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Metabolic Research Unit and Department of Medicine, University of California, San Francisco, California 94143
Address all correspondence and requests for reprints to: Dr. David G. Gardner, Box 0540, Metabolic Research Unit, University of California, San Francisco, California 94143. E-mail: gardner{at}itsa.ucsf.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ANP and BNP bind to a high affinity receptor, termed natriuretic peptide receptor A (NPR-A), on the surface of target cells, including vascular smooth muscle cells. NPR-A displays an extracellular, natriuretic peptide-binding domain at its amino-terminus. This is linked through a single transmembrane segment to a cytoplasmic kinase-like domain that regulates the effector portion of the molecule, the particulate guanylyl cyclase. NPR-A appears to be the primary receptor responsible for the natriuretic and vasorelaxant activities of circulating ANP and BNP in vivo (3, 4, 5).
Relatively little is known about the regulation of NPR-A gene
expression in target cells, and virtually nothing is known about the
molecular machinery that regulates its expression. With the initial
report of its genomic sequence, the rat NPR-A gene was noted to have a
TATA-less promoter, at least three putative Sp1-binding sites
(positioned between -341 and -50), and a paucity of other consensus
transcriptional regulatory elements in the proximal 5'-flanking
sequence (6). A number of genes expressed in vascular cells harbor Sp1
sites in their proximal promoters (7, 8, 9, 10, 11, 12, 13) implying that this particular
transcription factor may play an important role in regulating gene
expression in the vasculature. In fact, Sp1 levels have been shown to
be increased (
2-fold) in vascular smooth muscle cells of
hypertensive rats vs. their normotensive controls (14). With
this in mind, we have examined the role of Sp1 in the regulation of
NPR-A gene promoter activity. Our studies support a dominant role for
Sp1 or Sp1-like activity in establishing basal expression of this
vasorelaxant gene product.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Embryonic rat aortic smooth muscle (RASM) cells were obtained at
passage 19 from H. Ives at the University of California-San Francisco
and cultured at 37 C in a 5% CO2 humidified incubator in
DMEM-H21 medium containing 10% FBS, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 2% (vol/vol) broth, tryptose phosphate.
Drosophila Schneider cells (SL-2) were obtained from the
Cell Culture Facility at the University of California-San Francisco.
Cells were cultured in Schneider’s medium supplemented with 10% FBS,
100 U/ml penicillin, and 100 µg/ml streptomycin at 25 C.
Plasmid construction and site-directed mutagenesis
Rat NPR-A luciferase reporters have been described previously
(15). -387 NPR-A Luc contains 387 bp of the NPR-A 5'-flanking sequence
(relative to the transcription start site) and 280 bp of the
5'-untranslated region from the NPR-A coding sequence linked upstream
from the complete luciferase coding sequence. The Sp1 expression
vectors, pPacO, pPacSp1, pPadhSp1, and Copia ßGAL, were obtained from
R. Tjian at the University of California-Berkeley. pPacSp2 and pPacSp3
were provided by J. D. Noti at the Guthrie Foundation for Medical
Research (Sayre, PA). Site-directed mutagenesis was carried out
with the QuikChange kit (Stratagene, La Jolla, CA) using
conditions recommended by the manufacturer. In brief, mixtures
containing 10–50 ng -387 NPR-A luciferase, two complementary
mutagenic primers, deoxy-NTPs, and Pfu DNA polymerase were
added to the PCR buffer. PCR was carried out for 16–18 cycles using
30-sec denaturation at 95 C, 1-min annealing at 55 C, and 2-min/kb
extension at 68 C. After PCR, 1 µl DpnI was added to the
reaction to cut parental DNA template, and 5 µl of this digest were
used for transformation. Several candidate clones were identified by
restriction mapping and sequenced using a DNA sequence kit and
[
-35S]deoxy-ATP obtained from
Amersham (Arlington Heights, IL).
