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(Hypertension. 1997;30:1348-1355.)
© 1997 American Heart Association, Inc.

Articles

Regulation of the Rat Atrial Natriuretic Peptide Gene After Acute Imposition of Left Ventricular Pressure Overload

Torsten Cornelius; Stephan R. Holmer; Frank U. Müller; Günter A. J. Riegger; ; Heribert Schunkert

From the Medizinische Klinik und Poliklinik für Innere Medizin II, Universität Regensburg (T.C., S.R.H., G.A.J.R., H.S.); and the Institut für Pharmakologie und Toxikologie, Universität Münster, Germany (F.U.M.)

	Abstract

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Abstract The upregulation of left ventricular (LV) atrial natriuretic peptide (ANP) mRNA is a highly conserved marker of cardiac hypertrophy. The aim of this study was to further examine the pathway leading to ANP induction during pressure overload of the heart. Systolic wall stress was imposed acutely on isovolumetrically beating rat hearts in a Langendorff apparatus (=300x10³ dyne/cm²). Northern and Western blots revealed that elevated wall stress induced LV c-fos and c-jun mRNAs (3.5- and 3-fold, P<.05 after 60 minutes), c-Fos and c-Jun proteins (3.9- and 4.3-fold, P<.05 after 120 minutes), as well as ANP mRNA (2.2-fold, P<.05 after 120 minutes). ANP upregulation was prevented by inhibition of protein synthesis (cycloheximide). Electrophoresis mobility shift assays were performed to link c-Fos and c-Jun (ie, components of the heterodimeric transcription factor AP-1) and ANP induction. A putative AP-1 binding site within the rat ANP promoter (nucleotides -512 to -473) bound specifically to nuclear proteins of wall stress–stimulated hearts. Antibodies directed against c-Fos protein resulted in a shift of this DNA/protein complex, suggesting physical interaction between AP-1 and the ANP promoter. Myocardial transfection of promoter constructs revealed that after acute imposition of wall stress, this AP-1 site enhanced a reporter gene (8- to 10-fold compared with a minimal promoter, P<.05). Interestingly, nuclear extracts of stimulated hearts as well as pure AP-1 protein bound to a putative CRE site (nucleotides -613 to -584) as well. Like the AP-1 site, this cAMP-responsible element (CRE) site was found to enhance the transfected ANP promoter/reporter gene significantly (17.5-fold, P<.05). Mutation of either AP-1 or CRE sites did not decrease reporter gene activity, whereas mutation of both resulted in loss of inducibility. These experiments suggest that LV ANP regulation after acute wall stress includes the activation of AP-1 and/or CRE cis acting elements. However, the transient nature of c-fos and c-jun upregulation also suggests that AP-1 is not the only mediator of ANP induction in LV hypertrophy.

Key Words: atrial natriuretic peptide • gene regulation • activator protein-1 • c-fos • gene transfection • heart

	Introduction

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ANP is a cardiac peptide with natriuretic, diuretic, and vasodilatory effects. The expression of ANP is developmentally regulated in a characteristic fashion.^{1 2} In the fetus, atrial and ventricular myocardium synthesize ANP in equal amounts. In contrast, in adult atria, ANP constitutes as much as 1% to 3% of the entire mRNA, whereas ventricular expression decreases dramatically.^{3 4} Ventricular ANP expression in the adult is observed, however, in response to a great variety of mechanical and hormonal stimuli that have in common the ability to induce cardiac hypertrophy. Given its characteristic expression pattern, ANP upregulation has frequently been used as a genetic marker of the hypertrophic phenotype.

The characterization of the 5'-flanking sequence of the ANP gene gave hints on several regulatory elements that potentially mediate the reinduction of ANP in the hypertrophied ventricle.^{5 6} A DNA fragment that spans 3.4 kb upstream of the transcription start site (CAP) was found to contain all elements required for tissue and development-specific expression.⁵ In addition, SRE and SP-1 elements were found to confer the ₁-adrenergic stimulation of the promoter.^{7 8} Finally, two cis elements further distal from the transcription start site were identified to be putative recognition sites for the AP-1 and a CRE.⁹ However, neither the functional relevance of the AP-1 and CRE elements nor the precise nature of the elements that determine the reexpression of ANP in the hypertrophied heart are clarified yet.

