 1 mM DTT, 6% glycerol, and 0.05% Nonidet P-40] for 10 min at 4°C. Thereafter, one of the labeled probes was added and the mixture was incubated for a further 10 min at 4°C. For competition experiments and supershift assays, excess (50-fold) unlabeled oligonucleotides and antibody (2 μg) were added 10 min and 1 h before the addition of the labeled probe, respectively. The volume of the binding mixture was 20 μl throughout the experiment. The DNA-protein complex was then separated on a 4% polyacrylamide gel for 1.5 h at 200 V and room temperature in 0.5× Tris borate-EDTA buffer. Gels were dried and exposed to X-ray film for autoradiography.
Data analysis.
The results were expressed relative to pGL3-Basic and represent the mean ± SD of three replicates. Two or three experiments were conducted, and representative results are shown.
Determination of the transcription start site of OAT1.
To identify the transcription start site of human OAT1, 5′-RACE was performed. Sequencing of the longest RACE product showed that the terminal position of OAT1 cDNA was located 265 nucleotides above the start codon, which was 43 bp downstream of the 5′-end of OAT1 cDNA registered in the National Center for Biotechnology Information (NCBI) database (accession number NM_004790). No unreported intron was identified in the 5′-flanking region of OAT1, so the 5′-end of OAT1 cDNA registered in the NCBI database was numbered with +1 as the transcription start site in this study.
OAT1 promoter is transactivated by HNF-4α.
To perform the functional analysis of the human OAT1 promoter, the 2,747-bp flanking region upstream of the transcription start site of OAT1 was subcloned into the pGL3-Basic vector. At first, the OAT1 promoter construct −2747/+88 was transiently transfected into a renal epithelial cell line derived from OK, a cell line derived from human embryonic kidney (HEK293), and a human intestinal cell line (Caco-2), and luciferase activity was measured. As shown in Fig. 1, the −2747/+88 construct showed a significant increase in luciferase activity compared with pGL3-Basic in OK cells, which have many characteristics of renal proximal tubule cells, including an organic anion transport system (17). However, this construct had little promoter activity in HEK293 and Caco-2 cells, which lack an organic anion transport system. Therefore, OK cells were used in the subsequent experiments.
Next, to investigate whether the OAT1 promoter is regulated by HNF-1α/β or HNF-4α, the −2747/+88 construct was transiently transfected into OK cells simultaneously with the HNF-1α, HNF-1β, or HNF-4α expression plasmid. Only HNF-4α could transactivate the OAT1 promoter activity markedly; HNF-1α and HNF-1β could not (Fig. 2). These results suggested that the OAT1 promoter is regulated by HNF-4α.
Identification of HNF-4α response elements.
To determine the elements required for the transactivation of OAT1 promoter activity by HNF-4α, a series of deletion constructs of OAT1 were transfected into OK cells simultaneously with HNF-4α, and luciferase activity was measured (Fig. 3). With the −2747/+88 to −1191/+88 constructs, the promoter activity in the presence of HNF-4α increased seven- to ninefold compared with that in the absence of HNF-4α. Deletion of the fragment spanning −1191 to −700 bp reduced the activation of the OAT1 promoter by HNF-4α, and the −536/+88 to −140/+88 constructs showed the same level of activation by HNF-4α as the −700/+88 construct. Further deletion of the fragment spanning −140 to −79 bp almost abolished the responsiveness. These results suggested that the regions spanning −1191 to −700 and −140 to −79 bp contain the elements important for the transactivation of OAT1 promoter activity by HNF-4α.
HNF-4α binds as a homodimer to a DNA sequence consisting of a direct repeat of AGGTCA-like hexamers separated by one or two nucleotides, which were designated as DR-1 and DR-2, respectively (12, 21). We investigated whether the regions mentioned above had DR-1 and DR-2 sites using NUBIScan, an in silico tool to identify nuclear receptor response elements (29). As a result, a DR-2-like element was identified between −875 and −862 bp in the −1191/−700 fragment, while neither a DR-1 nor DR-2 site was found in the −140/−79 fragment. Between −123 and −104, however, the −140/−79 fragment contained an inverted repeat of hexamers separated by eight nucleotides (IR-8), which was recently identified as a HNF-4α response element in the human apolipoprotein AV promoter (32).
HNF-4α binds to DR-2 and IR-8 elements.
To confirm that HNF-4α was able to bind to the DR-2 and IR-8 elements within the OAT1 promoter, EMSA was performed using an OAT1 probe (−882/−853) containing the DR-2 element or OAT1 probe (−127/−100) containing the IR-8 element and nuclear extract from OK cells transiently transfected with HNF-4α (OK-HNF-4α NE). Both OAT1 probes formed a DNA-protein complex (Fig. 4B, lanes 2 and 9), and the formation of the complex was completely impaired by the addition of an excess amount of the unlabeled wild-type oligonucleotides (Fig. 4B, lanes 3 and 10). Mutations in DR-2 (Fig. 4A, mutDR-2-B, mutDR-2-AB) and in IR-8 (Fig. 4A, mutIR-8-A, mutIR-8-B, mutIR-8-AB) abolished the ability to compete with the formation of the complex, while mutDR-2-A weakly competed with the formation of the complex (Fig. 4B, lanes 4–6, 11–13). Furthermore, the DNA-protein complex was supershifted on the addition of the HNF-4α antibody (Fig. 4B, lanes 7 and 14). These results indicate that HNF-4α binds to DR-2 and IR-8 elements within the OAT1 promoter.
Mutagenesis of DR-2 and IR-8 elements.
To examine the contribution of the DR-2 and IR-8 elements to the responsiveness to HNF-4α, mutations at these sites were introduced into the −2747/+88 construct, and OK cells were transfected. Mutations in DR-2-B and IR-8-B reduced the transactivation of OAT1 promoter activity by HNF-4α more than mutations in DR-2-A and IR-8-A, respectively (data not shown); hereafter, mutations in DR-2-B and IR-8-B are regarded as mutations in DR-2 and IR-8, respectively. As shown in Fig. 5, mutations in DR-2 and IR-8 reduced the responsiveness to HNF-4α to two-thirds and one-half of the wild-type level, respectively. Furthermore, mutations in both DR-2 and IR-8 reduced the responsiveness to one-third that of the wild-type. These results suggest that the DR-2 and IR-8 elements in the OAT1 promoter play an important role in the responsiveness to HNF-4α and that IR-8 contributed to the activation by HNF-4α more than did DR-2.
HNF-4α is responsible for basal OAT1 promoter activity.
Focusing on the promoter activity in the absence of HNF-4α (Fig. 5), we found that mutations in IR-8 also reduced OAT1 promoter activity, suggesting that IR-8 is essential for basal promoter activity of OAT1. To investigate the influence of IR-8 on basal activity, we carried out a progressive deletion analysis and a mutational analysis. Transfection of the −2747/+88 construct resulted in a fivefold increase in luciferase activity compared with that of pGL3-Basic, and serial 5′-deletions of the construct from −2747 to −1851, −1191, −700, −536, −270, and −140 gradually increased luciferase activity (Fig. 6A). However, deletion of the fragment spanning −140 to −79 bp significantly reduced the luciferase activity (Fig. 6A). These results suggested that the −140/−79 region containing IR-8 is essential for basal transcriptional activity.
Next, a mutation in IR-8 was introduced into the −140/+88 construct, and luciferase activity was measured. The mutation reduced luciferase activity to one-third of the wild-type level, which was the same level of activity as the −79/+88 construct (Fig. 6B), suggesting that IR-8 is responsible for basal promoter activity of OAT1.
To examine whether endogenous HNF-4α binds to IR-8 without the overexpression of HNF-4α, EMSA was performed using an OAT1 probe (−127/−100) and nuclear extract from intact OK cells (OK-intact NE). Like OK-HNF-4α NE, OK-intact NE also formed a DNA-protein complex which was supershifted on the addition of the HNF-4α antibody (Fig. 7, lanes 2 and 7), suggesting that endogenous HNF-4α binds to IR-8 without the overexpression of HNF-4α. In competition experiments, mutIR-8-B and mutIR-8-AB did not impair the formation of the DNA-protein complex, while mutIR-8-A weakly competed with the formation of the complex (Fig. 7, lanes 4–6).
HNF-4α transactivates human OAT1 promoter activity, and this effect occurs mainly via DR-2 and IR-8 elements, as demonstrated by the deletion analysis, mutational analysis, and EMSA. Furthermore, it was found that HNF-4α is also responsible for basal promoter activity of OAT1 via IR-8 (Fig. 8). Prieur et al. (32) proposed that IR-8 is a novel HNF-4α response element, and our results support this. These findings may be useful for further understanding gene regulation by HNF-4α.
HNF-4α is an orphan member of the nuclear receptor superfamily that is expressed in the liver, kidney, intestine, stomach, and pancreas (27, 40). Liver-specific disruption of the HNF-4α gene indicated that HNF-4α is central to the maintenance of hepatocyte differentiation and lipid homeostasis (16). On the other hand, mutations in the gene encoding HNF-4α cause maturity-onset diabetes of the young (MODY), which is a genetically heterogeneous monogenic form of type 2 diabetes, associated with pancreatic β-cell dysfunction (46). In contrast to the roles of HNF-4α in the liver and pancreas, little has been reported on the physiological implications of HNF-4α in the kidney. In the present study, we clearly indicated that the renal OAT1 gene is transcriptionally regulated by HNF-4α. Because HNF-4α and OAT1 protein showed the restricted intrarenal localization, that is, in the renal proximal tubular epithelial cells (22, 26), it is strongly suggested that HNF-4α is a key factor regulating the intrarenal expression of OAT1 protein.
In the EMSA using OK-HNF-4α NE, the mutDR-2-A oligonucleotide containing an intact DR-2-B competed more with the formation of the DNA-protein complex than did the mutDR-2-B oligonucleotide containing an intact DR-2-A (Fig. 4B, lanes 4 and 5). Similarly, in the EMSA using OK-intact NE, HNF-4α bound to the mutIR-8-A oligonucleotide containing an intact IR-8-B with higher affinity than the mutIR-8-B oligonucleotide containing an intact IR-8-A (Fig. 7, lanes 4 and 5). These results indicate that DR-2-B and IR-8-B had greater activity to bind HNF-4α than did DR-2-A and IR-8-A, respectively, which are consistent with the finding that mutations in DR-2-B and IR-8-B reduced the responsiveness to HNF-4α more than mutations in DR-2-A and IR-8-A, respectively (data not shown). These findings suggest that DR-2-B and IR-8-B are important to the transactivation of the OAT1 promoter via HNF-4α compared with DR-2-A and IR-8-A. In the EMSA using OK-HNF-4α NE, both oligonucleotides mutIR-8-A and mutIR-8-B failed to impair the formation of the DNA-protein complex (Fig. 4B, lanes 11 and 12). It is possible that the abundance of HNF-4α caused the mutIR-8-A oligonucleotide to fail to completely compete with the formation of the complex.
