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Vol. 56, Issue 6, 1288-1297, December 1999
1B-Adrenergic Receptor Promoter
Department of Molecular Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio (M.J.Z., D.M.P.); and Department of Pharmacology and the Vascular Biology Research Group, University of Kentucky College of Medicine, Lexington, Kentucky (M.T.P.)
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Summary |
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 Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The functionality of a 3422-base pair promoter fragment from the mouse
1B-adrenergic receptor (1BAR) gene was
examined. This fragment, cloned from a mouse genomic library,
was found to have significant sequence homology to the known human and
rat 1BAR promoters. However, the consensus motif of
several key cis-acting elements is not conserved among
the rat, human, and mouse genes, suggesting species specificity.
Confirming fidelity of the murine promoter, robust in vitro expression
of a chloramphenicol acetyltransferase (CAT) reporter was detected in
known 1BAR-expressing BC3H1, NB41A3, and DDT1MF-2 cells transiently transfected with a
promoter-CAT construct. Conversely, minimal CAT expression was detected
in known 1BAR-negative RAT-1 and R3T3 cells. These
findings were extended by transfecting the same promoter-CAT construct
into various primary cell types. In support of the hypothesis that 1ARs are differentially expressed in the smooth muscle
of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT
expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic
bone-forming cells, which are known to be 1BAR negative,
showed minimal CAT expression. Indicating regulatory function through
cis-acting elements, RAT-1, R3T3, NB41A3,
BC3H1, and DDT1MF2 cells transfected with the
promoter-CAT construct all showed increased CAT production when
challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were
simultaneously challenged with both forskolin and hypoxia. These
results collectively demonstrate that a 3.4-kb PvuII
fragment of the murine 1BAR gene promoter can: 1) drive
tissue-specific production of a CAT reporter in both clonal and primary
cell lines; and 2) confer tissue-specific regulation of that CAT
reporter when induced by challenge with forskolin and/or hypoxic conditions.
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Introduction |
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Top
Abstract
 Introduction
 Materials and Methods
Results
Discussion
References
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1-Adrenergic
receptors (1ARs) are a group of heterogeneous
but related members of the G-protein-coupled receptor superfamily, which prototypically exhibits seven transmembrane-spanning domains. Extensive effort has been spent classifying the three known
1AR subtypes (1A,
1B, 1D) via molecular
cloning techniques (Cotecchia et al., 1988
; Lomasney et al., 1991;
Perez et al., 1991, 1994) and pharmacological analyses (Guarino et al.,
1996). Results from these studies show that, like other
1ARs, the 1B subtype
mediates the effects of the sympathetic nervous system evoked by the
catecholamine agonists epinephrine and norepinephrine. The best
characterized cardiovascular effects of 1BAR
activation include contraction, growth, and proliferation of vascular
smooth muscle (VSM) cells (Chen et al., 1995; Leech and Faber, 1996;
Hrometz et al., 1999) and increased cardiac contractility (Anyukhovsky
et al., 1992). In other 1BAR-expressing
tissues, including brain, liver, and kidney, the function of the
receptor is not so well defined. Limited data indicate that the
proposed function of the receptor is to regulate metabolic processes in
the liver (Kunos and Ishac, 1987), whereas its function is to regulate
sodium and water reabsorption in the kidney (Kopp and Dibona, 1992).
These responses, which are normally evoked by agonist binding to
1BARs, are believed to be transduced primarily
via receptor coupling to phospholipase C (Guarino et al., 1996), which
leads to the subsequent activation of downstream signaling molecules,
including protein kinase C and inositol-1,4,5-trisphosphate.
Because 1BARs are not ubiquitously expressed,
the development of appropriate physiological responses to activation of
second messengers depends in part on receptor tissue distribution. One way that this pattern of expression can be controlled is via regulation of the receptor's transcription by the gene's promoter. Therefore, to
examine the regulatory characteristics of the
1BAR promoter, several studies have been
performed to analyze its function in both humans and rats. The human
5'-flanking region, which has been sequenced as far 5' of the
translation start codon as 
923, was found to contain multiple Sp1
binding sites and a putative cAMP response element (CRE; Ramarao et
al., 1992). Comparatively, the more extensively characterized rat
promoter, which has been sequenced even further 5' to 2460, was found
to contain three distinct promoter regions (Gao and Kunos, 1994). The
middle promoter, denoted P2 by Gao et al., is located between 813 and
432 and is hypothesized to be responsible for driving transcription
of the 1BAR gene in most rat tissues,
especially in the liver (Gao et al., 1995). Multiple transcription
factor binding sites have been identified in this P2 promoter region
including a CRE, an hypoxia-inducible factor 1 (HIF-1) site, sites for
liver-specific factors [C/EBP, hepatic nuclear factor 1 (HNF-1), HNF-5], and sites for cardiac-specific factors (M-CAT and an
E-box) (Gao et al., 1995; Eckhart et al., 1997). Despite these studies
of the human and rat promoters, in neither species have cell-type
specificity issues been explored.
