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J Biol Chem, Vol. 275, Issue 11, 7604-7611, March 17, 2000
From the Department of Pharmacology, Emory University School of
Medicine, Atlanta, Georgia 30322
Cell surface receptor activation generally leads
to changes in mRNA abundance, which may involve regulatory targets
in processes working at the post-transcriptional level. Many types of
agonists down-regulate vascular smooth muscle angiotensin receptor
(AT1-R) gene expression, but it is unclear which of
these activate post-transcriptional mechanisms. To reconstitute
faithfully the normal AT1-R mRNA regulatory environment, tetracycline-suppressible promoters drive highly accurate
recombinant AT1-R mRNA mimics in vascular smooth muscle cells that co-express an endogenous AT1-R mRNA.
Down-regulation of the latter occurs shortly after stimulating
mitogenic receptors or by using forskolin, but only cAMP signaling
reduces expression of the recombinant AT1-R mRNA.
Transcription of the recombinant mRNA is unaffected by cAMP
signaling. Deletions of the AT1-R mRNA 3'-untranslated
region do not impair cAMP-mediated down-regulation. Both loss of
function and gain of function mutants show the response is mediated by
the 5'-untranslated region. These observations provide the first direct
functional evidence for modulation of vascular AT1-R gene
expression by a mechanism involving a protein kinase A-regulated
post-transcriptional process.
The vasoconstrictive and volume sparing hormone angiotensin II
acts through a cell surface G Down-regulation of AT1-R gene expression occurs within a
few hours following activation of vascular smooth muscle cells with many different types of stimuli. These include several growth factors
acting through receptor tyrosine kinases (4, 5), hormones, and
autacoids acting through either G A role for post-transcriptional regulation in agonist-mediated control
of AT1-R gene expression has wider implications. Current knowledge about regulated post-transcriptional processes lags considerably the more detailed understanding of how gene transcription rates are modulated by receptor signaling (11). Although there is a
growing appreciation that receptor-operated post-transcriptional control processes likely exist, gathering unambiguous functional evidence for this modality of gene expression regulation is not a
trivial matter (reviewed in Ref. 12). The typical approach involves
measuring how signaling changes mRNA decay rates, using general
transcriptional inhibitors like actinomycin D or DRB. The nonspecific
effects of these drugs are problematic and raise serious questions
about their utility and thus the reliability of many studies that
resort to their use. For example, actinomycin D can evoke several
stress-like responses in cells, potentially disrupting interfaces
between signaling and mRNA metabolism (13-18). Furthermore, the
mechanism of action of DRB is through inhibiting phosphorylation of RNA
polymerase, but its selectivity for the RNA polymerase kinases
versus signaling kinases is unknown (19, 20). Finally,
actinomycin D can directly interfere with basal mRNA turnover
mechanisms, raising concerns that this might be a more general problem
than is currently appreciated (12, 21, 22).
The present study surmounts these difficulties, providing direct
functional evidence that post-transcriptional processes regulated by
signaling contribute to modulating vascular AT1-R mRNA
abundance. We clarify the role of post-transcriptional regulation among
the many different factors that can down-regulate AT1-R
mRNA expression. We begin with the premise that an mRNA will be
regulated at the post-transcriptional level irrespective of whether it
is transcribed from a recombinant or a native locus. Mimicry by a
recombinant surrogate of a response affecting its native transcript is
essential direct evidence that a post-transcriptional regulatory
process exists. The predictive value of recombinant surrogates then
rests largely on how accurately mRNA expression dynamics have been
reconstructed. By using tetracycline-suppressible transcription factors
(23, 24) to express highly accurate AT1-R mRNA mimics
in vascular smooth muscle cells, we directly test whether
post-transcriptional mechanisms are involved in its modulation by
specific receptor-coupled signaling systems.
VSMC Cell Culture--
Rat aortic vascular smooth muscle (RASM)
cells (passages 8-25) and A7r5 smooth muscle cells were cultured as
described (3, 8). After reaching confluence, growth media containing
10% serum was replaced with serum-free media for an additional 16 h before initiating treatments.
Retrovirus Production and Infection--
Infectious retroviral
supernatants were generated by a helper virus-free, transient
transfection protocol and used to infect smooth muscle cells with
>99% efficiency as described elsewhere (25). To ensure stable
chromatin integration, cells infected with AT1-R mRNA
expression viruses were treated with 200 µg/ml G418 for 7-10 days.
Experiments were then performed on such recombinant cell batches over
the succeeding 3-5 passes through subculture, representing studies on
cells that have gone through approximately 12-20 population doublings
after retroviral infection.