It has been demonstrated that individual variation in the expression levels of drug transporters is responsible for the individual variation in pharmacokinetics by experiments using human tissue (15, 35, 36) and laboratory animals (11, 14, 20). Single nucleotide polymorphisms in the coding region [coding SNPs (cSNPs)] of drug transporter genes are also thought to be responsible for the variation in drug responses among individuals (19). Although there have been few reports on cSNPs of the human OAT1 gene, allele frequencies of these cSNPs were very low (5, 13, 45). In addition, Fujita et al. (13) reported that there was no significant decrease in the renal secretory clearance of adefovir in subjects with R454Q, which resulted in a complete loss of OAT1 function in vitro. These reports suggest that cSNPs of human OAT1 are unlikely to influence the interindividual variation in the drug responses and that OAT1 expression is an alternative candidate for the cause of the variation in pharmacokinetics among individuals. Recent studies suggest that SNPs in the promoter region [regulatory SNPs (rSNPs)] can alter the transcription of genes (7). In the OAT1 gene, Bhatnagar et al. (4) found one rSNP ∼3 kb upstream of the transcription start site, but it is unclear whether this rSNP affects the mRNA level of OAT1. We previously reported that the OAT1 mRNA level varies and is significantly lower in the kidney of patients with renal diseases than in the normal kidney cortex (36). Accordingly, it is important to clarify the cause of the interindividual differences in OAT1 mRNA expression and the reduction in renal diseases. The present study suggests that HNF-4α regulates OAT1 expression at the mRNA level. Regulatory SNPs of OAT1, the HNF-4α expression level, or cSNPs in the HNF-4α gene that led to a loss of function may be the cause of the interindividual variation in OAT1 mRNA levels.
Tissue-specific gene expression is generally regulated by transcription factors (48). For example, we previously demonstrated that the intestine-specific transcription factor Cdx2 is responsible for the intestine-specific expression of H+/peptide cotransporter 1 (38). OAT1 mRNA is mainly expressed in the kidney (1), and we found that HNF-4α is responsible for the transcriptional regulation of the OAT1 gene. Interestingly, the major expression site of HNF-4α is the liver, but OAT1 mRNA is not expressed in the liver. Furthermore, the human OCT1 gene is also under the control of HNF- 4α (34), but OCT1 mRNA is expressed in the liver but not in the kidney. Thus it is difficult to explain the renal-specific expression of OAT1 by HNF-4α itself. Other transcription factors that suppress the hepatic expression of OAT1 (or renal expression of OCT1) may contribute to tissue-specific expression. Alternatively, microRNA and DNA methylation of the promoter may be involved in the tissue-specific expression of genes (39, 44). Further studies are needed to elucidate the mechanism of tissue-specific expression of the SLC22A family.
In conclusion, the present study indicates that the human OAT1 gene is regulated by HNF-4α. This is the first report on the transcriptional regulation of OAT1. In addition, OAT1 is the first gene in the kidney whose promoter activity has been demonstrated to be regulated by HNF-4α.
This work was supported in part by the 21st Century COE program "Knowledge Information Infrastructure for Genome Science," a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and a Grant-in-Aid for Research on Advanced Medical Technology from the Ministry of Health, Labor and Welfare of Japan. J. Asaka is supported as a research assistant by the 21st Century COE program "Knowledge Information Infrastructure for Genome Science."
We thank Dr. Marco Pontoglio (Institute Pasteur, Paris, France) for kindly providing the human HNF-1α and HNF-1β expression vectors.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


text:
|Home | About | Journals | Submit | Contact Us | Français
The nuclear receptor CAR (NR1I3) regulates transcription of genes encoding xenobiotic- and steroid-metabolizing enzymes. Regulatory processes that are mediated by CAR are modulated by a structurally diverse array of chemicals including common pharmaceutical and environmental agents. Here we describe four in-frame splice variants of the human CAR receptor gene. The variant mRNA splice transcripts were expressed in all human livers evaluated. Molecular modeling of the splice variant proteins predicts that the structural effects are localized within the receptor's ligand-binding domain. Assays to assess function indicate that the variant proteins, when compared with the reference protein isoform, exhibit compromised activities with respect to DNA binding, transcriptional activation and coactivator recruitment.
CAR (constitutive androstane receptor, NR1I3) is a nuclear receptor that mediates the transcriptional activation of genes encoding phase 1 (e.g. CYP2B6 and CYP3A4) and phase 2 (e.g. UDP-glucoronysyltransferase 1A1) xenobiotic- and steroid-metabolizing enzymes, as well as a number of xenobiotic transporter proteins (e.g. multidrug resistance-associated proteins 2 and 3) (1–5). Nuclear receptors, such as CAR, the pregnane X receptor (PXR), peroxisome proliferator-activated receptor α (PPARα) and the aryl-hydrocarbon receptor, transcriptionally and coordinately regulate the expression of a battery of enzymes that function to metabolize a vast array of lipophilic molecules in mammalian tissues (6–9). These receptors are themselves activated or regulated by a similarly overlapping network of exogenous and endogenous substances. CAR is an essential mediator of the transcriptional response to a subclass of these structurally diverse chemicals, represented by the prototypical inducing agent, phenobarbital (PB) (10).
CAR is expressed primarily in the liver and to a lesser extent in the intestinal epithelium and the stomach (11). It is a member of the NR1I subfamily that also includes the vitamin D receptor (VDR) and PXR (12). Although most nuclear receptors have five domains, the human CAR (hCAR) protein contains three: a highly conserved DNA-binding domain (C domain); a hinge domain (D domain); and a divergent ligand-binding/dimerization/transcriptional activation domain (E domain). hCAR lacks an A/B domain that typically confers a ligand-independent transactivation response, and the more poorly characterized hypervariable F domain (13). CAR interacts with DNA as a heterodimer with retinoid X receptor (RXR) (14). The heterodimer can bind an array of prototypical nuclear receptor-binding sites, including DR-5 (direct repeat-5), DR-4, DR-3, ER-6 (everted repeat-6) and IR-8 (inverted repeat-8) motifs (2,15). A number of these response elements have been identified in the promoters of PB-responsive human genes (1,4,16). As with many nuclear receptors, CAR interacts with transcriptional coregulators, including p160 family members—steroid receptor coactivators (SRC-1, -2 and -3), small heterodimerization protein and nuclear receptor corepressors (11,17–21).
hCAR activity may be positively, or negatively, regulated by ligand interactions. For example, clotrimazole is an hCAR ligand than functions as an inverse agonist, an activity typical of most identified hCAR ligands (22), whereas 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime has been identified recently as an hCAR agonist (23). However, PB and other PB-like inducers do not function as direct CAR ligands, but rather activate hCAR by stimulating its nuclear translocation from the cytosol (24). The latter process appears ultimately important in governing receptor activity and remains relatively poorly understood (3).
In mice, the hepatocyte transcriptional response to PB is dependent on the CAR receptor and independent of PXR (10,25). However, due to significant species differences in the PB induction response, results from mouse studies do not necessarily represent the processes occurring in human hepatocytes. For example, studies suggest that PB is an activating ligand of human PXR (22,26,27) and that hPXR can control the transcriptional expression of a number of PB-inducible genes (7). Other results indicate that the human VDR also plays a role in regulation of the PB-inducible genes, CYP2B6, CYP3A4 and CYP2C9 (28). These findings suggest that an array of nuclear receptors are involved in regulating responses to PB-like inducing agents in humans, consistent with the idea that a collection of receptors probably function as a xenobiotic 'safety net' (29).
Considering the structural diversity of PB-like inducing agents, it appears unlikely that CAR, PXR and VDR exclusively mediate the biological responses to these agents. Alternatively spliced isoforms of such nuclear receptors may possess properties distinct from their reference counterparts, enabling expansion of receptor functional diversity and perhaps differential responsiveness to given subsets of PB-like inducing substances. Specifically, we hypothesized that splice variants of hCAR exist, possessing unique structures with altered receptor activities. In this investigation, we report the cloning and characterization of four distinct splice forms of the hCAR derived from human liver cDNA samples. All of the variants encode insertions or deletions of the ligand-binding domain. Examination of cDNA transcript levels from a human liver bank revealed that CAR transcript isoforms are commonly expressed in individuals. Functional characterization indicated that the variants possess altered capacities of DNA interaction, transcriptional activation and coactivator recruitment.
Human hepatocyte cDNA samples (30) were amplified for 30 cycles at an annealing temperature of 60°C using VENT DNA polymerase (New England Biolabs, Beverly, MA). DNA primers used for the PCR amplification were: hCAR FP, with an incorporated EcoRI site (5′-GATCGAATTCGTCATGGCCAGTAGGGAAGATGAG-3′), and hCAR RP, with an incorporated EcoRV + stop site (5′-GATCGATATCTCAGCTGCAGATCTCCTGGAGCCAG-3′). PCR cycling was performed at 95°C for 30 s, 60°C for 30 s and 72°C for 90 s. Purified products were separated by agarose gel electrophoresis, and fragments in the range of 800–1200 bp were purified using a Qiagen gel purification kit (Qiagen, Chatsworth, CA). Purified fragments were blunt-end ligated into the SmaI site of pBluescript and transformed into DH5α ultracompetent cells (Invitrogen, Carlsbad, CA). Colonies were screened by PCR to determine the presence of an insert within the vector. Positive clones were grown overnight in 2 ml cultures containing ampicillin, purified, and sequenced using an ABI 310 automated sequencer (Applera Corp., Foster City, CA).