As a prelude to the development of transgenic models, this report
describes our effort to clone, sequence, and analyze a 3.4-kb mouse
1BAR gene promoter fragment. Sequence analysis
of this murine promoter fragment and comparison with known rat and
human 1BAR promoter sequences has led us to
hypothesize that, whereas there is substantial similarity between the
three promoters, sufficient divergence has occurred to envision some
level of species specificity. We have also confirmed that the mouse
promoter fragment is able to regulate chloramphenicol acetyltransferase
(CAT) gene expression in response to the competency of a given cell to
express 1BARs. Furthermore, we have observed
regulation of promoter function by cAMP (via stimulation with
forskolin) and hypoxia (via exposure to 1.5%
O2). These data establish the fidelity of the
3.4-kb murine 1BAR promoter, suggesting that
it may be a powerful tool for driving selective expression of a target
gene in 1BAR-positive cells or tissues.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning and Sequencing of Murine 1BAR
Promoter.
A mouse genomic library (129SVJ female liver, 9- to
23-kb insert size; Stratagene, Inc., La Jolla, CA) was screened to
isolate putative promoter sequence from the mouse
1BAR gene. Screening by plaque hybridization
was performed with an [-32P]CTP probe
(Random Primed DNA Labeling Kit; Boehringer Mannheim, Mannheim,
Germany) derived from exon 1 of the human 1BAR
gene (Ramarao et al., 1992). Several rounds of screening facilitated identification of a 15-kb insert, which, after restriction mapping and
sequencing, was found to contain approximately 10 kb of 5'-flanking sequence plus exon 1 of the murine 1BAR gene
(Fig. 1). Based on work with the rat
1BAR promoter showing functionality of a 3.6-kb PvuII fragment just upstream of the gene's
transcription start site (Eckhart et al., 1997), an analogous 3.4-kb
PvuII fragment was isolated from our recovered mouse
promoter clone, which is located 66-base pairs (bp) upstream of the
murine coding region. This putative promoter fragment was subcloned
into the SalI site of the pCAT basic vector (Promega
Biotech, Madison, WI) to generate the 1BAR
promoter-pCAT plasmid. Because promoter insertion into pCAT was
accomplished via blunt-end ligation, clones with sense and antisense
promoter orientation were distinguished by sequencing with the
dideoxy-chain termination method (Sequenase kit; Amersham Corp.,
Arlington Heights, IL). Large-scale preparations of plasmid DNA were
purified with a kit (Wizard Maxipreps; Promega). Sequencing of the
entire 3.4-kb promoter fragment was carried out by the Cleveland Clinic
Foundation Sequencing Core with a series of nested primers with an
average run length of 600 bases (Fig. 1). All screening, subcloning,
transformation, and other molecular biology procedures were performed
with standard techniques described elsewhere (Sambrook et al., 1989).
Sequence alignments were performed with GeneWorks version 2.3, and
searches for possible promoter response elements were carried out using
Signal Scan, an on-line database and search engine designed and
maintained by the Advanced Biosciences Computing Center, University of
Minnesota.1
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Cell Culture.
All clonal cell lines used in this study
(BC3H1, DDT1MF-2, NB41A3,
RAT-1, and R3T3) were obtained from American Type Culture Collection
(Rockville, MD) and grown in a 37°C, 5% CO2
incubator with the culture medium recommended by the supplier. Primary
cultures of rat VSM cells derived from superior mesenteric, renal,
pulmonary, femoral, and iliac arteries were generated with a variation
of a previously published method (Gunther et al., 1982). Briefly, the
arteries were removed from the animal, aseptically dissected free from
fat and connective tissue, cut longitudinally, and spread out flat in a
sterile culture dish. Endothelial cells were removed by shearing with a
cotton swab, and the arteries were cut into small fragments. These
fragments were incubated with gentle agitation at 37°C for 90 min in
Hanks' balanced salt solution (HBSS; Sigma Chemical Co., St. Louis,
MO) containing 0.1% collagenase (lot 46A034; Worthington Biochemical
Corp., Freehold, NJ), 0.125 mg/ml elastase (Worthington), and 2 mg/ml
BSA (Sigma). After digestion, samples were centrifuged at
500g for 5 min, and the resulting cell pellets were washed
once by resuspension with HBSS. After another 5-min spin at
500g, cell pellets were resuspended in culture medium
composed of a Dulbecco's modified Eagle's medium base (BioWhittaker, Inc., Walkersville, MD) supplemented with 3.6 g/liter
NaHCO3, 1% penicillin/streptomycin
(BioWhittaker), and 10% fetal bovine serum (Gibco Laboratories, Grand
Island, NY). Suspended cells were dispersed in 250-ml tissue culture
flasks and were grown in a 37°C, 5% CO2
incubator. Culture medium was changed every 2 or 3 days, and cells were
split at confluency. Primary rat calvarial osteoblasts (obtained from
Dr. J. Edward Puzas, University of Rochester, Rochester, NY) were grown
in the same culture medium and under the same conditions described for
primary smooth muscle cells. Preparation of osteoblastic cells was
carried out with a previously described method (Martinez et al., 1995).