Transfection, luciferase, and ß-galactosidase assays
RASM cells were transiently transfected with 15 µg -387
rat(r)NPR-A luciferase and 2 µg Rous sarcoma
virus-ß-galactosidase (provided by W. Fong) by electroporation
(Gene-Pulser, Bio-Rad Laboratories, Inc.) at 250 mV and
960 microfarads. For Drosophila Schneider cells, 20 µg
-387 NPR-A luciferase, 2 µg Copia ßGAL, and 1–10 µg pPacO,
pPacSp1, pPacSp2, pPacSp3, or pPadhSp1 were cotransfected into the
cells by electroporation at 180 mV and 960 microfarads. After
transfection, cells were plated in six-well plastic plates and cultured
for 48 h. Cells were harvested and lysed in 100 µl cell culture
lysis reagent (Promega Corp., Madison, WI). The protein
concentration of each cell extract was measured using Coomassie protein
reagent (Pierce Chemical Co., Rockford, IL). Cell lysates
were processed (30 µg protein/sample) and assayed for luciferase as
described previously (14). Measurements of ß-galactosidase activity
were made using the Galacto-Light Plus kit from Tropix, Inc. (Bedford,
MA). For decoy experiments, RASM cells were transfected with 15 µg
-387 rNPR-A luciferase and 10–20 µg of the relevant double stranded
oligonucleotide containing wild-type or mutagenized Sp1-binding site
sequence. After 48 h of culture, cells were harvested, and
extracts were generated for luciferase assay.
Preparation of nuclear extracts
Nuclear extracts were prepared from RASM cells. Briefly, cells
were harvested and lysed by the addition of 0.5 ml lysis buffer
[containing 10 mM HEPES (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 0.5% Nonidet P-40, 1
mM dithiothreitol, 1 mM
phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, and 5 µg/ml
aprotinin] on ice for 10 min. Lysates were centrifuged, and the
pelleted nuclei were resuspended in buffer [containing 20
mM HEPES (pH 7.9), 420 mM NaCl, 1.5
mM MgCl2, 0.2 mM EDTA, 25%
glycerol, and the above protease inhibitors] and kept on ice for 30
min. Nuclei were centrifuged at 12,000 rpm for 15 min, and the
supernatant was saved. Extracts were stored at -80 C before use.
Sp1 was purified from nuclear extracts of rat pituitary GC cells by sequential wheat-germ agglutinin chromatography and DNA-binding site affinity chromatography, as described by Schaufele et al. (16). The final preparation was estimated to have been purified 10,000-fold.
Electrophoretic mobility shift assay (EMSA)
The oligonucleotides used for EMSAs were as follows: wild type 1
(WT1), 5'-GTCCCCCCCCCGCCCGCCTCCGGAA-3'; WT2,
5'-GGCCTAGCCGCCGCCCGCGGGTGCTG-3'; WT3,
5'-GCATAGGACAGAGGGCGGGGGGCAGCTTC-3'; M1,
GTCCCCCCCaaGttCGCCTCCGGAA-3'; M2,
5'-GGCCTAGCCGCtGaCCGCGGGTGCTG-3'; and M3,
5'-GCATAGGACAGAGGtCaGGGGGCAGCTTC-3' (only coding strand
sequence is provided; mutagenized bases are identified by
lowercase letters; Sp1 consensus sequences are
underlined). Nuclear extracts (10 µg) were incubated in
binding reaction buffer [25 mM HEPES (pH 7.5), 50
mM KCl, 1 mM dithiothreitol, 10 mM
ZnSO4, 0.2 mg/ml BSA, 10% glycerol, and 0.1% Nonidet
P-40] containing 0.5 µg poly(dI-dC) at room temperature for 10 min.
Purified 32P end-labeled, double stranded oligonucleotide
was added for an additional 10 min in a total volume of 20 µl. For
competition experiments, a 1- to 100-fold molar excess of unlabeled,
double stranded oligonucleotide was added to the binding reaction. For
immunoperturbation experiments, nuclear extracts were incubated on ice
for 1 h with 2 µg polyclonal antibody against Sp1, Sp3, Egr-1,
or ANP before the binding reaction. All samples were resolved on 5%
nondenaturing polyacrylamide gels. Gels were dried and exposed to film
for autoradiography.
Statistic analysis
Data were evaluated by one-way ANOVA with Newman-Keuls test for
significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Each
site, either alone or in combination, was mutated (specific mutations
are listed in the bottom portion of Fig. 1), within the
context of -387 NPR-A luciferase to a sequence that eliminated
Sp1-binding activity (discussed below). Each of these constructs was
then transfected into neonatal RASM cells.