Plasmid Construction--
The plasmid pXF42-eGFP was created by
removing the neomycin cassette in pXF40-eGFP (10) using BclI
and BstBI and first replacing it with a multicloning site
using
5'GATCAGGCCTTGTAGGCCTAGGCTCGAGGCCTACAAGGCCTT and its reverse complement (SfiI restriction sites are
underlined). The tetracycline transactivator (tTA) cDNA from
pUD15-1 (23) was excised with BamHI and EcoRI,
blunted, and adapted with the oligonucleotide pair 5'AGGCCTAG and
5'CTAGGCCTACA before cloning into these SfiI sites. All
retroviral AT1-R expression plasmids were constructed in
pTJM12, a derivative of pTJM9 (25) from which the internal CMV promoter
was removed using BamHI and HindIII before
blunting and self-ligation. To create pTSO33, which is the wild-type
AT1-R mRNA expression vector, eight copies of the tetracycline operator sequence (tetOp) derived from pUD10-3 (23) as a
XhoI-StuI fragment were fused upstream of a
PstI-KpnI fragment containing the minimal ( Ribonuclease Protection Assays and Data Analysis--
Samples of
total RNA were hybridized with a mixture of gel-purified,
[ Nuclear Run-on Assays--
Nuclei from confluent RASM cells were
isolated, and in vitro run-on transcription assays using 250 µCi of [32P]UTP (3000 Ci/mmol) were conducted in a
200-µl volume as described previously (8). Radiolabeled nuclear RNA
was isolated and split into 2 equivalent aliquots and each hybridized
in solution at 22 °C precisely as described (28), using either 0.21 nmol of biotinylated antisense HA epitope oligonucleotide (5'biotin-(18 carbon spacer)-AACGGCGTAGTCTGGGACGTCGTATGGGTAC) or 0.21 nmol of oligonucleotide antisense to the L32 ribosomal protein mRNA
(5'biotin-(18 carbon spacer)-TTCACATATCGGTCCGACTGGTGCCTGATGA) as an
internal control. After an overnight hybridization, streptavidin-coated ferrous beads (Promega, Inc. Madison, WI; 300 µg, ~1.25 nmol
capacity) were added. After a brief incubation, the samples were
clamped against a magnetic bar and washed six times with 1-ml aliquots of RNase-free 0.1× SSC. The samples were eluted in a sequencing gel
loading dye by heating to 90 °C for 10 min before resolution by
electrophoresis through 5% polyacrylamide sequencing gels.
Expression of recombinant AT1-R mRNAs in vascular
smooth muscle cells from tetracycline-regulated promoters exploits a
helper virus-free, non-replicating retroviral vector system (Fig.
1). The gene transfer efficiency is
routinely close to 100% using this, allowing for rapid derivation of
new recombinant cell populations compared with alternative methods.
Stable chromatin integration of the recombinant genes allows for
continued study on successive passages of cells following a single
infection protocol. Fluorescence-activated cell sorting (10) was used
first to create vascular smooth muscle cell lines enriched for
expression of the tetracycline transactivator (23) using the
self-regulating vector pXF42-eGFP (Fig. 1a). After expansion
and storage, these cells were used to provide a uniform genetic
background for subsequent transduction with recombinant
AT1-R retroviruses.
The vector used to express what is termed the wild-type recombinant
AT1-R mRNA (referred herein as wtAT1-R),
which represents the most accurate possible mimic of the native
transcript, is shown in Fig. 1b. This anhydrotetracycline
(Antet)-suppressible mRNA begins and ends, respectively, with
transcriptional initiation (minimal promoter) and termination
(polyadenylation) sequences derived from the native AT1-R
gene (26). In using these elements, rather than heterologous promoter
and poly(A)+ sequences, the composition of the 5' and 3'
ends of the recombinant mRNA precisely mimic those of the major
native 2.2-kb AT1-R transcript expressed in VSMC and other
cells (27). An in-frame hemagglutinin (HA) epitope within the amino
terminus of the AT1-R open reading frame is the only
difference between the recombinant wtAT1-R and the native
transcript, which was necessary to distinguish between them using
ribonuclease protection assays.
A7r5 smooth muscle do not to express the AT1-R gene
natively (3), but after sequential infection with tTA and
wtAT1-R mRNA viruses, they display an
anhydrotetracycline (Antet)-suppressible 2.2-kb AT1-R
mRNA (labeled "tetOp"). It migrates on agarose gels the same as
the more abundant native transcript in a second line of cells termed
RASM (Fig. 1c), demonstrating that the expression vector
produces the expected AT1-R mRNA product. Notably, this is slightly smaller than the 2.4-kb transcript produced from the vector
TSO31 (see below), which has additional heterologous 5'- and 3'-UTR
sequences derived from CMV and SV40 elements (3). The recombinant
wtAT1-R mRNA presumably migrates at 2.2 kb in recombinant RASM cells, inferred from the finding that none of the
detectable hybridization signals are suppressed by Antet treatment. Of
note, the weaker 3.2-kb transcript observed in RASM mRNA represents a previously cloned alternately spliced product of the native AT1-R gene, rather than a recombinant molecule (26, 27).
The barely detectable hybridization signals migrating at ~6 kb,
labeled "LTR," likely represent the transcript driven by the LTR
promoter, essentially reflecting a neomycin mRNA with antisense
AT1-R mRNA sequences in the 3'-untranslated region
(Fig. 1b).
Our ribonuclease protection assay discriminates recombinant from native
transcripts in a single sample, because the riboprobes used incorporate
sequence antisense to the HA epitope in addition to the flanking
AT1-R mRNA sequence (Fig.
2). The phosphorimage (Fig.