For promoter studies, confluent clonal or primary cells were split into
p60 dishes and grown to 70% confluency. Several experiments involved culturing of cells under hypoxic conditions. The hypoxic environment (1.5% O2/5% CO2, balance
N2) was established in an
O2-regulated incubator.
Transient Transfection.
Transient transfections were carried
out via a lipofection method with the commercially available
Transfectam reagent (Promega) according to the instructions provided by
the manufacturer. Briefly, 70% confluent cells in p60 dishes were
washed twice with serum-free culture medium and then bathed for 2 h in 1.5 ml serum-free culture medium containing DNA and the
Transfectam reagent. The 1.5-ml transfection solution for all clonal
cell lines contained 1 µg of sense or antisense (negative control)
1BAR promoter-pCAT plasmid, 1 µg
pSV-
-galactosidase (-gal) plasmid (Promega), and 4 µl
Transfectam reagent. Comparatively, the transfection solution for
primary cell types contained 1 µg of sense or antisense
1BAR promoter-pCAT plasmid, 1 µg -gal
plasmid, and 12 µl Transfectam reagent. After 2 h, the cells
were overlayed with 4 ml of complete medium. Cells were assayed 60 h after transfection.
Preparation of Cell Extracts.
Cell extracts were prepared
from cotransfected cells via cell lysis with a commercially available
Reporter Lysis Buffer (Promega). Briefly, culture medium was removed,
and the cells were washed twice with HBSS. After the second wash, HBSS
was removed, 0.4 ml Reporter Lysis Buffer was added to each dish, and
the dishes were slowly rocked at room temperature for 15 min. The cell
layer was then scraped, and cellular debris was transferred to a
microcentrifuge tube. Tubes were vortexed and then centrifuged at top
speed in a microcentrifuge for 2 min. Supernatants were recovered and
split into two aliquots, one for determination of -gal activity, the other for CAT activity. Lysates to be used for CAT determination were
heated to 60°C for 10 min to inactivate endogenous deacetylase activity.
Determination of -gal Activity.
Transfection efficiency
in each cell line was determined by measuring the amount of -gal
activity in cell extracts with a colorimetric assay system (Promega).
Aliquots of cell extract were mixed with 150 µl assay buffer
(supplied with kit) and sufficient Reporter Lysis Buffer to make a
final volume of 300 µl. Reaction mixtures were incubated between 0.5 and 2.5 h at 37°C and were terminated by adding 500 µl of 1 M
sodium carbonate. Absorbance was read at 420 nm, and absorbance
readings were translated into units of activity via a standard curve.
Standard curves for -gal enzyme activity were generated for between
0 and 6 mU of standard enzyme activity with purified -gal enzyme
according to instructions supplied by the manufacturer. The amount of
cell extract used in the experimental determination of -gal activity
was varied as necessary to be certain that 420-nm absorbance readings
were within the limits of the standard curve.
Determination of CAT Activity.
Subsequently,
1BAR promoter function was determined by
measuring the amount of CAT enzyme activity present in cell extracts via a liquid scintillation counting assay system (Promega). One hundred-microliter aliquots of cell extract were mixed with 3 µl
[14C]chloramphenicol (0.05 mCi/ml; New England
Nuclear, Boston, MA), 5 µl n-butyryl coenzyme A, and 17 µl distilled H2O. Reaction mixtures were
incubated for 1 to 5 h at 37°C and were terminated by adding 300 µl of mixed xylenes. The reaction product generated by CAT activity
in the cell extract, n-butyryl chloramphenicol, partitioned mainly into the xylene phase, whereas unmodified chloramphenicol remained in the aqueous phase. Samples were back-extracted twice with
100 µl of 250 mM Tris-HCl (pH 8.0) to completely remove unmodified chloramphenicol from the xylene phase. A fixed 250-µl volume of the
final xylene phase from each sample was transferred to a scintillation vial with 6 ml Ecoscint A (National Diagnostics, Inc., Manville, NJ).
The cpm measured in each sample was corrected for transfection efficiency (estimated by -gal activity measured in parallel
aliquots), and actual CAT activities were determined by extrapolation
from a standard curve. The amount of cell extract used in the
experimental determination of CAT activity was varied as necessary to
be certain that the measured radioactivity was within the limits of the
standard curve. One unit of CAT activity was defined as the amount of
CAT enzyme required to transfer 1 nM of the acetate moiety from
n-butyryl coenzyme A to chloramphenicol in 1 min at 37°C.
Statistical significance (p < .05) in all CAT assays
was identified via one-way ANOVA followed by a Neuman-Keuls
multiple-comparison test.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning and Sequence Analysis.
A positive plaque from the
murine genomic library, identified by hybridization with a probe from
exon 1 of the human 1BAR gene, was found to
contain an approximately 15-kb insert. After restriction mapping (Fig.
1) and Southern blot analysis with the same exon 1 probe, a 5-kb
BamHI fragment containing exon 1 was subcloned into
pBluescript. Sequencing of this BamHI fragment confirmed the
identity of the murine 1BAR gene and
identified the start of the 5'-untranslated region. Overall, the
recovered phage insert was found to contain approximately 10 kb of
5'-flanking DNA, 949 bp of the murine 1BAR
coding region in exon 1, and approximately 4 kb of intronic sequence.