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). Mutation of
the site closest to the core promoter (M1) was the most effective,
leading to a 75% reduction in reporter activity, whereas isolated
mutation of the second (M2) and third (M3) sites resulted in 65% and
50% reductions in activity, respectively. Mutation of any two of the
three sites resulted in further suppression of promoter activity,
whereas mutation of all three sites resulted in the lowest level of
activity (90% reduction relative to that of wild type).
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) similar to that
observed with Sp1 sites in other genomic contexts (7, 8, 11). This
complex was completely inhibited by competition with WT1, but not
mutated type 1 (M1), oligonucleotide.
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, A–C). In each instance, putatitve
Sp1/DNA complexes were effectively competed by unlabeled WT
oligonucleotide, but not by its mutated counterpart. Each complex was
also competed by unlabeled wild-type, but not mutated, oligonucleotide
sequence encoding each of the other sites (Fig. 5, A–C). Although detailed quantitative
analyses were not performed, WT1 and WT3 appeared, in general, to be
more effective Sp1 competitors than WT2.
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, A–C, the slower moving complexes on
the gels can be resolved into three or four individual bands. Based on
its elimination (or supershift in the case of WT2) with the anti Sp1
antibody, it appears that the most slowly migrating band near the top
of the gel is Sp1. Two additional bands migrating faster than Sp1 were
not recognized by the Sp1 antibody, but were disrupted with antibody
directed against Sp3. This is most clear in the analysis carried out
with the WT3 probe (Fig. 6C), but is apparent for the WT1 and WT2
probes (Fig. 6, A and B) as well. Neither antibody directed against
Egr1, a transcription factor that also recognizes GC-rich sequence, nor
antibody directed against ANP had any effect on the migration pattern
of the complexes formed between the extracts and radiolabeled
probes.
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Having confirmed that these DNA sequences are critical to NPR-A gene
expression and bind Sp1, we returned to a functional approach to
determine whether forced expression of Sp1 would activate the NPR-A
promoter. Sp1 is expressed at high levels in most cell types. Thus, it
has proven difficult to stimulate expression of Sp1-sensitive promoters
using conventional overexpression techniques. This was confirmed in our
own hands in that cotransfection of an expression vector placing Sp1
under the control of the cytomegalovirus promoter failed to activate
-387 NPR-A LUC in RASM cells (data not shown). To circumvent this
problem, we introduced the NPR-A-driven reporter constructs into
Schneider cells, a continuously replicating cell line from
Drosophila, which possesses very low levels of endogenous
Sp1 protein (19). The wild-type as well as each of the selectively
mutagenized promoter constructs were cotransfected in the presence of
pPadhSp1, an expression vector that positions the entire Sp1-coding
sequence downstream from the alcohol dehydrogenase promoter that is
active in Schneider cells (19). As shown in Fig. 7A, forced expression of Sp1 resulted in
almost an 8-fold increment in wild-type promoter activity. Mutation of
sites 1–3 individually effected a significant decrease in the
Sp1-dependent induction, and as in RASM cells, mutation of site 1 was
the most effective in this regard. Mutation of any two sites appeared
to lead to a slight further reduction in promoter activity, whereas
mutation of all three sites effected virtually a complete abrogation of
Sp1-dependent transcription. The relative contribution of each of these
sites, alone or in combination, is similar to that observed in RASM
cells, indicating that Sp1-supplemented Schneider cells faithfully
recapitulate NPR-A promoter activity.
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, forced expression of
Sp1, as expected, led to a concentration-dependent increase in
NPR-A-dependent reporter activity. Sp3 displayed very similar activity
with maximal induction equaling that seen with Sp1 (10-fold),
whereas neither the distantly related Sp2 gene product nor the empty
vector (pPacSp0) displayed any stimulatory activity. Interestingly, the
combination of Sp1 and Sp3 (5 µg each) provided a response that was
greater than that seen with 5 µg of either Sp1 or Sp3 alone but fell
short of that seen with 10 µg of either plasmid. Ten micrograms of
Sp1 and Sp3 gave the most robust response seen, with maximal induction
approaching a 13-fold increment over the control value. None of the
individual family members nor the Sp1+Sp3 combination proved capable of
activating the M1+M2+M3 mutant (Fig. 7C).
