©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Serum Response Factor Mediates AP-1-dependent Induction of the Skeletal -Actin Promoter in Ventricular Myocytes (*)

(Received for publication, January 16, 1996; and in revised form, February 23, 1996)

Pierre Paradis (1)(§) W. Robb MacLellan (1)(¶) Narasimhaswamy S. Belaguli (2) Robert J. Schwartz (2) (3) Michael D. Schneider (1) (2) (3)(**)

From the  (1)Molecular Cardiology Unit, Departments of Medicine, (2)Cell Biology, and (3)Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

``Fetal'' gene transcription, including activation of the skeletal alpha-actin (SkA) promoter, is provoked in cardiac myocytes by mechanical stress and trophic ligands. Induction of the promoter by transforming growth factor beta or norepinephrine requires serum response factor (SRF) and TEF-1; expression is inhibited by YY1. We and others postulated that immediate-early transcription factors might couple trophic signals to this fetal program. However, multiple Fos/Jun proteins exist, and the exact relationship between control by Fos/Jun versus SRF, TEF-1, and YY1 is unexplained. We therefore co-transfected ventricular myocytes with Fos, Jun, or JunB, and SkA reporter genes. SkA transcription was augmented by Jun, Fos/Jun, Fos/JunB, and Jun/JunB; Fos and JunB alone were neutral or inhibitory. Mutation of the SRF site, SRE1, impaired activation by Jun; YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred Jun activation to a heterologous promoter, as did the c-fos SRE. Deletions of DNA binding, dimerization, or trans-activation domains of Jun and SRF abolished activation by Jun and synergy with SRF. Neither direct binding of Fos/Jun to SREs, nor physical interaction between Fos/Jun and SRF, was detected in mobility-shift assays. Thus, AP-1 factors activate a hypertrophy-associated gene via SRF, without detectable binding to the promoter or to SRF.


INTRODUCTION

Mechanical load induces adaptive growth of cardiac muscle by cell enlargement, with little or no capacity for proliferative growth in terminally differentiated ventricular muscle cells (hypertrophy, not hyperplasia). Characteristically, this increase in myocyte mass is associated with qualitative and quantitative changes in cardiac-specific gene expression, including up-regulation of an ensemble of genes (including skeletal alpha-actin (SkA), (^1)beta myosin heavy chain, and atrial natriuretic factor (ANF)) that are highly expressed in embryonic but not normal adult ventricular myocardium, a phenomenon referred to as reinduction of a ``fetal'' phenotype(1, 2, 3) . SkA expression has been associated with increased contractility in the rodent heart (4) and with impaired contractility in patients with heart failure(5) . In addition to this set of genes, two others are induced, and to varying degrees have been implicated, in this response to mechanical load. First, ``immediate-early'' transcription factors including Fos and Jun potentially might couple trophic signals to long-term changes in growth and gene expression. Second, myocardial growth factors including angiotensin II and transforming growth factor beta (TGF-beta) evoke most aspects of the fetal/hypertrophic program in cultured cardiac muscle cells(1, 2, 3) . Induction of TGF-beta by load, passive stretch, alpha(1)-adrenergic agonists, and angiotensin II suggests that TGF-beta might participate in the onset, maintenance, or inhibition of cardiac hypertrophy, as an autocrine or paracrine factor(1) . We recently demonstrated that induction of the SkA promoter by TGF-beta requires the MADS box protein serum response factor (SRF) and the SV40 enhancer-binding protein, TEF-1(6) ; both also mediate activation of this promoter by alpha(1)-adrenergic agonists(7) . Dominant-negative mutations of the type II and type I TGF-beta receptor, which share related serine/threonine kinase domains, suffice to disrupt TGF-beta-dependent transcription(8, 9) ; however, the molecular circuit that confers signal from the TGF-beta receptor complex to SkA promoter-binding proteins is unknown.