The induction of AP-1 protein in response to an acute elevation of systolic wall stress, on the other hand, is a well-characterized phenomenon. Given the function of this transcription factor and the localization of a putative AP-1 binding site in the ANP promoter, it seemed attractive to investigate whether AP-1 plays a role in the upregulation of ANP after pressure overload of the heart. Because the inducibility of c-Fos and c-Jun (ie, the proteins that constitute AP-1) may be markedly altered in cultured myocytes,¹⁰ we attempted to perform these studies on intact adult hearts that were exposed to a sudden elevation of wall stress.

	Methods

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Perfusion of Isolated Rat Hearts
Protocols were approved by the institutional ethics committee responsible for supervision of animal rights. For anesthesia, male Wistar rats (250 to 300 g), obtained from Charles River Wiga Inc (Sulzfeld, Germany), were injected intraperitoneally with 6.25 mg methohexital per 100 mg body weight. The thorax was rapidly opened, and the hearts were quickly removed and placed in a constant temperature chamber within a modified Langendorff apparatus. Retrograde perfusion of the coronary arteries was restored through a short cannula inserted in the aortic root, using a modified Krebs-Henseleit buffer as previously described in detail.^{11 12} The perfusate was equilibrated with a 5% CO₂/95% O₂ gas mixture to maintain the perfusate pH at between 7.36 and 7.44 and the PO₂ was approximately 550 mm Hg. Temperature of hearts was kept constant at 35°C. Flow was adjusted to achieve a coronary perfusion pressure of 80 mm Hg and kept constant throughout the experimental protocol.

A collapsed latex balloon was placed in the LV chamber and expanded such that beating hearts were exposed to a LV end-diastolic pressure of 10 mm Hg and to a peak LV systolic pressure between 120 to 140 mm Hg (Statham P23 Db transducer; Statham Instruments). Measurement of balloon volume, peak LV systolic pressure, and LV wall thickness (obtained at the end of the experiment) allowed the calculation of systolic wall stress that was adjusted to approximately =300x10³ dyne/cm². This stimulation relates to an acute elevation of wall stress because the LV balloon permits only an isovolumetric mode of contraction. Thereby, the balloon apposes the physiologically occurring systolic increase in wall thickness as well as the geometric decrease in cavity size, resulting in an increase of stress per cross-sectional area of the LV. Control hearts were perfused with a deflated latex balloon inserted in the LV chamber. After perfusion, the LV tissue was immediately separated from the rest of the heart, frozen in liquid nitrogen, and stored at -80°C for subsequent analyses.

With these methods three protocols were carried out. First, we studied the time course of c-fos, c-jun, and ANP mRNA regulation (15, 30, 60,120, and 240 minutes; n=4 to 5 at each time point). In addition, we studied c-Fos, c-Jun, and ANP at the protein level at 120 minutes after exposure of the LV to elevated systolic wall stress. As part of this protocol, five hearts were stimulated with wall stress for 120 minutes with the addition of cycloheximide (5 µg/mL, Sigma), a blocker of translation, to the perfusion buffer.

In the second protocol, hearts were likewise perfused for 120 minutes with elevated wall stress (n=12) or collapsed balloons (control hearts, n=12). Cardiac tissue from these hearts was used for extraction of nuclear proteins. Finally, hearts that had been transfected with various reporter gene constructs 3 days before the isolated perfusion were exposed to elevated wall stress for 120 minutes (n=12, each group). In this group, reporter gene assays were carried out as described below.

RNA Measurement
RNA extraction and Northern blotting was performed as described before.¹² The blots were hybridized overnight with murine [³²P]-cDNA probes for the proto-oncogenes c-fos, c-jun,¹² a synthetic 84-bp oligonucleotide complementary to the coding region of the rat ANP gene,¹³ and GAPDH to control for possible sample variability, respectively. After hybridization, the membranes were stringently washed and exposed overnight to x-ray film (Kodak XAR, Eastman Kodak Co). Autoradiograms were scanned (Personel Densitometer No. 50301, Molecular Dynamics), and the scores for proto-oncogene mRNAs and ANP mRNAs were divided by the signal for GAPDH mRNAs for each sample, respectively. Data are reported as respective ratios of GAPDH.

Determinations of c-Fos, c-Jun, and ANP Proteins
Perfused hearts were homogenized and centrifuged. The protein content of the supernatant was assayed by the method of Lowry.¹⁴ Protein (50 µg) was separated by SDS–polyacrylamide gel electrophoresis by use of a 10% (wt/vol) acrylamide separating gel and a 6% (wt/vol) acrylamide stacking gel. Gels were electroblotted to a polyvinylidine difluoride membrane (Milipore, Inc). Detection of c-Fos and c-Jun was carried out by using corresponding anti-c-Fos (medac, Hamburg, Germany) and anti-c-Jun (Oncogene) antibodies and an ECL Western blotting analysis system (Amersham International) according to the manufacturers' guidelines.

