Identification of a Novel Set of Genes Regulated by a Unique Liver X Receptor-alpha -mediated Transcription Mechanism*

Leonard M. AndersonDagger , Sung E. Choe§, Rustam Y. Yukhananov, Rob L. HopfnerDagger , George M. Church§, Richard E. PrattDagger ||, and Victor J. DzauDagger ||

From the Dagger  Department of Medicine, Division of Cardiovascular Research, Laboratory of Genetic Physiology, the § Department of Medicine, Division of Genetics, and the  Department of Anesthesiology, Neurogenomic Laboratory, Pain Research Center, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Received for publication, August 22, 2002, and in revised form, January 23, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have reported previously that liver X receptor-alpha (LXRalpha ) can mediate a novel cAMP-dependent increase in renin and c-myc gene transcription by binding as a monomer to a unique regulatory element termed the cAMP-negative response element (CNRE). To determine whether this novel action of LXRalpha has global implications on gene regulation, we employed expression profiling to identify other genes regulated by this unique mechanism. Here we report the existence of a set of known and unknown transcripts regulated in parallel with renin. Querying the Celera Mouse Genome Assembly revealed that a majority of these genes contained the consensus CNRE. We have confirmed the functionality of these CNREs by competition for LXRalpha binding via electrophoretic mobility shift assays (EMSA) and by the use of CNRE decoy molecules documenting the abolishment of the cAMP-mediated gene induction. Taken together, these results demonstrate that the interaction between cAMP-activated LXRalpha and the CNRE enhancer element is responsible for widespread changes in gene expression and identify a set of LXRalpha /cAMP-regulated genes that may have important biological implications.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The transcription factor liver X receptor-alpha (LXRalpha ),1 a member of the nuclear hormone receptor superfamily, regulates the expression of genes involved in cholesterol homeostasis and bile acid synthesis (1, 2). The best known mechanism of LXRalpha -mediated transcriptional activation occurs through interactions with compounds such as oxysterols (22-cho) or retinoic acid (9cRA) and results in heterodimerization to other transcription factors including retinoid X receptor alpha  and peroxisome proliferator-activated receptor gamma  (3). Transcription of target genes such as the cholesterol 7alpha -hydroxylase gene occurs through a classical DR4/LXRE (5'-GGTTTAAATAAGTTCA-3') response element (4).

The aspartyl protease renin is synthesized in the kidney and secreted into the plasma. It is the rate-limiting enzyme in angiotensin biosynthesis and thus plays an important role in blood pressure and volume regulation. Renin gene expression and secretion are mediated in part by intracellular levels of cAMP in kidney juxtaglomerular (JG) cells (5). Previously, we have identified a cAMP-responsive element distinct from the classical cAMP-responsive element in the promoter region of the mouse renin gene and have termed this element CNRE (5'-TACCTAACTTGGCTCACAGGCAGAATTTATC-3') (6). Homologues of this element found at positions -619 to -588 of the mouse Ren-1D gene have been found in the mouse Ren-1C and Ren-2 genes as well as in the rat and human renin genes (7). Furthermore, using a yeast one-hybrid screening approach, we demonstrated that LXRalpha bound to this sequence, whereas functional studies using promoter/reporter gene chimeric constructs revealed that LXRalpha increased basal levels of renin expression and mediated the cAMP-dependent induction of mouse and human renin gene expression (8).

Interestingly, the previously reported LXRalpha ligands such as 22-cho had no effect on renin gene expression nor on the expression of renin gene promoter/reporter gene chimeric constructs. Moreover, the studies conducted with N-terminal deletion mutants of LXRalpha indicated that the binding mechanism and positive regulation of these genes through the CNRE element uniquely occurred as a monomer in contrast to the action at the classical LXRalpha /DR4 element. Further studies revealed that the c-myc gene, which also contains the CNRE element, was similarly regulated (9). The discovery of LXRalpha as a regulator of renin gene expression by a previously unknown mechanism lead to the global question as to whether a set of genes exists that could be regulated in response to cAMP by LXRalpha . To address this question, we performed expression profiling of a mouse kidney JG cell line (8) stably transduced with green fluorescent protein (10) or murine LXRalpha plus GFP and treated with cAMP or vehicle.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse Kidney As4.1 Cell Culture and cAMP Stimulation-- The mouse renin-expressing cell line, As4.1 (ATCC CRL2193), was previously isolated from the kidneys of transgenic animals harboring a chimeric renin gene promoter/SV40 T-antigen construct (11). Cells were cultured in high glucose DMEM (Invitrogen) supplemented with 10% fetal calf serum and antibiotics at 37 °C in 5% CO2. Stable cell lines infected with a bicistronic retroviral construct expressing either GFP alone or GFP plus mouse LXRalpha were generated as described previously (12). For cAMP stimulation, cells were first made quiescent in DMEM containing 0.1% fetal calf serum for 12 h. Fresh medium was added containing DMEM + 0.1% fetal calf serum plus either vehicle (PBS) or 1 mM 8-Br-cAMP and incubated for various lengths of time. Total cell RNA was harvested using the TriZOL reagent and was assessed for quality by spectrophotometric and electrophoretic analysis.