1BAR gene (3500
to +93, relative to the adenosine of the translation start codon) is
sufficient to drive expression of the promoterless
pGL3basic luciferase gene when transiently
transfected into primary rat aorta and vena cava VSM cells (Eckhart et
al., 1997). To accommodate a comparison of rat, mouse, and human
1BAR promoter sequences and to examine the
fidelity of the mouse promoter in vitro, an analogous 3422-bp PvuII fragment (3490 to 68) was isolated from the
original 15 kb-insert and subcloned into the SalI site of
the promoterless pCAT basic vector. Reported in Fig.
2 are sequence data from 1721 to the
PvuII site of the promoter fragment, along with sequence 3'
of the PvuII site to the start codon.
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1BAR gene
promoter fragment was aligned with the known rat and human
1BAR promoters, and the sequence was searched
for possible trans-acting factor binding sites. Of the
previously characterized 3.6-kb PvuII fragment of the rat
promoter, sequence has been determined as far 5' as 2460 (Gao and
Kunos, 1994; Eckhart et al., 1997). Alignment of this known rat
sequence with overlapping regions of the mouse promoter showed 78%
identity between the two species. Comparatively, alignment of the human
1BAR promoter, which has been sequenced as far
5' as 923 (Ramarao et al., 1992), showed 65% identity with
overlapping regions in the mouse promoter. It is also of interest that,
when aligning the 5'-flanking region of the mouse
1BAR gene with an overlapping 3500-bp domain
of the related human 1AAR gene, poor similarity (18% identity) was observed (Razik et al., 1997).
Subsequent sequence analysis of the mouse 1BAR
promoter revealed the presence of multiple potential sites for binding
of various factors (Table 1). Included
among those sites is confirmation of a site for HIF-1 and a CRE, both
identified in the rat 1BAR gene (Gao et al.,
1995; Eckhart et al., 1997).
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813 to
432 (Gao and Kunos, 1994), is believed to be essential for
transcription of the 1BAR gene in most rat
tissues (Gao et al., 1995). The alignment of this rat
1BAR gene P2 promoter with analogous
overlapping regions in the mouse and human gene showed 90% identity
between rat and mouse and 55% identity between rat and human (Fig.
3A). Several DNA consensus sequence
motifs, including the above-mentioned CRE, a GC box, a CACCC domain, an
activator protein 2 (AP-2) site, and binding sites specific for liver
factors (C/EBP, HNF-1, and HNF-5) and heart factors (M-CAT, E-box),
have been identified by Gao et al. (1995) in the rat
1BAR gene P2 promoter. Of these reported
sites, the CRE and the GC box are exactly conserved in both the mouse
and human, and the CACCC domain, the C/EBP site, the M-CAT site, and
the E-box are exactly conserved in the mouse (Fig. 3B). In addition to
these common transcription factor binding sites, Gao et al. (1995)
identified the binding of what was thought to be a novel factor
believed to be -adrenergic receptor specific. This factor, which
binds to two distinct sites in the rat 1BAR
gene P2 promoter, was later shown to be nuclear factor 1 (NF-1) (Gao et
al., 1996). The putative NF1 binding sites identified in these previous
studies, which comprise half the NF1 consensus sequence (see Table 1,
footnote b), are also exactly conserved between both the
mouse and human genes.
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Promoter Function in Clonal Cell Lines.
The ability of the
3.4-kb mouse 1BAR promoter to drive expression
of a CAT reporter gene was examined. This was accomplished by
cotransfecting the 1BAR promoter-pCAT plasmid
and the -gal plasmid into two 1BAR-negative
and three 1BAR-positive cell lines. The
ability of these cells to activate the promoter was then assessed by
measuring CAT enzymatic activity, which was corrected for transfection
efficiency (-gal). Figure 4 shows that
CAT activities were less than 3.3-fold over the antisense control in
1BAR-negative RAT-1 cells (rat fibroblast) and
R3T3 cells (mouse embryonic fibroblast). Comparatively, CAT activities
were greater than 15-fold over control in
1BAR-positive NB41A3 cells (mouse
neuroblastoma) and greater than 35-fold over control in
1BAR-positive BC3H1
cells (mouse brain tumor). The most robust production of CAT activity,
greater than 40-fold over the antisense control, was detected in
1BAR-positive DDT1MF-2
cells (hamster leiomyosarcoma). CAT activity produced by
DDT1MF-2, NB41A3, and BC3H1
cells was statistically different from activity produced by RAT-1 and
R3T3 cells (p < .05). Also, CAT activity produced by
DDT1MF-2 and BC3H1 cells
was statistically different from activity produced by NB41A3 cells
(p < .05).
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Promoter Function in Primary Cell Cultures.
