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Circulation Research. 1995;77:1060-1069

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(Circulation Research. 1995;77:1060-1069.)
© 1995 American Heart Association, Inc.

Articles

Involvement of Multiple cis Elements in Basal- and {alpha}-Adrenergic Agonist–Inducible Atrial Natriuretic Factor Transcription

Roles for Serum Response Elements and an SP-1–Like Element

Amy B. Sprenkle, Susan F. Murray, Christopher C. Glembotski

From the Department of Biology and Molecular Biology Institute, San Diego (Calif) State University.

Correspondence to Dr Christopher C. Glembotski, Department of Biology, San Diego State University, San Diego, CA 92182.E-mail cglembotski@sunstroke.sdsu.edu.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract In the present study, cis elements in the 5'-flanking sequence (FS) of the rat atrial natriuretic factor (ANF) gene involved in regulating basal and {alpha}1-adrenergic–inducible transcription were investigated. Truncation analyses using ANF-luciferase reporter constructs transfected into primary neonatal rat cardiac myocytes showed that an A/T-rich serum response element (SRE) at -114 bp of the ANF 5'-FS, which bound serum response factor (SRF), was required for basal and inducible transcription. In constructs composed of 134 bp of rat ANF 5'-FS driving luciferase (ANF-134Luc), mutations in the SRE at -114 bp disrupted SRF binding and ANF promoter activity. However, the same mutations in ANF-638Luc had little effect, suggesting a collaborating role for more distal sequences, such as the other SRE in ANF-638 at -406 bp. In ANF-638Luc, mutations in the SRE at -406 bp that disrupted SRF binding to that site decreased ANF reporter activity by only 25%; however, mutating both of the SREs completely blocked {alpha}1-adrenergic–inducible activity. Mutation analyses showed that an {bullet}{bullet}{bullet} (SP-1)–like site at -69 bp, shown previously to confer inducibility in reporters with 134 bp of ANF 5'-FS, was not required in ANF-638Luc. However, double mutants in the SP-1–like region and either SRE completely blocked {alpha}1-adrenergic–inducible ANF promoter activity. These findings emphasize that no single element is responsible for {alpha}1-adrenergic agonist–regulated ANF transcription but that the SREs at -114 and -406 bp and the SP-1–like sequence at -69 bp mediate the effect in collaboration.


Key Words: atrial natriuretic factor • {alpha}1-adrenergic agonist inducibility • transcription • serum response element • serum response factor


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
A great deal of interest has focused on the intracellular signals linking cell surface receptor activation with gene expression and the related hypertrophic cell growth of cardiac myocytes.1 A characteristic feature of treating primary neonatal ventricular myocytes with a variety of growth stimuli (eg, {alpha}1-adrenergic agonists, endothelin, transforming growth factor-ß, stretch, and electrical pacing of contractions) is the reactivation of certain cardiac genes, such as {alpha}-skeletal actin, ß-myosin heavy chain, myosin light chain-2, and ANF, which are normally expressed primarily during embryonic development.2 3 4 5 6 Recent studies have indicated that the induction of these genes is at least partly transcriptional and that their coordinate induction suggests the involvement of conserved signaling pathways.7 8 9

The present study was undertaken to gain a better understanding of the mechanisms by which {alpha}1-adrenergic agonists induce ANF transcription in primary neonatal ventricular myocytes. In cardiac myocytes, {alpha}1-adrenergic agonists, which activate ANF transcription (eg, see References 8 and 108 10 ), promote the conversion of membrane phosphoinositides to diacylglycerol and inositol 1,4,5-tris-phosphate, which results in the activation of PKC.11 12 13 14 In some cells, such PKC activation leads to the formation of fos/jun heterodimers, which can induce the expression of genes possessing cis-acting elements specific for AP-1 binding.15 16 17 Although {alpha}1-adrenergic agonists can activate myocardial cell PKC18 and although the resulting AP-1 may be required for some aspects of the hypertrophic growth response, it is unclear whether ANF transcription is stimulated via AP-1 binding at the ANF 5'-FS. For example, although the transfection of primary myocardial cells with fos and/or jun expression constructs or constitutively active PKC constructs can activate cotransfected ANF reporters,19 20 there has been no functional identification of AP-1–binding cis sequences or TREs in the ANF gene, and in the rat ANF 5'-FS, there are no true consensus TREs. Furthermore, there is also evidence that fos overexpression can repress ANF transcription.21 Also, one recent study has implicated the involvement of an SP-1–like protein binding at a promoter-proximal site of the ANF promoter in {alpha}1-adrenergic agonist–stimulated transcription.22 Therefore, it is possible that although {alpha}1-adrenergic agonist–activated ANF transcription may involve PKC, as previously suggested,21 it may not involve AP-1 as a transcription factor.