Among the secondary or tertiary messengers that might be involved in this signaling cascade, it is noteworthy that activation of nuclear oncogene transcription factors Fos, Jun, and JunB precedes growth and the up-regulation of ``fetal'' cardiac genes, in cultured myocytes (10, 11) and intact animals(12, 13, 14) . Fos and Jun proteins (Fos, FosB, Fra-1, Fra-2, Jun, JunB, and JunD) each possess a basic domain for DNA binding and a leucine heptad repeat (leucine zipper) as an interface for homo- or heterodimerization(15) . Each member of this AP-1 transcription factor family recognizes the 12-O-tetradecanoylphorbol-13-acetate response element (TRE: TGA(G/C)TCA), although noncanonical sites also are reported(16, 17, 18) . Three lines of evidence support the inference that Fos/Jun proteins might mediate TGF-beta signal transduction: TGF-beta up-regulates the expression of junB and c-fos in skeletal myocytes (19) , cardiac myocytes, (^2)and other cell types, and AP-1 sites mediate autoinduction of TGF-beta1 itself(20) . Moreover, in skeletal muscle, forced expression of either Fos or Jun reproduces the suppressive effect of TGF-beta on myogenic differentiation, although the issue of physical association between Fos/Jun with myogenic helix-loop-helix proteins is unresolved (21, 22, 23) .

Related co-transfection studies likewise support the premise that Fos/Jun mediates the induction of fetal cardiac genes by TGF-beta, as shown for ANF(24, 25) , beta myosin heavy chain, (^3)and SkA(18) . In the latter study, forced expression of Jun (or Fos plus Jun) up-regulated transcription of the human SkA promoter in cardiac myocytes from neonatal rats and in P19 teratocarcinoma cells. Deletion analysis of the SkA promoter indicated that nucleotides -153 to -36 were required for maximal trans-activation by Fos/Jun. Although no consensus AP-1 site was found within this region, sequence-specific binding to a noncanonical motif was believed to occur. Despite this suggestive information, the conclusion that Fos/Jun proteins augment the transcription of SkA and other fetal cardiac genes would be premature. Dichotomous results, repression (24) as well as activation by Fos/Jun(25) , have been reported for ANF. Mechanical load, ischemia, and isoproterenol each highly induce JunB in myocardium in vivo(13, 26, 27) , but few functional comparisons among Jun proteins are known for cardiac myocytes(24) . Although prior results pointed to direct binding of Fos/Jun near the first SRE of the human SkA promoter, it has not been demonstrated whether point mutations of this construct that abolish AP-1 binding remain susceptible, or not, to induction by AP-1 factors. Finally, given the emerging importance of SRF in concert with TEF-1, and given the displacement of SRF at the first SRE by a GLI-Krüppel protein, YY1, the relationship between control by these three factors and the induction by Fos/Jun merits study.

In the present report, we demonstrate that Jun, Fos plus Jun, Fos plus JunB, and Jun plus JunB all transactivate the SkA gene in cardiac myocytes, whereas JunB, like Fos, is ineffective individually. Notably, a SRE is necessary for AP-1 responsiveness of the SkA promoter, and suffices to confer induction by AP-1 to a heterologous promoter. Induction required full-length Jun protein, and did not involve measurable binding of AP-1 factors to the SkA SRE1, physical association of AP-1 and SRF, augmentation of SRF binding by AP-1, or potentiation of the SRF trans-activation domain, residues 266-508 (SRF(266-508)). Together, these results indicate that AP-1 factors can act through SRF to induce a hypertrophy associated gene, SkA, but trans-activate SRE reporter genes in the absence of direct binding to the promoter or DNA-bound SRF.


EXPERIMENTAL PROCEDURES

Plasmids

Luciferase reporter vectors driven by mouse c-fos core promoter (nucleotides -56 to +109, Delta56Fos), SkA SRE1-Delta56Fos, c-fos SRE-Delta56Fos, chicken SkA promoter (nucleotides -394 to +24), and linker-scanning BglII mutations of the promoter were detailed previously(6, 28) . The BglII mutations disrupt, respectively, the SRF binding site in SRE1 (M-94/-89), the overlapping YY1 site (M-81/-79), TEF-1 binding (M-70/-65), Sp1 binding (M-52/-47), and the TATA box (M-28/-23); nomencalture indicates the substituted nucleotides. A TRE reporter gene was constructed by subcloning a HindIII-BglII fragment containing nucleotides -71 to -65 of the human collagenase gene fused to nucleotides -105 to +51 of the herpes simplex virus thymidine kinase promoter (a gift from M. Karin), into the firefly luciferase expression vector pXP2 (29) . Nucleotides -456 to +23 of the mouse SRF promoter were cloned as a SmaI/XhoI fragment into the luciferase vector pGL2 (Promega, Madison, WI). Details of the sequence and construction will be described elsewhere. (^4)pUAS(GAL4)EpLuc3, containing four yeast GAL4 binding sites fused to nucleotides -72 to +7 of the rat elastase gene, was constructed by subcloning a HindIII-BamHI DNA fragment of G4ElpHGH (a gift from R. W. Moreadith)(30) , into the luciferase vector pGL3 (Promega).

