From the Departments of Pharmacology and Cancer
Biology, § Anesthesiology, and ¶ Surgery, Duke
University Medical Center, Durham, North Carolina 27710
Received for publication, November 25, 2002
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ABSTRACT |
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Recent studies reveal important and
distinct roles for cardiac Recent studies reveal an important and distinct role for
Surprisingly, given the importance of Library Screening
To isolate the 5'-UTR sequence of the rat Creation of Rat To determine the location of cis-elements important
in conferring cell-specific and hypoxia-mediated regulation of the rat Isolation of Total RNA
Total RNA was isolated by the Trizol method (Invitrogen).
Genomic DNA contamination was removed by digestion of RNA samples with
RNase-free DNase I (0.4 unit/µl of RNA sample) for 30 min at
37 °C, and degraded nucleic acid was removed using RNeasy spin columns (Qiagen; Valencia, CA). RNA concentrations were determined spectrophotometrically, and samples were stored at Northern Blot Analysis
A rat multitissue Northern blot containing 2 µg of
poly(A)+ mRNA in each lane was purchased from RNway
laboratories (Seoul, Korea). Membranes were hybridized with a
32P-labeled RNase Protection Assay (RPA) Methods
To identify the transcription initiation site of the rat
Primer Extension Assay
Primer extension assays were used to corroborate the
transcription initiation site results generated by RPAs. Primer
extension oligonucleotide Primer 1 was made complementary to the region +32/+4, and Primer 2 corresponded to +75/+48, relative to the ATG.
Oligonucleotides were end labeled with [ Quantitative Competitive RT-PCR
Competitor Construction--
Heterologous competitor DNA
constructs were amplified from pGEM-7Zf plasmid vector (Promega) using
chimeric primers containing both rat RT-PCR and Quantitation of General Cell Culture
To gain insight into mechanisms governing cell-specific
Rat Myocyte Isolation and Culture
Postnatal (1-2 days postbirth) rat pup hearts were
enzymatically dissociated with 73 units/ml collagenase and 0.6 mg/ml
pancreatin, and cardiomyocytes were purified by Percoll density
centrifugation as described previously (23). Myocytes were enriched
further by preplating cells for 15 min on standard tissue culture
dishes (Costar; Cambridge, MA) to remove fibroblasts. Cells were plated onto 30-mm laminin-coated culture plates at a density of 500,000 cells/well and were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, and insulin/transferrin/selenium (Invitrogen) and exposed to 90% ambient air and 10% CO2. Typical
myocyte yields were 2 × 106 cells/heart, with an
average viability of >98% (determined by trypan blue exclusion).
Transient Transfection of To examine Hypoxia Exposure
Myocytes were placed in a hypoxia chamber and exposed to 88.5%
N2, 10% CO2, and 1.5% O2 for the
lengths of time indicated in figure legends. Oxygen levels were
monitored by a Fyrite Gas Analyzer (Illinois Scientific).
Preparation of Nuclear Extracts
Nuclear cell extracts were prepared from cardiomyocytes as
described previously (24). Briefly, 1 × 107 cells
were washed twice with ice-cold phosphate-buffered saline and once with
a solution containing 10 mM Tris-HCl, pH 7.8, 1.5 mM MgCl2, and 10 mM KCl
supplemented with a protease inhibitor mixture containing 0.5 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each leupeptin, pepstatin, and aprotinin (Sigma). Cells were lysed with a Dounce homogenizer, and nuclei were
pelleted and resuspended in a solution containing 420 mM KCl, 20 mM Tris-HCl, pH 7.8, 1.5 mM
MgCl2, and 20% glycerol, supplemented with the protease
mixture described above and incubated at 4 °C with gentle agitation.
The extract was centrifuged at 10,000 × g, and the
supernatant was dialyzed twice against a solution of 20 mM
Tris-HCl, pH 7.8, 100 mM KCl, 0.2 mM EDTA, and
20% glycerol. Protein concentration was determined using BCA reagent
(Pierce) with bovine serum albumin as standard.
Electrophoretic Mobility Shift Assay (EMSA)
Oligonucleotide probe OL1, specific for region
Statistical Analysis
Linear regression analysis of cRT-PCR data was calculated by the
least squares method. Statistical analysis was performed using
two-tailed, unpaired Student's t tests with
p < 0.05 considered significant. Results are presented
as the means ± S.E.
Cloning of the Rat
Use of more distal overlapping RPA probes (Probes 5-9, Fig.
2A) revealed the presence of several closely clustered
upstream promoters, with major start sites located at Tissue-specific Expression of Rat
We next wished to determine absolute endogenous Cell-specific Promoter Activity--
To examine cell-specific expression of the proximal P1 promoter
directly, 1.5 kb of proximal sequence was fused upstream of the
luciferase reporter gene and transiently expressed in cell lines the
same cell lines described above. Consistent with results from the
full-length promoter fragment, the proximal promoter also directs
higher levels of reporter gene expression in cardiomyocytes versus PC12 cells and rat1-fibroblasts, in which promoter
activity decreases 61 and 69%, respectively (Fig. 4C,
left panel). Together these results demonstrate that both
proximal and distal promoter activity is consistent with high levels of
cardiac basal expression seen with the endogenous Hypoxia-mediated Modulation of
To identify cis-elements in the
Because hypoxia decreases Hypoxia-specific Binding of Nuclear Factors to an
To narrow the
To determine whether the regions identified by EMSA were functionally
relevant, these regions were mutated by site-directed mutagenesis in
the full-length 3.9-kb reporter construct and transfected into
cardiomyocytes under both normoxic and hypoxic conditions (Fig.
8B). Under normoxic
conditions, only mutant 1 had a statistically significant affect on
transcriptional activity, decreasing reporter gene levels 26% (Fig.
8B; p < 0.05). Importantly, under hypoxic conditions two sequential mutations within the Stimulation of cardiac Northern blot analysis confirms that there are at least two transcripts
in rat heart, with molecular weights of ~3.5 and ~1.7 kb. Using
5'-oriented probes, we saw no evidence of the higher molecular weight
9.5- and 11-kb transcripts reported previously to be expressed in rat
heart (19). Because the former study utilized a full-length cDNA
probe, including 3'-UTR sequence, the higher molecular weight forms may
represent 3'-UTR alternatively spliced variants. The smallest 1.7-kb
transcript was not detected in the former study, perhaps because of the
use of neonatal tissue in the current study versus adult
tissues reported earlier, suggesting developmental regulation of this
transcript. The difference in molecular weight of the two transcripts
( Once the rat 1a adrenergic receptors
(
1aARs). Surprisingly, given their importance in
myocardial ischemia/reperfusion, hypoxia, and hypertrophy as well as
frequent use of rat cardiomyocyte model systems, the rat
1aAR gene promoter has never been characterized.
Therefore, we isolated 3.9 kb of rat
1aAR
5'-untranslated region and 5'-regulatory sequences and identified
multiple transcription initiation sites. One proximal (P1) and several
clustered upstream distal promoters (P2, P3, and P4) were delineated.
