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INTRODUCTION |
The transcription factor liver X receptor-
(LXR
),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 LXR
-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
and
peroxisome proliferator-activated receptor
(3). Transcription of
target genes such as the cholesterol 7
-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 LXR
bound to this sequence,
whereas functional studies using promoter/reporter gene chimeric
constructs revealed that LXR
increased basal levels of renin
expression and mediated the cAMP-dependent induction of
mouse and human renin gene expression (8).
Interestingly, the previously reported LXR
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 LXR
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 LXR
/DR4 element. Further studies revealed that the
c-myc gene, which also contains the CNRE element, was
similarly regulated (9). The discovery of LXR
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 LXR
. 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 LXR
plus GFP and treated with cAMP or vehicle.
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EXPERIMENTAL PROCEDURES |
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 LXR
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,
-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 LXR
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 LXR
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/LXR
was incubated with 1.75 pM
-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-LXR
(Santa Cruz Biotechnologies), or gel shift buffer for an
additional 15 min.
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RESULTS |
Global Gene Expression Is Affected by LXR
in As4.1 JG Cell
Cultures--
The novel action of LXR
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.
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Serum-restricted mouse kidney juxtaglomerular cells (As4.1) stably
transduced with either GFP plus mLXR
(As4.1/LXR
) 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 LXR
in the presence of cAMP when compared with all other
conditions and thus demonstrate our previous observations of LXR
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/LXR 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 LXR /cAMP has a dramatic effect
on the profiles of expression in As4.1 juxtaglomerular cells.
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Identification of LXR
-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
LXR
is able to modulate the expression of genes with diverse
functional properties. These results indicate that at 6 h,
LXR
+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 LXR
+ 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 LXR
and cAMP. As4.1 cells that were stably transduced with a
retrovirus expressing either a bicistronic GFP/LXR 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 LXR -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.
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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%.
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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 LXR
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/LXR , 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/LXR cells. No significant difference
was observed with 18 S RNA signal
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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
LXR
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 LXR
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.
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As.4.1 cells transduced with either LXR
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 LXR
-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 LXR
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
LXR -regulated genes. As4.1/LXR 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.
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Assessment of Direct Interaction of LXR
to
Celera-identified CNRE Elements--
We utilized EMSA to determine
whether LXR
could directly interact with the identified CNRE
elements present in genes affected by decoy molecules. Nuclear extracts
were prepared from As4.1/LXR
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 LXR
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 LXR
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
LXR binding to novel CNRE enhancer elements
present in identified genes. Nuclear extracts were prepared from
cAMP-stimulated As4.1/LXR or As4.1/GFP cells, and 10 µg was used
to assess interaction of LXR 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-LXR antibody when compared with controls (IgG or no
antibody).
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To determine whether the competition observed was LXR
-specific, we
employed a LXR
-specific antibody (P-19) to neutralize the binding of
CNRE/LXR
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/LXR
or As4.1/GFP nuclear extracts in
the presence of anti-LXR
antibody. Fig. 7B indicates that
a strong shift is observed in As4.1/LXR
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-LXR
antibody, the CNRE/LXR
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 LXR
shift was observed in
control extracts (As4.1/GFP) or extracts incubated with
KIA0877 probe and/or antibody.
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DISCUSSION |
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 LXR
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 LXR
or
GFP expression vector. Specifically LXR
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 LXR
could transcriptionally regulate genes with
various cellular functions and implied a more ubiquitous role of
LXR
/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 LXR
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 LXR
in
global gene regulation and the implications to cellular physiology
(24-26).
Our previous reports demonstrate that in cells overexpressing LXR
,
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 LXR
, 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 LXR
transcript in these
cells are markedly elevated and hence probably result in
non-physiologically high levels of activated LXR
. 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 LXR
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
LXR
activation, i.e. the induction of expression of a
transcription factor or factors by LXR
that then induce the
expression of other genes secondary to the initial actions of LXR
.
Indeed, inspection of the genes induced by LXR
or cAMP-activated
LXR
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 LXR
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
LXR
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 LXR
to a sequence distinct from the CNRE or the regulation of gene
expression secondary to the induction of transcription factors by
LXR
.
EMSA assays were performed to determine whether LXR
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
LXR
binding, this finding indicates that these genes are
temporally regulated in part by the direct binding to LXR
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 LXR
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 LXR
. 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 LXR
remains to be determined.
The identification of a set of genes that are regulated by LXR
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, LXR
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 LXR
through
a specific DNA enhancer sequence (DR4/LXRE). Our data suggest LXR
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 LXR
/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 LXR
-regulated genes. Our
data taken together with previous reports indicate that LXR
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.
LXR
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
LXR
is an important transcriptional regulator of the cardiovascular
system and cellular physiology.