2a) shows a single large protected fragment representative of the recombinant wtAT1-R mRNA. The three smaller
fragments reflect the native mRNA because synthesis of the
riboprobe from a recombinant template crosses over both the
heterologous HA epitope and an exon that is alternatively spliced
within the native mRNA 5'-UTR (26, 27). Although the recombinant
transcript is suppressed after treatment with 1 µg/ml Antet, this
does not affect expression of the native AT1-R mRNA,
showing the selectivity of Antet (Fig. 2b). A comparatively
delayed decay occurs for the wtAT1-R in cells treated with
actinomycin D (20 µg/ml) relative to the effect of Antet, indicating
that actinomycin D strongly interferes with basal AT1-R
decay processes.
Reconstitution of Angiotensin Receptor mRNA
Down-regulation in Vascular Smooth Muscle
POST-TRANSCRIPTIONAL CONTROL BY PROTEIN KINASE A BUT NOT
MITOGENIC SIGNALING DIRECTED BY THE 5'-UNTRANSLATED REGION*
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ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
q-coupled
AT1-receptor
(AT1-R).1 A
long-standing observation both in vivo and in culture is
that AT1-R expression is dynamically regulated but by
cellular and molecular mechanisms that are not well understood (1, 2). Since modulation of AT1-R mRNA levels is a dominant
mechanism for determining target cell hormonal responsiveness (3),
understanding how this essential effector of the
renin-angiotensin-aldosterone endocrine axis is regulated might provide
new insights into how several cardiovascular diseases develop.
s- or
G
q-coupled receptors (6-8), and also direct activators
of protein kinase C or the cAMP-dependent protein kinase A
(PKA) (6, 8-10). We suspect that regulated post-transcriptional
mechanisms may account for some or all of the effects of these diverse
stimuli. Among the indirect evidence supporting this notion are
observations that cAMP-elevating agents do not affect AT1-R
gene transcription (8, 10). Although G
q-coupled and
growth factor receptor signaling suppresses AT1-R gene
transcription, the more rapid rate of AT1-R mRNA decay
in response to mitogens relative to transcriptional inhibitors raises
the possibility of superimposed post-transcriptional processes (4, 6).
Gaining tight and selective experimental control over AT1-R
gene transcription would help to clarify these matters.
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MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
54
to +104 base pair) rat AT1a-R gene promoter (26). A
KpnI-DraIII fragment was fused downstream of this, derived from the vector pTSO31 (3), providing rat vascular AT1-R cDNA sequence (27) between base pairs 104 and
849, wherein a hemagglutinin (HA) epitope was inserted at the fourth
amino acid of the receptor open reading frame. A 1.8-kb
DraIII-StuI fragment derived from a subclone of
the rat AT1-R genomic clone
RGL8 was cloned downstream
from this (26). This latter fragment encodes the remainder of the third
exon of the rat AT1-R gene, including its open reading
frame and 3'-UTR region and ~370 base pairs of additional
non-transcribed genomic sequence that encode downstream polyadenylation
signals. The vector pTSO41 uses the tetracycline-regulated minimal CMV
promoter, as a XhoI-EcoRI fragment from pUD10-3,
instead of the minimal AT1-R promoter, but is otherwise identical to pTSO33 downstream from this. The vector pTSO31 is the same
as pTSO41 at the 5' end but uses an SV40 pA+ signal, rather than
AT1-R pA+ signals as transcriptional terminators (3). Plasmids for the various mutant and deletion AT1-R mRNA
were constructed using shuttle vectors and standard oligonucleotide
site-directed mutagenesis or polymerase chain reaction protocols. The
plasmid pXF40-eGFP (10) served as a base to construct chimera
AT1-R/eGFP mRNAs, and unlike the AT1-R
mRNA constructs, these were infected into cells that express the
tTA through infection with the retrovirus pTSO5 (3). All mutations and
polymerase chain reaction-generated fragments were verified by
sequencing using the Emory University DNA sequencing core facility
before subcloning into the retroviral expression vectors. Plasmids and
their complete sequences are available on request.
-32P]UTP-labeled antisense AT1-R and
cyclophilin (Cyp) riboprobes and treated with RNase A and T1 before
resolution on sequencing gels as described in detail elsewhere (3). All
AT1-R riboprobes incorporate sequences antisense to the HA
epitope. The hybridization signals were quantified from phosphorimages
using a volume integration protocol in the ImageQuaNT software.
AT1-R mRNA hybridization signals were divided by those
for Cyp in each sample, and the data are normalized to this ratio in
untreated control samples, which is taken as 100%. Decay rate
constants were calculated after combining all sets of experimental data
for a given mRNA construct and protocol and performing an iterative
best-fit computation using the single-site exponential decay function
in GraphPad Prism.
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RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Fig. 1.
Recombinant AT1-R mRNA
expression strategy. a depicts the LTR region in the
tTA expression vector pXF42-eGFP. b depicts the LTR region
of the wtAT1-R mRNA expression vector pTSO33.
c is a phosphorimage from a Northern hybridization
experiment (10 µg of total RNA/lane from cells treated for 72 h
with (+) or without (
) Antet) using a double-stranded
AT1-R cDNA probe, labeled with [32P]dCTP
as described previously (8).

View larger version (37K):
[in a new window]
Fig. 2.