Sequences surrounding both proximal and distal promoters lack typical
TATA or CCAAT boxes but contain cis-elements for multiple
myocardium-relevant nuclear regulators including Sp1, GATA, and CREB,
findings consistent with enhanced cardiac basal
1aAR
expression seen in Northern blots and reporter constructs. Promoter
analysis using deletion reporter constructs reveals, in addition to a
powerful upstream enhancer, a key region (-558/
542) important in
regulating all
1aAR promoters with hypoxic stress. Gel
shift analysis of this 14-bp region confirms a hypoxia-induced shift
independent of direct hypoxia-inducible factor binding. Mutational
analysis of this sequence identifies a novel 9-bp hypoxia response
element, the loss of which severely attenuates hypoxia-mediated repression of
1aAR transcription. These findings for the
1a gene should facilitate elucidation of
1AR-mediated mechanisms involved in distinct myocardial pathologies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1ARs1 are
members of the larger family of G protein-coupled receptors that
mediate sympathetic nervous system responses such as smooth muscle
contraction (1) and increased myocardial contractility (2, 3); these
responses occur predominantly via activation of phospholipase C-
,
resulting in the stimulation of both protein kinase C and
inositol trisphosphate pathways (4). cDNAs encoding three
1AR subtypes (
1a,
1b, and
1d) have been cloned and pharmacologically characterized
in several expression systems, leading to the surprising finding that
1AR subtype tissue distribution is
species-dependent, with expression regulated at both gene
and protein levels (4). To gain insight into transcriptional pathways
governing
1AR expression, several laboratories have
initiated cloning and characterization of
1AR subtype
regulatory regions from different species, including the
1aAR (human (5), mouse (6)),
1bAR (mouse
(7), rat (8), human (9)), and
1dAR (rat (10)).
1aARs in the heart, including enhanced contractility (2,
3) and hypertrophy (11-14). Modulation of
1aAR
expression occurs during pathological states, such as myocardial
hypertrophy (13, 14) and hypoxia (15), the latter usually secondary to
ischemic injury. In hypertrophic models, classically performed in
cultured neonatal rat cardiomyocytes, direct chronic
1aAR stimulation results in increased transcription of
the
1aAR gene itself, providing a pathway for overall
sustained
1AR signaling in the heart (13, 14). In
contrast, both in vitro cardiomyocyte and whole animal models of hypoxia (1% O2, 72 h) reveal decreased
1AR signaling, caused in part by reduced levels of
1aAR mRNA and protein (15). Interestingly, these
studies also show that chronic hypoxia selectively attenuates
1AR-mediated hypertrophy (15), suggesting that
hypertrophy secondary to ischemic injury occurs via distinct,
uncharacterized mechanisms. A number of pathways have been implicated
in regulation of
1AR signaling including protein kinase
C, phosphoinositide 3-kinase, rho, ras, signal
transducers and activators of transcription (STAT), c-Jun
NH2-terminal kinase (JNK), p38, extracellular
signal-regulated kinase (ERK), and calcineurin-dependent
pathways (4). How these signals are integrated to regulate
1aAR expression also remains unknown.
1aARs in
myocardial pathology and the use of rat neonatal cardiomyocytes to
study hypertrophy, the rat
1aAR promoter has been
neither isolated nor characterized. Therefore, to facilitate our
mechanistic studies of myocardial
1aAR regulation, we
cloned and characterized rat
1aAR 5'-UTR and regulatory
sequences, investigated cell-specific expression, and characterized
basal transcription in the presence and absence of the physiologic
stress of hypoxia. Our data indicate that the rat
1aAR
gene is both similar and yet distinct from mouse and human
1aAR genes. The rat
1aAR gene is
transcribed from multiple promoters; a proximal promoter (
131 bp
relative to ATG) as well as several clustered upstream distal promoters
(P2, P3, P4, centered
2.1 kb relative to ATG). Both proximal and
distal promoters are TATA-less, containing consensus sequences for an
initiator element as well as cis-elements for transcription
factors Sp1, GATA, and CREB, shown to be important for cardiac
gene transcription (16, 17). These findings are consistent with
enhanced cardiac basal
1aAR expression seen in Northern
blots and reporter constructs. In addition to a powerful upstream
enhancer, reporter gene and gel shift analysis reveals the presence of
nuclear factor(s) able to associate with a 14-bp sequence of the rat
1aAR promoter in cardiomyocytes exposed to hypoxic
stress. Systematic mutation of sequences within this region identified
a potent novel 9-bp hypoxia response element (HRE). These findings
provide an important foundation for elucidating specific
1AR-mediated mechanistic pathways involved in distinct
myocardial pathologies.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1aAR
gene, a rat genomic library (
DASH, average insert size 15 kb,
Stratagene; La Jolla, CA) was screened using a liquid lysate approach.
The
1aAR gene was isolated from phage DNA (18) using
nested PCR amplification with two
1aAR-specific primers
corresponding to residues 836-860 and 776-800 from the published
cDNA sequence (19) (GenBank NM017191). T7 primer specific for the
DASH vector polylinker was used as the opposing primer, creating a
4.2-kb PCR-generated genomic fragment. PCR fidelity was assured by
subcloning and sequencing three independent amplification products.
1aAR 5'-UTR Deletion and Mutant
Constructs
1aAR gene, serial 5'-deletions of the full-length
promoter fragment were made. A HindIII-NcoI
digest of the 4.2-kb rat
1aAR fragment described above
was cloned into SmaI-HindIII pGL2-Enhancer
plasmid (Promega, Madison, WI) to generate the full-length rat
1aAR reporter construct. Serial deletions of the 5'-UTR
of the rat
1aAR gene were created using either
convenient restriction sites or a PCR-based approach; all PCR-generated
constructs were sequenced to ensure fidelity. Site-directed mutagenesis
was performed using the QuikChange XL kit (Stratagene) according to the
manufacturer's recommendations. A 634-bp
BamHI-NdeI fragment containing the desired
mutation was sequenced and reintroduced into the original full-length
reporter plasmid to ensure the integrity of all constructs.
80 °C.
1aAR RNA probe and washed as
described previously (20). The blot was stripped and reprobed with a
220-bp GAPDH probe specific for nucleotides 663-855 of the published
cDNA. Quantification was performed using ImageQuant software
(Molecular Dynamics, Sunnyvale, CA), normalized to GAPDH levels.
1aAR gene, RPAs were performed as described previously
(5) using 30 µg of total RNA isolated from postnatal day 8 rat heart
and >20-fold excess of radiolabeled rat
1aAR probe
(3 × 105 cpm/reaction). Nine RNase protection probes
spanning the 3.9-kb 5'-UTR were generated and cloned into pBluescript
vector (Stratagene): Probe 9, SpeI-PstI,
corresponding to the
3013/
2378; Probe 8, NcoI-HincII, corresponding to
2314/
1819;
Probe 7, PstI-PstI, corresponding to
2378/
2052; Probe 6, PflFI-NcoI, corresponding to
2100/
1819; Probe 5, PstI-ApaI,
corresponding to
2052/
1628; Probe 4, ApaI-BamHI, corresponding to
1628/
1091; Probe
3, BamHI-NdeI, corresponding to
1091/
457;
Probe 2, NdeI-NcoI, corresponding to
457/
2;
Probe 1, BstBI-NcoI corresponding to
430/
2
(all relative to ATG). A rat cyclophilin probe (Ambion, Austin, TX) was
used as a positive control (5 × 104 cpm/10 µg of
total RNA). Quantitation of
1aAR mRNA levels in different rat tissues was performed utilizing a previously described probe specific for nucleotides 556-860 of the rat
1aAR
gene (20).