For extraction of respective proteins, hearts were perfused for 120 minutes with elevated wall stress. The hearts were frozen in liquid nitrogen, and the frozen samples were pulverized, boiled for 5 minutes in 10 vol of acetic acid, and homogenized at high speed (Ultraturrax, Janke und Kunkel). After extraction on prewashed C18 columns (Sep-Pak, Waters), samples were washed with Tris HCl and eluted with acetonitril/ammonium acid.¹⁵ The radioimmunoassay was performed according to the manufacturers' instructions (BIOMAR). Samples were diluted to 1:500, and the protein content was assayed by the method of Lowry¹⁴ such that tissue ANP is expressed as nanomoles per milligram of protein.

Electrophoresis Mobility Shift Assays
After perfusion of hearts with or without imposition to elevated wall stress, LV tissue was frozen in liquid nitrogen and stored at -80°C. Ventricular nuclei and nuclear protein extracts were prepared as described elsewhere^{16 17} with the modification that nuclear proteins were extracted from a pool of three hearts that were treated identically. Purified AP-1 protein (4 µg, Promega) was used as a positive control for protein-DNA binding. Double-stranded synthetic oligonucleotides corresponding to the AP-1 binding site (5'-TCCACCCACGAG GCCAATGAATCAGGTGTGAAGGTAACT-3') or to the CRE site (5'-GCTTCCTGGCTGACTTCATACTCTAAAA-3') of the rat ANP promoter were labeled with [-³²P]-ATP (Amersham Buchler) by T4-polynucleotide kinase (Promega) and used for gel shift assays as described before.¹⁷ Nonlabeled double-stranded oligonucleotides corresponding to an AP-1 consensus sequence (5'-CTGTTGATGACTCAGCCGGAA-3', Promega)⁹ and a CRE consensus sequence (5'- GCTTCCT GGCTGACTTCATACTCTAAAA-3')⁹ were used as a specific competitor DNA in 200-fold molar excess. Nonspecific competitor DNAs included double-stranded oligos carrying the mutated binding sites for AP-1 or CRE (see mutagenesis) as well as an oligonucleotide corresponding to an SP-1 consensus site (Stratagene). For super shifts, increasing concentrations of monoclonal antibodies directed at c-Fos protein (c-FOS, medac) and ANP protein (Peninsula) were used. Polyacrylamide gels were dried and exposed to x-ray film (Kodak XAR).

Promoter Constructs
Different sections of the ANP promoter⁵ (Fig 4A) were cloned into a promoterless vector plasmid that carried the entire coding sequence of the reporter gene luciferase (pGL2Basic, Promega). Each promoter element was amplified via PCR by use of a standard protocol with oligonucleotide primers that included defined restriction enzyme sites (Sac I, Xho I, and Kpn I) at both ends. All PCR fragments were verified by complete sequencing. The PCR fragments were digested with the appropriate restriction enzymes and cloned into the luciferase vector via the corresponding restriction sites. The resulting constructs were named pANPluc, followed by the length in base pairs of the cloned ANP promoter fragment in relation to the CAP site. Two deletion constructs were cloned as follows. pANPluc-3418 (-690 to -138): a 557-bp HindIII fragment was excised from the rat ANP promoter plasmid. Next, the remaining plasmid was religated and used for PCR amplification with the same primer pair as described for pANPluc-3418. pANPluc-721 (-539 to -145): the segment from nucleotides -721 to -540 was amplified via PCR and cloned into the plasmid pANPluc-144. After large-scale plasmid DNA purification (Mega Plasmid Purification, QIAGEN), DNA was dissolved in 10 mmol/L Tris HCl and 1 mmol/L EDTA pH 8.0, with the nucleic acid concentration in the range of 2 to 3 mg/mL. DNA was stored at 4°C.