The full-length coding sequence for each hCAR isoform was subcloned into the mammalian expression vector pTracer CMV.2 (Invitrogen) using EcoRI–EcoRV restriction sites. To generate GST–hCAR fusions, the hCAR-coding sequence was subcloned in-frame with GST in the bacterial pGEX4T-1 vector using EcoRI–EcoRV sites. For bacterial expression, T7-hCAR fusion constructs were prepared by cutting the hCAR-coding sequence from the pTracer CMV.2 vector with EcoRI and NotI and subcloning the released fragment into the pET21a bacterial expression vector (Novagen, Madison, WI). Human RXRα (a gift from P. Chambon, IGBMC, Universite Louis Pasteur, Illkirch, France) was also subcloned into the EcoRI site in pTracer CMV.2. The GST–hRXRα bacterial expression plasmid was a gift from Dr Karen Swisshelm (University of Washington).
pGL-3 Basic PBREM-TK-Luciferase was generated by insertion of one copy of the CYP2B6 PB-responsive enhancer module (PBREM). The DNA primers CYP2B6 BS-FP (5′-TTGGTTCAGGAAAGTCCATGCTGCCACCTCTTCAGGGTCAGGAAAGTACAG-3') and CYP2B6 BS-RP (5′-ACTGTACTTTCCTGACCCTGAAGAG-3′) were annealed and extended with Klenow DNA polymerase. The fragment was then blunt-end ligated into the SmaI restriction site in pGL3-Basic Vector (Promega, Madison, WI), containing a thymidine kinase promoter (TK-luc3). TK-luc3 was prepared as described previously (16). Sequence analysis identified clones that were positive for a single full-length copy of the PBREM element.
The coding sequence of the SRC-1 receptor interaction domain (RID; amino acids 632–754) was amplified with Platinum Taq (Invitrogen), using a human SRC-1 FP that incorporated an EcoRI site (5′-GATCGAATTCAAACTAGTGCAGCTTTTGAC-3′), and human SRC-1 RP that included a translation stop signal (5′-GAATTCTCATCAATCATCCAGGCTCAGGTTTGGAG-3′). The PCR fragment was cloned in pGEM-T (Promega). Clones were characterized by PCR and by DNA sequencing. To generate a GST-fused construct, the SRC-1 RID fragment was subcloned into the EcoRI restriction site of pGEX4T-1 (Amersham Pharmacia Biotech AB, Piscataway, NJ).
A rabbit polyclonal antibody was generated against an epitope of the hCAR ligand-binding domain, N-KGQQRRPRDRFLYAKL-C (Quality Control Biochemicals, Hopkinton, MA). Antibody was affinity purified and dialyzed against phosphate-buffered saline (PBS) as described in the supplier's protocol. The specificity of the antibody was determined by western blot of crude bacterial lysates containing recombinantly expressed hCAR. The antibody reacted with all isoforms of hCAR, but not with any components present in crude lysates.
CAR was modeled based on the reporter structure of the VDR, 1DB1(31). Models were generated with the HOMOLOGY module of INSIGHTII software (Accelrys, San Diego, CA) using established protocols. Protein backbone coordinates were taken from VDR for all helices, strands and loops with identical lengths.
Access to a human liver tissue bank was generously provided by Dr Kenneth Thummel, University of Washington. cDNA fractions were prepared from sample livers and subjected to PCR amplification with primer pairs designed to generate amplicons that spanned the exon 6–7 splice junction, the exon 7–8 junction, or both splice junctions combined. The amplicons were separated by agarose gel electrophoresis and stained with ethidium bromide. Primers used for detection of the reference (wt) and 12 bp insertion at the exon 6–7 junction were Exon 6 FP (5′-GACCAGATCTCCCTTCTCAAG-3′) and Exon 7 RP (5′-CTCAGGCTCTTGGAGCTGCAG-3′). Primers used for detection of the wt and 15 bp insertion at the exon 7–8 junction were Exon 7 FP (5′-GGAGTTGCTCTTTCACTTCCA-3′) and Exon 8 RP (5′-CCTTCGCTGCTGGCCCTTGATG-3′). Primers used for detection of the exon 7 deletion were Exon 6 FP and Exon 8 RP. PCR amplifications were performed in a 50 µl reaction volume containing 3.0 mM MgCl2, 25 pmol of each primer and 0.5 µl of cDNA. Cycling was conducted at 95°C for 30 s, 60°C for 30 s and 72°C for 30 s. For detection of the 12 bp (exon 6–7 junction) and 15 bp (exon 7–8 junction) insertions, the amplification was carried out for 32 cycles, whereas detection of the exon 7 deletion variant was completed in 35 cycles.
COS-1. Approximately 400 000 COS-1 cells were plated in each well of a 6-well plate and the cells were allowed to attach overnight. Cells were transfected using Lipofectamine (Invitrogen) with either 2 µg of hCAR expression plasmid or empty vector control, in accordance with the manufacturer's protocol. Whole-cell lysates were generated 24 h post-transfection by application of RIPA buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) containing a protease inhibitor cocktail (Calbiochem, La Jolla, CA).
BL21. The GST or GST-fused proteins (GST–hCAR, GST– SRC-1-RID) were expressed using the pGEX4T-1 bacterial expression plasmid (Amersham Pharmacia Biotech AB) in the Escherichia coli strain BL21(DE3). In brief, 3 ml of overnight bacterial growth was added to 27 ml of 2× YT broth containing 100 µg/ml ampicillin and allowed to reach an optical density of 0.6–1.0 (1–2 h) at 37°C. Bacteria were then induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to 0.5 mM and incubated for an additional 3 h at room temperature. Cells were collected by centrifugation and lysed by sonication in PBS containing protease inhibitors (Calbiochem). Cell debris was removed by centrifugation, and the supernatant was added to 300 µl of glutathione– Sepharose 4B (Amersham Pharmacia Biotech AB). GST fusion proteins were bound to the resin during rotation at room temperature and then washed four times with PBS, in a total wash volume of 4 ml. Depending on the procedure, proteins were either eluted in glutathione elution buffer (10 mM reduced glutathione in 50 mM Tris–HCl pH 8.0) or were equilibrated with GST interaction buffer (2 mg/ml bovine serum albumin, 25 mM HEPES pH 7.6, 100 mM NaCl, 20% glycerol and 1 mM EDTA) (32). Eluted proteins were visualized with PAGE and subsequent Coomassie staining. The hCAR isoforms were also expressed in bacteria as T7-tagged proteins using the pET21a vector. T7-tagged proteins were expressed using the same procedure; however, no affinity purification followed the sonication step.
PCR products were generated that contained an open reading frame encoding hCAR isoforms or RXR, in addition to a 5′ T7 promoter and a region directing 3′ polyadenylation. The PCR products were then used as templates for transcription in the TNT-coupled reticulocyte lysate (Promega Corp.). Protein expression was carried out in the presence of [35S]methionine as described in the manufacturer's protocol.
Soluble fraction proteins from 30 µg of total transfected COS-1 cell lysate, 20 µg of crude bacterial lysate (soluble fraction) or 200 µg of whole human liver lysate were separated by denaturing PAGE and transferred to a PDVF membrane. The membrane was incubated with an hCAR antibody (1:5000) generated in our laboratory, as described above. The primary antigen reaction was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (Biorad, Hercules, CA). Proteins were visualized using the enhanced chemiluminescence reagent kit (Amersham Pharmacia Biotech AB).
EMSAs were performed in a similar manner to the procedures already described (16) with the exception that bacterially expressed and purified GST-fused proteins were used. Briefly, GST, GST–hCAR isoforms and GST–hRXRα were expressed as described above and proteins were incubated in combination with double-stranded 32P-labeled oligonucleotides encoding either the CYP2B6 NR1 or NR2 element (100 000 c.p.m. per reaction). DNA–protein complex formation was conducted at room temperature in the presence of the previously described binding buffer and 2 µg of poly(dI/dC). Complexes were resolved in 5% non-denaturing polyacrylamide gels. The gels were dried and mobility shifts were visualized by autoradiography.
Cells were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). One day prior to transfection, cells were trypsinized and plated onto 24-well plates (100 000 cells/well). For determination of transcriptional activity of the variant hCAR forms, cells were transfected using Lipofectamine with 50 ng of pRL (Renilla luciferase; Promega) for normalization, 50 ng of pTracer CMV.2-hRXRα, 200 ng of pGL-3-PBREM-TK-Luciferase and 200 ng of either pTracer CMV.2-hCAR or variant hCAR constructs. Transfections were performed according to the manufacturer's recommendations.
Dual-luciferase assays were performed as per the manufacturer's protocol (Promega, Dual-Luciferase Reporter Assay System).
GST 'pull-down' assays were performed as previously described (32). After the last wash, complexes were suspended in 2 ml of interaction buffer (described above), and 150 µl of the mixture was used for each reaction. A total of 350 µl of addition interaction buffer was then aliquoted into each tube. An additional 150 µl of the GST and GST–SRC-1 RID suspension was eluted from the beads in 10 mM reduced glutathione and separated in a pre-cast 10% Tris–HCl gel (Biorad). The gel was Coomassie stained to visualize expression of the proteins. To each reaction, 5 µl of 35S-labeled in vitro translated hCAR or hCAR variant was added and the complexes were incubated overnight at 4°C with rotation. The complexes were washed four times with a total of 4 ml of interaction buffer and were eluted in 50 µl of 10 mM glutathione. Eluted protein was separated in a pre-cast 10% Tris–HCl gel (Biorad), and the gel was dried and exposed to film.
To identify novel isoforms of hCAR, we designed oligonucleotide primers targeted to the amino acid-coding sequence of the reference mRNA (14). PCR was performed on an individual human liver cDNA sample and the products were subcloned into pBluescript. Twenty clones were selected at random and subjected to DNA sequencing. A distribution of five reference, four 12 bp insertion, eight 15 bp insertion and one 12/15 bp double insertion clones was obtained (Fig. (Fig.1B1B and C). Two additional clones contained complete intron 8 insertions, probably a product of incompletely processed mRNA. The exon 7 deletion plasmid was detected in a similar manner from a separate set of cDNA clones. The nomenclature of the hCAR variants that is used throughout this manuscript is presented in Table Table11.
The reference isoform of hCAR is a 348 amino acid protein with a theoretical molecular weight of 39.558 kDa. The genomic sequence of human CAR is located in contig NT_026945.9. As shown in Figure Figure1A1A and B, the reference mRNA isoform is composed of nine exons (NCBI LocusLink Report number 9970) (Fig. (Fig.1A1A and B) and the DNA-binding domain is encoded by exons 2, 3 and 4 (Fig. (Fig.1A).1A). A small hinge domain is localized to a portion of exon 4, while the ligand-binding domain is encoded by exons 4–8 together with the 5′ portion of exon 9 (Fig. (Fig.11A).