Construction of the 19,064 Element Mouse cDNA Microarray-- The cDNA microarrays generated in our laboratory consisted of 19,064 elements that included 2,432 cDNAs from three mouse libraries donated by colleagues at the Brigham and Women's Hospital (neonatal kidney and brain library from David Beier and nucleated erythroid cell library from C. C. Liew). Genes and ESTs were identified using sequence comparison of the mouse Unigene EST data base. An additional 15,264 cDNA clones derived from the NIA15k mouse developmental set were spotted, resulting in a total of 18,296 cDNA clones available for expression analysis (13). For use in the determination of nonspecific hybridization, cDNAs corresponding to several bacterial genes were also spotted in various regions of the array for a total of 768 control elements, thus resulting in the generation of a 19,064-element cDNA array.

Amplified cDNA inserts were obtained from purified plasmid DNA templates, lambda -phage clones, or directly from bacteria-harboring plasmids containing cDNA inserts. Purified plasmids were amplified using T7 (5'-GTAATACGACTCACTATAGGGC-3') and T3 (5'-AATTAACCCTCACTAAAGGG-3') or SP6 (5'-ATTAGGTGACACTATAG-3') primers with the cycling parameters of 94 °C for 1 min, 37 °C for 1 min, and 73 °C for 1 min, 30 s for 40 cycles with an additional extension cycle of 73 °C for 8 min. Libraries consisting of bacteria or phage received an initial incubation cycle of 94 °C for 5 min before entering the previously mentioned cycling parameters. Amplified clones were analyzed for single band product by agarose gel electrophoresis. PCR products were purified by ethanol precipitation in 96-well format as described previously (14). Purified inserts were spotted in linear format at 250 µm center-to-center distance on CMT-GAPS (Corning, NY) slides using the GMS417 Arrayer (Affymetrix).

Probe Labeling and Hybridization-- Total RNA (70 µg) was used as template to generate fluorescently labeled cDNA probes by a single round of oligo(dT)-primed reverse transcription in the presence of either Cy3-dUTP or Cy5-dUTP. First strand cDNA derived from RNA extracted from As4.1 cells that were transduced with the GFP retrovirus and treated with vehicle was used as reference cDNA in all of the experiments and was labeled with Cy3, whereas cDNA derived from RNA isolated from cells treated with cAMP and/or transduced with LXRalpha was labeled with Cy5. The labeled probes were purified using G50 spin columns (Amersham Biosciences), combined, and resuspended in a hybridization buffer containing 50% formamide and hybridized to the array in the presence of blocking DNAs (mouse cot-1 genomic DNA, oligo(dA), tRNA) for 16 h at 42 °C under a coverslip. After hybridization, slides were washed in 1% SSC plus 0.1% SDS to remove the coverslip and remove non-hybridized probe and then washed sequentially in 0.5% SSC followed by 0.1% SSC. The slides were dried by centrifugation at 2000 rpm for 2 min and were immediately scanned, and Cy3/Cy5 signal intensities were measured using a GMS418 Scanner (Affymetrix).

Microarray Data Collection and Analysis-- The intensities Cy3 and Cy5 fluorescence of each spot were measured by overlaying a quantitation grid over the scanned image (ScanAlyze). Local background was subtracted from the overall spot intensity in both channels. Nonspecific cross-hybridization signal was also determined. Signals from each bacterial clone were measured, and the values were averaged and subtracted from the murine cDNA spots within that subarray. Spots that did not meet a value >1.5 × background in either channel at all of the experimental time points were flagged or excluded. Calculated pixel-by-pixel correlation coefficients (Ch1GTB2 and Ch2GTB2) for Cy3 and Cy5 fluorescent intensities in each spot were used to determine overall spot quality, and spots that contained values of <0.6 in both channels were also flagged or excluded.

For total array conditional comparisons, ratios (Cy5/Cy3) for each slide were calculated, and to account for differences in overall hybridization and labeling efficiencies, ratios were log2-transformed and median-centered. The resulting data were then normalized using a lowest-fit intensity-dependent algorithm and subjected to significance filtering software (www.stat.berkeley.edu/users/terry/zarray/html/matt.html) based on observed versus expected values (15, 16). All of the conditions were combined in one large data set, and hierarchical clustering using Pearson-centered correlative and complete linkage-clustering algorithms were then employed to generate a tree containing gene expression clusters with similar temporal expression profiles in all of the conditions using GeneSpring Software, version 4.13 (SI Genetics). A subset of genes was then obtained, which contained an expression profile similar to mouse renin for subsequent analysis.