One case of transcriptional activation that conditionally involves PKC, but not AP-1, is the induction of the human c-fos gene through SRF binding to an SRE in the 5'-FS. In several cell lines, it has been shown that some growth factors (eg, phorbol esters) augment c-fos expression through the activation of MAPK23 ; this is followed by the MAPK-mediated phosphorylation of an accessory factor (eg, p62TCF, also known as Elk-1 or Sap-1) that binds to SRF/SRE to form the ternary complex that enhances transcription (for recent review, see Reference 2424 ). Alternatively, some growth factors (eg, serum) augment c-fos transcription through the MAPK- and pp90rsk-mediated phosphorylation of SRF itself,25 apparently independently of ternary complex formation.26 The recent demonstration that {alpha}1-adrenergic agonists activate myocardial cell MAPK,27 along with the finding that the blockade of MAPK inhibits {alpha}1-adrenergic agonist–induced ANF transcription,28 supports the hypothesis that SRF could participate in the PE-inducible transcription of at least some of the myocardial cell genes activated during the hypertrophic program.

To evaluate the possible involvement of SRF in ANF induction, the FSs of the rat ANF gene most proximal to the transcriptional start site were tested for the ability to mediate basal and {alpha}1-adrenergic agonist–inducible transcription. In the present study, we report on the identification of several SRE-like elements in the ANF 5'-FS that bind a cardiac-derived protein that is antigenically indistinguishable from SRF and that participate together in PE-inducible ANF transcription.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Plasmid Constructs and Oligonucleotide Probes
An EcoRI–Spe I fragment of the plasmid pANF 30038 was cloned into pGeneLight2-Promoter (pGL2-P, Promega), replacing the simian virus 40 promoter of the vector with rat ANF 5'-FSs from -638 to +65, to produce ANF-638Luc. Similar truncations were produced with HindIII–Spe I (ANF-134Luc) and Nla IV–Spe I (ANF-65Luc) fragments. Other truncations between -134 and -65 were created by using the Promega Erase-a-base kit according to the manufacturer's instructions. Site-directed mutagenesis of ANF-134Luc and ANF-638Luc was performed by using the Promega Altered Sites in vitro mutagenesis kit according to the manufacturer's instructions and the appropriate oligonucleotides shown in Fig 1Down. The ANF sequences in all of the expression constructs used in the present study terminate on the 3' end at ANF +65. All plasmid constructions were verified by dideoxy sequencing. Plasmids used for electroporation were purified according to manufacturer's instructions on a pZ523 column (5-Prime 3-Prime) and transfected at a concentration of 10 µg luciferase vector per 3 µg ß-gal vector (CMV-ß-gal, Clontech) per 35-mm well.



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  		Figure 1. Synthetic oligonucleotides. The sequences shown represent oligonucleotide pairs used as labeled probes and/or competitors in EMSAs. The boxes identify the positions of either the core SRE sequence or the core GC-rich SP-1–like sequence, and mutated bases are bold/underlined. The ANF SRE/114 group includes oligonucleotides containing regions of the SRE-like sequence at -114 bp of the rat ANF 5'-FS, with ANF SRE/114 as the native sequence and the others as cluster mutations. The ANF SRE/406 group includes oligonucleotides containing regions of the SRE at -406 bp of the rat ANF 5'-FS, with ANF SRE/406 as the native sequence and SRE/C406 as a cluster mutation. The SRE group includes the c-fos and chicken {alpha}-SkA SRE-1/YY-1, which are previously reported oligonucleotides known to bind SRF. The PERE group includes the ANF SP-1–like element located at -69 bp of the rat ANF 5'-FS, previously named a PERE.22 The other group includes oligonucleotides that contain enhancers other than an SRE. The c-fos SRE, chicken {alpha}-SkA SRE-1/YY-1, collagenase TRE, chicken cardiac troponin T MCAT, rat ANF PERE, and human endothelin GATA sequences have been previously published.15 22 29 30 31 32

Synthetic oligonucleotides (Fig 1Up), which were used as probes and/or competitors in EMSAs, were prepared by the CSUPERB Microchemical Core Facility, San Diego (Calif) State University. Each pair of oligonucleotides was prepared with the addition of Psp A1 sequences to the 5' of the positive (+) strand and Sal1 sequences to the 5' of the negative (-) strand to facilitate labeling.