The 12 bp insertion hCAR is localized between exon 6 and exon 7 and is produced by an alternative 3′ acceptor site that incorporates 12 nucleotides of intron 6 (Fig. (Fig.1B).1B). The transcript encodes an in-frame insertion of 4 amino acids (SPTV) in the ligand-binding domain (Fig. (Fig.1C).1C). The theoretical molecular weight of this isoform is 39.942 kDa. It is noteworthy that an isoform of CAR was cloned recently from rhesus monkey (GenBank accession: AY116212) that contains a 12 bp insertion identical to that identified for hCAR, perhaps indicating that the respective 4 amino acid CAR isoform is primate specific. A similar 4 amino acid insertion (SLSR), produced by apparently identical splicing events, occurs in both human and mouse RXRβ3 (33–35).
The hCAR 15 bp insertion occurs between exon 7 and exon 8 and is produced by an alternative 3′ acceptor site that incorporates 15 bp of intron 7 (Fig. (Fig.1B).1B). The transcript encodes an in-frame insertion of 5 amino acids (APYLT) (Fig. (Fig.1C).1C). The theoretical molecular weight of this isoform is 40.103 kDa. Independent confirmation of the existence of the transcript is found in the AceView database (LocusID 9970, mRNA variant c and d). Following sequence alignment and structural analysis, a similar insertion point of 8 amino acids (GVYTFLSS) was identified in estrogen receptor α, and shown to be involved in receptor dimerization (36).
CAR transcripts that encode a deletion of exon 7 are predicted to result in a protein containing 39 fewer amino acids. The theoretical molecular weight of this CAR isoform is 35.042 kDa. Independent confirmation of the exon 7 deletion transcript is indicated by examination of the Aceview database (LocusID 9970, mRNA variant c).
Clones of the variant hCAR isoforms were observed in cDNA derived from four human livers. However, the question still existed as to whether the mRNAs encoding the isoforms occur at large in the human population. To evaluate this issue, PCR amplifications using primer sets designed to detect each of the identified different isoforms of hCAR were performed on cDNA samples derived from a tissue bank of 25 human livers. Each of the hCAR isoforms was detected readily by PCR analyses in all of the 25 liver samples (data not shown). Data from representative groups of liver samples are presented in Figure Figure2.2. Different livers are represented in the respective panels due to limitations in the available tissue resource. The amplification of the exon 6–7 junction produced two bands that were sequence verified to correspond to cDNA encoding the hCAR-REF isoform and the cDNA encoding the hCAR-4aaINS isoforms, respectively (Fig. (Fig.2A).2A). Amplification of the exon 7–8 junction produced three bands, two of which corresponded to the anticipated hCAR-REF and hCAR-5aaINS clones (Fig. (Fig.2B).2B). When the third lower mobility band was subjected to DNA sequence analysis, the data demonstrated that the fragment encoded only the 15 bp CAR insertion. This same banding pattern was reproduced by amplification with different primer pairs and different cDNA preparations and, therefore, its migration is anomalous. The exon 7 deletion isoform of hCAR was detected by use of an exon 6 forward primer and exon 8 reverse primer. In Figure Figure2C,2C, a doublet band is present in the position of the exon 7 deletion amplicon, probably consisting of an mRNA isoform containing the exon 7 deletion plus 15 nucleotides of intron 7 (as found in the mRNA encoding the hCAR-5aaINS). The existence of the latter isoform was supported by data contained within Aceview database (LocusID 9970, mRNA variant c).
We assessed whether hCAR variants were detectable as stable protein isoforms in human liver samples. Western immunoblot analyses were conducted using the soluble fractions of human liver whole-cell lysates (Fig. (Fig.3).3). The antibody used in these experiments targets an epitope external to the regions of polymorphism and is therefore capable of detecting all hCAR isoforms described in this report (Fig. (Fig.1D).1D). In addition, the antibody produces only minimal background immunoreactivity within the 30–50 kDa size range (Fig. (Fig.4B4B and C, empty vector lanes). However, the antibody reacted with four distinctive immunoreactive bands, apparent in Figure Figure3.3. The strongest band (no. 2) migrates at ~40 kDa, corresponding to the estimated size of the CAR REF isoform, as well as the 4aaINS, 5aaINS and dblINS isoforms. Because these isoforms differ from one another by only a few amino acids, their unambiguous resolution is difficult using 10% PAGE. However, additional immunoreactive protein bands were detected in several liver samples, including band no. 3, migrating at ~35 kDa, a size anticipated to correspond to the hCAR-39aaDEL isoform. Immunoreactive bands were also apparent just below 50 kDa (no. 1) and just below 35 kDa (no. 4). Despite the difficulty inherent in resolving proteins differing by <1 kDa, the western immunoblot data provided suggestive evidence for an array of immunoreactive CAR protein species in human liver.
We questioned whether the cloned variant hCAR cDNAs were capable of producing stable full-length proteins. To this end, hCAR protein isoforms were expressed in three separate systems, in E.coli, in a rabbit reticulocyte lysate system and in mammalian cells transfected with the respective expression constructs. Figure Figure4A4A presents the results of the TNT- coupled transcription and reticulocyte translation assays, conducted in the presence of [35S]methionine with subsequent visualization of the proteins separated by SDS–PAGE. The data demonstrate that all of the identified hCAR isoforms are expressed and resolved in this system. The full-length and 4, 5 or 9 (dbl) amino acid variant hCARs migrated at ~40 kDa, with slight, yet discernible separation of the bands as predicted by theoretical molecular weight analysis. The hCAR-39aaDEL isoform was also clearly apparent at its correspondingly lower molecular weight. In addition, Figure Figure4B4B illustrates that each of the respective hCAR protein isoforms is expressed stably in E.coli. Similarly, Figure Figure4C4C further demonstrates the expression of each hCAR variant in mammalian cells, using transfection approaches in COS-1 cells.
We conducted computer homology modeling studies to better assess the potential structural impact of the various amino acid insertions and to predict their possible functional effects. Currently, a crystal structure does not exist for any mammalian CAR protein. Therefore, our modeling approach required the use of homologous structures in the crystal database, and several criteria were taken into consideration. First, since all structural variation identified in our study mapped to the hCAR ligand-binding domain (amino acids 102–348), it was required that the homologous template structure must align to that region. Secondly, in order to generate a model of significant accuracy, the template needed to exhibit a sequence identity of >30% (37). Thirdly, since CAR is constitutively active, it was desirable to identify a template that was crystallized in the presence of an agonist and therefore likely to exhibit the structural state of an active receptor. A Psi-blast of the protein sequences database derived from the Brookhaven Protein Data Bank was performed with the hCAR amino acid sequence as the query. The iterative search revealed two possible model structures that fit the above criteria. hPXR (1ILH) (38) exhibited 57% sequence identity to hCAR, while hVDR (1DB1) (31) exhibited 37% sequence identity. Relative to the CAR structure, hPXR and hVDR contain large insertions between helix 1 and helix 3 of their respective ligand-binding domains that do not align with hCAR. Although the amino acid sequence of the crystallized hPXR protein retains this insertion, the insertion was largely removed from the sequence of the crystallized hVDR protein (31) (Fig. (Fig.5C).5C). Therefore, despite the higher overall sequence identity with hPXR, hVDR (1DB1) was selected as the most appropriate template structure to model the hCAR variant receptors.
Figure Figure5A5A illustrates a model of hCAR-REF indicating features of a fold that are typical of a nuclear receptor ligand-binding domain, including 10 α-helices and a single β-sheet composed of three β-strands. The root mean square deviation between our model and hVDR over 245 superimposed Cα positions was 0.61 Å. Visual evaluation of the superimposed structures of hCAR-REF and 1DB1 indicated little deviation within the core of the structure (helix 3–helix 10/11) with the exception of the loop between helix 9 and 10, the loop between helix 3 and 4, and the β-turn between S2 and S3. In regions corresponding to the loop between helix 1 and helix 3, and the loop between H10/11 and H12, there was substantial deviation of the model from the template due to additional amino acids present in 1DB1 that are not present in hCAR. In addition to this deviation predicted for the hCAR-REF structure, the 4 or 5 amino acid insertions embedded in the hCAR isoforms produced marked deviation from the local structural alignment.
Figure Figure5B5B depicts the predicted effects resulting from the incorporation of the 4 amino acid insertion, SPTV CAR variant. The predicted structural impact of this insertion includes a C-terminal extension of helix 6 producing an alteration in the structure of the ligand-binding cavity (Fig. (Fig.5C).5C). This prediction is supported by modeling studies of hCAR performed by others that noted the integral role of helix 6 in the formation of the ligand-binding pocket (5).
Insertion of the five amino acids (APYLT), corresponding to the 15 bp insertion hCAR variant, expands the loop between helix 8 and helix 9 (Fig. (Fig.5B5B and C). Superimposing our hCAR model containing the 5 amino acid insertion over that of PPARγ, within the PPARγ–RXRα heterodimer structure, 1FM9 (39), enables the prediction that the 5 amino acid insertion in hCAR would produce a bulged loop within this domain, probably resulting in steric hindrance of the dimerization potential of CAR with RXR.
Removal of the amino acids encoded by the exon 7 deletion variant characterized in this report is consistent with the removal of α-helices 7 and 8 of the CAR reference protein structure, and therefore is likely to result in the collapse of the hCAR ligand-binding domain.
A well-characterized property of the reference form of hCAR is its ability to bind a subset of DNA response elements as a heterodimeric complex with RXRα (14,16). The predicted structural variation inherent in the identified hCAR isoforms predominantly impacts the protein domain participating in heterodimerization, a prerequisite for DNA binding. Therefore, it is possible that the variant isoforms of hCAR exhibit altered DNA-binding capacities.
To examine this latter issue, we performed EMSAs using NR1 and NR2 elements derived from the CYP2B6 PBREM (16) using expressed, affinity-purified, GST fusion proteins of hRXRα and the hCAR isoforms (Fig. (Fig.6A).6A). GST alone did not bind to either element (Fig. (Fig.6B,6B, lane 1). We were surprised to find that all of hCAR isoforms interacted with DNA in the absence of RXR (Fig. (Fig.6B,6B, lanes 2–5), in particular the NR2 element. These results indicate that hCAR may possess a limited ability to homodimerize. Similarly, hRXRα alone interacted with the NR1 element, although not with NR2 (Fig. (Fig.6,6, lane 6). hCAR-REF–RXR bound robustly to both the NR1 and NR2 elements (Fig. (Fig.6,6, lane 7). The hCAR-4aaINS–RXR complex interacted with both the NR1 and NR2 elements, albeit at much weaker levels than the hCAR-REF–RXR complex (Fig. (Fig.6,6, lane 8 versus lane 7). The two remaining hCAR isoforms also appeared to bind weakly to NR1 elements in the presence of RXR. Since GST itself may dimerize and therefore mask apparent dimerization of GST–CAR with GST–RXR, we conducted additional studies in the presence of an excess (50×) of free GST. Inclusion of excess free GST did result in a reduced NR1 DNA interaction for the hCAR-5aaINS–RXR and hCAR-dlbINS–RXR complexes, but excess GST did not affect any of the remaining protein–DNA interactions (data not shown). As might be predicted from the extent of the deletion, results from additional experiments employing the hCAR-39aaDEL construct demonstrated that it lacked the ability to bind either the NR1 or NR2 element as a heterodimeric complex with RXR (data not shown).