Multiplex Reverse Transcriptase (RT)-PCR-- A semi-quantitative RT-PCR was employed for validation as follows. Gene-specific primers were generated based on the known EST sequence. SpliceView and ExonPCR programs were utilized to generate primers that had a high probability of spanning an intron (17, 18). Primers specific for 18 S RNA (forward 5'-CGG CTA CAT CCA AGG AA-3'; reverse 5'-GCT GGA ATT ACC GCG GCT-3') were added as an internal positive control. Template RNA (500 ng), gene-specific primers (20 pM), 18 S RNA primers (20 pM), and oligo(dT) (500 ng) were added to a RT-PCR kit (Amersham Biosciences), and reactions were subjected to low cycle PCR as follows: first strand synthesis (1 cycle of 60 °C for 30 min and 94 °C for 2 min), amplification (18-25 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min), and extension cycle (1 cycle of 72 °C for 5 min). Aliquots of the resultant products (5 µl) were subjected to 2% agarose gel electrophoresis for further analysis. All of the RT-PCR experiments were conducted in triplicate using at least three separate RNA samples.

Double-stranded Decoy Experiments and Electrophoretic Mobility Shift Assays (EMSA)-- A stock solution (150 µM) of complementary phosphorothiate-modified single-stranded oligomers (Invitrogen) was incubated at 65 °C for 10 min and allowed to anneal at 25 °C for 2 h in annealing buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA in diethyl pyrocarbonate-treated water). For experiments, annealed oligomers were diluted to various concentrations using annealing buffer, whereas excess stock was stored at -20 °C for future use.

Assessment of decoy transduction efficiency was conducted as previously reported with some adjustments (19). As4.1 cells were grown to 70% confluence and transduced with 5-100 µM FITC-labeled double-stranded decoy oligonucleotides containing the following sequences from previously published data (20): CNRE decoy (sense 5'-TAC CTA ACT TGG TCT CAC AGG CTA GAA TTT ATC-3'); cAMP-response element (CRE) decoy (sense 5'-GCT TAC CCA CAG TCC CCC GTG ACG TCA CCC GGC-3'); and scrambled DNA (5'-GTC AGC TAG TGT TGA CAG GCC AGT TAG GTC TCG AG-3') using OligofectAMINE reagent (Invitrogen). After 48 h, detection of positively transduced cells was conducted by fluorescent microscopy using a conventional fluorescein detection filter. Untransduced cells and phase-contrast microscopy were utilized as a control.

For assessment of decoy effect on target gene transcription, As 4.1 cells containing either GFP or LXRalpha were transduced with 40 µM non-FITC labeled decoy as previously mentioned. After 24 h, cells were washed with PBS and then grown in serum-reduced media (DMEM + 0.1% FBS) for 12 h. Fresh growth medium then was added containing 0.1% FBS (Invitrogen) and either 1 mM 8-Br-cAMP or PBS (vehicle) and allowed to incubate at 37 °C in 5% CO2 for a period of 6 h. After treatment, total RNA was isolated using TriZOL (Invitrogen) and an aliquot was subjected to quality assessment by spectrophometric and agarose gel analysis.

EMSA assays were performed as described previously with some modifications (7). As4.1 cells were serum-restricted and treated with 1 mM 8-Br-cAMP or vehicle as described previously. Nuclear protein extracts were prepared using the NuclearPrep Kit (Pierce), and protein concentration was assessed by spectrophotometric assay. Nuclear extracts were then aliquoted and stored at -80 °C for future use. For competition experiments, 10 µg of extract from As4.1/LXRalpha was incubated with 1.75 pM gamma -32P-labeled probe containing the ren-1D CNRE sequence (5'-CTA ACT TGG TCT CAC AGG CTA GAA-3') and 100-fold molar excess of unlabeled probe containing the CNRE and flanking 6-bp sequences from each identified gene. After incubation at 37 °C for 30 min, the reaction was loaded onto a 6% polyacrylamide gel and electrophoresed in 1× Tris borate EDTA buffer for 2 h at 200 V. After electrophoresis, the gel was dried and exposed to autoradiography film overnight at -80 °C. For antibody neutralization assays, the reaction was carried out as described previously; however, after incubation with lysate, reactions were further incubated with anti-goat IgG (Santa Cruz Biotechnologies), anti-LXRalpha (Santa Cruz Biotechnologies), or gel shift buffer for an additional 15 min.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Global Gene Expression Is Affected by LXRalpha in As4.1 JG Cell Cultures-- The novel action of LXRalpha in the regulation of renin and c-myc genes leads us to ask whether other genes are regulated in a similar manner. To address this issue, we initiated a cDNA-based transcript profiling study. A mouse cDNA microarray was generated that contained a total of 19,064 spotted elements. Table I indicates the results of querying the cDNA sequences against the Unigene data base (build number 88), which revealed that 6,446 (35.2%) cDNAs matched to known genes and 5,990 (32.7%) matched to other reported ESTs, resulting in a total of 12,436 (67.9%) previously reported sequences. An additional 5,910 (32.3%) were unable to be matched and thus were considered novel. Additionally, 768 (4.2%) bacterial control spots were distributed throughout the array as control elements for normalization.