-32P]ATP using
T4 polynucleotide kinase, and primer extension assays were performed as
described previously (21) using 30 µg of total RNA isolated from
postnatal day 8 rat heart and isolated rat neonatal cardiomyocytes.
Products from primer extension assays were size fractionated on a 6%
polyacrylamide gel; a sequencing reaction using Primer 1 was used as a
bp marker.
1aAR cDNA
and pGEM-7Zf plasmid sequence. Chimeric primers were as follows:
forward, 5'-GTAGCCAAGAGAGAAAGCCGGACGCTCAAGTCA GAGGTGGCGAAACCCGA-3', and reverse 5'-CAACCCACCACGATGCCCAGCTT CTAGTGTAGCCGTAGTTAGGCCACCAC-3'; bold sequences are specific for the rat
1aAR coding sequence (residues 663-682 and
874-855, respectively, from ATG (19)) whereas non-bold sequences
correspond to residues 601-630 and 971-1000 of pGEM-7Zf plasmid
sequence, respectively (GenBank X65310). The resulting 440-bp PCR
product was subcloned into pCRII vector (Invitrogen) and competitor
constructs verified by sequencing. Competitor cRNA for RT-PCRs was
synthesized by in vitro transcription using commercially
available reagents (Ambion). RNA concentrations were determined spectrophotometrically.
1aAR mRNA
Levels--
Because of the accuracy of competitive RT (cRT)-PCR over
other RT-PCR methods and excellent target sensitivity, this method was
used to quantitate
1aAR mRNA levels in several
assays. Total RNA and competitor RNA were synchronously reverse
transcribed and amplified using a commercially available kit
(PerkinElmer Life Sciences). Control reactions with no RT were
performed to ensure absence of contaminating genomic DNA. Samples were
amplified as described (22). GAPDH was used to normalize mRNA
levels using primers corresponding to residues 120-137 and 338-320 of
the published cDNA sequence, respectively. Final PCR products were
size fractionated on a 1.5% Tris borate-agarose gel bands, and
normalized net band intensities were transformed into an amplification
ratio and plotted logarithmically versus competitor amount.
The x axis of the linear regression (least squares method)
reflects the equivalence point at which the competitor concentration
equals the initial mRNA concentration. This method provides the
absolute amount of target transcript in starting samples.
1aAR expression, rat1-fibroblasts, rat PC12
pheochromocytoma cells, and primary neonatal rat cardiomyocytes were
used as model systems. Rat1-fibroblasts were grown in Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with 10% fetal
bovine serum, 100 units/ml penicillin/streptomycin, and 2 mM
L-glutamine. PC12 cells (ATCC; Rockville, MD) were maintained in
RPMI medium (Invitrogen) supplemented with 10% fetal bovine serum and
100 units/ml penicillin/streptomycin. Cells were plated onto 30-mm
culture plates at a density of 200,000 cells/well in the appropriate medium.
1aAR Reporter Constructs
into Cultured Cells
1aAR promoter activity in different
cellular contexts,
1aAR reporter constructs were
transiently expressed in neonatal rat cardiomyocytes, PC12 cells, and
rat1-fibroblasts followed by measurement of luciferase reporter gene
activity. 24 h after initial plating, cells were washed with
phosphate-buffered saline and fed with fresh medium. Cultures were
transfected with 1 pmol of double CsCl-banded reporter plasmid and 0.5 pmol of
-galactosidase normalization plasmid (Promega) using the
calcium phosphate precipitation method, as described previously (23). 24 post-transfection, cells washed twice with phosphate-buffered saline
and maintained in appropriate medium for an additional 24 h.
Transfected cells were harvested, and luciferase and
-galactosidase activity was assayed using the Dual-Light Assay System according to the
manufacturer's recommendations (Tropix; Bedford, MA). Transfection efficiency was 5-10%, measured by visual
-galactosidase staining.
573/
515, used for EMSA was 5'-GATC CTC CTC AAA TAT TTC AGA CCC ATG
TCA CTT AGC CAG AAC TCC TAG ACG CTG GAG CTA GC-3'. The sense and
antisense strands were annealed and end labeled with
[32P]ATP (PerkinElmer Life Sciences) using T4
polynucleotide kinase (Invitrogen). To measure DNA-protein
interaction, 20 fmol (2-5 × 104 cpm) of
oligonucleotide probe was incubated with 5 µg of nuclear extract and
0.1 µg of sonicated, denatured salmon sperm DNA (Invitrogen) in 10 mM Tris-HCl, pH 7.8, 50 mM KCl, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol, and 5% glycerol
for 20 min at 4 °C in a total volume of 20 µl. The reaction
mixture was size fractionated on 6% nondenaturing polyacrylamide gels
at 4 °C. Dried gels were subjected to autoradiography for 16-48 h.
For competition experiments, a 1,000-fold molar excess of unlabeled oligonucleotide was added to the binding reaction just before the
addition of radiolabeled probe. Competitor oligonucleotides were
5'-CTCCTCAAATAT TTCAGA-3', 5'-AGACCCATGTCACTTAG-3',
5'-AGCCAGAACTCCTAGACG-3', 5'-TAGACGCTGGAGCTAGC-3', corresponding
to sequential, overlapping 18-mers within the
573/
515 region, as
described in the Fig. 6B legend. For gel supershift
analysis, 1 µl of monoclonal antibody against HIF-1
(StressGen
Biotechnologies, Victoria, B. C., Canada) was added after the initial
20-min incubation, and the solution was incubated further for 10 min at
4 °C before electrophoresis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1aAR 5'-Regulatory Sequence and
Identification of Proximal and Distal Promoters--
To initiate
studies on transcriptional regulation of the rat
1aAR
gene, a 4.2-kb EcoRI-EagI fragment was subcloned
from a genomic library and sequenced (3.9 kb of regulatory sequence is present upstream of the ATG shown in Fig.
1).
To determine the location of 5'-regulatory sequence, the transcription
initiation site (TIS) was identified by RPA using several overlapping
probes that spanned the entire region, resulting in the identification of multiple promoters (RPA Probes 1-9; Fig.
2A). The proximal promoter was
identified using Probe 1 (Fig. 2A); four protected fragments
are present (three minor, one major) ~85, 125 (major), and 140 bp in
length (Fig. 2B). These products correspond to initiation sites of approximately
145,
130, and
85, respectively, relative to the ATG. Primer extension analysis was used to confirm RPA results
(Fig. 2C). Two antisense oligonucleotide primers,
corresponding to bp +32/+4 (Primer 1, relative to ATG) and +77/+50
(Primer 2) were utilized to map the TIS. In both cases, one major
product was detected (163-bp product for Primer 1 and 208-bp product
for Primer 2) corresponding to the 125-bp major product detected by RPA
analysis. A bp sequencing ladder provides the exact site of transcription initiation as adenosine/-131
(CCAGCTG, relative to the ATG) in a region
conforming to a loose initiator sequence (initiator; PyPyA
(1)NT/APyPy(25)). Neither the shortest 85-/83-bp, nor the longest
140-bp RPA products were detected by primer extension analysis. Because
the major TIS RNA product was confirmed by two separate primers, it is
possible that unconfirmed RPA products may be enzymatically generated
because of inherent folding properties of the RPA probe used or that
these potential TISs are below the threshold of detection by primer
extension. We designate the main TIS located 131 bp upstream from the
translation start site as the proximal (P1)
1aAR
promoter.