The reference form of hCAR transactivates gene expression through a number of response elements observed in PB-responsive genes, with the human PBREM response element of CYP2B6 representing a well-characterized region (16). We generated a PBREM-TK-luciferase reporter and evaluated the transactivation capacity of the different hCAR isoforms in co-transfection studies. Reporter expression was assessed following co-transfection with an RXRα expression plasmid, the Renilla luciferase reporter (for transfection normalization), and either hCAR reference, hCAR variants or empty vector expression plasmids. The reference isoform induced the reporter expression ~3-fold over the empty vector control (Fig. (Fig.7A).7A). The only other isoform noted to exhibit activity was hCAR-4aaINS, which activated the reporter ~2-fold (Fig. (Fig.7A).7A). A TK-luciferase reporter without the PBREM sequence was not activated by any of the hCAR isoforms.
We reasoned that hCAR variants may function to transactivate reporter expression in a secondary manner, e.g. acting in concert with hCAR-REF, or perhaps through dominant-negative mechanisms. Remarkably, the hCAR-4aaINS isoform appeared to act in concert with the reference isoform when transactivating the reporter (Fig. (Fig.7B).7B). However, when the reference isoform of hCAR was co-transfected sequentially with a 2-fold molar excess of each of the other hCAR variants, no further alterations of hCAR-REF activity were observed (Fig. (Fig.77B).
Another property of reference hCAR that can be evaluated using the transfection approach is repression of its transactivation potential by the ligand clotrimazole (22). Clotrimazole additions repressed hCAR-REF transactivation, and also repressed transactivation stimulated by the hCAR-4aaINS isoform. Clotrimazole additions produced no observable effect on any of the other hCAR isoforms (Fig. (Fig.77C).
It has been reported that CAR interacts with SRC-1 in the absence of ligand (15). Analysis of our model, whereby hCAR was superimposed upon 1FM9 (the 1FM9 structure was derived by co-crystallization with an SRC-1 coactivator fragment), led us to predict that the hCAR variant insertions would not disrupt the interaction with SRC-1. To evaluate the SRC-1 interactions more directly, we performed protein interaction assays with in vitro translated 35S-labeled hCAR protein (Fig. (Fig.8A)8A) and the human SRC-1 RID fused to GST (Fig. (Fig.8B).8B). The interaction of GST–SRC-1 with hCAR-REF was robust (Fig. (Fig.8C,8C, lane 2), with no detectable interaction apparent for hCAR-REF and GST alone (Fig. (Fig.8C,8C, lane 1). Interactions of the hCAR variant isoforms with GST–SRC-1 were weaker, but specific (Fig, (Fig,8C;8C; lanes 4, 6, 8 and 10).
Previous reports have documented alternative splicing events in the mouse and human that likely impart structural and functional diversity within the NR1I subfamily (39–44). PXR and VDR splice variants have been described that appear to possess altered receptor activities (40,42–44). An alternatively spliced isoform of mCAR was identified in earlier studies (39). More recently, data obtained from the use of ribonuclease protection assays suggested alternative splicing within the ligand binding domain of hCAR (41). Combined, these observations lend support to the concept that alternative splicing may serve as an important mechanism for expanding the repertoire of xenobiotic receptors available to the mammalian cell.
In this investigation, we have identified and partially characterized activities associated with four novel isoforms of hCAR. These isoforms are encoded by alternatively spliced transcripts of the hCAR gene. One transcript incorporates 12 nucleotides of intron 6 through an alternative splice acceptor site, encoding an in-frame 4 amino acid insertion product, the hCAR-4aaINS isoform. Another transcript incorporates 15 nucleotides of intron 7 through an alternative splice acceptor site, encoding an in-frame 5 amino acid insertion variant, the hCAR-5aaINS isoform. A third transcript incorporates both splicing alterations simultaneously, encoding hCAR-dblINS. The fourth variant transcript, encoding hCAR-39aaΔ, results from a complete deletion of exon 7, also translationally in-frame. Each of the respective hCAR isoforms are predicted to possess altered structure, residing in the receptor's E domain. The E domain, commonly referred to as the ligand-binding domain, performs integrated functions of dimerization, coregulator recruitment and transactivation (45). In this report, these functions were evaluated for each of the variant hCAR receptors.
An important issue in defining receptor function involves demonstration of its stable expression, both in vivo and in vitro. We have presented evidence supporting the biological relevance of variant hCAR receptors. First, mRNA transcripts encoding each of the hCAR isoforms characterized herein were detected using RT–PCR analysis in 25 human liver tissues sampled to date. Secondly, each of the hCAR protein transcripts was efficiently and stably generated in multiple in vitro expression systems, including transformed bacteria, reticulocyte lysates and transfected mammalian cells. Thirdly, each of the identified hCAR isoforms codes for in-frame translation variants and therefore would not be expected to be targeted by nonsense-mediated mRNA decay mechanisms that degrade premature termination products (46). Fourthly, western blots derived from human liver extracts suggest the presence of multiple CAR protein isoforms.
Amplification of cDNA derived from liver samples indicated that variant isoforms of hCAR are likely to be present in all humans. However, the RT–PCR analysis presented here does not yield an accurate account of the abundance and ratios of these transcripts. These values may exhibit a large interindividual variability. It is already known that hCAR mRNA transcript levels vary significantly between liver samples (47), and that activation of certain signaling cascades alters levels of CAR mRNA (41,48). Consequently, one intriguing question is whether genetic variables play a role in expression of hCAR mRNA and/or the ratio of CAR transcripts. A precedent for signal transduction cascades that directly impinge on pre-mRNA splicing events has been presented (49). Hence, a further intriguing question is whether treatments with dexamethasone or interleukin-6 affect the ratio of CAR transcripts and, in turn, what effect such exposures may have on responsiveness to inducing agents.
EMSA analysis revealed that of the hCAR variants assessed, the hCAR-4aaINS isoform was uniquely capable of interacting with both NR1 and NR2 elements, when heterodimerized with RXR. Based on our results from computer homology modeling, a potential explanation for the latter result is advanced. Insertion of the 5 amino acid sequence, APYLT, between helices 8 and 9 was predicted to stericly hinder the dimerization interface with RXR. Even more dramatically, removal of 39 amino acids, as encoded by the exon 7 deletion variant, would be likely to lead to the collapse of the dimerization interface.
In our studies, the DNA interaction of the hCAR-4aaINS–RXR was weak relative to the reference hCAR isoform complex. The hCAR modeling analysis predicts that the 4 amino acid insertion point for this variant receptor lies immediately adjacent to the dimerization interface and probably results in a C-terminal extension of helix 6, extending the helix in the direction of the dimerization interface. Thus the 4 amino acid insertion is also predicted to impact the receptor's heterodimerization and DNA-binding functions, supporting the EMSA results presented here. Studies of hRXRβ3 that contains a 4 amino acid insertion in a structurally homologous position also reveal a compromised ability to form heterodimers with the retinoic acid receptor (35). It is of interest to note that Mahajna et al. (35) also noted that the hRXRβ variant possessed an enhanced capacity to homodimerize, suggesting an alternative means by which the hCAR-4aaINS might interact with DNA (35). In addition, it is possible that the rather weak interaction of the hCAR-4aaINS–RXR heterodimer with DNA in vitro may be more robust in vivo. For example, accessory factors may bind the PBREM sequence to form an enhanceosomal architecture, facilitating the interaction of the variant hCAR-4aaINS complex with its cognate response element. It is also possible that the hCAR-4aaINS isoform may be subjected to unique post-translational modifications in COS-1 cells, modifications that may modify its capacity to bind DNA. A number of nuclear receptors undergo post-translational modification by phosphorylation, consequently modifying receptor function (50). Preliminary analysis of the of hCAR-4aaINS amino acid sequence using NetPhos 2.0 (51) indicates that the inserted amino acids possess a high-scoring putative phosphorylation site (0.992) at the serine residue of the peptide GARVSPTVG. Thus, alternative regulation strategies may impact hCAR receptor function and need to be evaluated further.
Studies conducted with the thyroid hormone receptor have suggested that structural alterations of the ligand-binding domain can alter the repertoire of DNA-binding elements involved in receptor interaction (52). It therefore seems reasonable to speculate that variant isoforms of hCAR may bind to a different subset of response elements, distinct from previously defined hCAR–RXR response elements, as tested here. The biological implications of this suggestion may be important, as variant isoforms of hCAR may then be involved in the regulation of different subsets of human genes, possibly distinct from that of the reference form of hCAR. Efforts are underway in the laboratory to evaluate these possibilities.
Since all the hCAR isoforms assessed in the current studies were capable of interacting specifically with nuclear receptor coactivator SRC-1, two possibilities are suggested: (i) that the isoforms of hCAR may be capable of dominant-negative activity by acting to squelch coactivator recruitment; and/or (ii), as suggested above, that the variant hCAR isoforms may be capable of interacting with currently undefined DNA response elements, or might be recruited to promoters through other bridging factors, thereby modulating the transcriptional process through recruitment of coregulatory factors to target promoters. Results of transfection experiments presented in Figure Figure7B7B indicated that the former suggestion is not likely to be correct. In order to test the latter suggestion, experiments are currently underway examining the effects of artificial recruitment of hCAR isoforms to DNA using mammalian two-hybrid strategies that allow the evaluation of coactivator recruitment.
The variant isoforms of hCAR are predicted to encode structurally unique ligand-binding domains. Molecular modeling analysis reveals that the ligand-binding pocket structure would probably not be impacted in the hCAR-5aaINS insertion variant. However, the hCAR-4aaINS variant is predicted to impart a direct extension of helix 6, probably resulting in structural alteration of the ligand pocket. Receptors with this amino acid insertion would also be likely to possess an alteration in the pocket's side chain composition, potentially resulting in altered ligand binding specificity of the isoform, an observation that would be agreement with studies of mouse RXRβ3 (34). This prediction raises an important question: does this hCAR isoform respond to a set of ligands/pharmacophores that are distinct from the reference CAR isoform? One ligand evaluated in the current investigation, clotrimazole, exhibited inverse agonist activity with both the reference and the 4aaINS isoforms of hCAR. It remains curious that, unlike clotrimazole, PB and a number of PB-like inducing agents have been identified that are not ligands for the reference CAR isoforms (15,19,22). This observation leads to the compelling question as to whether certain inducing compounds may interact alternatively with the ligand-binding pocket of hCAR-4aaINS, or other CAR isoforms. Assays are being developed to evaluate this intriguing question.