                              
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Table I
EST distribution of spotted clones on mouse array
Sequences from the in-house clones or from the NIA 15k clone set were blasted against the Unigene data base (Build number 88) to generate a distribution based on match to known genes or other ESTs.

Serum-restricted mouse kidney juxtaglomerular cells (As4.1) stably transduced with either GFP plus mLXRalpha (As4.1/LXRalpha ) or GFP (As4.1/GFP) were treated with either 8-Br-cAMP or PBS (vehicle) for 1-24 h (1, 6, 12, or 24 h). Total RNA was used as template to generate fluorescently labeled cDNA probes by a single round of oligo(dT)-primed reverse transcription in the presence of either Cy3-dUTP or Cy5-dUTP. Standard background subtraction and normalization protocols were then employed, and clustering analysis was performed (Fig. 1). These results indicate that the greatest amount of differential gene expression occurred in cells expressing LXRalpha in the presence of cAMP when compared with all other conditions and thus demonstrate our previous observations of LXRalpha as a cAMP-responsive transcription factor.


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Fig. 1.   Hierarchical cluster analysis of differentially expressed genes in mouse As4.1 JG cells. As described under "Results," As4.1/GFP or As4.1/LXRalpha cells were treated with 1mM 8-Br-cAMP or vehicle (PBS) for 1, 6, 12, or 24 h. Profiling was performed using a custom 19,064-element array produced in-house. The data was filtered to include only genes (11,528), which demonstrate differential expression in at least one condition. The results indicate that LXRalpha /cAMP has a dramatic effect on the profiles of expression in As4.1 juxtaglomerular cells.

Identification of LXRalpha -modulated Transcripts That Exhibit a Ren-1D Expression Profile-- We next asked whether there existed a subset of genes with an expression pattern similar to that observed for renin. Utilizing the temporal expression profile of mouse Ren-1D where expression peaks at 6 h (Fig. 2, A and B) to query the entire set of expressed genes with a correlation coefficient cutoff of >= 0.92, a subset of genes was then obtained for subsequent cluster analysis (Fig. 3, A and B). Table II indicates a list of 41 genes that exhibit an increase in transcriptional activity peaking at 6 h. The gene descriptions in this table suggest that LXRalpha is able to modulate the expression of genes with diverse functional properties. These results indicate that at 6 h, LXRalpha +cAMP is able to induce the expression of these genes with an observed range of induction of 3.4-51.7-fold with 38 (92.6%) genes at a p <=  0.05 and a subset of 24(58.5%) genes at a p <=  0.01 when compared with all other conditions. The average fold induction (Fig. 3D) in LXRalpha + cAMP-treated cells at the 6-h time point was 8.4-fold higher when compared with all other conditions.


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Fig. 2.   Mouse renin (Ren-1d) expression in As4.1 cells in presence of LXRalpha and cAMP. As4.1 cells that were stably transduced with a retrovirus expressing either a bicistronic GFP/LXRalpha construct or GFP were treated with 1 mM 8-Br-cAMP or vehicle (PBS) for the times indicated. Total RNA was isolated and used as template for RT-PCR amplification using primers specific for mouse renin. Results by either agarose gel analysis (A) or average luminosity signal intensity (B) indicate that renin is induced with a maximal peak at 6 h poststimulation in LXRalpha -transduced As4.1 cells when compared with control cells (As4.1/GFP + Vehicle).


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Fig. 3.   Hierarchical cluster of genes that exhibit a renin expression profile. Hierarchical clustering (A) of genes that exhibit a highly similar (correlation >= 0.92) temporal expression profile to mouse renin (B and C) was done using a Pearson centered correlation coefficient distance metric (separation ratio = 0.9; minimum distance = 0.001). The results indicate the existence of groups of genes that tend to cluster separately from the full list.


                              
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Table II
Expressed genes that exhibit a temporal expression profile similar to mouse renin
The entire list of expressed genes (11,529) were queried using the temporal expression profile of mouse renin (Ren-1d) and resulted in the identification of 41 genes, which displayed a profile similar to that of renin (correlation >= 0.92). Gene function annotation is derived from GeneSpring and is based on Gene Ontology consortium classification.