To take the analysis one step further, we attempted to suppress
endogenous Sp1-like activity in RASM cells. To accomplish this we
employed a decoy nucleotide strategy similar to that used previously to
inhibit E2F-dependent expression of the c-myc,
cdc2, and proliferating cell nuclear antigen genes in
vascular smooth muscle cells (20) and angiotensinogen gene expression
in hepatocytes (21). In this approach high concentrations of double
stranded oligonucleotide encoding the recognition element of interest
are introduced into target cells together with the reporter. These
decoy oligonucleotides compete with the elements present in the
promoter region of the reporter for binding to the cognate
transcription factor, leading to a reduction in promoter activity. As
shown in Fig. 8, cotransfection of double
stranded oligonucleotide encoding wild-type sequence for WT1, WT2, or
WT3 resulted in a 50–60% reduction in -387 NPR-A LUC activity,
whereas cotransfection of mutant oligonucleotides, which fail to bind
the Sp1 or Sp3 proteins in vitro, was completely
ineffective.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Overexpression of either Sp1 or Sp3 in Schneider cells resulted in induction of the wild-type NPR-A promoter, confirming that these factors serve as positive regulators of the NPR-A gene. This induction was impaired after introduction of Sp1-binding site mutations into the promoter. Finally, using a decoy approach, we showed that depletion of endogenous Sp1 or Sp1-like proteins (e.g. Sp3; see below) in RASM cells resulted in a reduction in NPR-A promoter activity. Collectively, these data argue convincingly for an important functional role for Sp1 or Sp3 in supporting NPR-A promoter activity.
Sp3 has been shown to either activate (22, 23) or lack activity (24, 25) at conventional Sp1-binding sites in other systems. In selected instances, Sp3 has been shown to reverse Sp1-dependent transcriptional activity (24, 25, 26), implying that these two proteins may function in antagonistic fashion to control expression of shared target genes. On the NPR-A promoter, Sp3 functions predominantly in a stimulatory mode, and the combination of Sp1 and Sp3 provided an only slightly less than additive effect. Thus, it would appear that either Sp1 or Sp3 can bind at the recognition sequences identified above and activate transcription of the NPR-A gene, each without seriously impacting on the functional activity of the other protein.
The use of the decoy strategy to selectively antagonize Sp1 activity in transfected RASM has, to our knowledge, not been reported previously. This approach has been employed successfully to reduce angiotensinogen gene expression in hepatocytes (21) and to decrease c-myc, cdc2, and proliferating cell nuclear antigen gene expression in vascular smooth muscle cells (20). In the latter instance this resulted in a suppression of mitogenesis in the transfected cell population. Such approaches may prove useful as alternatives to antisense methodology for delivery of oligonucleotide inhibitors of gene expression to selected vascular distributions in vivo.
Genes lacking TATA boxes include those in the housekeeping gene category (27, 28, 29, 30) and receptor genes (31, 32, 33, 34) including NPR-A (18). TATA-less promoters, in general, have been shown to be particularly sensitive to regulation by Sp1 (35, 36). The mechanism(s) underlying this phenomenon remain poorly understood, but may involve the ability of Sp1 to form particularly strong contacts with individual components of the basal transcriptional machinery. Such contacts might help to compensate for the loss of the stabilizing effect of the TATA-binding protein in establishing the preinitiation complex (35, 36, 37). Unlike other transcriptional activators, Sp1 does not require the classic TATA-binding protein-TFIIB interaction to demonstrate its full potential in stimulating transcriptional activity (37). This supports the idea that Sp1 possesses the unique ability to bypass selected steps in assembly of the core transcriptional complex, a property that it may employ to advantage in activating TATA-less promoters.
The nature of the smaller DNA protein complexes (i.e. those
with faster electrophoretic mobility than that noted for the Sp1 or Sp3
complexes) found with each of the oligonucleotides in Figs. 4 and 5
remains undetermined; however, there are a number of lower mol wt
proteins that represent reasonable candidates. One of these, basic
transcription element binding protein (BTEB), recognizes the same
GC-rich sequence as Sp1, but is considerably smaller in size (38). Of
interest, while both BTEB and Sp1 are ubiquitously expressed, they show
preferential expression in different tissues, and they can display
divergent regulatory activity in different promoter contexts. Thus,
BTEB (or a closely related protein) could prove to play a unique role
(vs. Sp1) in the regulation of NPR-A gene transcription.