Antet selectively causes recombinant
AT1-R mRNA decay. a, representative RPA
phosphorimage of RASM cells expressing both the recombinant
wtAT1-R mRNAs, which were treated for the indicated
times with either Antet (1 µg/ml) or actinomycin D (20 µg/ml).
Protected fragments representing the recombinant transcript (wtAT1-R)
and the endogenous transcript (native AT1-R) are noted.
Cyp, cyclophilin mRNA internal control. b,
quantified effects of Antet (closed symbols) or actinomycin
D (open symbols) on the recombinant wtAT1-R
(circles) or the native transcript (triangles)
are quantified from three experiments (mean ± S.E.). Control
represents Cyp-normalized AT1-R mRNA hybridization
volumes in untreated cells.
Previous studies have established that forskolin, a direct activator of
adenylyl cyclase, down-regulates the AT1-R mRNA through activation of PKA, because this response is inhibited in cells expressing a highly selective polypeptide inhibitor of the
cAMP-dependent kinase (10). A bolus (5 or 10 µM) of forskolin was used to stimulate PKA in RASM cells,
and recombinant wtAT1-R mRNA levels were measured at
various times thereafter (Fig. 3).
Treatment with forskolin causes down-regulation of the mRNA, with
an apparent half-life of 1.7 h (Table
I). Notably, decay rates following Antet
treatment or Antet plus forskolin treatment are similar (Table I).
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Because Antet suppresses transcription by a mechanism that relieves
transactivation, rather than through direct inhibition (23), nuclear
run-on assays were performed to ensure that forskolin does not directly
inhibit transcription of the wtAT1-R gene. Nuclei were
collected after treating cells for 45 min with forskolin, reasoning
that any transcriptional suppression caused by forskolin by this point
should be quite pronounced to account for its affect on steady-state
mRNA levels. For comparison, cells were also treated with Antet to
assess transcriptional suppression and with Antet plus forskolin to
test whether forskolin interferes with the mechanism of action for
Antet. The recombinant AT1-R transcripts were selectively captured by solution hybridization using a biotinylated antisense oligonucleotide directed against its HA epitope (28). Capture of the
ribosomal L32 protein mRNA similarly from aliquots of the same
samples served as an internal control. Antet treatment for 45 min
suppresses the transcripts captured by the wtAT1-R probe, with transcription to about 10% of control levels consistent with the
"leak" that occurs in this system (3) (Fig.
4). Similar results were obtained in
other run-on experiments in which the Antet treatment period was 30 min, suggesting Antet-mediated transcriptional suppression occurs
rapidly (data not shown). Co-treatment with forskolin and Antet does
not interfere with the transcriptional suppression, and forskolin has
no effect on transcription compared with vehicle-treated cells. These
observations establish that transcriptional suppression cannot account
for the forskolin-mediated reduction of the recombinant
wtAT1-R mRNA.
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To determine if mitogenic receptor signaling affects recombinant
wtAT1-R mRNA expression, the cells were stimulated for
up to 6 h following a bolus of either angiotensin II or the growth factor PDGF-BB. As shown in Fig.
5a, their effects are not
comparable to that of forskolin, wherein angiotensin II has no effect
and PDGF reduces the recombinant mRNA only at the latest time point tested. Whereas the recombinant mRNA is largely unaffected by mitogens, the native AT1-R mRNA is down-regulated in
the same samples, which establishes that the agonists nevertheless
evoked their regulatory effects on AT1-R gene expression
(Fig. 5a). The modest suppressive effect of PDGF on the
recombinant wtAT1-R mRNA at the latter time point might
be due to a transcriptional effect. To test this, the PDGF response was
measured in cells expressing a recombinant mRNA from the vector
TSO41, which is identical to the wtAT1-R mRNA except
that it is driven from a tetracycline-suppressible minimal CMV
promoter. This renders an mRNA with ~60 additional bases of
heterologous CMV sequence in the 5'-UTR. As shown in Fig.
5b, the TSO41-derived recombinant AT1-R mRNA
is down-regulated by forskolin but unaffected by PDGF-BB treatment. We
conclude that the reduction of the recombinant wtAT1-R
mRNA in response to PDGF-BB is most likely due to a modest degree
of transcriptional suppression at the minimal AT1-R
promoter.
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To test whether the AT1-R mRNA is regulated in another
smooth muscle cell phenotype, levels of the recombinant
wtAT1-R mRNA levels were measured after infection of
A7r5 cells with the display system. Like RASM, A7r5 cells are derived
from rat thoracic aorta (29) but differ in morphological and growth
characteristics (30).2 As
shown in Fig. 6, 8-h treatments with
either Antet or forskolin reduce wtAT1-R mRNA
expression in each cell line. In contrast, angiotensin II has no effect
in either cell line, and PDGF-BB treatment only reduces the mRNA
when expressed in RASM cells. Thus, both cell lines appear to support
PKA-specific down-regulation.
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We compared four possible combinations of minimal AT1-R or
minimal CMV promoters and AT1-R or SV40 polyadenylation
signals to determine if heterologous sequences influence forskolin
responsiveness or intrinsic decay rates. These nominally full-length
mRNAs (wtAT1-R, KX38, TSO41, and TSO31) show generally
equivalent Antet-mediated decay rates, although forskolin-stimulated
down-regulation of the TSO31 mRNA is slightly delayed compared with
the others (Fig. 7; see also Table I).