Identification of Genes That Contain an Upstream CNRE Element and RT-PCR Validation-- We next asked whether these genes identified, by virtue of having an expression profile similar to renin, contained a CNRE element. The EST sequences corresponding to each of the 41 genes were compared with the Celera Mouse Genome Assembly to obtain the surrounding genomic sequence for each gene. 50 kilobases of flanking sequence was queried for the presence of the consensus CNRE element (5'-TNN(T/G)TC(C/T)CA(C/G)AGG-3'). Because most of the ESTs to be queried are not matched to known genes and are thus considered novel, a 50-kilobase distance was utilized to ensure the probability of analyzing a promoter region. The 5' end of the EST sequence was used as an "anchor," and all consensus CNRE hits are reported in terms of the distance to their respective anchors. The results of this search indicated 16 genes (Table III) that contain a consensus CNRE within 50 kilobases of the anchor. The total number of genes could further be divided into groups that contain CNRE elements within a short (0-15 kb, 7 genes), moderate (16-30 kb, 9 genes), and long distance (31-50 kb, 7 genes) from the anchor sequence.


                              
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Table III
List of genes with CeleraTM database identified CNRE element
Using the Celera data base 50-kb, of flanking sequence were queried for the presence of the consensus CNRE element. Because most of the ESTs to be queried are not matched to known genes, a 50-kb distance was utilized to ensure the probability of analyzing a promoter region. The upstream region of the 41 EST sequences were used as an "anchor" and resulted in 16 genes that contained a consensus CNRE within a 50-kb upstream region. The total number of genes could further be divided into groups that contained CNRE elements within a short (2), moderate (4), and long distance (4) from the anchor sequence. A comparison of the CNRE sequences to mouse Ren-1d CNRE resulted in an identity range of 61.5-84.6%.

RT-PCR assays were conducted to validate the temporal expression profile results by using EST-specific primers generated with a high possibly of spanning an intron using splice prediction software (SpliceView and ExonPCR). An internal control set of primers generated to amplify 18 S RNA was added to each reaction to account for initial template concentration, and all of the amplification reactions were conducted under low cycle number conditions. The results from these experiments (Fig. 4) indicate 15 out of 16 genes that contained a CNRE validated by a marked increase in amplified product present in cells transduced with LXRalpha and treated with cAMP when compared with cells treated with vehicle or cells transduced with GFP and/or treated cAMP for a period of 6 h.


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Fig. 4.   Multiplex RT-PCR validation of genes that contain a CNRE element. Total RNA was isolated from As4.1/GFP and As4.1/LXRalpha , which were treated with vehicle or 8-Br-cAMP for 6 h. Multiplex RT-PCR amplification was performed using gene-specific and 18 S RNA primers. Results indicate that 15 of 16 genes analyzed and displayed the expected increase in mRNA abundance in cAMP-treated As4.1/LXRalpha cells. No significant difference was observed with 18 S RNA signal

Effects of Oligonucleotide Decoy Molecules on cAMP-induced Gene Expression in Mouse As4.1 Cells-- To determine whether the temporal expression profile exhibited by the identified genes was the result of LXRalpha directly modulating transcription levels through binding a CNRE enhancer element within the promoter region or through some other indirect mechanism, we employed a decoy strategy. Double-stranded decoy molecules that correspond to the CNRE element present in the mouse renin (Ren-1d) promoter were generated to determine whether this decoy would be able to "sequester" endogenous LXRalpha and hence suppress target gene inducibility. Control double-stranded DNA representing decoy to the classical cAMP-response element-binding protein (CREB) enhancer sequence and a scrambled DNA containing the CNRE nucleotides were utilized to assess specificity of the results. FITC-labeled decoy was used to analyze the transduction efficiency in As4.1 cells. Various concentrations (0-100 µM) of DNA molecules were utilized to determine an optimal concentration that would yield high transduction efficiency with low cytotoxicity. Efficiency was assessed by the percentage of positive nuclei under a fluorescent microscope after 24 h. The results indicated 40 µM to be sufficient for 100% cellular transduction with no visible cytotoxicity. Fig. 5 indicates that all three DNA molecules exhibited a similar efficiency when employed at 40 µM. No visible fluorescence was observed in cells that were mock-transduced.


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Fig. 5.   Assessment of decoy transduction efficiency in As4.1 cells. Double-stranded FITC-labeled oligonucleotide (FITC-Labeled Decoy) containing CNRE, CRE, or scrambled DNA sequence was transduced (40 µM) into As4.1 JG cells. After 48 h, assessment of transduction efficiency was conducted using fluorescent and phase-contrast microscopy. The number of positively transduced cells (fluorescent), transduced with CNRE, CRE, or scrambled DNA, was significantly higher than mock-transduced cells (No Decoy). No significant difference was observed between cells transduced with CNRE, CRE, or scrambled decoy.