View larger version (34K): [in this window] [in a new window]   Figure 4. ANP promoter activity after exposition to elevated wall stress (120 minutes). Top, Different segments of the ANP promoter were fused to a luciferase reporter gene. On top, the entire cloned rat ANP promoter is displayed. Relevant restriction sites and nucleotide positions in accordance to the transcription start site (CAP) are shown. The region of the distal promoter is not drawn to scale. The positions of the putative CRE and AP-1 binding sites are indicated. Deletions within the promoter are indicated as single lines. The right column displays reporter gene activity (luciferase) corrected for transfection efficiency (ß-galactosidase) in percentage of the full-length promoter. *P<.05 vs pANPluc-3418 (-690 to -138), pANPluc-483, and pANPluc-144; #P<.05 vs all other constructs; +P<.05 vs pANPluc-3418. Bottom, Site-directed mutagenesis was performed to destroy the transcription factor binding sites for CRE and/or AP-1. Mutated sites are shown in open capital letters, whereas wild-type sites appear in filled capitals. Results are displayed as percentage of the pANPluc-3418 reference construct. *P<.05 vs pANPluc-3418 mut CRE mut AP-1; #P<.05 vs all other constructs.

In Vitro Site-Directed Mutagenesis
Site-directed mutagenesis to the sequences of the AP-1 and the CRE sites were performed with the QuikChange Site-Directed Mutagenesis Kit according to the manufacturer's instructions (Stratagene). Starting with the pANPluc-3418 clone, mutagenic primers were designed such that they contained the desired mutation and annealed to the same sequence on opposite strands. The mutagenic primer for the AP-1 site (5'-TGAATCA-3') was 5'-GGCCAAgGAtcCAG GTG-3' and the primer for the CRE site (5'-TGACTTCA-3') was 5'-CTTCCTGGCTGAtcaCATACTCTA-3'. The transcription-factor binding sites are shown in uppercase and the mutated bases are shown in lowercase italic characters. The resulting plasmids were named pANPluc-3418 mut AP-1 for the mutated AP-1 binding site and pANPluc-3418 mut CRE for the mutated CRE site. After the mutations were performed on the single sites, the pANPluc-3418 mut AP-1 clone was used to perform a second mutation, now in the CRE site (pANPluc-3418 mut CRE mut AP-1).

In Vivo DNA Transfection
The DNA solution was mixed as follows: 100 µg of luciferase promoter construct and 100 µg of pSV-ß-galactosidase vector (Promega) were mixed with 200 µL of the liposomal transfection reagent DOTAP (Boehringer Mannheim) in glass tubes. Parasternal thoracotomy was performed on anesthetized rats, and small fractions of the DNA mixture were injected by syringe through a 27 Gx1" cannula into several regions of the beating LV wall. Thoracotomy was closed, and animals were housed individually for an additional 3 days. Rats were then injected intraperitoneally with 6.25 mg methohexital per 100 g body wt, the thorax was rapidly opened, and the hearts were quickly removed and placed into the Langendorff apparatus for perfusion and imposition of wall stress as described above.

Reporter Assays
The LV was separated from the rest of the heart and homogenized in 1 mL of homogenization buffer containing 25 mmol/L diglycine pH 7.8, 15 mmol/L MgSO₄, 4 mmol/L EGTA, and 1 mmol/L DTT.¹⁸ The homogenate was centrifuged at 6000g for 15 minutes at 4°C. The supernatant was transferred to a new tube. A sample of 20 µL was placed into a luminometer (LB 9501 Lumat, Berthold GmbH), and after automatic injection of 100 µL luciferase assay reagent (Luciferase Assay System, Promega), light emission was counted as relative light units (RLU). The assay for the ß-galactosidase reporter gene was performed according to the manufacturer's instructions (Galacto-Light, Tropix) with slight modifications. The supernatant (5 µL) was diluted with 15 µL of lysis buffer with 0.2 mmol/L PMSF and 5 µg/mL leupeptin and incubated at 48°C for 1 hour to decrease the endogenous ß-galactosidase activity.¹⁹ Next, reaction buffer (200 µL) was added, and incubation was performed at room temperature for 1 additional hour. After automatic injection of 300 µL light accelerator buffer, RLUs were counted as described above. To standardize the results for potentially variable transfection efficiency, luciferase RLUs were divided by ß-galactosidase RLUs and expressed as a ratio. The results were compared with the reference promoter construct pANFluc-3418.

Statistical Analyses
All results are expressed as mean±SEM. Multiple comparisons between two groups were performed with unpaired t tests; between three or more groups they were carried out with two-way ANOVA and Fisher's exact tests for post hoc analyses. Significance was accepted at a value of P<.05.