Most significantly, each is down-regulated by forskolin treatment, and
like the wtAT1-R mRNA, these each respond to
co-treatment with forskolin and Antet without an accelerated decay
compared with Antet alone. Because a combination of minimal AT1-R gene promoter and SV40 poly(A)+ signals
(KX38) share response characteristics with the wtAT1-R mRNA, a series of 3'-UTR deletion mutants were expressed using SV40
poly(A)+ signals for termination. As seen in Fig. 7, these
mutants show a tendency for increasingly slower rates of decay as a
function of deletion, following Antet treatment. This suggests elements within the 3'-UTR confer basal decay characteristics. Again most notably, forskolin treatment alone causes down-regulation of each construct, indicating the 3'-UTR does not mediate this response to PKA
activation. Furthermore, co-treatment with forskolin and Antet does not
lead to an accelerated rate of decay for any of these eight mRNAs
compared with the effect of Antet alone. These results indicate that
the 3'-UTR does not mediate PKA-stimulated down-regulation (Table
I).
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We next tested if the 271-base long AT1-R mRNA 5'-UTR
determines responsiveness to PKA signaling. Deletion of the 5'-UTR from the wtAT1-R mRNA background making KX30 completely
impairs forskolin-stimulated down-regulation, and the down-regulation
by Antet shows this is not attributable to intrinsic stabilization of
the mRNA (Fig. 8, see also Table I).
Furthermore, forskolin clearly evoked the regulatory process in these
cells as shown by down-regulation of the internal control native
mRNA in the same samples (Fig. 8). Because this mRNA does not
have any predictable 5'-UTR sequence, we also deleted it within the
context of the KX38 mRNA background to make KX63. This would have
an ~60-base heterologous 5'-UTR remnant derived from the CMV minimal
promoter region. The KX63 mRNA is also insensitive to forskolin yet
is not intrinsically stabilized (Fig. 9,
see also Table I)). To control for length effect, the 5'-UTR was
inverted to make KX65, and this is also shown to be insensitive to
forskolin signaling but also not intrinsically stabilized (Fig. 9).
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We next asked if the 5'-UTR region could confer PKA-mediated regulatory
characteristics upon a heterologous mRNA. It was next cloned
upstream of an eGFP coding region in either the forward (KX41) or
reverse (KX41(
)) orientations and compared with a control eGFP
mRNA (XF40). The XF40 mRNA is unaffected by forskolin treatment but decays with a half-life of ~5.4 h after Antet treatment,
representing the most stable mRNA used in this study (Fig. 9; see
also Table I). Forskolin treatment causes down-regulation of the
forward orientation 5'-UTR AT1-R/eGFP mRNA chimera but
not that with a reverse orientation 5'-UTR. Intriguingly, the forward
5'-UTR chimera but not the reverse also acquires the more rapid basal
decay kinetics associated with AT1-R mRNA, which we
suspect may be attributable to basal levels of PKA activity. Together,
these observations indicate that the 5'-UTR region functions as a
cis-acting element that directs post-transcriptional
down-regulation of AT1-R mRNA expression in response to
PKA activation.
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DISCUSSION |
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Difficulties in deriving direct and unambiguous evidence that specific mRNAs are susceptible to regulated post-transcriptional control has impeded progress in developing an understanding of this potentially important interface between signaling and gene expression. A careful reconstitution of the normal cellular environment for an mRNA ensures that a suspected regulatory circuitry remains intact and provides a stringent approach to test this concept. Current "circular" models of mRNA structure argue that the 5' and 3' ends of an mRNA are co-engaged in a complex of interacting proteins linking the 5'-cap structure with the 3' poly(A)+ tail (reviewed in Ref. 31). Reasoning that post-transcriptional regulation of the AT1-R mRNA might very well depend upon accurate formation or function of these or other context-specific complexes, which may also be cell type-specific, we were cautious to recreate a recombinant mRNA as precisely as possible. Coupled with the comparatively selective tetracycline-regulated system, as opposed to general transcriptional inhibitors, and expressing these recombinants in their native cell system, the most accurate possible reconstitution of the normal AT1-R mRNA regulatory environment has been achieved. From the behavior of the recombinant mRNAs in such a system, solid inferences can be drawn about how the native AT1-R mRNA is regulated.
A primary goal of this study was to establish a direct functional test for whether post-transcriptional regulation contributes to vascular smooth muscle AT1-R mRNA down-regulation. Finding this, a secondary goal was to ascertain which of the receptor signaling systems that down-regulate AT1-R gene expression do so through control by post-transcriptional mechanisms. Three lines of evidence developed in this study indicate a PKA-regulated post-transcriptional process targets the vascular AT1-R mRNA. First, the experimental design is predicated on recapitulating a reduction in steady-state expression of the recombinant wtAT1-R mRNA should a stimulus evoke a post-transcriptional regulatory process. We show this not only for the stringently designed wtAT1-R mRNA but also with great consistency on seven additional independent recombinant AT1-R transcripts (see Fig. 7). The second line of evidence is that nuclear run-on analysis shows that the cAMP-mediated effect is not attributable to interference with tTA-mediated transactivation of the recombinant gene. We found this important to evaluate because of the formal possibility that cAMP signaling might unexpectedly inhibit the tTA protein or its mechanism of action. Third, both loss of function and gain of function mutants establish that the 5'-UTR is an obligate component of PKA-mediated AT1-R mRNA down-regulation. Acquired from analysis of five independent recombinant mRNAs, they argue quite strongly that the response to PKA signaling is an intrinsic feature of the mRNA molecule. Taken together, these observations provide the first direct and largely unambiguous functional evidence that this post-transcriptional process exists.