It is particularly noteworthy that two other vasodilatory/antimitogenic genes expressed in vascular cells (i.e. endothelial nitric oxide synthase and endothelial prostaglandin synthase H, an enzyme linked to prostacyclin production) are also TATA-less genes that have been shown to require intact Sp1 sites in their proximal promoter for optimal activity. Interestingly, the gene responsible for the vasoconstrictor thromboxane is negatively regulated by Sp1 (13) as is the smooth muscle myosin heavy chain gene (9), a gene important for contractile function in the vascular smooth muscle cell. This may suggest a general theme for the activation of vasodilatory/antimitogenic gene products in the vasculature. Sp1 expression has been shown to be increased in vascular smooth muscle cells from hypertensive vs. normotensive rat models (14). Thus, up-regulation of Sp1 in response to hemodynamic overload could result in activation of NPR-A, endothelial nitric oxide synthase, or PG synthase and suppression of thromboxane synthase. Such responses might be viewed as reactive, providing a physiological brake that restrains the uncontrolled vasoconstriction and growth activity found in the walls of hypertensive vessels.
In summary, we have identified three Sp1-binding sites in the proximal 5'-flanking sequence of the NPR-A gene that both bind the Sp1 protein and convey an Sp1-dependent signal to the core transcriptional machinery. Manipulation of this signal in vivo may provide us with important clues to the role NPR-A plays in the maintenance of normal vascular tone and its function in different vascular beds.
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Footnotes |
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Received October 2, 1998.
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1B adrenergic receptor gene in DDT1MF-2 cells. J Biol
Chem 272:23144–23150This article has been cited by other articles:
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  D. G. Gardner, S. Chen, D. J. Glenn, and C. L. Grigsby Molecular Biology of the Natriuretic Peptide System: Implications for Physiology and Hypertension Hypertension, March 1, 2007; 49(3): 419 - 426. [Full Text] [PDF]
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D. Rahmutula, J. Cui, S. Chen, and D. G. Gardner Transcriptional Regulation of Type B Human Natriuretic Peptide Receptor Gene Promoter: Dependence on Sp1 Hypertension, September 1, 2004; 44(3): 283 - 288. [Abstract] [Full Text] [PDF]
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A. R. Nawrocki, C. E. Goldring, R. M. Kostadinova, F. J. Frey, and B. M. Frey In Vivo Footprinting of the Human 11beta -Hydroxysteroid Dehydrogenase Type 2 Promoter. EVIDENCE FOR CELL-SPECIFIC REGULATION BY Sp1 AND Sp3 J. Biol. Chem., April 19, 2002; 277(17): 14647 - 14656. [Abstract] [Full Text] [PDF]
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 B. C Kone Molecular biology of natriuretic peptides and nitric oxide synthases Cardiovasc Res, August 15, 2001; 51(3): 429 - 441. [Abstract] [Full Text] [PDF]
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 K. A. Lucas, G. M. Pitari, S. Kazerounian, I. Ruiz-Stewart, J. Park, S. Schulz, K. P. Chepenik, and S. A. Waldman Guanylyl Cyclases and Signaling by Cyclic GMP Pharmacol. Rev., September 1, 2000; 52(3): 375 - 414. [Abstract] [Full Text] [PDF]
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J. H. Yu, G. Schwartzbauer, A. Kazlman, and R. K. Menon Role of the Sp Family of Transcription Factors in the Ontogeny of Growth Hormone Receptor Gene Expression J. Biol. Chem., November 26, 1999; 274(48): 34327 - 34336. [Abstract] [Full Text] [PDF]
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F. Liang, F. Schaufele, and D. G. Gardner Functional Interaction of NF-Y and Sp1 Is Required for Type A Natriuretic Peptide Receptor Gene Transcription J. Biol. Chem., January 5, 2001; 276(2): 1516 - 1522. [Abstract] [Full Text] [PDF]
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