Rat c-fos cDNA, provided by T. Curran(31) , was subcloned as an EcoRI-XhoI fragment into the SV40-driven eukaryotic expression vector, pSV-SPORT1 (Life Technologies, Inc.). Mouse c-jun and junB cDNAs, provided by E. Olson(19) , were subcloned as EcoRI fragments into pSV-SPORT1. Mouse JunDeltaRK and JunDeltaLZ, a gift from I. M. Verma(32) , were subcloned as XhoI-SacI and PstI DNA fragments, respectively, into pSV-SPORT1; JunDeltaRK and JunDeltaLZ are mutations of Jun in which the DNA binding and dimerization domains have been deleted, respectively (amino acids 251-276 and 281-313). TAM67 (a deletion mutant of human Jun lacking amino acids 3-122 in the trans-activation domain, driven by the CMV promoter) and an insertless CMV vector derived from pCMV-beta-gal (Clontech, Palo Alto, CA) were kindly donated by M. J. Birrer(33) . Human SRF plasmids were kindly provided by R. Prywes. SRF (residues 1-508, wild type), SRF (residues 1-338), and SRFpm1 (34) were, respectively, subcloned as XbaI-BamHI, XbaI-PvuII, and XbaI-BamHI DNA fragments into pSV-SPORT1. SRF(1-338) harbors a deletion of the transcriptional activation domain, and SRFpm1 contains three point mutations in the basic region that abolish DNA binding. GAL4-SRF(266-508) comprises the DNA-binding domain of yeast GAL4 (amino acids 1-147), fused to residues 266-508 in the SRF trans-activation domain(35) , and was subcloned into the CMV-driven expression vector pCGN. Plasmid DNAs were purified using Quiagen Maxiprep columns (Chatsworth, CA).

Cell Culture and Transfection

Primary cultures of cardiac myocytes and cardiac fibroblasts were prepared from 1-2-day-old Sprague-Dawley rats with modifications to previously described methods (8, 9) . Ventricles of 100 hearts were digested four times, 15 min each, in 20 ml of phosphate-buffered saline without Ca and Mg, containing 1% dextrose, 0.1% collagenase (CL2, 250 units/mg), 0.05% trypsin (TRL3, 250 units/ml), and 0.05% deoxyribonuclease I (D, 3200 units/mg); enzymes were from Worthington Biochemical Corp. (Freehold, NJ). Cardiac myocytes and cardiac fibroblasts were first purified using a Percoll step gradient comprising (Pharmacia, Uppsala, Sweden) in Ads buffer (116.4 mM NaCl, 5.4 mM KCl, 5.6 mM dextrose, 10.9 mM NaH(2)PO(4), 405.7 µM MgSO(4), 20 mM HEPES, pH 7.3), adjusted to final densities of 1.082, 1.061, and 1.051 g/ml. The enriched myocytes (banding between the 1.082 and 1.061 g/ml layers) and fibroblasts (banding between the 1.061 and 1.051 layers) were washed with medium DF (Dulbecco's modified Eagle's medium:Ham's nutrient medium F-12, 17 mM HEPES, pH 7.4, 3 mM NaHCO(3), 2 mML-glutamine, 50 µg/ml gentamicin) containing 5% horse serum (Hyclone, Logan, UT). Ventricular myocytes were further purified by preplating to remove residual non-myocytes by differential adhesiveness, then were plated at a density of 1 times 10^6 cells/35-mm dish (Primaria, Falcon) and cultured 24 h in DF containing 5% horse serum. Fibroblasts were cultured for 1-2 days in DF supplemented with 5% horse serum, then were passaged once in the same medium using 0.5 times 10^6 cells/35-mm dish (Falcon).