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Fig. 1.
5'-Regulatory and 5'-UTR DNA sequence from
rat 1aAR gene. The rat
genomic DNA sequence is shown with consensus sequences for
transcription factor binding sites (highlighted in bold
above the sequence). Where cis-elements overlap, the
5'-element is underlined and 3'-element in bold.
TIS for P1, P2, and P3 are highlighted in bold and
underlined; major initiation sites in P2 and P3 are labeled
with arrowheads, and the P4 cluster is boxed.
Initiation sites identified by primer extension are indicated by a
bold vertical line above the nucleotide. A sequence
encompassing P1 conforming to a consensus initiator element
(Inr) is also boxed. The novel HRE is
double underlined.
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Fig. 2.
Identification of TIS of the rat
1aAR gene. A, schematic
representation of RPA probes used for identification of the TIS.
Restriction sites utilized for the generation of RPA Probes 1-9 are
indicated. Primer locations for primer extension analysis are
labeled and indicated by arrows. B,
identification of the proximal promoter TIS by RPA. Total RNA from
postnatal day 8 rat hearts (lanes 1-3) was hybridized to
Probe 1 at 55 °C. Yeast tRNA was used as a negative control
(lane 4). The molecular weight marker (MW) was a
5'-end-labeled HaeIII digest of
X174
bacteriophage. Four protected fragments were identified
(arrows), with the major 125-bp product indicated by the
wide arrow (n = 3). C, primer
extension assays were performed with antisense oligonucleotides Primer
1 (lanes 4-6) and Primer 2 (lanes 1-3) using 40 µg of total RNA from postnatal day 8 rat heart or cultured neonatal
cardiomyocytes; tRNA served as a negative control (lanes 1 and 4). A DNA sequencing reaction (utilizing Primer 1 and
rat
1aAR genomic DNA) is shown on the right
for a bp marker. One major product was detected for each primer, with
the difference between product lengths correlating precisely with their
distance apart (
43 bp). Collectively, these data confirm a TIS at
A(
131), relative to ATG. D, identification of the distal
promoter TIS by RPA. Total RNA from 18-day gestation and 8-day
postnatal rat hearts was hybridized to Probes 6 (lanes
5-8), 7 (lanes 1-4) and Probe 9 (lanes
9-12) at 60 °C. Unprotected probes along with fully protected
Probe 6 are labeled. Protected products for P2 (Probe 6, lanes 5 and 6), P3 and the P4 cluster (Probe 7, lanes 2 and 3) are labeled (minor sites indicated
by inverted v). The molecular weight marker (MW)
is described in A above (n = 3). Cyclophilin
was used as a positive control probe, indicated by an arrow
and labeled (inset). Yeast tRNA was used as a negative
control for each probe (lanes 4, 7, and
12).
2.2,
2.1 kb,
and
1.9 kb, relative to ATG (Fig. 2D). No protected
fragments were detected using Probe 9, indicating that the
2.2-kb
cluster represents the most distal
1aAR transcription
start points in heart tissue (Fig. 2D, right
panel). These initiation sites were also confirmed using primer
extension (data not shown), and the initiation sites are shown in Fig.
1. Thus, the rat
1aAR gene utilizes both proximal and
distal promoters in heart located at
131 bp,
1.9 kb,
2.1 kb, and
less abundantly at
2.2 kb, relative to the ATG; we designate these
the proximal (P1) and distal (P2-P4) promoters.
1aAR
mRNA--
As a first step in examining regulation of
1aAR gene expression, we determined the relative
abundance of each transcript. Northern analysis was performed using
mRNA isolated from rat heart and revealed two major products with
molecular weights of ~3.5 and 1.7 kb (Fig.
3, lane 2). The difference in
molecular weights of the two forms correlates well with the difference
in length between the major products of the proximal and distal
promoters (1.8 kb), corresponding to alternate promoter usage. Thus,
the 1.7 kb product likely corresponds to P1-initiated transcripts. Similarly, the 3.5 kb band likely corresponds to the distal P2-P4 transcript cluster that comigrates closely because of poor resolution of products in this molecular weight range. Quantification of the two
different forms shows that the higher molecular weight transcripts
predominate, comprising 71% of the total
1aAR
expression in rat heart. Interestingly, this transcript is expressed in
heart and brain but is absent in other tissues examined (Fig. 3),
suggesting that although P2-P4 promoters are important for
cardiovascular and neural expression, P1 represents the key promoter in
most cell types.
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Fig. 3.
Northern analysis of
1aAR expression in various
tissues. 2 µg of poly(A)-enriched mRNA was probed
successively for
1aAR and GAPDH as described under
"Experimental Procedures." The molecular weights (in kb) of the two
1aAR forms are indicated. Transcript heterogeneity
within the 3.5-kb cluster is indicated by horizontal
lines.
1aAR
mRNA levels in our model system (neonatal rat cardiomyocytes) using cRT-PCR. Results from cRT-PCR were compared with RPA results in a
number of control cell lines and tissues (neonatal heart (8-day postnatal), adult heart, liver, kidney, rat1-fibroblasts, and PC12
pheochromocytoma cells) to ensure the fidelity of the technique (Fig.
4A). Both 8-day
postnatal and adult rat heart have robust
1aAR
expression; in contrast, rat kidney displayed much less
1aAR mRNA and PC12 cells even less (Table I).
Rat liver and rat1-fibroblasts contain no detectable
1aAR mRNA. RPA results are highly consistent with
cRT-PCR, with neonatal heart > adult heart
adult kidney
(Fig. 4B). Quantitation of
1aAR mRNA from these experiments demonstrates (Table I) a high degree of
correlation between cRT-PCR and RPA methodologies.
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Fig. 4.
Cell-specific expression of the rat
1aAR gene. a,
quantitation of
1aAR mRNA by cRT-PCR. Total RNA from
various sources was coamplified with serial dilutions of competitor RNA
for 35 cycles. 25 ng of total RNA from 8-day postnatal (PN)
and adult rat heart were synchronously amplified with 24, 4.8, 0.96, and 0.19 pg of cRNA (lanes 1-4 and lanes 6-9,
respectively); 200 ng of adult rat liver (lanes 11-14),
adult rat kidney (lanes 16-19), rat1-fibroblasts
(lanes 21-24), and PC12 cells (lanes 26-29)
were amplified synchronously with 4.8, 0.96, 0.19, and 0.038 pg of
cRNA. GAPDH was amplified with each sample set in a separate reaction
using 200 ng of total RNA for normalization purposes (lanes
5, 10, 15, 20, 25, and
30). Competitor concentrations were used to obtain a
standard line to calculate the initial amount of target RNA in each
sample. b, an RPA confirms quantitation of
1aAR
mRNA levels by cRT-PCR. Total RNA (30 µg) was hybridized to
radiolabeled rat
1aAR RPA probe (upper panel)
and cyclophilin control probe (lower panel). tRNA
(lane 7) was used as a negative control. The results are
representative of four independent experiments. c,
cell-specific expression of rat proximal and distal
1aAR
promoters. Either the full-length 3.9-kb
1aAR gene
(containing both proximal and distal promoters) or a 1.5-kb P1 fragment
was cloned upstream from the luciferase reporter gene and cotransfected
with pSV-
-galactosidase reference plasmid into indicated cell lines.