Because the
promoter-reporter studies discussed above confirm the fidelity of the
3.4-kb mouse 1BAR promoter in established cell
lines, the 1BAR promoter-pCAT plasmid was used
as a tool to compare the competency of several primary cell types to
activate CAT expression. As in the above experiments with clonal cell
lines, primary cell cultures were cotransfected with the
1BAR promoter-pCAT plasmid and the -gal
plasmid. The ability of primary cells to activate the promoter was then
determined by measuring CAT enzymatic activity, which was corrected for
transfection efficiency. Whereas 1BAR-negative
rat calvarial osteoblasts (bone cells) did not significantly activate
the promoter compared with the antisense promoter control, several rat
VSM cell types exhibited competency (Fig.
5). VSM cells derived from vessels such
as iliac, femoral, and pulmonary artery produced CAT activity that was
3- to 6-fold over antisense controls. Comparatively, VSM cells derived
from vessels such as renal and superior mesenteric artery showed more robust production of CAT activity that was greater than 10-fold over
antisense controls. Not only did all of the VSM cells tested produce a
significantly greater amount of CAT activity compared with the
osteoblasts (p < .05), but VSM cells from renal and
superior mesenteric artery produced significantly greater CAT activity than VSM cells from iliac, femoral, or pulmonary artery
(p < .05).
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Promoter Regulation by cAMP and Hypoxia.
The presence of the
aformentioned CRE and HIF-1 binding site on the mouse 3.4-kb
PvuII promoter fragment suggests possible regulatory roles
for cAMP and hypoxia in the mouse 1BAR gene. To examine these putative regulatory functions, RAT-1, R3T3, NB41A3, BC3H1, and DDT1MF2 cells
that were transiently cotransfected with the -gal plasmid and the
1BAR promoter-pCAT plasmid were challenged for
12 h with 100 µM forskolin, hypoxic conditions, or both. After the 12-h challenge, cell extracts were harvested for determination of
CAT activity. Compared with control cells that were transfected with
the promoter-reporter construct but not treated, CAT production was
significantly stimulated in all five cell types by forskolin or by
exposure to hypoxia (Fig. 6). Forskolin
and hypoxia evoked a roughly similar percent increase in all cell
types, with R3T3 cells and RAT-1 cells exhibiting the most robust
response (190 and 145% increases, respectively; Fig. 6A).
Comparatively, NB41A3, BC3H1, and
DDT1MF2 cells showed less robust but significant
increases in CAT production (83, 34, and 49% increases, respectively;
Fig. 6B). Interestingly, when cells were challenged with both forskolin and hypoxia, cell-type-specific regulation was observed. In both the
RAT-1 and R3T3 lines, dual challenge evoked a significant increase in
CAT production relative to levels measured when either one challenge or
the other was applied (107 and 70% increases, respectively; Fig. 6A).
However, NB41A3, BC3H1, and
DDT1MF2 cells did not exhibit this additive
effect. Dual exposure in these cells resulted in a level of CAT
production that was not significantly different from levels seen with
either forskolin or hypoxia treatment alone (Fig. 6B). These novel
findings establish the functionality and cell-type specificity of CRE
and HIF-1 consensus sites in the 3.4-kb PvuII fragment of
the 1BAR gene and combine with our earlier
data to confirm the in vitro fidelity of this promoter.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Genomic Structure.
The genomic structure of the murine
1BAR gene is believed to be similar to the
reported rat (Gao and Kunos, 1993) and human (Ramarao et al., 1992)
1BAR gene structures. Specifically, a large
intron was found in the murine gene located at the end of the
transmembrane 6 coding region, the exact location of which is conserved
in the human and rat 1BAR genes as well as in
the human and bovine 1AAR genes (Razik et al.,
1997). Because we did not clone exon 2 of the murine
1BAR gene, we do not know the size of the
putative intervening intron, which we predict could be as extensive as
10 to 20 kb in length. Because other adrenergic receptors are either
intronless or have not conserved their intronic structure, current
theories pertaining to the origin of genes (Gilbert et al., 1997)
suggest that the conservation of intronic structure in the
1AR family make it the youngest phylogenetic
member of the adrenergic receptor superfamily (Feng et al., 1997).
Comparison of Mouse, Rat, and Human 1BAR Promoter
Regions.
The sequence of the 3.4-kb mouse
1BAR gene promoter fragment was compared with
previously published promoter sequence in both human and rat
1BAR genes. This comparison showed that the mouse fragment was 65% identical with overlapping domains in the human
gene and 78% identical with overlapping domains in the rat gene. These
alignments indicate that, although there is significant similarity
between overlapping putative promoter domains from each species, there
is sufficient sequence divergence to raise the possibility that
regulation of promoter function may vary from species to species.
). The proximal P1
promoter and the distal P3 promoter (455 to 49 and 1608 to
1107, respectively), which were shown to be strong enough to evoke
expression of a CAT reporter, were thought to be responsible for
driving production of 2.3- and 3.3-kb mRNA transcripts (Gao and Kunos,
1994). These two transcripts, found to be present only in the liver,
have unknown functional relevance. Compared with P1 and P3, the P2
promoter was more extensively characterized in this earlier study.