	Results

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Acute imposition of LV systolic pressure overload resulted in induction of the proto-oncogenes c-fos and c-jun. Maximal mRNA levels of c-fos and c-jun were 3.4±0.5-fold (P<.05) and 3.0±0.7-fold (P<.05) higher, respectively, than those in hearts perfused for the same length of time but without inflation of the LV balloons. The time-course experiment revealed a transient maximum of proto-oncogene mRNA levels at 60 minutes of stimulation (Fig 1A and 1B). Corresponding proteins were detected by Western blotting at 120 minutes of elevated wall stress (Fig 1C). Compared with control hearts, 3.9±1.2-fold (c-Fos) and 4.3±1.4-fold inductions (c-Jun) were detected (P<.05 for both).

View larger version (34K): [in this window] [in a new window]   Figure 1. Levels of c-fos and c-jun mRNA in rat hearts after acute imposition to elevated LV systolic overload. Rat hearts were imposed to increased LV wall stress for 0, 15, 30, 60, 120, and 240 minutes. A, Representative Northern blot for the proto-oncogenes c-fos and c-jun and for the housekeeping gene GAPDH. B, Corresponding time-course experiment as line graph. Four to five hearts were perfused for each time point. c-fos as well as c-jun expression reached highest levels after 60 minutes of exposure to elevated wall stress. C, Representative Western blot for the proteins c-Fos and c-Jun without stimulation (ns) and after 120 minutes of perfusion with elevated wall stress (120 minutes). Results are mean±SEM. *Significant differences, P<.05.

Likewise, we observed an induction of ANP mRNA after imposition of elevated wall stress (Fig 2A). The time-course experiment revealed a transient maximum of ANP mRNA after 120 minutes of stimulation with levels that were 2.2±0.35-fold (P<.05) higher than those in control hearts (Fig 2B). To determine whether ANP induction was facilitated via newly synthesized proteins (eg, transcription factors), hearts were perfused with cycloheximide, a translation inhibitor, that was added to the perfusion buffer. Interestingly, cycloheximide completely prevented the increase of ANP mRNA levels in hearts exposed to acute wall stress (Fig 2C). In addition, ANP was quantified at the protein level using radioimmunoassays. In contrast to ANP mRNA levels, elevation of wall stress for 120 minutes resulted in a decrease of LV ANP peptide levels (0.286±0.091 nmol ANP/µg protein versus 0.535±0.042 nmol ANP/µg protein, P<.05).

View larger version (19K): [in this window] [in a new window]   Figure 2. Levels of ANP mRNA after elevation of wall stress; effects of cycloheximide. A, Representative Northern blot; B, line chart. Hearts were imposed to elevated systolic overload for 0, 15, 30, 60, 120, and 240 minutes. The ANP mRNA levels reached a maximum after 120 minutes of stimulation. Results are given as mean±SEM. C, The effect of the translation blocker cycloheximide is demonstrated. Hearts were perfused for 120 minutes with elevated LV wall stress with or without coadministration of cycloheximide (5 µg/mL). ANP levels were significantly higher after wall stress and were reduced again after cycloheximide treatment. *Significant differences, P<.05.

To determine whether the putative AP-1 binding site at -512 to -473 may play a role in ANP mRNA induction after the imposition of wall stress, we performed electrophoresis mobility shift assays. AP-1 protein was able to bind the AP-1 site in the ANP promoter (Fig 3A, lane 2). Likewise, incubation of nuclear extracts derived from hearts stimulated for 120 minutes with elevated wall stress showed binding to the ANP AP-1 oligo (Fig 3A, lane 4), whereas nuclear extracts from unstimulated hearts did not shift the oligo (Fig 3A, lane 3). Molecular excess of unlabeled ANP AP-1 oligo (50- to 200-fold) prevented the binding of nuclear proteins of stimulated hearts to the labeled oligo in a dose-dependent manner (Fig 3A, lanes 5, 6, and 7). Coadministration of a 200-fold molar excess of the consensus AP-1 oligo also prevented the shift with nuclear extracts of stimulated hearts (Fig 3A, lane 8). Unspecific competitor DNA (200-fold excess) corresponding to either the mutated AP-1 binding site or to an SP-1 consensus sequence had no effect on the binding of the nuclear extracts at the AP-1 site of the ANP promoter (Fig 3A, lanes 9 and 10). Interestingly, we also observed binding of the nuclear extracts of stimulated hearts as well as purified AP-1 protein to the CRE binding site within the ANP promoter (Fig 3B). Nonlabeled ANP CRE oligo acted also as a specific competitor (50- to 200-fold molar excess) and prevented the binding of nuclear protein to a labeled ANP AP-1 oligo, whereas the mutated CRE oligo (200-fold excess) did not (Fig 3C).