This study significantly extends previous findings that predicted post-transcriptional control by PKA signaling and clarifies the mechanism of AT1-R mRNA down-regulation caused by mitogenic signaling (4, 6, 7, 10). The post-transcriptional response has features of inherent specificity, in that recombinant mRNA down-regulation occurs following cAMP but not mitogenic signaling. This is consistent with previous nuclear run-on observations, wherein AT1-R gene transcription is suppressed some 50-80% by mitogenic signaling, but has never been observed in response to cAMP signaling (4, 6, 8). We infer from this that mitogens likely down-regulate the endogenous transcript predominantly, if not entirely, by a mechanism that involves transcriptional suppression rather than post-transcriptional control. The present direct, function-based approach and the acquisition of reliable AT1-R mRNA half-life estimates made possible by using the tetracycline-regulated system highlights previous uncertainties that have plagued us regarding mechanisms of mitogen-induced down-regulation. Despite the evidence showing mitogen-induced transcriptional suppression, the more rapid rate of AT1-R mRNA down-regulation caused by mitogens compared with actinomycin D or DRB treatment raised the possibility of post-transcriptional regulation because the mRNA did not seem capable of decaying fast enough. We now show just how inaccurate the toxin chase-based half-life estimates were. By using Antet instead, a <2-h half-life shows that a significant reduction in steady-state mRNA can follow inhibiting AT1-R gene transcription, even if only partially. Additionally, the failure of angiotensin II treatment to reduce levels of the recombinant wtAT1-R mRNA raises qestions about the relevance of previously described angiotensin II-stimulated AT1-R mRNA binding protein activity (7). They now seem unlikely to reflect factors involved in post-transcriptional regulation.
We are cautious not to describe this post-transcriptional response as PKA-regulated AT1-R mRNA "destabilization." Of the several mRNAs created for this study, none are reduced at a faster rate by combined treatment with forskolin and Antet, compared with transcriptional suppression alone. Destabilization or "regulation of mRNA stability" implies a different kinetic behavior than this. As the evidence shows that a regulated post-transcriptional mechanism must account for down-regulation of the mRNA caused by forskolin, we speculate either of two general mechanisms could explain the behavior of the AT1-R mRNA. One possibility is that PKA stimulates AT1-R mRNA degradation but through a mechanism that functions upstream of the mRNA decay processes. For example, PKA may prematurely "uncouple" pre-existing mRNA from its normal state and shuttle it into a degradation process wherein decay per se is rate-limiting. Thus, mRNAs would show equivalent decay kinetics irrespective of whether they enter the degradation process through a default or PKA-regulated process.
A more attractive possibility is that separate pools of the AT1-R mRNA exhibit differential sensitivity to PKA signaling. By the following logic, our data might be more consistent with this latter speculation. At steady state, the rates of transcript formation and degradation must be equivalent. The similarity between Antet and forskolin-stimulated decay rates are consistent with a common failure to regenerate the cellular pool. The pool is not refilled following Antet obviously because transcription is suppressed. Because we show that forskolin does not affect transcription, we speculate that PKA might activate a process that selectively targets only newly transcribed AT1-R mRNA molecules. This would have the kinetic appearance of failing to regenerate the cellular pool much like for Antet but through a distinct mechanism. A period might exist in which the mRNA is susceptible to this PKA-regulated process. At latter points in its maturation pathway, it either acquires a defense against it or is no longer associated with the components that PKA presumably regulates. Testing this notion will involve developing schemes that selectively measure nascent mRNAs and distinguish them from more mature mRNAs.
The trans-acting iron-responsive element mRNA-binding proteins connect both iron availability and intracellular oxide concentrations to the stability and translation of specific mRNAs by interacting with specific mRNA cis-acting elements (21, 32-34). To the extent that oxidative metabolites serve as second messengers (35), the iron response element-binding proteins remain one of the few unambiguous interfaces between mRNA stability regulation and signal transduction or changes in the extracellular environment.
Other models systems for the concept that the more traditional receptor-regulated signaling pathways control post-transcriptional processes are not without ambiguity. Interestingly, several of these describe cAMP-regulated processes, including down-regulation of adrenergic receptor mRNAs (36-39) and a plasminogen activator inhibitor mRNA (40, 41) and up-regulation of a lactate dehydrogenase mRNA (42, 43). Concomitant transcriptional regulation by the stimuli that affect steady-state levels of these mRNA and the various toxin-based pulse-mRNA chase paradigms used in these studies are the basis for most uncertainty regarding the role of post-transcriptional processes. Nevertheless, they have compelling biochemical findings that largely correlate mRNA protein binding activity and mRNA structure relationships with responses to stimuli. Curiously, they all implicate 3'-UTR adenylate/uridylate (AU)-rich domains as signaling responsive mRNA elements. On this basis, they seem to differ fundamentally from those likely affecting the AT1-R mRNA, because its 5'-UTR is devoid of AU-rich elements. Also, because AU-rich elements function as powerful epitopes driving the intrinsic decay of many highly unstable mRNAs (44), it remains unclear how they might function as conditional stability determinants responsive to cAMP signaling or other pathways (45, 46).