Located between 813 and 432, P2 was implicated as the domain
responsible for generating the major 2.7-kb transcript in most rat
tissues, particularly in the liver. Figure 3A shows sequence alignments
of the rat P2 domain with overlapping domains in the mouse and human
promoter. Analysis of these alignments indicated that there is 90%
identity between rat and mouse and 55% identity between rat and human. These findings support the previously mentioned possibility that regulation of 1BAR promoter function may be at
least partially species specific. As shown in Fig. 3B, this hypothesis
is further strengthened by the finding that several
cis-acting elements present in the rat
1BAR gene P2 promoter are not exactly
conserved in the mouse or human genes.
Contrary to our search for rat-specific DNA elements in the mouse P2
promoter, the full rat promoter was examined for elements that are
found to be present in the mouse. A computer-based search of the entire
3.4-kb mouse 1BAR gene promoter fragment
revealed the presence of multiple possible regulatory elements, a
subset of which is shown in Table 1. Whereas several of these sites are
exactly conserved between overlapping regions of the mouse and rat
sequence, several sites are mouse specific. Conserved sites include a
CRE and an HIF-1 site, supporting the hypotheses that cAMP (Gao et al.,
1997) and induction of hypoxia (Eckhart et al., 1997) are important
universal regulators of 1BAR promoter function. Note that both of these sites are conserved in the human promoter as well. Suggestive of some divergence between the rat and
mouse, several mouse-specific elements were identified, including two
possible sites for binding of the liver, factor C/EBP and an NF-E1
site. Not noted in the table, several AP-1, AP-2, HNF-5, and leader
binding protein 1 (LBP-1) sites found in the mouse are also not
exactly conserved in overlapping regions of the rat. Note that our
identification of the presence/absence of possible response-element
consensus motifs does not imply their functionality; functional
analysis of such sites must be performed to confirm the transcriptional
role of any such putative element.
Because 1ARs are thought to be involved in
cell growth and proliferation, a search of our murine promoter fragment
for elements known to control gene transcription during growth or
proliferative phases was performed. In particular, an
1AR regulatory sequence denoted as a
phenylephrine response element (PERE), which has been identified in the
hypertrophy-specific atrial natriuretic factor promoter region (Ardati
and Nemer, 1993), is not present in the 3422-bp
1BAR promoter. Furthermore, neither the
cardiac hypertrophy-specific NF-AT3/GATA4 binding site
(Molkentin et al., 1998) nor the developmental regulator of smooth,
skeletal, and cardiac muscle proliferation referred to as the CArG box
(Karns et al., 1995) was found to be conserved in the
1BAR promoter. However, proliferative-specific
elements such as AP-1 (Angel and Karin, 1991) and NF-
B (Baeuerle and
Baltimore, 1996) and hypertrophy-specific elements M-CAT and Sp1 (Karns
et al., 1995) are present (see Table 1).
1BAR Promoter Function in Clonal Cell Lines.
To
determine whether our 3.4-kb mouse promoter fragment was sufficient to
maintain appropriate tissue-specific expression, CAT reporter studies
were performed in 1BAR-positive and -negative clonal cell lines. Presumably, if sufficient promoter was recovered from our genomic screen to conserve fidelity of expression,
1BAR-negative cells transfected with the
1BAR promoter-pCAT plasmid should not
significantly activate the promoter or show expression of the CAT gene.
Conversely, 1BAR-positive cells should be able to activate the promoter, which in turn should evoke strong expression of CAT activity. Clonal cell lines utilized for this control experiment that were 1BAR negative included RAT-1
fibroblasts and R3T3 mouse embryonic fibroblasts. The fact that these
fibroblastic lines are adrenergic receptor negative has made them
common candidates for stable expression of various adrenergic subtypes.
For comparison, three 1BAR-positive clonal
cell lines were also tested, including BC3H1
mouse brain tumor cells, NB41A3 mouse neuroblastoma cells, and
DDT1MF-2 hamster leiomyosarcoma cells. In
DDT1MF-2 and BC3H1 cells,
previously reported saturation binding experiments showed Bmax values of 396 and 118 fmol/mg of
protein, respectively (Han et al., 1992). This report also showed
competition binding data from each line that were consistent with the
presence of a single population of 1BAR sites.
Similar binding experiments in NB41A3 cells showed a
Bmax of 23 fmol/mg of protein, with
competition binding data also consistent with the presence of a single
population of 1BARs (Esbenshade et al., 1993).
Based on these combined data, all three of these cell lines are
considered to express a pure population of the
1B subtype.
1BAR gene
promoter fragment, RAT-1 and R3T3 cells produced significantly less CAT
activity than 1BAR-positive NB41A3,
BC3H1, and DDT1MF-2 cell
lines (Fig. 4). In addition to this ability to drive tissue-specific
expression, the promoter also exerted regulatory control over CAT
expression that paralleled the aformentioned differences in
1BAR content seen among the three
1BAR-positive lines tested.
DDT1MF-2 and BC3H1 cells,
which express a large number of 1BARs,
produced the largest amount of CAT activity. NB41A3 cells, which
express relatively fewer receptors, produced more CAT activity than the fibroblastic lines but significantly less activity than
DDT1MF-2 and BC3H1 cells.