Cells were transfected by a modified DEAE-dextran method. DNA (2.5 µg/ml reporter and 0-10 µg/ml expression vectors) was mixed with 150 µg/ml DEAE-dextran (average molecular weight, 500,000; Sigma) in DF supplemented with 2.5% Cosmic calf serum (Hyclone). For all comparisons, DNA and promoter content were kept constant using equivalent amounts of vector. Cells were washed once, were incubated with 1 ml/35-mm dish of DNA-DEAE-dextran complex for 1 h, and were then shocked for 30 s with 10% dimethyl sulfoxide in DF. Cells were cultured overnight in DF supplemented with 5% horse serum, after which the medium was replaced by DF supplemented with 5 µg/ml transferrin, 1 nM Na(2)SeO(3), 1 nM LiCl, 25 µg/mL ascorbic acid, and 100 µg/ml bovine serum albumin (fatty acid free). Twenty-four h later, cells were harvested and assayed for luciferase activity (6) and for protein content using the Bradford assay (Pierce). Luciferase activity for each promoter was corrected for protein content of each extract and was normalized to the activity of each promoter in parallel cultures of control, vector-transfected cells. Co-transfection with a constitutive lacZ gene was omitted for three reasons. The brief half-life of luciferase compared to other reporter proteins can lead to misleading interpretation, especially for transcriptional repression(36) ; commonly used viral promoters contain functional AP-1 or SRF sites (37, 38, 39) and are up-regulated by expression vectors used here; (^5)neutral core promoters that are unaffected by Fos/Jun, such as the c-fos -57/+109 core promoter, are insufficiently active in cardiac myocytes for accurate quantitation of lacZ activity,^5 but can be utilized to drive luciferase in parallel cultures, as an ostensibly constitutive control. Protein content was not significantly altered by any of the inteventions, compared to the corresponding vehicle-treated, vector-transfected cells. Where indicated, serum-free medium was supplemented with 1 ng/ml of TGF-beta1 purified from porcine platelets (R& Systems, Minneapolis, MN) or the vehicle (4 µM HCl, 1 µg/ml bovine serum albumin, fatty acid free).

Electrophoretic Mobility Shift Assays

Jun, Fos, and SRF were produced in vitro using the SP6 TnT coupled reticulocyte lysate system (Promega). Gel mobility shift assays were performed as described previously(40) : 6 µl of the coupled transcription/translation reaction mixture was incubated with 20,000 cpm of P-end-labeled DNA probe in 20 µl of 20 mM HEPES, pH 7.9, 25 mM KCl, 0.1 mM EDTA, 10% glycerol, 10 mM MgCl(2), 0.2 mM dithiothreitol, 0.025% Nonidet P-40, 50 µg/ml of poly(dG-dC) or poly(dI-dC), 50 µg/ml of bovine serum albumin. Poly(dG-dC) was used for assays involving the SkA SRE1-TATA probe and poly(dI-dC) for the TRE probe. DNA-protein complexes were incubated at room temperature for 45 min. The reaction mixtures were then loaded on a 4% low ionic strength polyacrylamide gel (acrylamide:bisacrylamide, 80:1) containing 45 mM Tris-borate, 1 mM EDTA, and 0.05% Nonidet P-40, and were electrophoresed in 45 mM Tris-borate, 1 mM EDTA at 4°C and 250 V for 2 h. The SkA SRE1-TATA probe, comprising SkA SRE1 (nucleotides -100 to -73) fused to mouse c-fos nucleotides -56 to -19, was generated by digesting the SkA SRE1-Delta56Fos luciferase expression vector with HindIII and PvuII. To ensure high specific activity, the probe was double labeled with the Klenow fragment of DNA polymerase I using [alpha-P]dCTP and with polynucleotide kinase using [-P]dATP. Competing double-stranded oligonucleotides encompassing the M-94/-89 and M-81/-79 mutations of the SkA SRE1, which abolish SRF and YY1 binding respectively, were detailed previously(6) . A double-stranded oligonucleotide containing a consensus TRE was subcloned into the SacI/NheI sites of pGL3 (5`-AGCTCGCTTGATGACTCAGCCGGAAGCTAG-3`); the sense strand is shown, and the consensus sequence is underlined. The probe was end-labeled using the Klenow fragment of DNA polymerase and [alpha-P]dCTP. AP-1 consensus and mutant oligonucleotides were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Statistics

For each promoter tested, luciferase activity is shown as the mean ± S.E., relative to parallel cultures of vehicle-treated, vector-transfected cells. Data were compared by analysis of variance followed by the Student-Newman-Keuls multiple comparison test, using a significance level of p < 0.05.