Cell lysates were harvested and luciferase activity measured and
normalized to
-galactosidase activity. Values are expressed as -fold
change over basal expression of pGL2 enhancer plasmid (mean ± S.E.; results are representative of three to five independent
experiments).
Quantitation of 1aAR mRNA levels from rat tissues
1aAR mRNA levels (column 1) was performed
using cRT-PCR in each rat tissue
listed.
1aAR promoter
activity was measured in different cell types by transient luciferase
reporter gene expression driven by the
1aAR 3.9-kb
promoter fragment. Results show that rat
1aAR promoter activity is robust in cultured cardiomyocytes versus
rat1-fibroblasts and PC12 cells, with promoter activity decreasing 38%
in the latter two cells compared with cardiomyocytes (Fig.
4C, right panel). This is consistent with higher
myocardial expression seen with the endogenous gene compared with PC12
or rat1-fibroblasts in which endogenous gene expression is either
extremely low or absent (Fig. 4, A and B). Thus,
although the latter two cells express little endogenous
1aAR mRNA, they do express factors capable of
supporting high levels of
1aAR transcription, suggesting
that post-transcriptional mechanisms may be in place to repress
1aAR mRNA expression in vivo.
1aAR gene.
1aAR mRNA in Rat
Neonatal Myocytes--
After determining basal
1aAR
expression in rat heart, we wished to examine the overall effect of
hypoxia on
1aAR myocardial expression. Total RNA was
isolated from neonatal rat cardiomyocytes exposed to either normoxic
(23% O2) or hypoxic (1.5% O2) conditions for
2, 7, and 24 h and
1aAR mRNA levels quantified
by cRT-PCR (Fig. 5A). GAPDH
mRNA was used to normalize mRNA levels between samples (Fig.
5A, bottom panel) and
1aAR levels
expressed as pg
1aAR mRNA/µg total RNA. As,
expected,
1aAR mRNA levels do not change with time
under normoxic conditions (2 h, 23.9 ± 2.1 pg/µg total RNA;
7 h, 23.2 ± 2.0 pg/µg; 24 h, 25.7 ± 2.8 pg/µg); however, when cardiomyocytes are exposed to hypoxic
challenge,
1aAR mRNA levels decrease 11% after
2 h (21.3 ± 2.1 pg/µg), 16% after 7 h (19.4±.2.1
pg/µg; p = 0.01), and 77% after 24 h (5.78 ± 0.61 pg/µg; p < 0.001) versus normoxic
control at each time point (Fig. 5B); these results are in
agreement with previous findings (15). No further change in
1aAR mRNA expression occurs with prolonged hypoxia
challenge (72 h; 6.91 ± 0.70 pg/µg), thus, mechanistic
experiments described below were performed at 24 h.
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Fig. 5.
1aAR gene
expression decreases in response to hypoxia. a,
isolated neonatal cardiomyocytes were cultured for 2, 7, or 24 h
under either normoxic (23% O2) or hypoxic (1.5%
O2) conditions. Total RNA was isolated, and
1aAR mRNA levels were quantitated by cRT-PCR.
Competitor concentrations for each sample set were 24, 4.8, and 0.96 pg. b, summary of hypoxia experiments. cRT-PCR bands were
digitized and normalized for GAPDH levels (mean ± S.E.; results
are representative of three independent experiments).
1aAR Promoter Activity under Normoxic and Hypoxic
Conditions--
To identify important regions governing
1aAR cardiac basal gene expression with normoxia and
hypoxia, full-length and serial 5'-deletion reporter constructs were
transiently expressed into neonatal rat cardiomyocytes. As shown in
Fig. 6, the full-length 3.9-kb reporter
construct confers a 32-fold increase in luciferase levels over pGL2
empty vector under basal conditions in cardiomyocytes (Fig.
6A, white bars). Deletion of the most distal 430 bp of the cloned promoter results in the loss of 95% of basal reporter
gene activity (1.5-fold over empty vector; p < 0.05)
suggesting that a powerful enhancer is in the
3861/
3431 region.
With further deletion to
3013 and
2100, reporter gene activity
remain at low levels until the segment that contains P2 (
1926) is
removed, where luciferase levels again increase in the
1538 construct to 3.6-fold over basal pGL2 enhancer vector. Removal of the
1538/
1233 region results in a 13% decrease in reporter gene levels
(p < 0.05). Deletion to
950 results in a 33%
increase in reporter activity (p < 0.01), consistent
with the presence of basal repressor element(s) in this
1233/
950
region. Further deletion to
457 and
179 reduces
1aAR
basal transcription to 27% and 47%, respectively (p < 0.01), relative to the full-length fragment, suggesting the presence
of independent enhancer elements in each of these latter regions,
important for maximal
1aAR basal transcription. Finally, transcription occurs with as little as 48 bp of 5'-regulatory sequence
upstream of P1 (
179 construct), suggesting that the proximal 48 bp
are necessary and sufficient to confer promoter activity of the
1aAR gene, thus constituting a minimal promoter.
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Fig. 6.
Basal expression of
1aAR mRNA under normoxic and
hypoxic conditions. Full-length
1aAR 3.9 kb and
deletion mutant reporter constructs were cotransfected with
pSV-
-galactosidase reference plasmid into rat neonatal
cardiomyocytes. 6 h post-transfection, cells were exposed either
to normoxic (23% O2) or hypoxia (1.5% O2) for
24 h; cell lysates were prepared and luciferase and
-galactosidase activities measured. Luciferase levels were
normalized to
-galactosidase levels and expressed as -fold over
basal pGL2 enhancer plasmid (mean ± S.E.; results are
representative of three or four independent experiments). On the
left side of the schematic, shaded areas indicate
5'-UTR, and hatched areas represent a 5'-regulatory
sequence. On the right side of the schematic, luciferase
activities for normoxia (white bars) and hypoxia
(black bars) are shown. b, luciferase activity of
smaller deletion constructs under hypoxic stress was used to define the
1aAR HRE further. Two additional deletion constructs
were created (
564,
374), and luciferase activity was measured and
normalized to
-galactosidase activity as described above. *,
p < 0.05 new deletion construct versus next
longest reporter construct.
1aAR promoter
region conferring hypoxia-mediated decreases in
1aAR
expression, we next characterized the full-length and deletion reporter
constructs under hypoxic conditions (Fig. 6A, dark
bars). Hypoxia induces a 59 ± 8.2% reduction in
transcriptional activity of the full-length reporter construct versus normoxic conditions (Fig. 6A compare
dark versus white bars), correlating fairly well
with the 77 ± 10.6% repression of endogenous gene expression
demonstrated previously in Fig. 5. Similar to results under normoxic
(basal) conditions, deletion of the
3861/
3431 region results in a
sharp reduction in
1aAR promoter activity under hypoxic
conditions, consistent with the loss of the powerful basal enhancer in
this region. Hypoxic transcription for subsequent deletion constructs
parallels normoxic results, with hypoxic reporter gene activity largely
remaining 30-40% less than normoxic until P1 regulation is examined
in the
520 construct. Deletion from
681 to
520 results in an 85%
increase over the previous deletion construct, increasing
reporter gene activity 4.1-fold over empty vector. Additional deletion
to
457 again reduces transcription 77% (p < 0.01)
versus the previous deletion construct with hypoxia,
consistent with loss of an enhancer in this region. Finally, removal of
sequence to
179 generates a construct with virtually identical
activity under normoxic and hypoxic conditions, indicating creation of
a constitutively active minimal promoter fragment. Deletion to +88
abrogates all promoter activity, consistent with loss of all initiation
sites (data not shown).