In summary, this study has general importance by showing a stringent
approach useful for understanding roles of post-transcriptional processes in regulation of mRNA abundance. More specifically, it
clarifies the general mechanisms that are involved in agonist-induced down-regulation of vascular smooth muscle AT1-R gene
expression. Future studies will be directed at achieving improved
insight into the factors that mediate this process. We also hope to
gain some insight into how it contributes to the well known plasticity of smooth muscle cell gene expression and its relevance to the generally suppressive role of PKA in smooth muscle cell proliferation and migration (47).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Rick Bright, Tracy Obertone, Xiaofei Wang, and Brian Adams for their preliminary contributions to this study.
| |
FOOTNOTES |
|---|
* This work was supported by NHLBI Grants HL52810 and HL56107 from the National Institutes of Health.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.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Dept. of Pharmacology, Emory
University, 5031 O. W. Rollins Research Bldg., Atlanta, GA 30322. Tel.: 404-727-2467; Fax: 404-727-0365; E-mail:
medtjm@bimcore.emory.edu.
2 K. Xu and T. J. Murphy, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
AT1-R, type I angiotensin II receptor;
DRB, 5,6-dichloro-1-
-D-ribofuranosylbenzimidazole;
UTR, untranslated region;
Antet, anhydrotetracycline;
eGFP, enhanced green
fluorescent protein;
wtAT1-R, a recombinant
AT1-R mRNA mimic;
PKA, cAMP-dependent
protein kinase A;
VSMC, vascular smooth muscle cells;
RASM, rat aortic
vascular smooth muscle;
tTA, tetracycline transactivator;
kb, kilobase
pair;
HA, hemagglutinin;
CMV, cytomegalovirus;
Cyp, cyclophilin;
LTR, long terminal repeat;
PDGF, platelet-derived growth factor;
RPA, ribonuclease protection assay.
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REFERENCES |
|---|
|
|
|---|
| 1. | Matsusaka, T., and Ichikawa, I. (1997) Annu. Rev. Physiol. 59, 395-412[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Regitz-Zagrosek, V., Neuss, M., Holzmeister, J., Warnecke, C., and Fleck, E. (1996) J. Mol. Med. 74, 233-251[CrossRef][Medline] [Order article via Infotrieve] |
| 3. |
Adams, B.,
Obertone, T. S.,
Wang, X.,
and Murphy, T. J.
(1999)
Mol. Pharmacol.
55,
1028-1036 |
| 4. | Nickenig, G., and Murphy, T. J. (1994) Mol. Pharmacol. 46, 653-659[Abstract] |
| 5. |
Ullian, M. E.,
Raymond, J. R.,
Willingham, M. C.,
and Paul, R. V.
(1997)
Am. J. Physiol.
273,
C1241-C1249 |
| 6. | Lassegue, B., Alexander, R. W., Nickenig, G., Clark, M., Murphy, T. J., and Griendling, K. K. (1995) Mol. Pharmacol. 48, 601-609[Abstract] |
| 7. | Nickenig, G., and Murphy, T. J. (1996) Mol. Pharmacol. 50, 743-751[Abstract] |
| 8. |
Wang, X.,
Nickenig, G.,
and Murphy, T. J.
(1997)
Mol. Pharmacol.
52,
781-787 |
| 9. | Chen, X., Nishimura, J., Hasna, J., Kobayashi, S., Shikasho, T., and Kanaide, H. (1994) Eur. J. Pharmacol. 267, 175-183[CrossRef][Medline] [Order article via Infotrieve] |
| 10. |
Wang, X.,
and Murphy, T. J.
(1998)
Mol. Pharmacol.
54,
514-524 |
| 11. | Hill, C. S., and Treisman, R. (1995) Cell 80, 199-211[CrossRef][Medline] [Order article via Infotrieve] |
| 12. |
Ross, J.
(1995)
Microbiol. Rev.
59,
423-450 |
| 13. | Cherry, L. M., and Hsu, T. C. (1982) Environ. Mutagen. 4, 259-265[Medline] [Order article via Infotrieve] |
| 14. |
Lee, K. C.,
Crowe, A. J.,
and Barton, M. C.
(1999)
Mol. Cell. Biol.
19,
1279-1288 |
| 15. | Beard, S. E., Capaldi, S. R., and Gee, P. (1996) Mutat. Res. 371, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
| 16. |
Jackman, J.,
Alamo, I., Jr.,
and Fornace, A. J., Jr.
(1994)
Cancer Res.