As.4.1 cells transduced with either LXRalpha or GFP were transduced with either the CNRE decoy, CRE decoy, or CNRE-scrambled DNA. After a period of 24 h, all of the cells were serum-restricted for 12 h and then treated with vehicle or 8-Br-cAMP for 6 h. Total RNA was harvested, and RT-PCR was conducted using gene-specific primers as mentioned before. RT-PCR assays conducted with mouse Ren-1d in the presence of CNRE decoy or control DNAs indicate that the CNRE decoy is indeed able to inhibit LXRalpha -mediated induction of transcription (Fig. 6A). However, no inhibition of Ren-1d gene expression was observed when cells were treated with the classical CREB protein binding element (CRE) decoy or with a molecule containing the scrambled CNRE sequence. Results from RT-PCR experiments using primers specific for genes that contained a CNRE element (Fig. 6B) revealed that 11 out of 16 genes whose cAMP-inducible expression was suppressed specifically by the CNRE decoy, suggesting that these genes are regulated by LXRalpha and cAMP through a cis-element CNRE binding sequence located in close proximity to the target gene.


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Fig. 6.   Effects of decoy administration on LXRalpha -regulated genes. As4.1/LXRalpha cells were transduced with the indicated double-stranded DNA as described under "Experimental Procedures." Cells were then exposed to cAMP or vehicle for 6 h and then harvested for RNA isolation. The isolated RNA was subjected to multiplex RT-PCR using primers for the indicated transcripts. A, the Ren-1d PCR product was greatly diminished in cells treated with the CNRE decoy but not in cells treated with the CRE decoy or with scrambled double-stranded DNA. B, 11 of 16 transcripts were decreased following exposure of the cells with the CNRE decoy but not the CRE decoy or with scrambled double-stranded DNA.

Assessment of Direct Interaction of LXRalpha to Celera-identified CNRE Elements-- We utilized EMSA to determine whether LXRalpha could directly interact with the identified CNRE elements present in genes affected by decoy molecules. Nuclear extracts were prepared from As4.1/LXRalpha or As4.1/GFP cells, which were treated with 8-Br-cAMP for 6 h. Double-stranded EMSA probe(s) for each gene was generated, which contained the Celera-identified CNRE sequence, with an additional 6 nucleotides of flanking sequence to assess direct LXRalpha interaction. Competition assays were conducted using a double-stranded 32P-labeled oligomer probe containing the CNRE element from mouse renin (ren-1d) in the presence of 100-fold molar excess of each unlabeled probe containing the CNRE sequence present in each specific gene. Results in Fig. 7A demonstrate that the CNRE elements present in 9 of the 11 decoy-affected genes queried were able to effectively compete the labeled renin CNRE probe for LXRalpha binding as evidenced by abolishment in signal. No competition was observed from the CNREs in the remaining 5 of the total 16 genes assayed.


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Fig. 7.   Electrophoretic mobility shift assays of LXRalpha binding to novel CNRE enhancer elements present in identified genes. Nuclear extracts were prepared from cAMP-stimulated As4.1/LXRalpha or As4.1/GFP cells, and 10 µg was used to assess interaction of LXRalpha to novel CNRE elements by competition or direct binding assays. A, effects of 100-fold molar excess of unlabeled probe containing the cis-element CNRE sequence with flanking sequences to compete with a 32P-labeled probe containing the CNRE sequence present in mouse renin promoter. The CNRE element(s) present in 9 of the 16 identified genes was able to effectively compete for binding (noted with an asterisk). B, nuclear extracts were incubated with 32P-labeled probe containing the CNRE element present in either mouse ren-1d or the newly identified psx-1 or KIAA0877 genes. A strong shift is observed using the psx-1 probe, which is comparable with the renin CNRE probe. No gel shift was observed from the KIA0877 probe. Moreover, abolishment of gel shift was observed when using an anti-LXRalpha antibody when compared with controls (IgG or no antibody).