1aAR expression in all
reporter constructs (excluding the
520 construct), this suggests that
both P1 and P2-P4 promoters may be affected by an element in this
region. Therefore we investigated this further by creating smaller
deletion constructs and analyzing them for transient reporter gene
activity. These deletion constructs were designed either to remove an
additional regulatory sequence or to mutate a consensus MCAT site at
512; MCAT is an element shown to be involved in basal regulation of the murine
1aAR gene (6). None of the smaller deletion
constructs or the mutant MCAT construct had a statistically significant
change in reporter gene levels under normoxic levels (data not shown), thus only data for reporter constructs is presented for hypoxic conditions (Fig. 6B, dark bars). Interestingly,
mutation of the MCAT site did not have a significant effect on
luciferase activity, suggesting differential regulation of the
1aAR gene in rat versus murine myocardium
(data not shown). Removal of the
1aAR regulatory sequence to
564 had no significant affect on luciferase activity versus the
681 construct, whereas deletion to
374
results in a 54% increase in luciferase activity over the preceding
reporter construct. Thus, deletion reporter gene analysis revealed the presence of three
1aAR HREs within the
564/
520,
520/
457, and
457/
374 regions. These regions were subsequently
analyzed for direct protein binding by EMSAs.
1aAR HRE--
Transient reporter expression experiments
indicate that the
564/
374 region is important for hypoxia-mediated
regulation of the proximal promoter, thus we wished to determine
whether soluble nuclear factor(s) are able to bind specifically to this sequence. Oligonucleotide probes were generated (Fig.
7A, OL1, OL2, and OL3) which
encompassed this region and subjected to gel shift analysis using
normoxic and hypoxic extracts to determine whether a correlation exists
between soluble nuclear factor binding and promoter activity. Only OL1,
specific for region
573/
515, generated a new, specific complex
induced under hypoxic versus normoxic conditions in extracts
prepared from cardiomyocytes (Fig. 7A, lanes
6-9, shift 1). This shift is competed by excess unlabeled self-oligonucleotide (Fig. 7A, lane 7) but is not
competed by a nonspecific oligonucleotide (Fig. 7A,
lane 8). Additionally, a specific constitutive shift is
present in normoxic extracts (Fig. 7A, lanes
2-5, shift 2), which increases with low oxygen stress. Again,
this shift is competed by self-oligonucleotide (Fig. 7A,
lane 3) but unaffected by a nonspecific oligonucleotide (Fig. 7A, lane 4). Finally, although no consensus
sequence is present in the rat
1aAR promoter for the
hypoxic transcriptional regulator HIF-1 (TACGTGCT), potential direct
binding was examined. None of the shifts was affected by polyclonal
antibody to HIF-1
, suggesting that HIF is not a component of the
complex that directly binds to this region of the rat
1aAR regulatory sequence (Fig. 7A,
lanes 5 and 9). Together, these results suggest
the presence of soluble nuclear factors that are able to associate with
the
573/
515 region to confer hypoxia-mediated responsiveness to the
rat
1aAR proximal promoter.
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Fig. 7.
Binding of nuclear
factors to rat 1aAR HRE in
cardiomyocyte nuclear extracts exposed to hypoxic stress.
A, isolated cardiomyocytes were cultured and exposed either
to normoxic (20% O2) or hypoxic (1.5% O2)
conditions for 24 h. Cells were then harvested and nuclear
extracts prepared. 5 µg of extract was used in each sample with 20 fmol of 5'-end labeled oligonucleotide (OL1) and incubated
on ice for 20 min. Shifts were competed with either 1,000-fold molar
excess of self (S) oligonucleotide or nonspecific
oligonucleotide (NS). HIF-1 in the binding complex was
tested using a rabbit polyclonal antibody raised against HIF-1
(
HIF). Samples were loaded on a 6% native polyacrylamide
gel and resolved at 250 volts at 4 °C. The gels were dried and
exposed to autoradiography. Two specific shifts (1 and
2) are indicated by arrows. B, an EMSA
was performed as described in A. Nuclear extracts from
cardiomyocytes exposed to hypoxic stress (5 µg) were incubated with
20 fmol of 5'-end-labeled OL1. Cold, competing oligonucleotides that
encompass the
573/
515 region were added in either in a 20-fold or
500-fold molar excess. Cp1 is specific for
573/
555; Cp2,
558/
542; Cp3,
544/
528; and Cp4,
531/
515.
1aAR HRE further, the specific hypoxia
shift was competed with even smaller overlapping oligonucleotides
within the
573/
515 region (Fig. 7B). Only Cp2 was able
to compete the binding in a dose-dependent manner,
suggesting that protein binding occurs within the
558/
542 region of
the rat
1aAR promoter (Fig. 7B, lanes
5 and 6). Interestingly, the highest concentration of Cp4 was able to compete to a lesser degree than Cp2, suggesting that
proteins within the hypoxia-binding complex may be making contacts with
the DNA in the
531/
515 region as well (Fig. 7B, lanes 9). Together, EMSA data suggest that nuclear factors
associate with the
558/
542 and
531/
515 regions of the rat
1aAR promoter and modulate transcription under both
constitutive and hypoxic conditions.
558/
542 region resulted in a marked attenuation of hypoxia responsiveness. Mutants 1 and 4 had little effect on hypoxia-mediated repression of reporter gene
activity, but mutants 2 and 3 were able to increase
1aAR promoter responsiveness 1.9-fold versus the wild-type
construct under hypoxic conditions. Thus, compared with the wild-type
fragment under normoxic conditions, mutants 2 and 3 resulted
in an attenuated 27 and 28% hypoxic decrease in reporter gene
activity, respectively, versus the 59% decrease for the
wild-type fragment (Fig. 8B; p < 0.05).
Thus, the 9-bp element (
555 CCCATGTCA
547) is able to modulate
1aAR responsiveness to hypoxia, although having little affect under normoxic, basal conditions. Coupled with evidence of a
soluble nuclear factor binding to this region, inducible with hypoxia,
this is strong evidence that this element is a potent HRE regulating
the
1aAR gene under conditions of low oxygen stress.
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Fig. 8.
A novel HRE regulates both proximal and
distal 1aAR promoters in response
to hypoxic stress. A, site-directed mutagenesis was
used alter the full-length 3.9-kb reporter construct to create four
independent reporter gene constructs (m1-m4), each
containing a 3-bp transition mutation in the
558/
542 region.
Mutated bp are indicated in bold and underlined.
The sequence of all HRE mutants was confirmed by sequence analysis.