These findings suggest that, in the case of mouse-derived R3T3, NB41A3,
and BC3H1 cells, our 3.4-kb
1BAR gene promoter fragment is sufficient to
not only confer tissue-specific expression but also exert regulatory
control that mirrors the function of the endogenous promoter. Note that
in the case of RAT-1 and DDT1MF2 cells, which are
derived from rat and hamster, respectively, our finding of apparent
tissue-specific expression and regulatory control exerted by the
promoter could be partially influenced by both tissue-specific and
species-specific factors.
Screening VSM Cells with the 1BAR Promoter-pCAT
Plasmid.
As a means to understanding the role of
1ARs in the regulation of blood pressure and
hypertrophic cell growth, significant effort has been exerted by
numerous investigators to determine the pattern of vascular expression
for each of the three 1AR subtypes. Regarding
the pattern of 1BAR expression in particular, some studies have approached this question by quantitating mRNA levels
in several different rat blood vessels. However, these RNase protection
and reverse transcriptase-polymerase chain reation experiments showed
no significant difference in 1BAR mRNA content between various vessels (Guarino et al., 1996). Other recent
experiments have involved immunohistochemistry along with antisense
oligonucleotide techniques in aortic, caudal, femoral, iliac,
mesenteric resistance, renal, and superior mesenteric arteries. In
agreement with the earlier mRNA experiments, these studies showed that
antisera raised against the 1BAR (Fonseca et
al., 1995) exhibited roughly equal immunoreactivity in each of the
blood vessels examined (Piascik et al., 1997; Hrometz et al., 1999).
From a functional perspective, however, the magnitude of developed
tension evoked by phenylephrine or naphazoline in
1BAR antisense-treated vessels was not found to be uniform, with only the mesenteric resistance artery exhibiting an
antisense-evoked reduction in contractile responses to agonist (Piascik
et al., 1997; Hrometz et al., 1999). Because these results suggest a
contractile role for 1BARs in mesenteric
arteries only, the possibility arises that
1BARs in general may be more functionally relevant in other aspects of vascular function. As an example of this
idea, 1ARs are also thought to be involved in
catecholamine-induced proliferation and hypertrophy of VSM (Nakaki et
al., 1990).
1BAR promoter-pCAT reporter plasmid in clonal
cell lines, we attempted to use this reporter plasmid to: 1) assess the
competency of various primary VSM cell types to activate transcription
of the 1BAR gene; and 2) compare these
promoter-reporter results with the pharmacological and functional
results discussed above. Although all primary VSM cell types tested
produced significantly more CAT activity than the negative control, VSM
cells from renal and superior mesenteric arteries produced the most
robust CAT activity (Fig. 5). The fact that these two VSM cell types
produced CAT activity that was significantly greater than that seen in
VSM cells from iliac, femoral, or pulmonary artery suggests that they are the most competent of the cell types tested to transcribe the
1BAR gene. The
1BAR-negative osteoblasts, which did not produce a significant amount of CAT activity, served as a negative control for this experiment. Collectively, these data are consistent with previous RNA and immunohistochemical analyses, which showed that
the 1BAR is widely expressed in all vascular
tissue. However, the more robust CAT activity seen in renal and
superior mesenteric arteries suggests differential regulation of the
1BAR subtype in various vessels. Although
contraction of renal and superior mesenteric artery does not appear to
be mediated by the 1BAR, proliferative and/or
growth responses in these cells may be regulated by this subtype.
Indeed, the mouse 1BAR promoter contains an M-CAT site and numerous AP-1, Sp1, and NF- sites (Table 1), suggesting receptor regulation by factors that control hypertrophic and
proliferative responses. Therefore, we propose that measuring the
competence of a cell to activate transcription of our promoter-reporter plasmid may have predictive value when attempting to screen tissues for
1BAR content or regulation. Given the lack of
highly selective ligands for the various adrenergic subtypes, similar
promoter-reporter constructs could be valuable tools when screening
numerous tissues for adrenergic competency.
Regulation of 1BAR Gene Promoter by cAMP and
Hypoxia.
Because the rat 1BAR promoter
has already been shown to possess functional CRE and HIF-1 sites, our
goal became to test for similar response element function in the murine
promoter. Thus, the functional fidelity of the 3.4-kb murine
1BAR gene promoter fragment was examined by
applying forskolin and/or hypoxic challenge to cells transfected with
the 1BAR promoter-pCAT plasmid. As can be seen
in Fig. 6, all five cell lines showed increased CAT production after
forskolin or hypoxic challenge compared with the unchallenged control.
These findings, which are similar to previously published results with
the analogous rat promoter (Eckhart et al., 1997; Gao et al., 1997),
provide important support for the hypothesis that our
1BAR gene promoter fragment possesses functional fidelity.