Cell Culture and Transfections
Myocardial cells were prepared as described previously.5 Briefly, 1- to 4-day neonatal rat hearts were dissected, the atrial and ventricular portions were subdissected, and the apical one third of the ventricle was removed for trypsin dissociation. Cultures prepared by this method contain >98% cardiac myocytes. For transfections, freshly dissociated ventricular cells at a density of 10 million cells per milliliter minimal medium (DMEM/F-12 [GIBCO/BRL] containing 1 mg/mL BSA) were mixed with DNA, electroporated in a BioRad (Hercules) gene pulser (600 V, 25 µF, 100 {Omega}, 0.2-cm gap cuvette), and plated on fibronectin-coated wells. Nonmyocardial cells obtained by preplating dissociated tissue, when electroporated under the conditions described above, demonstrated no luciferase or ß-gal activity after 48 hours in serum-containing medium, suggesting that any nonmyocardial cells present in the cultures do not directly contribute to reporter enzyme activity. Viability of electroporated ventricular myocytes 12 to 14 hours after plating in DMEM/F-12 (1:1) plus 10% FBS (Gemini Bioproducts) was empirically determined to be {approx}30%; therefore, final density was 750 000 to 1 million cells per 35-mm well (78 000 to 100 000 cells per square centimeter). After 12 to 14 hours after electroporation in 10% FBS, cells were washed thoroughly, and the medium was replaced with minimal medium. Twenty-four hours later, the medium was again replaced with minimal medium with or without the additions as described in the figure legends. Luciferase and ß-gal assays were performed as described previously.33 34 Luciferase activity was measured for 30 seconds on a Bio Orbit 1251 Luminometer (LKB/Pharmacia). Data are expressed as "relative luciferase" (arbitrary integrated luciferase units per ß-gal unit), representative of at least three independent experiments performed with two different plasmid preparations, and represent the mean±SEM of triplicate 35-mm wells.

Preparation of Nuclear Extracts
Nuclei from ventricular tissue were obtained as described previously.35 Nuclei were extracted essentially as described previously36 in high salt buffer C (20 mmol/L HEPES [pH 7.9], 25% glycerol, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, and 2 µg/mL aprotinin) and then dialyzed (12 000– to 14 000–molecular weight cutoff) against buffer D (20 mmol/L HEPES [pH 7.9], 20% glycerol, 100 mmol/L KCl, 0.2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.5 mmol/L dithiothreitol). Extracts were stored at -70°C.

EMSA
EMSA was performed as described previously,37 with minor modifications. Probes were prepared by Klenow fragment–mediated filling of the sticky ends of double-stranded oligonucleotides. A typical binding assay contained 20 000 cpm double-stranded probe and 5 to 10 µg protein in 1x binding buffer (30 mmol/L NaCl, 0.1 mmol/L EDTA, 8 mmol/L Tris-HCl [pH 8.2], 8% glycerol, 1 nmol/L dithiothreitol, and 0.2 mmol/L ZnCl2). After a 10-minute preincubation of extract and 0.1 µg nonspecific competitor (poly dI-dC, Pharmacia), the probe was added. Binding was allowed to proceed at room temperature for 30 minutes before separation of bound and free probe on a 4% native polyacrylamide gel (29:1 bis/acrylamide) in 0.25x Tris-borate-EDTA buffer at room temperature. For supershift experiments, 1 µL of the appropriate antiserum was preincubated for 30 minutes with 5 to 10 µg of extract before the addition of nonspecific competitor, followed by probe. The antisera, which were raised against human SRF and rat SRF, have been previously described.39 DNA-protein complexes were detected by autoradiography. The autoradiograms of some gels in this manuscript were scanned with a Molecular Dynamics Personal Densitometer, and the resulting image was imported to Adobe Photoshop and Claris MacDraw Pro II for final figure preparation.

Interference Analyses
Methylation interference analysis was performed as described previously,40 as was carbethoxylation interference,41 42 with minor modifications. Glycogen was used in place of tRNA as a precipitation carrier. DMS was used to treat singly end-labeled double-stranded probe, whereas DEPC modification was performed on labeled single-stranded oligonucleotide before annealing to produce double-stranded probe. For all probes, labeled, modified, and double-stranded product was purified from a 15% nondenaturing polyacrylamide gel by electroblotting to NA-45 paper (Schleicher & Schuell) and eluting according to the manufacturer's instruction before use in EMSA. The EMSA was scaled up five times with respect to nuclear extract and nonspecific competitor (dI-dC), with 300 000 cpm of probe. Specific complexes 1 and 2 and free probe were isolated from the EMSA gel and also electroblotted to and eluted from NA-45 paper. Piperidine-cleaved probe was fractionated on a 10% denaturing polyacrylamide gel and visualized by autoradiography.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Truncation Analysis of SREs and ANF Induction
In the proximal 638 bp of the rat ANF 5'-FS, consensus SREs, or near-consensus (9 of 10 bases match) SREs (ie, SRE=CC[A/T]6GG; eg, see Reference 4343 ), are located at -406 and -114 bp relative to the transcription start site.3 To gain a better understanding of the potential roles for these SREs in basal and {alpha}1-adrenergic agonist–inducible ANF transcription, a group of reporter constructs was prepared by fusing various lengths of the ANF 5'-FS to a luciferase reporter gene, transfecting primary myocardial cells, and determining basal and PE-inducible levels of reporter enzyme activity.