View larger version (29K): [in this window] [in a new window]   Figure 3. A, Representative electrophoresis mobility shift assay. An oligo corresponding to the AP-1 site within the ANP promoter was incubated with nuclear proteins of nonstimulated rat hearts (ns), wall stress–stimulated hearts (120 minutes), or with purified AP-1 protein. In addition, unlabeled competitor oligos were used as mentioned in the Table. Competitor oligos included the unlabeled ANP AP-1 binding site as well as an AP-1 consensus oligo. Ineffective competitor oligos included an oligo carrying a mutated AP-1 binding site as well as a consensus SP-1 sequence. Unlabeled oligos were used in 50-, 100-, and 200-molar excesses. The purified AP-1 protein as well as nuclear proteins of stimulated hearts were able to bind the labeled AP-1 oligo, whereas nuclear extracts of nonstimulated hearts did not. This shift could be prevented by coadministration of increasing amounts of unlabeled AP-1 ANP oligo and a AP-1 consensus sequence but not with a mutated AP-1 site. B, The CRE site of the ANP promoter was used as labeled oligo. Purified AP-1 protein as well as stimulated nuclear extracts bound to the CRE site, whereas nuclear protein derived from nonstimulated hearts did not. C, Experiment using cold CRE sequences of the ANP promoter (CRE ANP), a CRE consensus (CRE con), or a mutated CRE site (CRE mut) for competition. The CRE site was also able to prevent the shifting of the labeled oligo. D, Protocol with coadministration of a c-Fos antibody in different concentrations. This antibody was able to bind the AP-1 oligo/nuclear protein complex resulting in a supershift. ANP antibody had no effect.

Studies performed with a monoclonal antibody directed against c-Fos protein showed a further shifting of the labeled ANP AP-1 oligo in a dose-dependent manner (Fig 3D), whereas antisera directed against ANP protein (negative control) had no effect at the same concentrations.

To study the functional significance of the AP-1 binding site of the ANP promoter in the setting of acute imposition of cardiac wall stress, we constructed several ANP-luciferase fusion genes that were transfected into the LV wall of beating rat hearts. Three days after cardiac DNA injection, hearts were exposed for 120 minutes to LV systolic overload. The 3.4-kb 5'-untranslated region of the rat ANP gene fused with the luciferase gene served as a reference construct, and its reporter gene activity was set to 100% (Fig 4A). A promoterless luciferase construct (pGL2Basic) served as a negative control (Fig 4A). Luciferase reporter gene constructs that included the AP-1 binding site (pANPluc-721 and pANPluc-560) showed reporter activity that was similar to that observed after injection of the entire known (pANPluc-3418) ANP promoter sequence. In contrast, constructs covering the proximal sequence of the ANP promoter (pANPluc-483 and pANPluc-144) were less effective. In other words, the region covering -560 to -483 conferred a 10-fold increase of reporter gene activity compared with the more proximal promoter segment. In addition, experiments with a plasmid pANPluc-3418 deleted between nucleotides -690 to -138 [pANPluc-3418 (-690 to -138)] displayed a significant decrease in reporter gene activity compared with the untruncated sequence. The experiments suggest that a functional element is harbored within the region between nucleotides -560 to -483. The AP-1 binding site (nucleotides -496 to -489) is the prime cis regulatory element candidate in this area. Interestingly, injection of an ANP reporter fusion gene lacking the AP-1 site but harboring the CRE site [pANPluc-712 (-539 to -45)] led to significant induction of the reporter gene as well. Compared with the reference construct (pANPluc-3418), the promoter activity of pANPluc-712 (-539 to -145) reached 223.4±46.8%; when compared with the proximal promoters (pANPluc-483 and pANPluc-144), RLUs were elevated 17.5-fold.