1BAR gene
promoter is dependent on a combinatorial mechanism that is comprised of
the regulatory effect of multiple factors. Precedent for this idea was
established in an earlier study that reported the interaction of an
AP-2 site with the CRE in the regulation of both basal and cAMP-evoked
activity of the rat 1BAR promoter (Gao et al.,
1997). Our initial test of this hypothesis was attempted in this study
by exposing the mouse 1BAR gene promoter
fragment to a simultaneous challenge with forskolin and hypoxia. In
1BAR-negative RAT-1 and R3T3 cells, this dual
challenge of the promoter evoked a significant increase in CAT
production above the level of production seen with either challenge
alone (Fig. 6A). The effect was additive in both cell lines, with the
sum of each individual treatment roughly equaling the level of
production observed with the dual treatment. Interestingly, in
1BAR-expressing NB41A3,
BC3H1, and DDT1MF2 cells,
this additive effect was not present, with dual challenge producing a
level of CAT activity that was similar to levels seen with forskolin or
hypoxia alone. These results support our hypothesis that
1BAR promoter control is dependent on a
combinatorial mechanism by demonstrating that cells competent to
express 1BARs (i.e., NB41A3, BC3H1, and DDT1MF2 cells)
can exhibit a different promoter response than
1BAR-negative cells to the same conditions. We
suspect that the lack of promoter response to the dual challenge seen
in 1BAR-competent cells may be due to the net
effect of a complement of 1BAR-related positive and negative transcriptional regulators that are always present and functional due to the endogenous expression of the 1BAR gene. In
1BAR-negative fibroblasts, this complement of factors may not be present, leading to divergent responsiveness. Whereas our current data cannot sort out this issue, the novel responses seen to dual challenge of the murine
1BAR gene promoter fragment with forskolin and
hypoxia confirm our assertion that this promoter is capable of
regulating tissue-specific transcription apparently via the action of
one or more functional cis-acting domains.
Concluding Remarks.
Experiments described in this article 1)
compared known sequences from mouse, rat, and human
1BAR promoters and 2) examined the function of
the mouse promoter in various clonal and primary cell lines. Sequence
analyses indicated that, whereas there is substantial similarity among
mouse, rat, and human 1BAR promoter regions,
there is some sequence divergence, with several putative response
elements not being conserved between species. CAT reporter studies of
the mouse 1BAR promoter showed that CAT
production was restricted to 1BAR-positive
clonal cell lines, supporting our hypothesis that the promoter can
regulate tissue-specific expression. Also demonstrating differential
regulation of the promoter, VSM cells from vessels such as renal and
superior mesenteric artery showed robust CAT production, whereas VSM
cells from vessels such as iliac, femoral, and pulmonary artery showed
a significantly lower competency to produce CAT activity. Finally,
functionality of a CRE and an HIF-1 site in this mouse
1BAR gene promoter was confirmed, and novel
tissue-specific effects on promoter function were observed when both of
these sites were simultaneously activated. Based on these collective
results, we propose that the 1BAR
promoter-pCAT plasmid, when used in CAT reporter studies like the ones
described herein, may prove to be a valuable tool when screening cell
types for competency to activate the 1BAR
gene. Our 3.4-kb mouse promoter could also be useful to target a gene
systemically to 1BAR-expressing tissues in a
transgenic model.
| |
Acknowledgments |
|---|
We thank Dr. Derek Damron of the Department of Anesthesiology Research, Cleveland Clinic Foundation (Cleveland, OH), for kindly providing primary cultures of rat pulmonary artery vascular smooth muscle cells. We also thank Dr. J. Edward Puzas of the Department of Orthopaedics, University of Rochester, Rochester, NY, for kindly providing primary cultures of rat calvarial osteoblasts. Finally, we thank Stephanie Edelmann for excellent technical assistance in isolating the various rat vascular smooth muscle cells examined in this study.
| |
Footnotes |
|---|
Received August 12, 1999; Accepted September 21, 1999
This work was supported by National Institutes of Health Grants RO1HL52544 (D.M.P.), RO1HL61438 (D.M.P.), RO1HL31820 (M.T.P.), and F-32-HL-10004-02 (M.J.Z.); and an unrestricted grant from Glaxo Wellcome (D.M.P.). The work was performed under the tenureship of an Established Investigator Award from the American Heart Association (D.M.P.). The sequence reported in this article has been deposited in the GenBank database (accession no. AF116943).
1 Signal Scan, a search engine maintained by the Advanced Biosciences Computing Center, University of Minnesota, is available on the Internet at a site maintained by the Bioinformatics and Molecular Analysis Section of the National Institutes of Health: http://bimas.dcrt.nih.gov/molbio/signal/.
Send reprint requests to: Prof. Dianne M. Perez, Department of Molecular Cardiology, NB5, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. E-mail: perezd{at}ccf.org
| |
Abbreviations |
|---|
1AR, 1-adrenergic
receptor;
CAT, chloramphenicol acetyltransferase;
VSM, vascular smooth
muscle;
HBSS, Hanks' buffered salt solution;
-gal, -galactosidase;
CRE, cAMP response element;
HIF-1, hypoxia-inducible
factor 1.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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). The PvuII site, which marks the 3'
end of the recovered promoter fragment, is indicated by a shaded box.
Regions of this mouse promoter that overlap with important domains
identified in the rat 