To determine whether the competition observed was LXRalpha -specific, we employed a LXRalpha -specific antibody (P-19) to neutralize the binding of CNRE/LXRalpha direct interactions. Labeled probes containing the cis-element CNRE sequence from ren-1d, placental-specific homeobox (psx-1), or KIA0877 genes were incubated with As4.1/LXRalpha or As4.1/GFP nuclear extracts in the presence of anti-LXRalpha antibody. Fig. 7B indicates that a strong shift is observed in As4.1/LXRalpha control (-Ab or +goat IgG) cell lysates with the ren-1d or psx-1 probes when compared with the KIAA0877 probe. However, in the presence of anti-LXRalpha antibody, the CNRE/LXRalpha interaction is abolished from the ren-1d probe. Likewise, reduced binding but not complete abolishment is also observed between the psx-1 and CNRE probe. No LXRalpha shift was observed in control extracts (As4.1/GFP) or extracts incubated with KIA0877 probe and/or antibody.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear orphan receptor-mediated transcriptional regulation can be divided into two major sequences of cellular events. First, the inactivated nuclear receptor becomes activated by a conformational change induced either by ligand binding or phosphorylation at key sites within the receptor (21). Second, activated nuclear receptor is then translocated to the nucleus to regulate target gene transcription either as a monomer or complexed with other transcription modulatory proteins (10). Previously, we identified LXRalpha as one of the key modulatory proteins that control the expression of renin in both cultured mouse kidney juxtaglomerular (As4.1) (8) and human lung (Calu-6) cells (22), both of which were transduced with an LXRalpha or GFP expression vector. Specifically LXRalpha in the presence of cAMP up-regulates renin gene expression by directly binding as a monomer to a unique CNRE located in close proximity to the transcriptional start site. Furthermore, we also demonstrated the c-Myc was regulated in a similar manner. Taken together, the previous data suggested that cAMP-activated LXRalpha could transcriptionally regulate genes with various cellular functions and implied a more ubiquitous role of LXRalpha /CNRE-mediated gene regulation in cellular physiology. Indeed, the results of this current study strongly support this contention.

The clones spotted on the mouse 19,064 element array were generated from various cDNA libraries that consisted of cDNA clones from mouse brain, kidney, and erythroid blood cell libraries. The functional distribution of known genes on the array correlated with the expected division of expressed protein functions involved in normal cellular physiology and indicated that although the various clones were obtained from different sources, the cumulative clone set maintained a representative functional allocation with respect to cellular physiology (23). Moreover, because a moderate number of spotted clones were unable to be matched in the Unigene data base and thus were considered novel, our mouse array provides a useful gene discovery source with which to identify novel genes that are regulated by LXRalpha in the presence of small molecule activators such as cAMP. It is certain that because the human and mouse genomes are now sequenced and the era of functional genomics is becoming fully realized, more of these novel genes will eventually become known and functionally annotated and will provide further information to the role of LXRalpha in global gene regulation and the implications to cellular physiology (24-26).

Our previous reports demonstrate that in cells overexpressing LXRalpha , renin mRNA levels increase as early as 1 h in response to cAMP that peaks at 6 h (8). This expression "signature" of mouse renin was also confirmed by our RT-PCR results in this study. To identify genes that are potentially regulated by activated LXRalpha , we queried the entire data set of expressed genes for genes that exhibited an expression profile similar to that of renin. Some of the listed genes such as Hsc70, P450, and Ufd1 have been reported to be involved in the signal transduction pathway of nuclear receptor inactivation and activation, respectively, whereas the P450 family of genes has been reported to be transcriptionally regulated by various nuclear receptors (27-29). Another subgroup of genes from this list (Psx-1, MM-1, and OxyR) have been reported to be involved in modulating gene transcription by directly interacting with the promoter regions of other target genes (30-32). It should be noted that the intracellular levels of cAMP and LXRalpha transcript in these cells are markedly elevated and hence probably result in non-physiologically high levels of activated LXRalpha . Thus, some of the genes identified, demonstrating a significant temporal change in transcript abundance in vitro, may not change in a temporal manner by conventional detection methods in vivo. It will be interesting to compare the changes observed in vitro under a more physiologically relevant condition; however, it is not within the scope of this communication.

We used a conservative algorithm to identify genes that contained a consensus CNRE binding sequence (33). This strategy would allow us to identify genes that contained this enhancer motif within a 50-kb region upstream of the transcriptional start site because previous reports indicate the CNRE element as being located upstream of other identified genes (7). The results of the analysis for genes containing a CNRE enhancer element within this subset indicated yet a smaller subset of genes that contained a sequence that matched the consensus LXRalpha binding site. Not all of the genes exhibiting a profile of expression similar to that of renin contained a CNRE enhancer. Several possible explanations exist for this finding. One likely explanation is that we have not eliminated secondary effects of LXRalpha activation, i.e. the induction of expression of a transcription factor or factors by LXRalpha that then induce the expression of other genes secondary to the initial actions of LXRalpha . Indeed, inspection of the genes induced by LXRalpha or cAMP-activated LXRalpha reveals several known or putative transcription factors. Furthermore, although we have searched the 50-kb upstream of the EST with our best estimate of a consensus CNRE, it is possible that other sequences that deviate from the consensus sequence are functional but ignored in the bioinformatics-screening process. Moreover, it is likely that LXRalpha binds and exerts action through other response elements distinct from the CNRE such as the previously described DR4 sequence (34). However, it is important to note that although not all of the genes that possessed an expression profile similar to that of renin was shown to have a CNRE in the 50-kb region, of those that did, a vast majority (15 of 16) had profiles of expression that were verified by an independent assay (RT-PCR) to be similar to renin.