To verify these findings on the AP-1 and CRE binding sites and to exclude artifacts related to prior deletions and subsequent changes in three-dimensional DNA structure, site-directed mutagenesis was performed. In particular, the binding sites of AP-1 and CRE were destroyed without changing the immediate proximity of the gene. The resulting constructs either contained the mutated CRE site (pANPluc-3418 mut CRE) or the mutated AP-1 site (pANPluc-3418 mut AP-1) or mutations at both sites (pANPluc-3418 mut CRE mut AP-1). After transfection and wall stress stimulation of the rat hearts, the pANPluc-3418 mut AP-1 plasmid (with an intact CRE site) and the pANPluc mut CRE (with an intact AP-1 site) displayed reporter gene activities that were significantly higher (Fig 4B) than those of the minimal promoter. Indeed, RLUs were similar to those for promoters that carried either the intact CRE or the intact AP-1 site. However, when both binding sites were mutated, the resulting promoter activity was markedly decreased (Fig 4B).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The induction of c-fos and c-jun proto-oncogenes has often been used as a surrogate end point of the initial growth-related signaling cascade.^{12 20 21 22 23} Since respective proteins form the transcription factor AP-1, it seemed attractive to speculate that c-fos and c-jun also play a functional role in the subsequent mediation of phenotypic changes in cardiac hypertrophy.^{2 9 24} Yet, direct evidence in favor of this hypothesis is lacking. A potential paradigm to demonstrate such AP-1–mediated changes in cardiac gene expression is the regulation of the ANP gene.^{7 25} In particular, Seidman and colleagues⁵ located by sequence analysis a putative AP-1 binding site in the ANP promoter that was subsequently found to bind exogenous AP-1 protein.⁹ However, there is no evidence that this process may take place in the intact heart or that a physiological stimulus enhances the ANP promoter activity via this cis-acting element.

In the present investigation, the potential implications of c-fos and c-jun induction were studied early after imposition of wall stress. This strategy was selected because the stimulation of these proto-oncogenes is transient and short-lived.^{12 26} The results suggest that AP-1 and ANP mRNA are consecutively induced in intact hearts after the acute imposition of elevated wall stress. Cycloheximide prevented the early peak of ANP mRNA but not the induction of c-fos or c-jun mRNAs.¹² Thus, in contrast to c-fos and c-jun, intact protein synthesis seemed to be a prerequisite for wall stress–related stimulation of ANP. To identify some of the nuclear proteins that potentially stimulate ANP expression after an elevation of wall stress, mobility shift assays were performed. Both wall stress–induced nuclear proteins as well as synthetic AP-1 were found to bind to the ANP promoter at the putative AP-1 binding site (-496 to -489). Interestingly, bands that were shifted either by nuclear proteins from stimulated hearts or synthetic AP-1 migrated at an identical level. Furthermore, anti-c-Fos antibodies as well as excess concentrations of AP-1 consensus sequence interfered with the binding of cardiac nuclear proteins to the ANP promoter oligonucleotide. Taken together, the data strongly suggest that AP-1 protein is at least one member of the nuclear fraction that is synthesized as a rapid response to wall stress and that consecutively binds to the ANP promoter.

The potential of the AP-1 binding site to modulate the ANP expression was further examined in beating hearts that had been transfected with promoter reporter gene constructs before the imposition of wall stress. Deletion or mutation of the AP-1 binding site in these constructs decreased the activity of the transfected reporter gene significantly. Thus, the data suggest that the AP-1 binding site carries functional significance related to the induction of the ANP promoter early after imposition of elevated wall stress.

Initially designed to serve as a negative control, the CRE binding site was found to substantially affect the expression of promoter reporter gene constructs as well. Indeed, using site-directed mutagenesis, we found that either the CRE or the AP-1 site was sufficient to induce ANP promoter activity after elevation of cardiac wall stress. In contrast, mutation of both cis-acting elements resulted in a substantial loss of promoter function. It is unclear which element binds to the CRE site under these circumstances; however, previous studies²⁷ have documented that in addition to the CRE, AP-1 may bind to this site as well. In agreement with these findings, we observed that synthetic AP-1 protein shifted an oligonucleotide with the sequence of the CRE binding site at position -602 to -596 of the ANP promoter. Likewise, nuclear proteins extracted from wall stress–stimulated hearts shifted the CRE site. Thus, c-fos and c-jun may potentially affect the activity of the ANP promoter via binding to the CRE site as well. However, the findings also allow the interpretation that redundant pathways may contribute to the early induction of ANP after elevation of wall stress. Interestingly, transfection of the reporter gene construct that carried only the CRE site [pANPluc-712 (-539 to -145)] resulted in higher luciferase activity than pANPluc-721, which included both the CRE and AP-1 site. These data allow us to speculate that, under certain conditions, the AP-1 site may also downregulate the promoter activity, a notion that was previously observed in human and rat cardiac myocytes in culture.^{28 29}