Reporter activity was similar with plasmids containing 638 and 134 bp of ANF 5'-FS (Fig 2Down), suggesting that sequences proximal to -134 bp were sufficient for maximal PE-inducible transcription. Whereas truncation to 122 bp of 5'-FS resulted in an {approx}2-fold loss of both basal and PE-inducible reporter activity, further truncation to -109 bp resulted in decreases of {approx}10-fold, suggesting that sequences between -134 and -109 bp were required for optimal basal and PE-inducible transcription. PE-inducible reporter activities were between {approx}5- and 7-fold for ANF-638GL, ANF-134GL, and ANF-122GL and then decreased to between 3.5-fold and 2-fold for the shorter constructs. This suggested that although sequences between -638 and -122 bp were required for maximal PE inducibility, more proximal sequences residing between -122 and -65 bp remained capable of conferring some degree of PE-inducible reporter activity. Since there was a dramatic decrease of reporter activity observed between -122 and -109 bp and since there is an A/T-rich SRE-like sequence centered at -114 bp (Fig 3Down), further studies were focused on determining whether and how this SRE-like region could participate in ANF transcription.



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  		Figure 2. Truncation analysis of the rat ANF 5'-FS and PE-inducible reporter expression. Myocardial cells were transfected with constructs prepared by fusing various lengths of the rat ANF 5'-FS (-638 to -65 bp) to a luciferase reporter. After 24 hours in serum-free media, the cultures were treated for 6 hours with either serum-free media and no additions (control [Con]) or with 50 µmol/L phenylephrine+1 µmol/L propranolol (PE) and harvested for reporter enzyme assays. In the bar graph, the results are shown as relative luciferase (Rel Luc) activities (ie, luciferase/ß-gal) ±SEM obtained in the control and PE-treated cultures; fold PE induction of each construct (PE Rel Luc/control Rel Luc±SEM) is shown in table form at the right of the construction diagram. In the diagram the asterisks mark the approximate locations of SREs (consensus=GG[A/T]6CC) or near SREs (9 of 10 matches) in the 5'-FS; the SRE-like sequences in the proximal 638 bp of the ANF 5'-FS are located at -406 and -114 bp. Similar results were found upon exposures to PE of up to 48 hours (not shown). These data represent the average of six independent experiments performed with two different plasmid preparations and are expressed as the mean±SEM of triplicate 35-mm wells. In the bar graph, symbols are as follows: *P<.01 and §P<.01 vs other values obtained with PE treatment; {dagger}P<.01 vs other control values using ANOVA followed by Newman-Keuls post hoc ANOVA.



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  		Figure 3. Diagram of the proximal rat ANF 5'-FS and mutations. Portions of the rat ANF 5'-FS from -134 to -26 bp are shown. An SRE-like sequence, the SP-1–like PERE,22 and the TATA box are boxed and labeled. Beneath the native sequences are shown the nucleotide substitutions prepared as the cluster mutations used in the present study. Details of the mutant preparations are described in "Materials and Methods." The arrows mark the locations of the truncation mutants used in the present study.

Nuclear Proteins That Bind to the A/T-Rich Region of the ANF 5'-FS
The nature of factors that might bind to the A/T-rich sequence in the ANF 5'-FS was studied by EMSAs using a double-stranded oligonucleotide containing ANF SRE/114 (ANF [-134 to -95]; see Fig 1Up) as a probe and neonatal ventricular tissue nuclear extracts. A single major complex was obtained that could be inhibited by the addition of 100-fold molar excess of the unlabeled probe, c-fos SRE, or YY-1 double-stranded oligonucleotides, whereas TRE,15 MCAT, PERE,22 and GATA double-stranded oligonucleotides were not effective competitors (Fig 4Down). Competition by the c-fos SRE and YY-1 oligonucleotides, both of which have canonical SREs and bind SRF, is consistent with the putative identification of an SRE-like site within ANF SRE/114.



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  		Figure 4. EMSA of myocardial cell nuclear protein binding to ANF SRE/114. A double-stranded oligonucleotide consisting of ANF (-134 to -95 bp) (ANF SRE/114) was used as a probe in a binding reaction with 10 µg ventricular tissue nuclear extract and 0.1 µg nonspecific competitor (dI-dC) per lane. Optimal levels of dG-dC were determined over a concentration range of 0 to 8 µg (not shown). Unlabeled self (ANF SRE/114), c-fos SRE, {alpha}-SkA YY-1 (YY1), collagenase TRE, troponin C MCAT (MCAT), ANF PERE (ANF SP-1/69), and human endothelin GATA (GATA) competitors were added at a 100-fold molar excess before addition of the probe. To ensure that the complex formed required a double-stranded DNA probe, a control experiment was carried out where single-stranded DNA, either + or - strands, was end-labeled and then used as probe; no specific binding was observed. Refer to Fig 1Up for oligonucleotide sequences.