B, wild-type and mutant reporter constructs were transiently
expressed in neonatal cardiomyocytes as described in Fig. 5. **,
p
0.05 hypoxia versus full-length
construct under hypoxic conditions (dark bars).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1aARs has been shown to play
an important role in myocardial hypertrophy (13, 14), contractility (3), arrhythmias (26), and ischemic preconditioning (27). In the
present study, we report molecular cloning and functional characterization of the rat
1aAR gene, including
identification of multiple transcription initiation sites, with
transcription initiation sites located proximally at 131 bp and a
distal (upstream) cluster located 1.9, 2.1, and 2.2 kb upstream from
the ATG. In addition to identification of a powerful enhancer/control
element
3.6 kb upstream from the ATG necessary for optimal basal
gene expression, we have demonstrated cell-specific transcription of both the proximal and distal promoters. Promoter activity is highest in
cardiomyocytes versus other cell types examined, consistent with enhanced cardiac expression of the endogenous gene. Further, we
demonstrate a correlation between endogenous gene levels and promoter
activity with hypoxic stress and identify specific regions of the rat
1aAR gene capable of modifying basal and hypoxia-induced regulation of heterologous reporter constructs. This region appears to
modulate hypoxia-induced decreases in expression for all promoters. Results from site-directed mutagenesis experiments support in vitro binding data and reveal a novel 9-bp HRE located 555 bp upstream from the ATG as a potent regulator of
1aAR
transcription with hypoxic stress. These new findings provide clues to
mechanisms underlying modulation of myocardial gene expression with
disease and stress.
1.8 kb) correlates with the difference in length between the
proximal and distal promoters. Thus, the distal P2-P4 promoter cluster
initiated transcripts (which were not well resolved in our Northern
analysis) likely correspond to the 3.5-kb transcript seen in Northern
analysis with P1-initiated transcripts corresponding to the 1.7-kb product.
1aAR transcription start points were
determined, alignment with published mouse and human
1aAR regulatory sequences was performed to gain insight
into possible species-specific differences. Comparison with human
1aAR 5'-regulatory sequence reveals that although
overall nucleotide identity is only 61%, several of the cis-elements such as MCAT, GATA, and AP1 within regions of
increased identity are conserved (Fig.
9). Thus, although rat (and mouse) promoter sequence is quite divergent from human, the presence of
similar putative cis-elements suggests that they can be
transcribed under similar conditions. Indeed, unlike the murine
promoter, the human
1aAR promoter is highly active in
rat cardiomyocyte reporter gene assays (11-fold, Fig. 9).
Interestingly, although rat and mouse promoters are highly similar with
large stretches of identity (90%) and a number of conserved
cis-elements, including CRE, GATA, MCAT, and Sp1, the two
promoters display large differences in promoter strength in the
myocardium (32-fold versus 2.3-fold for full-length reporter
constructs, Fig. 9). Further examination reveals that the most
significant region of divergence falls in the segment containing the
powerful rat basal enhancer, which contains putative binding sites for
MCAT, GATA, and AP1 (light blue box, Fig. 9). This region
shares little similarity to known sequences and may play a key
regulatory role in the observed species-specific differences in
1aAR gene expression. The absence of this enhancer region in the human
1aAR gene appears to be compensated
for with multiple other enhancers because rat and human promoters
result in very high overall expression, 32-fold and 11-fold over base line, respectively (compared with 2.3-fold for mouse).
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Fig. 9.
Comparison of rat, mouse, and
human 1aAR promoters. The rat
promoter sequence was aligned with published mouse and human promoter
sequences using MacVector software (MacVector 7.0; Kodak Biosciences).
5'-UTR and 5'-regulatory sequences are indicated in blue,
and coding region sequences are in green (ATG is labeled).
The locations of cis-elements are referenced to the ATG in
each gene. Transcription initiation sites are indicated by
arrows. -Fold changes for full-length reporter constructs
versus empty vector in rat cardiomyocytes are listed for
rat, mouse, and human
1aAR promoters. Shaded
regions indicate the percent identity to the rat promoter; the
light blue box indicates a region of divergence between rat
and mouse promoters. Consensus sequences designated in the
key are based on perfect matches to the following sequences:
AP1(CTGACTCA), CRE(CTGCA), CTF/NF1(AGCCAAT), GATA(A/TGATAA/G),
MCAT(CATG/CCCA (6)), and Sp1(GGGCGG).
The large difference in promoter strength between rat and mouse is
consistent with markedly different regulation of 1aAR transcription between the two species in response to regulatory signals. For example,
1AR agonist stimulation of rat
cardiomyocytes results in up-regulation of endogenous rat
1aAR mRNA (13, 14), whereas mouse
1aAR reporter constructs are virtually inert to
1AR stimulation in these same cells (6). Also unlike the
endogenous rat
1aAR gene, the mouse
1aAR
promoter is unresponsive to stimulation by hypertrophic agonists such
as endothelin-1, phorbol 12-myristate 13-acetate, and prostaglandin
F2
(6). Additionally, rat and mouse
1aAR genes
display very different responses to hypertrophy in vivo
induced by aortic constriction pressure overload; such intervention
results in up-regulation of
1aAR in rat models (14) but
no change in mouse
1aAR levels (6, 28). Finally, recent studies reveal that mouse
1ARs also fail to activate
phospholipase C
, extracellular signal-regulated kinase, p38, or
stimulate cardiomyocyte hypertrophy, indicating a generalized
impairment of
1AR signaling in murine myocardium (29).
This is consistent with other studies demonstrating that several well
characterized trophic effectors induce hypertrophy in rat
cardiomyocytes (e.g. endothelin-1, phorbol 12-myristate
13-acetate, and prostaglandin F2
) but have no effect on
cultured mouse myocytes (30). Collectively, clear species-specific differences exist between mouse and rat cardiomyocytes in addition to
differences in
1aAR promoter/gene structure which
contribute to divergent regulation of
1aAR transcription
in rat and mouse heart. Our current characterization of the rat
1aAR gene now makes it possible to address these
differences mechanistically and should therefore provide insight into
species-specific modulators of myocyte function.
Similar to human and mouse homologs, the rat 1aAR P1 and
distal promoters lack both CAAT and TATA boxes. The distal promoters (P2-P4) occur within atypical initiation sites, with the predominant P2 transcript initiating within a GC box, P4 transcripts initiating at
several sites within a 50-bp cluster, and the predominant P3 transcript
initiating proximally to a 30-bp polypyrimidine tract. These tracts
have been shown to regulate transcription at many levels including
direct DNA binding (31, 32), post-transcriptional processing of nascent
transcripts (33), and alternate DNA structure/topology (34). The lack
of typical regulatory or promoter elements for the distal promoters may
explain the presence of multiple promoters in this region. Conversely,
the P1 initiator conforms to a loose initiator sequence that is an
identical match to a functional initiator element in the
cdc25 gene (CCCAGCT) (35). From our serial
deletion experiments, it is apparent that basal
1aAR promoter activity resides in most distal cloned 400-bp fragment, with
the loss of this region abrogating 95% of basal transcriptional response. Additionally, P1
1aAR transcription is
modulated by multiple independent repressor (
681/
520,
520/
457
region) and activator (
1233/
950) elements in rat neonatal
cardiomyocytes, containing a number of putative binding sites for
transcription factors known to be relevant in heart, such as GATA,
MCAT, and Sp1. The latter two sites have been shown to be important for basal expression of the human (5) and mouse (6)
1aAR
genes, respectively, and may provide additional layers of regulatory control for the rat gene.