The regulation of ANP in the first 4 hours after exposure to growth factors has not been studied systematically previously.^{30 31 32 33 34} It seems interesting to note, however, that Kinnunen et al³⁵ observed a substantial release of ANP from the rat ventricle 30 minutes after imposition of myocardial stretch. In agreement with these findings, ANP protein levels were decreased in LVs subjected to 120 minutes of wall stress in the Langendorff apparatus. Thus, it is conceivable that the ANP mRNA induction discussed above serves to rebuild or increase the LV ANP peptide levels after imposition of wall stress. With regard to ANP mRNA levels, most studies analyzed the expression at later time points^{2 22 36 37 38 39} and reported ANP induction starting at 12 hours and at up to 7 days after respective stimulation.^{2 22 37} In fact, ANP upregulation may be maintained by load-independent mechanisms in chronic pressure-overloaded hearts.^{38 39} Likewise, the regulation of brain natriuretic peptide, another natriuretic peptide induced in the pressure-overloaded LV, was mainly investigated after 35 and 42 days of aortic banding.⁴⁰ Thus, our present observation of an early, transient peak of ANP mRNA induction does not contradict previous work. Nevertheless, to further confirm the potential of transient ANP induction early after growth stimulation and given, as noted above, the potential that CRE might stimulate ANP, we examined the effects of forskolin perfusion on ANP mRNA regulation (Holmer and Schunkert, data not shown, 1996). In agreement with the rapid ANP induction after elevation of wall stress, we also found a transient increase of ANP mRNA 90 minutes after forskolin stimulation.

Some study limitations need to be considered before the conclusion is drawn that AP-1 and/or CRE are indeed responsible for the rapid induction of ANP. First, other cis-acting elements may be involved in the regulation of the rat ANP promoter as well. In particular, SRE, SP-1 element, and vitamin D3 receptor have been previously demonstrated to enhance ANP expression after -adrenergic–or vitamin D–related stimulation.^{8 41} The ANP promoter construct that carried the AP-1 binding site, used in the present study, also carried the SRE (nucleotides -400 to -409 and -117 to -108) and SP-1 sites (nucleotides -74 to -67) such that their requirement for wall stress–related ANP induction cannot be completely ruled out. However, a construct that carried the proximal SRE and SP-1 sites but lacked both AP-1 and CRE sites (pANPluc-483 and pANPluc-144) failed to stimulate the reporter gene. Thus, we have no reason to believe that these elements in the proximal region of the ANP promoter play a significant role in wall stress–mediated ANP induction.

It should be specifically mentioned that our data apply only to the initial hours following imposition of wall stress. At steady state conditions, hearts with established hypertrophy usually do not express c-fos and c-jun mRNA.⁴² In fact, we recently noted that inducibility of these proto-oncogenes is impaired in hypertrophied hearts.¹² Thus, the slow and steady rise of ANP mRNA levels that peaks in hearts with severe hypertrophy is likely to be mediated by different mechanisms. Indeed, this second and more pronounced induction of ANP seems to require elements outside the presently characterized ANP promoter region. In particular, Sadoshima et al²² showed that neonatal cardiomyocytes transfected with the 3.4-kb ANP promoter do not stimulate a reporter gene after 12 or more hours of stretch. Similarly, Knowlton et al² demonstrated in transgenic mice that the same construct does not carry the elements required for ANP induction during chronic pressure overload of the heart subjected to aortic banding (7 days). A potential explanation for the apparently divergent results between the previous and the present study may be the different methodological approaches. Alternatively, the data provide evidence that the induction of AP-1 has functional relevance only early after the imposition of elevated wall stress and is lost in the chronic phase of cardiac pressure overload. The question of whether cardiac genes other than ANP are regulated by c-fos and c-jun needs to be addressed in future studies.

	Selected Abbreviations and Acronyms

 

ANP = atrial natriuretic peptide AP-1 = activator protein-1 CAP = site of initiation of transcription CRE = cAMP-responsible element LV = left ventricle, left ventricular PCR = polymerase chain reaction RLU = relative light unit SRE = serum response element<.>

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG Schu 672/3-1, 9-1, and 10-1) and the Bundesministerium fr Bildung, Wissenschaft, Forschung und Technologie (KBF 01 GB 9403).

	Footnotes

 
Reprint requests to PD Dr H. Schunkert, Klinik und Poliklinik für Innere Medizin II, Universität Regensburg, Franz-Josef-Strauss-Allee, D-93042 Regensburg, FRG.

Received February 13, 1997; first decision March 17, 1997; accepted July 3, 1997.

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