Alternative splicing of mRNA is one of the primary avenues for diversifying the proteome, and this process presents itself as a representation of nature's efficiency, enabling an array of functions to be encoded in a single gene. The isoforms of hCAR presented in this study may be an example of this efficiency. Diversifying the proteome of the xenosensor system by mechanisms such as alternative splicing would probably facilitate an organism's ability to adapt to its chemical environment. Further characterization of the variant isoforms of hCAR in animal models will be likely to lead to deciphering specific biological functions in the adaptive xenobiotic induction pathway.
The authors would like to acknowledge Samir Kalada, Heather Klintworth and Sean Boyle for their technical contributions and fruitful discussions. This paper is dedicated to the memory of Dr Christopher Hassett, a loyal friend, outstanding colleague and meticulous scientist. This study was supported by a grant from the National Institute of General Medical Sciences, GM66411 (C.J.O.), a Center Grant from the National Institute of Environmental Health Sciences (ES07033) and an NIEHS Training Grant, ES07032 (S.S.A.).
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.

subset: commoncrawl


text:
Methodic 1.5 Trailer (2009)
"His evil is spreading!!!" 23 October 2009 Horror 22 mins
Tells the story of a parolee, given a second chance at life, who falls victim to the viral, infectious control of the evil Dollman.
Watch the official Methodic 1.5 2009 trailer in HD below or find more Methodic 1.5 videos on Vidimovie.


text:
Catalogue Style Website Design
A Catalogue Style Website is perfect for when you have products in your catalogue you would like to display on the Internet, more about but not sell online.
The benefits of a Catalogue Style Website
- Display your products 24 hours a day 7 days a week to a worldwide audience
- Update your products online, link without having to reprint and redistribute
- Customers can contact you to ask questions about your products
- Display useful information such as Terms and Conditions, Shipping prices and Help Guides
- Advertise additional services
Once you have a website you can submit your site to various search engines and free directories to increase your presence on the Internet and increase your visibility.
IT Skills
You will need to be able to update your catalogue. This will involve you resizing, cropping and uploading images, writing product descriptions, creating categories and responding to e-mail queries.
You are welcome to use our FREE tutorials to help you with the administration of your website and if you need help we do operate a Website Administration Service were we can update your website for you.
More information:
To promote a special offer consider a Microsite Design
If your product range is fairly static you could consider a Brochure Style Website.
To sell products online direct from your website consider an E-commerce Website.


text:
Letter to the Editor: Just getting started
Fifty years is a good start, not a good finish.
The 50th anniversary of the Arctic National Wildlife Refuge should be a time to celebrate the historic conservation achievement for "America's wildest refuge," not plan its epitaph. Unfortunately, the latter is what some members of Congress are calling for, most recently Rep. Fred Upton (R-Mich.), who is in the running to chair the powerful House Energy and Commerce Committee.
Story Continued Below
In " Fortifying our energy Security" (POLITICO, Dec. 6), Upton, writing with former Energy Secretary Spencer Abraham, calls opening the pristine Arctic National Wildlife Refuge to drilling part of an "all of the above" energy policy. But it should be called "all of the failed energy policies that got us here," focused on destroying iconic landscapes like the Arctic Refuge, while leaving renewable resources, like wind and solar, as a sideshow.
In addition, the estimated amount of oil beneath the Arctic Refuge would have limited effect on gas prices or U.S. energy independence. According to a U.S. Energy Information Administration report, production would peak at just a tiny fraction of the 21 million barrels used every day in America. Meanwhile, the oil industry's history on the adjacent North Slope has been marred by an average of a spill a day, according to the Alaska government.
Introducing drilling to the Arctic Refuge means roads, pipelines, airfields, wells and all the risks that come with them. It not only threatens iconic wildlife, but it also threatens the way of life for cultures, like the Gwich'in, whose history with the Arctic Refuge stretches back millennia.
Marking the 50th anniversary, President Barack Obama said in a proclamation: "Today, the Arctic National Wildlife Refuge remains distinct in the American landscape, and we must remain committed to making responsible choices and ensuring the continued conservation of these wild lands."
We, as a nation, shouldn't say that we are willing to sacrifice our heritage and our future for a few barrels of oil. We should instead say that we will protect a place as special as the Arctic National Wildlife Refuge — and pursue better energy options instead.
William H. Meadows
The Wilderness Society
Cindy Shogan
Executive director
Alaska Wilderness League


text:
Kingston (Ontario, Canada) SELLER MANAGED Fundraising Online Auction - Princess Street
A max bid would make it more difficult for others to outbid you.
A max bid is the highest amount you are willing to pay for an auction item.
In response to other bids, the auction system will automatically increase your bid in the smallest increment possible (up to your max bid amount) in order to maintain your lead position.
To get a max bid you simply enter the maximum amount you are willing to pay into the bid form just as you would any other bid. If your bid is more than one bidding increment above the current high bid you will get a max bid unless another bidder has a max bid in place that is higher than the amount you enter.
At the end of the auction, the winning bidder pays only the lowest winning bid possible. That means that you can win an auction item at a price less than your max bid amount.
Your max bid amount is kept confidential from other bidders.
You can lower or delete a max bid by entering a lower amount.
Note: After watching hundreds of auctions and talking to thousands of bidders, we think using max bids in your bidding strategy works best for most people. It eliminates some of the pressure and reduces the risk of "sniping", where other bidders slip in slightly higher bids in the last seconds of bidding.
You now have a max bid.
You can raise or lower your max bid at any time by entering a new amount.
If you wish to remove your max bid you can do so by entering an amount equal to the current bid into the bid form.
You can adjust this max bid by using the form above.
If you want to remove your max bid click here.
You can enter the minimum bid of %s or a greater amount.
If you bid more than the minimum bid you will get a max bid that the system will use to bid for you against other bidders, up to the amount you enter. So if you enter a bid of %s for this item, the system will place bids on your behalf in response to bids from other users until someone bids more than %s.
British artist signed George Henry Jenkins (1843 to 1914). Frame measures 21 x 17.
92 x 72 inches double si. Marks visible in picture.
Two off white canvas and wood lounges. 21 inches wide x 22 inches deep x 28 inches tall at back. Canvas is worn.
Large backpack. Looks new. Blue and black in color
Set of dishes. Set originally for 12. Some pieces missing.
Stamped western Germany.
Camera, various lenses and flash. Bag and lots of small misc items included. Untested.
Five misc sized crocks. Tallest is 13 inches.
9 pieces misc bunnykins dishes.
Set 900, light weight mobile sound booster and infrared stereo tv listening system. Lithium polymer battery lasts up to 12 hours continuous use. Untested.
Comes with 4 controllers and 5 games. Untested.
30 inches long x 9.5 inches wide. Made of wood.
Includes 32 games and 1 each video touch pad and controller. Controller name Coleco. Untested
42 x 11.5 inches. Note: Jersey not included (see lot 11).
6 plates, 7 misc sized steins, 2 cream and sugar sets, 1 sugar bowl with lid, 2 cups and saucers, one mug.
Misc small to med sized round and square tin boxes - 22 in total
Five pieces of cranberry glass. Tallest piece is 10 inches high. Tag states Canadiana Cranberry Glass - genuine cranberry glass. Distributed by Chalet Artistic Glass. N.C. Cameron & Sons, LTD. Toronto, Canada.
Small coffee / decorative spoons
2 pieces, one with lid. Larger one is 8 inches tall and smaller is 2 1/2 inches tall.
The Grand Alliance, Their Finest Hour, The Hinge of Fate, Triumph and Tragedy, Closing the Ring and The Gathering Storm. Published by Thomas Allen in 1950.
Keystone 8mm movie projector. Measures 12 x 9 inches. Untested
Hand push cart with 12 right handed clubs. Dark green in color. Zippers closed.
Label states: blade exclusively for Bruce Hood. No size visible. Some marks.
No makers name. Solid piece. Handle may be new. 9 inches wide by 5 inches tall
Whites brand. 61 inches tall from neck to boot.
Volume one is autographed JR Smallwood - premier of Newfoundland and Labrador. Published in 1981. Volume one is A to E. Volume 2 is FAC to HOY.
One 18 inches and one 15 inches. Not matching.
White jersey. Small stain on front by emblum. Size XL
Size large. Extra weights included. Fitness tech power suit. Used for Resistance power training.
No marks to determine maker. Head broken at neck area. Measures 14 x 5 1/2 body. 3 inches high
9 inches wide x 5 inches tall. No chips. Some internal cracks.
Wooden pipe with face. 18 inches long x 6 1/2 inches deep. Looks to be a useable pipe. Note: iphone not included. Cast Iron Kettle in other lot.
Standing wooden Rocking cradle. 34x20x31 inches. Wooden with material insert. Not safety tested for use with children.
Size small
9 knives, 8 butter knives, 8 forks, 10 dessert forks, 8 small forks, 13 teaspoons, 4 serving spoons, 6 soup spoons, 8 coffee spoons. 1 serving fork, butter knife, 8 other misc pieces. Community plate is stamped on the back.
30.5 inches high. Bronze and gold coloured.
No specific markings. Metal mesh casing over glass bottles. Paint mark on one.
States expo 2010 Shanghai China. Picture of 4 pandas. Stored in fabric covered box. Measures 46x12 inches.
Vintage -Rough with scratches and marks. Measures 16 1/4 x 13 1/4 inches
Size 12. Bare brand. 54 inches tall from leg to neck
Wooden rocking cradle with material insert. 37x18x19 inches. Not safety tested for use with children.
Off white color. 3.5 x 2.5 x 1 inch high.
2 juicers. Smaller one has chips
Large glass container with lid. Measures 9 1/2 x 7 inches. Perfect condition
2 are 11 inches tall. One is 6 inches tall. Wrapped in a leather like mache.
Royal Adderley Multicoloured Bone China flower bowl. 5 inches high and 6 inches wide.
Smith-Corona Memory Correct II. In plastic case. Electric. Untested
Glass container with lid. Lid has chips on underside. Measures 4 1/4 x 3 1/2 inches.