RESULTS

AP-1 Factors Differentially Activate the Skeletal alpha-Actin Promoter in Ventricular Myocytes

In preliminary Northern blot analyses, we observed a marked increase in c-fos and junB in ventricular myocytes treated with TGF-beta, with little or no change in c-jun,^5 as shown previously in skeletal muscle(19) . To compare the potential for these three immediate-early genes to trans-activate the SkA promoter, ventricular myocytes were co-transfected with the SkA luciferase reporter gene and Fos, Jun, or JunB expression vectors, singly or in combination (Fig. 1). Whereas Fos and JunB alone were neutral, Jun activated transcription via the SkA promoter in a dose dependent fashion, up to 3.4-fold (p < 0.01). No further increase in Jun-dependent transcription was seen with a 5-fold larger, 2-kilobase pair segment of the promoter.^5 Although Fos and JunB singly had no effect, in tandem they transactivated the SkA promoter synergistically (2.2 ± 0.16; p leq 0.01). More than additive induction likewise was seen using Fos plus Jun (3.1 ± 0.32; p leq 0.01), but not Jun cotransfected with JunB. Thus, co-expression of Fos and JunB, AP-1 factors that are highly induced by TGF-beta, can suffice to increase transcription of the SkA promoter.


Figure 1: Fos/Jun proteins transactivate the skeletal actin promoter in ventricular muscle cells. Cardiac myocytes were transfected with 2.5 µg of the luciferase reporter genes and 0-10 µg of the Fos, Jun, or JunB vectors. Control cultures were transfected with 2.5 µg of reporter plasmid and 10 µg of the empty SV40-driven vector. For each reporter plasmid, luciferase activity is expressed relative to that in vector-transfected cells. Results are the mean ± S.E. for at least four transfections with two exceptions; n = 3 for the SkA reporter co-transfected with Fos plus JunB, and for the TRE reporter co-transfected with Jun plus JunB. *p < 0.05, versus vector-transfected control cells; p < 0.05, versus parallel cultures co-transfected with 5 µg of Jun.



To verify that induction of the SkA promoter by forced expression of Fos, Jun, and JunB is specific, a canonical AP1-responsive element and a constitutive neutral promoter were examined, as positive and negative controls, respectively. Control of the SkA promoter by the various permutations of AP-1 factors accurately resembled activation of the human collagenase TRE1, although the TRE1 was more highly induced. By contrast, little or no activation was seen using the c-fos neutral core promoter, Delta56Fos. Jun, Fos/Jun, and Fos/JunB up-regulated the Delta56Fos reporter by no more than 40, 70, and 60%, respectively (p leq 0.01). Indeed, slight inhibition was observed at high concentrations of Fos or JunB (p leq 0.01).

Trans-activation of the alphaSkA by the AP-1 Factors Requires SRE1 in Concert with the TATA Box

To test the hypothesis that induction by Fos/Jun might map to one or both TGF-beta response elements of the SkA promoter (i.e. SRE1 and a TEF-1 site), cardiac myocytes were co-transfected with 10 µg of the Jun expression vector or the empty vector control, together with a luciferase reporter gene driven by the wild-type SkA promoter versus linker-scanning mutations as summarized in Fig. 2. Detailed previously (6) , mutation of the YY1 site weakly activates basal activity; mutations of the SRF, TEF-1, Sp1, and TATA motifs inhibit basal activity of the promoter by up to 90%. Of the five linker-scanning mutations tested, three had no significant effect on induction by Jun. The mutation specific for YY1 (M-81/-79) lies immediately 3` to the proximal SRF binding site, and disrupts a potential TRE-like sequence (18) . Mutation of the YY1 site did not alter trans-activation by Jun (5.09 ± 0.42, relative to activity of this promoter in vector-transfected cells), nor did a mutation that destroys the binding site for TEF-1 (M-70/65; 4.97 ± 0.76). The mutation that blocks the binding of Sp1 (M-52/-47) also had no significant effect (3.88 ± 0.41). M-94/-89 is a substitution in the 5` arm of the SRE1 palindrome, which specifically disrupts SRF binding while sparing YY1: by contrast to activation of the wild-type promoter and these three mutations by Jun, mutation of the SRF binding site reduced Jun-dependent transcription of the promoter by one-half (2.34 ± 0.40; p leq 0.01). A similar decrease in induction by Jun resulted from mutation of the TATA box (M-28/-23; 2.00 ± 0.07; p leq 0.01). Together, these mutations suggest that SRF, but not TEF-1 or Sp1, is necessary for full trans-activation of the SkA promoter by Jun. That a mutation of the TATA box also inhibited trans-activation by Jun is intriguing, as both SRF (40, 41) and Jun (42) have been proven to associate with basal transcription factors.