54,
5656-5662 |
| 17. | Asakuno, K., Kohno, K., Uchiumi, T., Kubo, T., Sato, S., Isono, M., and Kuwano, M. (1994) Biochem. Biophys. Res. Commun. 199, 1428-1435[CrossRef][Medline] [Order article via Infotrieve] |
| 18. | Dubois, M. F., Bellier, S., Seo, S. J., and Bensaude, O. (1994) J. Cell. Physiol. 158, 417-426[CrossRef][Medline] [Order article via Infotrieve] |
| 19. |
Dubois, M.-F.,
Nguyen, V. T.,
Bellier, S.,
and Bensaude, O.
(1994)
J. Biol. Chem.
269,
13331-13336 |
| 20. |
Yankulov, K.,
Ymashita, K.,
Roy, R.,
Egly, J.-M.,
and Bentley, D. L.
(1995)
J. Biol. Chem.
270,
23922-23925 |
| 21. | Mullner, E. W., and Kuhn, L. C. (1988) Cell 53, 815-825[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Seiser, C.,
Posch, M.,
Thompson, N.,
and Kuhn, L. C.
(1995)
J. Biol. Chem.
270,
29400-29406 |
| 23. |
Gossen, M.,
and Bujard, H.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
5547-5551 |
| 24. |
Gossen, M.,
and Bujard, H.
(1993)
Nucleic Acids Res.
21,
4411-4412 |
| 25. |
Boss, V.,
Abbott, K. L.,
Wang, X.-F.,
Pavlath, G. K.,
and Murphy, T. J.
(1998)
J. Biol. Chem.
273,
19664-19671 |
| 26. |
Takeuchi, K.,
Alexander, R. W.,
Nakamura, Y.,
Tsujino, T.,
and Murphy, T. J.
(1993)
Circ. Res.
73,
612-621 |
| 27. | Murphy, T. J., Alexander, R. W., Griendling, K. K., Runge, M. S., and Bernstein, K. E. (1991) Nature 351, 233-236[CrossRef][Medline] [Order article via Infotrieve] |
| 28. |
Rasmussen, E. B.,
and Lis, J. T.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7923-7927 |
| 29. | Kimes, B. W., and Brandt, B. L. (1976) Exp. Cell Res. 98, 349-366[CrossRef][Medline] [Order article via Infotrieve] |
| 30. | Firulli, A. B., Han, D., Kelly-Roloff, L., Koteliansky, V. E., Schwartz, S. M., Olson, E. N., and Miano, J. M. (1998) In Vitro Cell & Dev. Biol. Anim. 34, 217-226[Medline] [Order article via Infotrieve] |
| 31. | Sachs, A. B., Sarnow, P., and Hentze, M. W. (1997) Cell 89, 831-838[CrossRef][Medline] [Order article via Infotrieve] |
| 32. |
Casey, J. L.,
Hentze, M. W.,
Koeller, D. M.,
Caughman, S. W.,
Rouault, T. A.,
Klausner, R. D.,
and Harford, J. B.
(1988)
Science
240,
924-928 |
| 33. |
Pantopoulos, K.,
and Hentze, M. W.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
1267-1271 |
| 34. | Pantopoulos, K., Weiss, G., and Hentze, M. W. (1996) Mol. Cell. Biol. 16, 3781-3788[Abstract] |
| 35. |
Hentze, M. W.,
and Kuhn, L. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8175-8182 |
| 36. |
Hadcock, J. R.,
Wang, H.-Y.,
and Malbon, C. C.
(1989)
J. Biol. Chem.
264,
19928-19933 |
| 37. |
Huang, L. Y.,
Tholanikunnel, B. G.,
Vakalopoulou, E.,
and Malbon, C. C.
(1993)
J. Biol. Chem.
268,
25769-25775 |
| 38. |
Danner, S.,
Frank, M.,
and Lohse, M. J.
(1998)
J. Biol. Chem.
273,
3223-3229 |
| 39. |
Port, J. D.,
Huang, L. Y.,
and Malbon, C. C.
(1992)
J. Biol. Chem.
267,
24103-24108 |
| 40. |
Heaton, J. H.,
Tillmann-Bogush, M.,
Leff, N. S.,
and Gelehrter, T. D.
(1998)
J. Biol. Chem.
273,
14261-14268 |
| 41. |
Tillmann-Bogush, M.,
Heaton, J. H.,
and Gelehrter, T. D.
(1999)
J. Biol. Chem.
274,
1172-1179 |
| 42. |
Tian, D.,
Huang, D.,
Brown, R. C.,
and Jungmann, R. A.
(1998)
J. Biol. Chem.
273,
28454-28460 |
| 43. |
Tian, D.,
Huang, D.,
Short, S.,
Short, M. L.,
and Jungmann, R. A.
(1998)
J. Biol. Chem.
273,
24861-24866 |
| 44. | Chen, C.-Y. A., and Shyu, A.-B. (1996) Trends Biochem. Sci. 20, 465-470 |
| 45. | Ming, X. F., Kaiser, M., and Moroni, C. (1998) EMBO J. 17, 6039-6048[CrossRef][Medline] [Order article via Infotrieve] |
| 46. |
Chen, C. Y.,
Del Gatto-Konczak, F.,
Wu, Z.,
and Karin, M.
(1998)
Science
280,
1945-1949 |
| 47. | Bornfeldt, K. E., and Krebs, E. G. (1999) Cell. Signal. 11, 465-477[CrossRef][Medline] [Order article via Infotrieve] |
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