To map more precisely the nature of the DNA-protein interactions, carbethoxylation and methylation interference analyses were carried out. DEPC-mediated modification of adenines at positions -113, -112, -111, -110, and -105 on the + strand of the ANF 5'-FS interfered with complex formation (Fig 5ADown, DEPC +), as did modification of adenines -114 and -116 on the - strand (Fig 5ADown, DEPC -). DMS-mediated modification of guanines at -109, -108, and -107 of the + strand of the ANF 5'-FS interfered with complex formation (Fig 5ADown, DMS +), as did modification of the guanines at -117 and -106 of the - strand (Fig 5ADown, DMS -). The contact sites made between the protein responsible for the band shift in Fig 4Up and the ANF promoter sequences are diagrammed in Fig 5BDown. The importance of the A/T-rich core and the adjacent pyrimidines in this region suggests a binding event similar to the binding of SRF to canonical SREs (eg, see Reference 4343 ). Accordingly, the characteristics of the protein(s) that binds to ANF SRE/114 to form the major complex on the EMSA were analyzed further by using several antisera raised against transcription factors that bind to A/T-rich elements.



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  		Figure 5. Carbethoxylation and methylation interference of nuclear protein binding to the ANF SRE/114. A, Singly end-labeled ANF SRE/114 oligonucleotides (either positive [+] or negative [-] strands) were modified with either DEPC or DMS before use in a preparative EMSA. The complex shown in Fig 4Up (C1) as well as free probe (F) were isolated, cleaved, and fractionated on a 10% denaturing gel, as described in "Materials and Methods." The numbers indicate the positions of each band of interest; + and - beneath each gel represent analyses of either labeled + or - strand, respectively. B, Diagram of the bases important for binding as determined by DEPC, which modifies adenines ({bullet}) or as determined with DMS, which modifies guanines ({circ}).

Among the nuclear proteins known to bind to A/T-rich elements are MEFs 2A through 2D,44 45 some of which are also known as related to rat SRF46 and recognize the consensus CTA[A/T]4TAG and CArG binding factor,47 now known to be SRF.39 To characterize the protein(s) binding to the SRE-like region in ANF SRE/114, antisera specific for MEF 2 and SRF were used in the EMSAs. Using either ANF SRE/114 or the c-fos SRE probe (see Fig 1Up), ventricular nuclear extracts produced a major band with the same mobility (Fig 6Down). Only the SRF antiserum produced a supershifted complex with either probe, demonstrating that this complex is the result of the binding of an SRF-related protein to this region of the ANF 5'-FS. Indeed, the A/T-rich sequence in the ANF promoter (CT[TTAAAA]GG) is similar to the canonical SRE core sequence (CC[A/T]6GG) but differs significantly from the consensus MEF 2 binding site (CTA[A/T]4TAG).



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  		Figure 6. Supershift of ANF SRE/114 binding proteins. EMSA was carried out with either ANF SRE/114 or c-fos SRE oligonucleotides as probes and ventricular tissue nuclear extract preincubated with antisera to human SRF, MEF 2A/2B, MEF 2C/2D, or preimmune serum (Pre-imm) (see Reference 39 for antisera characterization). The open arrowhead indicates supershifted complex; the closed arrowhead indicates the original complex.

Mutations that were predicted to disrupt the binding of an SRF-like protein to ANF SRE/114 were prepared. Whereas the native ANF SRE/114, c-fos SRE, and YY-1 oligonucleotides each blocked band formation in the EMSA, as expected, unlabeled oligonucleotides containing cluster mutations made at the putative SRE-like site (eg, ANF/C114 or ANF/C121; see Figs 1Up and 3Up) were ineffective competitors of complex formation (Fig 7Down), consistent with the need for an intact SRE for efficient complex formation. An unlabeled oligonucleotide harboring a similar-sized cluster mutation to the 3' region of the putative SRE-like site in ANF (ANF SRE/C101; see Figs 1Up and 3Up) served as an effective competitor, as expected, since the region affected lies outside the SRE-like area. As expected, the TRE, MCAT, PERE, and GATA oligonucleotides were ineffective competitors. These data support the interference and supershift analyses indicating that the complex represents the binding of an SRF-like protein to ANF SRE/114.



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  		Figure 7. Effects of mutations on complex formation. EMSA was carried out by using ventricular tissue nuclear extract with ANF SRE/114 as the probe and mutant or control unlabeled competitors, as shown in Fig 1Up and described in Fig 4Up. Unlabeled competitors were used at 100-fold molar excess in each binding reaction.