Celebrity 12. In hard case. Case measures 18 x 13 inches. Electric. Untested
Benedict Proctor Canada 47 printed on the bottom.


text:
Summary: Intel Cpu Ucode Loading Error problems might be caused by many reasons, especially bad registries or Trojan infection. And it's easy to find that your Opening programs will be slower and response times lag as well. Most Importantly, when you have multiple applications running, you may come across PC crashes and freezing. The main reason for Intel Cpu Ucode Loading Error problems are startup clutter, registry errors, RAM decline, fragmented files, unnecessary or redundant program installations, etc.
Solution: Fixing Intel Cpu Ucode Loading Error errors is a really difficult task for most computer users. In order to fix your errors and speed up PC, it is recommended that your download the 'Intel Cpu Ucode Loading Error Repair Tool'. It's an amazing software which is designed for users to troubleshoot Intel Cpu Ucode Loading Error errors efficiently..
Install Intel Cpu Ucode Loading Error Error Fixer Now
In order to repair Intel Cpu Ucode Loading Error problem, use the SmartPCFixer software program. This system optimizer software is already proven to locate, identify, and remove Intel Cpu Ucode Loading Error errors. Speed up your PC with SmartPCFixer at once
What Else Does SmartPCFixer Offer You?
SmartPCFixer™ is not only able to troubleshoot Intel Cpu Ucode Loading Error error, but also help identify and fix Windows' invalid registry entries. By running Scan & Cleaner as part of scheduled maintenance, it will keep your PC from freezing or frequent crashes . Using it will reduce the probability of you getting a "blue screen", program not responding or lock up.
SmartPCFixer is designed to scan, diagnose and repair your operating system. Using it results in better optimization, manages startup and desktop, assists you with maintaining browser objects, internet options, system service, and repairs file extensions. With this arsenal of powerful, sophisticated utilities your system is tuned to run at its optimal state. Included are Easy Repair Wizard, Error Utilities, File Association fixer, Register ActiveX, Shortcuts Fixer, Winsock2 Repair toolkit, Dll Fixer and more.
Intel Cpu Ucode Loading Error error can never be ignored when you come across it on your PC. If Intel Cpu Ucode Loading Error error cannot be troubleshooted in a effective way, you may received more severe computer problems. Therefore, for the sake of computer security and personal information, you need to get rid of Intel Cpu Ucode Loading Error error as soon as possible with the guide in this post. SmartPCFixer can be your best choice to remove Intel Cpu Ucode Loading Error error in time.
Related: Unable To Load Odbcji32.dll Windows 7,"windowsupdate_00000643" "windowsupdate_dt000",0x80070428,"windowsupdate_c1900101-30017,Tdmservice.exe
Read More: ,It And Forget It Lightly,Avscan Error Code,Microsoft Word Plug In,Driver Software,Mcshield Hig Cpu Usage Window Xp


text:
Have a dino-mite birthday with 20 of your closest friends!
Water, nature, space, and more! Explore the Discovery Room with your young scientist. Each area of the room allows you to immerse yourself in science learning through play. The Discovery Room is open to everyone, but is specially designed for young learners ages 1-8.
Water Table
Use the tiles to redirect the flow of water. Design a path to channel it from one end to the other. Test it out with floating toys.
Space Rocket
Climb aboard and pretend to be a NASA engineer or astronaut. Prepare for liftoff and imagine you are flying to the moon, Mars, or anywhere in space!
Live Animals
See a variety of live animals from around the world including an axolotl (Mexican salamander), saltwater species, and hold a rainforest cockroach!
Animal Clinic
Imagine being a veterinarian and care for the animals in our hollow tree clinic.
And More!
See in tiny details with the microscope, create constellations in the Star Room, play with your shadow in the Shadow Theater, and explore air movement at the Wind Tunnel. Math and engineering activities are changed regularly. Get creative in our Makerspace and learn to use a variety of materials in unique ways.
Sessions Start on the hour and last for 45 minutes
Monday through Thursday: 10 am, 11 am, 12 pm, 1 pm
Friday: 10 am, 11 am, 12 pm, 1 pm, 2 pm, 3 pm
Saturday: 10 am, 11 am, 12 pm, 1 pm, 2 pm, 3 pm, 4 pm
Sunday: 12 pm, 1 pm, 2 pm, 3 pm, 4 pm
Saturday, May 27th through Sunday Aug. 13th:
Monday-Saturday 10am, 11am, 12pm, 1pm, 2pm, 3pm, 4pm
Sunday 12pm, 1pm, 2pm, 3pm, 4pm
Monday, Aug 14th to Sunday Aug 20th:
Monday-Friday 10am, 11am, 12pm, 1pm, 2pm, 3pm
Saturday 10am, 11am, 12pm, 1pm, 2pm, 3pm, 4pm
Sunday 12pm, 1pm, 2pm, 3pm, 4pm
Monday Aug 21, regular hours resume
Space is limited to 50 people. One adult chaperone is required for every 4 children.
$4.00 Ages 1 and Up.
Babies need a free "Under 1" ticket.
$3.00 Military
FREE for Members – must get free tickets at the Box Office or Membership Desk.
Groups can reserve the Discovery Room for private sessions.
Private rate: $120 per session (up to 50 people-maximum 40 children and 10 adults)
Call 314-289-4400 for reservations.
Click here (Program Guide) for preschool through 2nd grade program information.
Celebrate your big day at the Science Center! Choose your party package, and enjoy the Discovery Room with friends. Click here for more information.
Preschool Science Series
These classes are designed for preschool aged children to explore science with an adult partner. Click here to learn more about class availability and topics.
Science Storytime
Free story time presented by Ready Readers. Click here for more information and schedule.


text:
Don't worry – this dress is not actually see-through, haha. It's honestly one of the least sheer, light-colored dresses that I own. There's a nude slip under it (and even a tank-top as well)! I know some of these photos made that a bit hard to tell, though. (And my husband said it's quite the optical illusion in-person too, haha.) With that being said, however, I am really coming around to the whole white, sundress thing! And I couldn't have… Read more »
Posts Tagged: sundress
Wear More Pink
If asked what my favorite color is, I will always say pink. However, I don't actually own all that much for pink clothing…or pink anything, really. I tend to gravitate more toward black clothing. Doesn't exactly make sense, does it? So, when Hot Kiss Clothing was so kind as to send me this cute, pink (and incredibly soft) sundress… I realized this was something that needed to change – immediately. I started with picking up this hot pink shade of… Read more »
First Time for Everything
Shop the look: There's a first time for everything! And this is the first time I've ever worn a sun hat. I was planning on waiting until I was at the beach this summer to break it out, but the weather has been sunny and in the 80s (and is supposed to get up to 90 today!)…so, it seemed silly to wait any longer, when it appears summer has already arrived. This sun hat I bought at Target…when I was grocery… Read more »


text:
Butterfly Dial Floral Pattern Leather Band Women Casual Quartz Watch
Availability: Out of Stock
$ 5.17
Ends On Nov.26
- BUYER PROTECTION Learn More >>
- 30 Day Returns Guarantee
- Full or Partial refund if the iterm is not as described
Similar Recommendation:
Waterproof Watch Automatic Watch Pocket Watch Vintage Watch Gold Band Black Digital Watch Mechanical Watch Flash Watch
• Fresh style quartz watch for women
• Dial face inhabit butterflies, brims the beauty of nature
• Watch band with floral pattern, gives you a concise, elegant and aesthetic feeling
• A joker style, suitable for your dress
Available Color: Pink,White,Red,Green,Gray
Style: Fashion&Casual
Shape of the dial: Round
Display type: Analog
Case material: Alloy
Clasp type: Pin buckle
The dial diameter: 4.0 cm / 1.57 inches
The band width: 2.1 cm / 0.83 inches
Wearable length: 20 - 24.5 cm / 7.87 - 9.65 inches
Product weight: 0.039 kg
Package weight: 0.089 kg
Product size (L x W x H): 27 x 4 x 1.2 cm / 10.61 x 1.57 x 0.47 inches
Package size (L x W x H): 28 x 5 x 2.2 cm / 11.00 x 1.97 x 0.86 inches
We ship to over 200 countries worldwide through fast and reliable delivery partners. You can choose your preferred shipping method on the Order Information page during the checkout process.
Total Delivery Time = Processing Time + Shipping Time
Processing Time: Before we ship your order, we have to prepare your products, perform strict quality-control tests and package items carefully.
● Expedited Shipping: 3-7 business days
● Standard Shipping: 6-8 business days
● Flat Rate Shipping: 10-30 business days
Products with quality-related issues may be returned for a refund or exchange by contacting Customer Service within 30 days of receiving your order. Our Customer Service staff will arrange it for you after the confirmation.
Products (excluding Made-To-Order, disposable, lingerie items, and Clearance items) with no quality-related issues may be returned for a refund or exchange by contacting Customer Service within 7 days of receiving your order. You will be responsible for all return shipping fees incurred.
Any product that has been used does not qualify for a return or exchange. All returned items must be in brand-new condition, unused and with original tags and packaging.
4 BeautifulDelivery was prompy. Beautiful watch
by Barbara Jun-02/2016 02:43:39
5 Butterfly Floral WatchMy Aunt is 97 years "young" and she loves butterflies. I purchased this watch for her as a recent Mother's Day gift. She is thrilled with it and has enjoyed showing it off to her friends. Quality watch and it is a very pretty design.
by Debbie Apr-07/2016 19:39:45
5 This dress is gorgeous love itJust cannot stop buying them
by kathy brown Feb-25/2016 06:34:12
5 butterfly floral watchEvery time I wear this watch I get complements,and I love wearing it since I have a butterfly tattoo on my right wrisk only thing is the watch doesn't have a second hand
There are no questions yet.ASK A QUESTION
Bulk Order Discount:
|2+ Units
|10+ Units
|Dropship Price
|Please submit your inquiry below
New Arrival
Related Searches
Popular Picks


text:
North Penn House Indianapolis, IN
This modern 3,500 square foot house is defined by the juxtaposition of its rectilinear roof and the dramatic topography of the wooded site. We designed the house on a well-defined plinth of limestone pavers and introduced tall sliding glass doors to create the illusion that the long, flat roof floats over its base. The sliding glass doors also encourage movement and views between the interior and exterior. Zinc panels and mahogany doors and windows combine with the light gray stone pavers in a modern, yet warm palette of materials.