Although the majority of genes containing the CNRE exhibited reduced expression levels in the response to CNRE decoy administration, a few genes remained unaffected. Perhaps the CNRE sequence found in the genes that were non-responsive to the decoy might have a greater affinity for LXRalpha than the renin CNRE sequence used in the decoy molecules. Alternatively, although positive for the presence of the consensus CNRE sequence, these genes might be regulated by other transcriptional mechanisms described above such as the binding of cAMP-activated LXRalpha to a sequence distinct from the CNRE or the regulation of gene expression secondary to the induction of transcription factors by LXRalpha .

EMSA assays were performed to determine whether LXRalpha was directly interacting with the identified cis-element CNRE sequences present in the genes. Because the majority of the CNRE elements present in the identified genes were able to effectively compete the previously described renin CNRE element in the majority of genes (9 of 11) for LXRalpha binding, this finding indicates that these genes are temporally regulated in part by the direct binding to LXRalpha to these cis-elements in As4.1 cells. Interestingly, the identified CNRE element(s) present in two genes (RP11-492E24 and Mrpl9) was unable to compete for LXRalpha binding. One likely explanation could be that these particular genes are indirectly regulated by other genes, which contain a functional CNRE element, and thus are directly regulated by LXRalpha . Other possible explanations could be that the sequences that flank these particular CNRE elements reduce the affinity to effectively compete the renin CNRE probe or that the functional CNRE element lies outside of the queried region (50-kb). Whether this occurs under physiological conditions with endogenous levels of LXRalpha remains to be determined.

The identification of a set of genes that are regulated by LXRalpha through the CNRE element suggests that this mechanism of cAMP-mediated induction may be a specific gene regulatory system when compared with the widespread effects of CRE-mediated induction. Indeed, LXRalpha has been reported to be a key mediator in the regulation of expression of genes that tend to maintain cellular or systemic physiological homeostasis (1). Moreover, these genes are regulated by LXRalpha through a specific DNA enhancer sequence (DR4/LXRE). Our data suggest LXRalpha as being a key mediator in the induction of other genes in mouse kidney cells though the CNRE element and indicate that modulation of transcription via CNRE is most likely a temporal and/or cell type-specific mechanism of cAMP induction when compared with CRE-mediated induction. The human renin gene also contains a CNRE in addition to a CRE element, which indicates the need for a more detailed regulatory system in response to cAMP. It will be interesting to determine how these newly identified LXRalpha /CNRE-regulated genes play a role in the overall cellular physiology in kidney cells in response to cAMP and the teleological importance of gene modulation exerted by this specific mechanism.

In summary, in this communication, we have utilized a genomics approach by querying a 19,064-element mouse cDNA microarray generated in our laboratory for potential cAMP-activated LXRalpha -regulated genes. Our data taken together with previous reports indicate that LXRalpha can regulate the expression of different sets of genes using multiple mechanisms (i.e. heterodimerization at the DR4 element, monomer at the CNRE) to regulate different physiological functions. LXRalpha not only regulates cholesterol metabolism and bile acid production but also renin expression, a major mediator of blood pressure homeostasis and kidney function. These data would suggest that LXRalpha is an important transcriptional regulator of the cardiovascular system and cellular physiology.

    ACKNOWLEDGEMENTS

We thank our colleagues, Drs. David Beier (Genetics Division, Brigham and Women's Hospital) and C. C. Liew (Cardiovascular Division, Brigham and Women's Hospital), for providing cDNA clones for arraying brain and kidney libraries (D. B.) and nucleated erythroid cell library (C. C. L.). We also thank Dr. C. C. Liew and members of his laboratory for helpful discussion.

    Note Added in Proof

Following the acceptance of this article, we became aware of several other articles describing profiling of LXRalpha -dependent genes (35-38). Genes reported in these studies, which mainly involved differential expression between LXRalpha agonist and vehicle-treated tissue or cells or between tissues from LXRalpha -/-, LXRbeta -/- and wild type mice, were different from those reported in our article. This emphasizes the unique mechanism of action described in our article.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL35610 and HL58516 (to V. J. D.) and HL61661 (to R. E. P.).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.

|| To whom correspondence should be addressed. E-mail: rpratt@rics.bwh.harvard.edu or VDZAU{at}Partners.org.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M208644200

    ABBREVIATIONS

The abbreviations used are: LXRalpha , liver X receptor-alpha ; CNRE, cAMP-negative response element; JG, juxtaglomerular; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; 8-Br-cAMP, 8-bromo-cyclic AMP; kb, kilobase; PBS, phosphate-buffered saline; CRE, cAMP-inducible and negative response element; CREB, cAMP-response element-binding protein; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; FITC, fluorescein isothiocyanate; EST, expressed sequence tag.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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