Although the functional relevance of multiple initiation sites is not
yet clear, use of multiple promoters is common among several
characterized 1AR genes (rat
1bAR (8),
human
1bAR (9), rat
1dAR (10)). For
example, the rat
1bAR gene generates transcripts of 2.3, 2.7, and 3.3 kb in length via multiple promoters (8). Recent studies
show that the proximal
1bAR promoter directs expression
in a tissue-specific manner (36), with DNase footprinting experiments
revealing that the proximal promoter p1f3 region binds a
tissue-restricted factor in liver and hamster smooth muscle extracts,
distinct from the predominant P2 promoter (36). In contrast, the
recently identified murine
1aAR ortholog is transcribed from a single promoter located at
588 relative to the ATG (6). The
human
1aAR ortholog, unlike either rat or
mouse, is transcribed from a dominant promoter at
696 (relative to
ATG), with several minor initiation sites clustered proximally (5).
Given these data, a potential reason for multiple promoter
utilization may be to aid in cell-specific regulation of a given gene,
consistent with the well documented tissue- and species-specific
distribution of
1AR subtypes (4, 20, 37). Alternate
transcripts might also be used to modulate
1aAR mRNA
stability as has been reported recently for the rat
1AR
gene in response to agonist-independent and dependent down-regulation
of
1AR mRNA (38).
Myocardial hypertrophy occurs via several mechanisms. Well
characterized among these mechanisms are pathways resulting from sympathetic (norepinephrine) stimulation (14) secondary to
chronic hypoxia and/or ischemic insult (39). Recent studies aimed at elucidating mechanisms governing hypertrophy in response to these stimuli in rat heart reveal that 1aAR expression is
modulated differentially by hypoxia (versus
norepinephrine stimulation) in a chamber specific manner. This
suggests that norepinephrine-stimulated hypertrophy proceeds via
distinct pathway(s) from hypertrophy secondary to hypoxic stress (40)
and is consistent with in vitro studies demonstrating
hypoxia results in decreased
1aAR mRNA and receptor
levels coupled with attenuation of
1aAR-mediated signaling and hypertrophy (15). Interestingly, this is in contrast to
increased
1aAR mRNA levels with norepinephrine or
phenylephrine stimulation in the same model (14). To begin to elucidate
these distinct mechanisms, we analyzed transcriptional activity of the rat
1aAR gene in neonatal cardiomyocytes under normoxic
and hypoxic conditions. Initially, we examined a potential role for the
well characterized HIF-1, a heterodimeric, basic helix-loop-helix
transcription factor expressed in response to cellular hypoxia which
mediates multiple cellular and systemic homeostatic responses to
hypoxia (41). In the rat
1aAR gene, although several
regions are able to confer hypoxia-mediated regulation in transient
reporter assays, the 5'-regulatory region is devoid of HIF-1 consensus
sequences (42), suggesting that direct HIF-1 binding may not be the
primary mechanism for altered (decreased) rat
1aAR
expression with hypoxia. This is supported by in vitro gel
shift analysis using anti HIF-1
antibody. Results showed that the
558/
542 region binds to nuclear factors under hypoxic conditions,
and these shifts are not affected by the presence of anti-HIF.
Functional analysis of mutant reporter constructs that alter this
region in sequence reveals the presence of a novel 9-bp HRE (
555
CCCATGTCA
547) Further examination of these sequences reveals no
obvious candidates known to confer hypoxia responsiveness, suggesting
potentially novel pathways for hypoxia-mediated transcriptional control
in rat
1aAR gene.
Although our findings do not preclude a potential indirect role of
HIF-1, recent studies demonstrate that other factors modulate transcription during hypoxia, independent of direct HIF-1 binding. For
example, it has been demonstrated that cAMP and subsequent CREB levels
decrease under low oxygen exposure, contributing to hypoxia-elicited
induction of epithelial tumor necrosis factor- (43).
Mechanistically, this may occur via phosphorylation of serine 133 by an
unidentified kinase (44), resulting in CREB ubiquitination and
proteosomal degradation (45). Given the presence of a cAMP response
element at
756, CREB degradation could explain hypoxia-mediated
repression of
1aAR transcriptional activity in the
950/
681 region (i.e. loss of an activator). Indeed,
previous studies in our laboratory have shown that the human
1aAR gene is induced by increased cAMP levels (5).
Although direct binding of proteins to these regions was not seen in
our gel shift analysis, it is possible that factors associating with
the
1aAR HRE aid in nucleating complexes at these other
sites. Such is the case with the yeast homothallic switching
endonuclease gene promoter where transient Swi5 binding (<5 min)
initiates a cascade of sequential binding of Swi/Snf and SAGA complexes
at distal sites (46). This in turn recruits swi4/swi6-dependent
cell-cycle box (SBF) to the promoter, which then initiates
homothallic transcription in early G1 phase (46). If a
similar process regulates the
1aAR proximal promoter
during hypoxia, no binding at the more proximal sites would be seen by
gel shift analysis. Alternatively, another explanation is that deletion
of more proximal sites alters helical phasing of factor binding within
the
558/
520 region to the promoter, resulting in a decrease in
transcriptional activity. Indeed, phasing, or location of protein
binding on a face of the DNA helix, has been shown to be important in
the collagenase-3 promoter where insertion of nucleotides to disrupt
helical phasing between the AP-1 and runt domain sites results in
abrogation of AP-1/runt domain binding and decreased collagenase-3
promoter activity (47). Studies are under way in our laboratory to
determine which of these potential processes may be at work regulating
1aAR transcription with hypoxia.
In summary, we present the first cloning and characterization of the
rat 1aAR gene, identifying a number of cardiac enriched putative cis-element binding sites that can direct robust
expression from proximal and distal promoters. Heterogeneity of
1aAR mRNAs in rat heart appears to be the result of
transcription initiation from multiple promoters. We have correlated
functional activity of a novel 9-bp HRE between P1 and P2 to
HIF-independent hypoxia-induced binding activity to this region. Loss
of this region attenuates
1aAR responsiveness to
transcription, and the factor(s) associated with this region may help
nucleate other factors such as CREB to the
1aAR
promoter. These novel findings should facilitate studies designed to
elucidate
1AR-mediated mechanisms involved in distinct
myocardial pathologies as well as aid in elucidation of differential
1aAR regulation across species.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Daniel Morris and Marshall Brinkley for helpful discussion and critical reading of the manuscript as well as Zarrin Brooks for administrative assistance.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AG00745 and HL49103 (to D. A. S.).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.
Senior fellow in the Center for the Study of Aging and Human
Development, Duke University Medical Center. To whom correspondence should be addressed: Dept. of Anesthesiology, Box 3094, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-4781; Fax:
919-681-4776; E-mail: schwi001@mc.duke.edu.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M211986200
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ABBREVIATIONS |
---|
The abbreviations used are:
1AR(s),
1-androgen receptor(s);
CREB, cAMP response element-binding protein;
cRT, competitive reverse
transcription;
EMSA, electrophoretic mobility shift assay;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HIF, hypoxia inducible
factor;
HRE, hypoxia response element;
OL, oligonucleotide;
P1, proximal promoter;
P2-P4, distal promoters;
RPA(s), RNase protection assay(s);
RT, reverse transcription;
TIS, transcription initiation site(s);
UTR, untranslated region.
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REFERENCES |
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1. |
Leech, C. J.,
and Faber, J. E.
(1996)
Am. J. Physiol.
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