3,500 square feet
Completed 2016
Key People
Deborah Berke
Noah Biklen, Project Lead
Caroline Wharton Ewing, Project Lead – Interiors
Collaborating Firms
Deborah Berke Partners – Architect and Interior Designer
Silver Creek Engineering, Inc. – Structural Engineer
MSA Professional Services – MEP Engineer
PHT Lighting Design, Inc. – Lighting Designer


text:
Search Results for "red tape"
Search Filters
- 2017
- 2016
- 2015
- 2014
- 2013
- 2012
- 2011
- 2010
- 2009
- 2008
- 2007
- 2006
- 2005
- 2004
- 2003
- 2002
- 2001
- 2000
- 1999
- 1998
- 1997
- 1996
- 1995
- 1994
- 1993
- 1992
- 1991
- 1990
- 1989
Sunday March 20,
Tags #technology, #coding, #code, #control, #efficiency, #purpose, #job, #red tape
Mordac: Step away from that open source code! Dilbert: Why? Mordac: Because I am Mordac, The Preventer of All Efficient Solutions in the Information Technology Realm. Dilbert: That isn't an actual job. Mordac: I was hoping it was. I lost the file with my job description. That was five years ago. I've been winging it since then. My parents taught me that I could be anything I wanted to be. And I wanted to be this. So don't use that code! Dilbert: Not even when you turn around?
Thursday May 25,
Tags #exercise & fitness, #run, #workout tape, #scare
Dilbert arrives at home and asks, "Okay, where are these so-called upholsterygeist?" Dogbert replies, "In the living room." Dilbert says, "You turn on the VCR, I'll get the Jane Fonda workout tape!" The chair runs away screaming, "Aaagh! The Exercist!"
Thursday September 09,
Tags #cleaning, #janitor, #crime scene, #overalls, #assistant, #feet in air, #dead body, #mop, #duct tape
Crime Scene Cleaning Dilbert says, "There's no budget for a mop or cleaning supplies." Dilbert says, "All I have is this pole and you." Ratbert says, "You could duct tape me to the pole." Dilbert says, "Yup. If we had duct tape."
Wednesday September 29,
Tags #new boyfriend, #coworker, #angry, #jack, #clench fist, #restaurant, #waiter, #bent fork, #red flag, #relationship, #bad choice
Alice says, "Carol, this is my new boyfriend, Angry Jack." Alice says, "I met him in a restaurant after he beat up a busser for bringing a bent fork." Carol says, "In thie white trash community, we call that a red flag." Alice says, "You weren't there. That fork was a mess."
Thursday January 19,
Wally's Red File Gets Him Out Of Work
Tags #laziness, #ruse, #work ethic, #deception, #excuse
Wally: I'd love to help you, but I'm busy working on the red file. Woman: Is the red file a real thing or just a thing you say to get out of work? Wally: It's all the same on your end.
Saturday January 21,
Secret Red File
Tags #work ethic, #laziness, #deception, #con, #stalemate, #bluffing, #forgetful
Boss: What did you accomplish this month? Wally: I made a lot of progress on the secret red file project that you gave me. Boss: Remind me what project that is. Wally: You made me promise I wouldn't tell you.
Wednesday July 05,
Tags #cross word, #challenge
Dilbert and Dogbert sit at a table playing Scrabble. As Dogbert arranges his letters on the game board, Dilbert asks, "What the heck is 'jequirity?'" Dogbert replies, "You know - the poisonous, red and black seed of the Indian Licorice plant. Want to challenge?" Dilbert replies, "No. At least this one has vowels." Dogbert says, "That's 704 for me."
Friday July 28,
Dilbert and Dogbert sit at the table listening to a tape player. Dilbert says, "I do NOT snore, and I do NOT believe you made this recording of me last night." Animal-like sounds come from the tape player. Dilbert looks at a cassette box and says, "In fact, this tape box says 'National Geographic's Songs of the Whale.'" Dogbert says, "So, you admit that even National Geographic can't tell the difference between your snoring and a twenty-ton kelp-scarfing mammal."
Monday October 23,
Dilbert sits on the floor hugging his knees and watching television. Dogbert says, "You've been watching this video tape over and over for days." Dilbert says, "These tennis instruction tapes are great. I can just feel my game improving as I watch." Dilbert continues, "In fact, I see no need to actually physically play the game ever again."
Monday March 19,
Dilbert stands at the "Happy Airline" ticket counter. The ticket agent says, "I'm sorry, sir, but you've been 'bumped.'" Dilbert says, "What?!" Dilbert puts his hands on his hips and says, "I've got a ticket! I demand satisfaction! I'll call the president of your stupid company!!" Dilbert is strapped to the wing of a plane. Dilbert thinks, "I wonder if there's really such a thing as the 'duct tape section.'"


text:
Norcross, GA (PressExposure) September 17, 2009 -- We have launched some new product in our category with more advance features and high quality. Sony clock radio self recording spy camera which supports the GB card of sizes 1GB, 2GB, 4GB, 8GB, and 16GB. It has the capability of Recording Length of 10 seconds â 2 minutes per clip or per motion and has a Recording Capacity of 4 hours per G. It comes with a 2GB SD Cards. It displays the exact date and time when the video or the picture was created.
SALE! SALE! SALE! Only for this month: Item code: SCR320 Regular price: $479.00 Sale price: $249.00
The Sony Clock Radio Spy Cameras comes in 4 modes:
320x240, 5FPS, 2GB Max SD, $299
Hi Res 640x480, 12FPS, 16GB Max SD, $399
320x240, Realtime 30FPS, 16GB Max SD, $429
704x488, Realtime 30FPS, H.264 32GB Max SD, COMING SOON!.
The 320x240 Spy Cameras records at a medium quality, displays a medium size video, plays at medium video speed and uses low storage space. But the best thing about this model is it's the least expensive Self Recording Spy Camera on the market ever!
Our Spy Cameras are built in our office and can be customized and shipped daily.


text:
QVC Accessorizes The All Stars on Lifetime®'s Project Runway
Leading Multimedia Retailer Collaborates on Season Three of Project Runway All Stars
QVC Host Lisa Robertson Will Serve as Guest Judge
Oct 22, 2013, 12:09 ET from QVC, Inc.
WEST CHESTER, Pa., Oct. 22, 2013 /PRNewswire/ -- With a repertoire of brands from well-known designers and stylists, QVC has announced it will participate in the upcoming season of Lifetime®'s Project Runway All Stars. The leading multimedia retailer is scheduled to sponsor the famed Accessory Wall on the highly popular design competition series, and it will host a design challenge for the contestants. Additionally, the season's grand-prize winner will have the opportunity to design a collection to be sold on QVC.
"We were eager to collaborate on this season of Lifetime's Project Runway All Stars since it targets the interests of women that are central to our shopping community," said Karen Fonner, vice president of content marketing for QVC. "By aligning with the popular series, we have the opportunity to increase exposure as a fashion authority in a culturally relevant way."
The challenge, sponsored by QVC, will feature Host Lisa Robertson as a guest judge. With the retailer's reputable position in the fashion industry, Robertson has become a relatable style authority and is set to join returning judge Isaac Mizrahi, "Mad Men" actress Elisabeth Moss and former Project Runway winner Mondo Guerra. The contestant who wins the QVC challenge will have a look inspired by their design sold through the multimedia retailer following the episode.
"Given the notable fashion and accessories brands QVC offers, they were a natural fit to sponsor this season's Accessory Wall and host a challenge," commented Meryl Poster, executive producer of Project Runway All Stars. "QVC also works with a range of design and style experts, including the series' judge Isaac Mizrahi, making it well respected in the industry."
The third season of Project Runway All Stars is scheduled to air on Lifetime beginning October 24. QVC plans to promote the program on its website via multimedia content featuring the designers, challenges and products inspired by the show.
For more information about QVC's involvement with the series, visit www.qvc.com/ProjectRunwayAllStars.
About QVC
QVC, Inc., a wholly owned subsidiary of Liberty Interactive Corporation (NASDAQ: LINTA, LINTB), is the world's leading video and ecommerce retailer. QVC is committed to providing its customers with thousands of the most innovative and contemporary beauty, fashion, jewelry and home products. Its programming is distributed to approximately 290 million homes worldwide through operations in the U.S., Japan, Germany, United Kingdom, Italy and a joint venture in China. West Chester, Pa.-based QVC has shipped more than a billion packages in its 26-year history and the company's website, QVC.com, is ranked among the top general merchant Internet sites. QVC, Q, and the Q Ribbon Logo are registered service marks of ER Marks, Inc.
About The Weinstein Company
The Weinstein Company (TWC) is a multimedia production and distribution company launched in October 2005 by Bob and Harvey Weinstein, the brothers who founded Miramax Films in 1979. TWC also encompasses Dimension Films, the genre label founded in 1993 by Bob Weinstein, which has released such popular franchises as SCREAM, SPY KIDS and SCARY MOVIE. TWC also boasts an active television production division led by former Miramax Films Co-President of Production Meryl Poster. TWC garnered nine Emmy nominations in 2013. The Weinstein Company Television produces the reality powerhouse PROJECT RUNWAY; with its spin-off series PROJECT ACCESSORY and the critically acclaimed PROJECT RUNWAY ALL STARS, hosted by Alyssa Milano in its upcoming third season. TWC recently launched MIAMI MONKEY on VH1, and MILLION DOLLAR SHOPPERS and SUPERMARKET SUPERSTAR on Lifetime. TWC is currently in production on MOB WIVES Season 4 for VH1; and WELCOME TO MYRTLE MANOR Season 2 for TLC. Upcoming TWC series premieres include RODEO GIRLS for A&E. Announced scripted projects in development include John Fusco's historical action-adventure epic MARCO POLO and an adaptation of Daniel Stashower's bestselling Civil War-era spy thriller THE HOUR OF PERIL. TWC also developed Amy Sherman Palladino's THE NANNY DIARIES for ABC and produced the award-winning HBO series THE NO. 1 LADIES' DETECTIVE AGENCY, directed by Anthony Minghella.
About Lifetime
Lifetime is a premier female-focused entertainment destination dedicated to offering the highest quality original programming spanning scripted series, non-fiction series and movies. In 2012, as a result of its aggressive programming strategy that doubled the hours of original programming, Lifetime posted its strongest year-on-year growth among the key demographics in 10+ years, while also reaching its youngest median age in 16 years. Lifetime Television®, LMN®, Lifetime Real Women® and Lifetime Digital™ are part of Lifetime Entertainment Services, LLC, a subsidiary of A+E Networks. A+E Networks is a joint venture of the Disney-ABC Television Group and Hearst Corporation.
SOURCE QVC, Inc.