Effects of Mutations on PE Inducibility
To evaluate further the importance of the SRE at -114 bp in ANF transcription, the effects of the cluster mutations were determined in transfection experiments. Beginning with ANF-134Luc, which consists of 134 bp of ANF 5'-FS driving luciferase expression, three mutations were prepared, two of which spanned the putative SRE (C114 and C121; see Fig 3Up) and one of which was located outside this region (C101). Compared with the native sequence, both of the SRE-directed mutants possessed {approx}5-fold lower basal transcription and {approx}8-fold lower PE-inducible transcription (Fig 8ADown). The C101 mutation, which has no effect on SRF binding, also had no effect on transcription.



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  		Figure 8. Effects of cassette mutations on ANF reporter expression. A, Myocardial cells were transfected with native or cassette-mutated ANF-134Luc (see mutant sequences in Fig 3Up) and maintained for 6 hours in either serum-free medium alone (control [Con]) or PE+propranolol (PE) as described in the legend to Fig 2Up. Similar results were found upon exposures to PE of up to 48 hours (not shown). B, Effect of cassette mutations prepared in ANF-638Luc on PE-inducible transcription. Data (mean±SEM) are presented as luciferase/ß-gal for control and PE-treated cultures and are representative of at least three experiments using two different plasmid preparations. In the top panel, symbols are as follows: *P<.01 and §P<.01 vs 134 control values using ANOVA followed by Newman-Keuls post hoc ANOVA. In the bottom panel, symbols are as follows: *P<.01, §P<.01, {bullet}P<.01, and {dagger}P<.01 vs all other values using ANOVA followed by Newman-Keuls post hoc ANOVA.

While these studies were in progress, an article was published demonstrating that a GC-rich region at -69 bp, which binds an SP-1–like protein, was important for PE-inducible ANF transcription.22 Since this element, which was named PERE, was intact in the ANF SRE/C114 and ANF SRE/C121 mutants, we sought to determine whether the SRE-like sequence at -114 bp and the SP-1–like sequence at -69 bp both participated in regulating ANF transcription. Accordingly, a mutation was prepared in the PERE on ANF-134Luc (see Fig 3Up); this was identical to the cluster mutation used previously to identify the element.22 Like the C114 and C121 mutations, the C69 mutation within ANF-134Luc resulted in a coordinate 6- to 8-fold decrease in both basal and PE-inducible transcription (Fig 8AUp). Thus, it was apparent that in the context of ANF-134Luc, both ANF SRE/114 and the previously identified ANF SP-1/69 sites were required for optimal basal and PE-inducible transcription; however, neither one alone was sufficient to confer induction.

Given the dramatic impact of the SRE/114 and SP-1/69 mutations in ANF-134Luc, we wished to confirm the importance of these elements in a larger context of the ANF promoter. Accordingly, both the C114 and C69 mutations were prepared in ANF-638Luc. Although the C114 mutation in ANF-638Luc resulted in a drop in both basal and PE-inducible transcription, the decrease was modest, amounting to only a 50% reduction (Fig 8BUp). Thus, it was clear that although the SRE at -114 bp was important for optimal induction using ANF-638Luc, regions of the ANF 5'-FS distal to -134 bp could serve as enhancers and in part they could "rescue" mutations in more promoter-proximal regions of the 5'-FS. We also investigated the effects of the PERE cluster mutation in ANF-638Luc. The PERE mutation resulted in a small but significant decline in basal and PE-inducible transcription of {approx}8% to 9%; this was unexpected given the dramatic effects of this mutation in the -134 context in the present study (Fig 8AUp) as well as the previous report.22 Continuing with the hypothetical involvement of SRF, we next evaluated whether mutating the SRE at -406 bp, which is the only other SRE in ANF-638Luc, effected ANF transcription. Although basal transcription was not affected significantly in the ANF SRE/C406 mutant, PE-inducible transcription decreased, but by only 25% (Fig 8BUp). This result was consistent with the possibility that SRF binding to both ANF SRE/406 and ANF SRE/114 might be required for maximal basal and PE-inducible transcription.

To evaluate whether an SRF-like protein could bind to ANF SRE/406, EMSA was carried out. Using a probe that included the ANF SRE/406 and some FS (see Fig 1Up), a nuclear protein/DNA complex was observed that possessed the same mobility as that obtained with the ANF SRE/114 probe (Fig 9Down). Indeed, both complexes were efficiently supershifted by using the SRF antiserum. Moreover, the competition profile for the ANF SRE/406 complex was consistent with SRF binding, since it could be competed by ANF SRE/114, c-fos SRE, and YY-1; however, it could not be competed by ANF SRE/C406 or ANF SRE/C114 (which are SRE-directed cluster mutations; see Fig 1Up), the native PERE, or an MCAT consensus sequence.



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  		Figure 9. EMSA of myocardial cell nuclear protein binding to the ANF SRE/406. A double-stranded oligonucleotide consisting of ANF (-413 to -392 bp) (ANF SRE/406) was used as a probe in a binding reaction with 10 µg ventricular tissue nuclear extract and 0.1 µg nonspecific competitor (dI-dC) per lane. Optimal levels of dG-dC were determined over a concentration range of 0 to 8 µg (not shown). Unlabeled self (ANF SRE/406), c-fos SRE, {alpha}-SkA YY-1 (YY-1), collagenase TRE, troponin C MCAT (MCAT), ANF PERE, and human endothelin GATA (GATA) competitors were added at a 100-fold molar excess before addition of the probe. To ensure that the complex formed required double-stranded DNA probe, a control experiment was carried out where single-stranded DNA, either positive (+) or negative (-) strands, was end-labeled and then used as probe; no specific binding was observed. Refer to Fig 1Up for oligonucleotide sequences.

Effects of Double Mutations on PE Inducibility
Since either the ANF SRE/C114 or the ANF SP-1/C69 mutants alone blocked transcription in ANF-134Luc but had minimal effects in ANF-638Luc, as did ANF SRE/C406, we determined whether these potential cis elements might participate combinatorially to regulate ANF transcription. Accordingly, double mutations were prepared in ANF-638Luc; interestingly, mutating any two of the three elements severely inhibited transcription, both basal and PE inducible (Fig 10Down). When any two of the SRE/C114, SRE/C406, and/or SP-1/C69 mutations were present in ANF-638Luc, basal transcription decreased by {approx}2-fold, whereas PE-inducible transcription decreased by {approx}5-fold. This amounted to a decrease in PE-inducible transcription from 3.5-fold in ANF-638Luc to {approx}1.5-fold in any of the three double mutants.



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  		Figure 10. Effects of double cassette mutations on ANF-reporter expression. Myocardial cells were transfected with native or cassette-mutated ANF-638Luc (see mutant sequences in Fig 3Up) and treated as described in the legend to Fig 2Up. Data (mean±SEM) are presented as luciferase/ß-gal for control (Con) and PE+propranolol (PE)–treated cultures and are representative of at least three experiments using two different plasmid preparations. *P<.01 and §P<.01 vs 638 Con values using ANOVA followed by Newman-Keuls post hoc ANOVA.


*    Discussion
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 *Discussion
down arrowReferences
 
Taken together, these results demonstrate that multiple regions of the ANF 5'-FS are involved in regulating both basal and PE-inducible transcription and that two of these are A/T-rich SRE-like sequences at -406 and -114 bp, which likely bind an SRF-related protein. Although a GC-rich region at -69 bp, previously identified as a PERE, is also important for both basal and inducible transcription in promoter/reporter constructs possessing 134 bp of ANF 5'-FS, as shown previously,22 when tested in the context of larger stretches of the ANF 5'-FS and promoter, the SP-1–like region appears to be less important, much like the ANF SRE at -114 bp. It is possible that this SP-1–like element accounts for the partial PE-inducible reporter activity obtained by using constructs possessing -109 bp or less of the ANF 5'-FS (see Fig 2Up). The results of the present study emphasize the value of carrying out mutational analyses of putative cis elements in both small and large promoter contexts; this approach revealed that elements distal to -134 bp can apparently contribute to basal and PE-inducible ANF transcription such as to complement or duplicate the functions of proximal elements. Indeed, a previous study identified the potential importance of sequences residing between -134 and -638 bp of the ANF 5'-FS in PE inducibility,8 and a very recent study has indicated the probable involvement of a non–SRF-binding A/T-rich sequence at -530 bp of the ANF 5'-FS in ANF transcription.1 Thus, it is very probable that an array of cis elements residing in the ANF 5'-FS, and perhaps elsewhere in the gene, contribute to the regulation of basal and inducible transcription.

Although this is the first report indicating that {alpha}1-adrenergic receptor activation can enhance ANF transcription through an SRF-related molecule(s), several previous studies have identified SREs in other genes as potential enhancers of cardiac gene expression. For example, an SRE in the cardiac {alpha}-SkA gene was shown to confer basic FGF– but not acidic FGF–inducible transcription to reporter expression.48 More recently, the characterization of the transcription factors that interact with this SRE has shown that in addition to SRF, TEF-1 and SP-1 also participate in basal and inducible transcription,49 and {alpha}1-adrenergic induction of {alpha}-SkA in cardiac myocytes also requires all three elements.50 In another example, transfection experiments showed that stretch-induced c-fos transcription in cardiac myocytes maps to the SRE in that gene51 ; however, no direct evidence was presented to address the nature of the protein(s) binding to that element. Also, a report from Argentin et al52 indicated that the SRE at -406 bp of the rat ANF 5'-FS contributed to basal ANF transcription in ventricular myocytes; however, the protein(s) that bound to this element, which did not apparently bind to the c-fos SRE, was not positively identified.

What is the mechanism through which SRF mediates cardiac gene induction? In part, the answer to this question may depend on the identity of the gene under study as well as the stimulus. Most previous studies relating to the mechanism of SRF action have investigated the induction of the c-fos gene in non–muscle cell lines. In general, stimuli that induce c-fos transcription via SRF fall into two categories: (1) the PKC/ternary complex–dependent stimuli (eg, phorbol ester), which require MAPK-mediated p62TCF phosphorylation,53 and (2) the PKC/ternary complex–independent stimuli (eg, serum),26 which require pp90RSK-mediated SRF phosphorylation.25 Although a variety of stimuli can mediate myocardial cell hypertrophy and transcriptional activation, a potential role for SRF has been indicated for stretch-induced c-fos, basic FGF–induced {alpha}-SkA expression, and (from the present study) ANF induction. Myocardial cell stretch is known to activate PKC and MAPK,49 suggesting that it may fall into category 1. Indeed, mutations of the c-fos SRE, which are known to block ternary complex formation,26 also block stretch-activated c-fos transcription.51 It is also clear that the 5'-FS of the chicken {alpha}-SkA gene possesses several SRE-like sequences, one of which confers basic FGF induction of promoter/reporter constructs in rat myocardial cells.48 49 Interestingly, the {alpha}-SkA SRE-1 possesses no flanking Ets-domain sequence, and coupled with the belief that FGF operates through flg-mediated activation of tyrosine kinase and not PKC, it would appear that SRF may mediate induction through the PKC/ternary complex–independent mechanism. We hypothesize that SRF-regulated ANF transcription falls into the PKC/ternary complex–independent category. Our preliminary studies have shown that no ternary complex is ever formed when either the ANF SRE/114 or ANF SRE/406 is used as a probe. This finding would indicate that in myocardial cells, PE-activated MAPK may lead to pp90rsk activation and that the subsequent phosphorylation of SRF at Ser-103 would lead to transcriptional activation, as described for the c-fos gene in a different cell type.25 We have preliminarily demonstrated that SRF is phosphorylated at Ser-103 in myocardial cells, lending credibility to this hypothesis.

In summary, the present study provides direct evidence that SRF participates in both basal and PE-inducible ANF transcription, providing the framework for a complete signaling pathway from the cell surface {alpha}1-adrenergic receptor to transcriptional activation of the ANF gene. Interestingly, recent studies on ß-myosin heavy chain induction in primary myocardial cells have demonstrated that the binding of a TEF-1–like protein to an MCAT sequence in the 5'-regulatory region of that gene confers PE-inducible transcription.54 Thus, it appears that in cardiac myocytes there are multiple enhancers through which {alpha}1-adrenergic receptor activation can lead to apparently coordinated transcriptional activation of a collection of genes that are typically expressed primarily during early development. These factors include but are not limited to TEF-1,54 a cardiac SP-1–like protein,22 and an SRF-like protein (present study). Future studies focusing on the molecular mechanisms by which these proteins are ultimately activated as enhancers by {alpha}1-adrenergic receptor activation will be of interest, as will an analysis of how they collaborate to confer not only hormone-inducible transcription but also stage- and tissue-specific expression.


*    Selected Abbreviations and Acronyms
 
{alpha}-SkA = {alpha}-skeletal actin
ß-gal = ß-galactosidase
ANF = atrial natriuretic factor
AP-1 = activator protein-1
DEPC = diethylpyrocarbonate
DMS = dimethylsulfate
EMSA = electrophoretic mobility shift assay
FGF = fibroblast growth factor
FS = flanking sequence
MAPK = mitogen-activated protein kinase
MEF = myocyte enhancement factor
PE = phenylephrine
PERE = PE response element
PKC = protein kinase C
SP-1 =
SRE = serum response element
SRF = serum response factor
TEF-1 = simian virus 40 enhancer binding factor
TRE = TPA-responsive elements


*    Acknowledgments
 
This study was supported in part by National Institutes of Health grants NS-25073 and HL-46345. A.B. Sprenkle was supported by a predoctoral fellowship from the American Heart Association, California Affiliate, Inc (grant 92-418). We gratefully acknowledge the expert technical assistance of Donna Thuerauf. We thank R. Prywes (Columbia University, New York, NY) for the SRF and MEF 2 antisera.

Received August 16, 1995; accepted September 25, 1995.


*    References
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 up arrowIntroduction
 up arrowMaterials and Methods
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 *References
 

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