Figure 2: Trans-activation of the SkA promoter by Jun requires SRF binding and the TATA box. A, schematic representation of the proximal SkA promoter, from SRE1 to the TATA box. B, cardiac myocytes were co-transfected with 10 µg of the Jun expression vector versus the empty vector control, and 2.5 µg of the luciferase reporter genes. Nucleotides altered by the linker-scanning BglII mutations and the binding site affected are indicated at the left. For each reporter plasmid, luciferase activity (mean ± S.E.) is shown for Jun-transfected cells relative to vector-transfected control cells; n = 6 for each condition tested. *p < 0.05, versus induction of the wild-type SkA promoter.



A SRE Confers Jun-dependent Transcription to a Heterologous Promoter

Conversely, to establish whether the SkA SRE1 suffices for trans-activation by Jun, we co-transfected Jun with the SkA SRE1 upstream of the neutral promoter, Delta56Fos (Fig. 3A). Luciferase expression driven by Delta56Fos was not significantly changed by Jun (1.39 ± 0.09). By contrast, Jun trans-activated the SRE1-Delta56Fos construct 5.02 ± 0.48 fold (p leq 0.01). To distinguish whether AP-1 dependent activation was specific to the SkA SRE1, this element was exchanged for the c-fos SRE: Jun induced the c-fos SRE-Delta56Fos reporter to the same extent (5.11 ± 0.47; p leq 0.01). By contrast, Jun decreased transcription of these isolated elements in parallel cultures of cardiac fibroblasts under the same transfection conditions; where indicated, cardiac fibroblasts were cultured in the presence of TGF-beta1, to demonstrate that the isolated SREs are functional in this cell background (Fig. 3B). Together with mutagenesis of the full-length SkA promoter, these findings indicate that SRF is both necessary and sufficient for activation of the SkA promoter in ventricular muscle cells by AP-1 transcription factors.


Figure 3: SRF binding sites confer AP-1 responsiveness to a heterologous promoter. A, cardiac myocytes were co-transfected with the luciferase reporter genes, 0 or 10 µg of Jun, and 0-100 ng of the SRF vector. Mutations altering the DNA binding domain of Jun (JunDeltaRK) and SRF (SRFpm1), Jun dimerization domain (JunDeltaLZ) and trans-activation domain of Jun (TAM67) and SRF (SRF91-338) also were tested, as indicated. Luciferase activity (mean ± S.E.) is expressed relative to the activity of each reporter, respectively, in parallel cultures of vector-transfected cells. n geq 6 for each condition tested, except n = 3 for the c-fos SRE co-transfected with SRF or SRF plus Jun. *p < 0.05, versus vector-transfected control cells; p < 0.05, versus cells co-transfected with 10 µg of Jun; **p < p0.05, versus cells co-transfected with 10 µg of Jun plus 25 ng of SRF. B, cardiac fibroblasts were co-transfected with the luciferase reporter genes and 10 µg of Jun versus the empty vector control. Luciferase activity (mean ± S.E.) is expressed relative to the activity of each reporter, respectively, in parallel cultures of vector-transfected cells. n geq 6 for each condition tested. *p < 0.005 versus vector-transfected control cells. C, schematic diagram of wild-type and mutant Jun and SRF.



To test what domains of Jun might be necessary or sufficient for these effects, we co-transfected cardiac myocytes with 10 µg of the Jun expression vector, SRE reporter genes, and 0-100 ng of an SRF expression vector (Fig. 3, A and C). This concentration of SRF by itself did not significantly activate any of the three reporter genes. However, exogenous SRF was synergistic with Jun, up-regulating both the SkA SRE1- and c-fos SRE-Delta56Fos constructs, up to 14-fold, nearly three times the level of induction produced by Jun alone. When wild-type SRF was co-transfected with Jun mutants, no trans-activation was observed with a deletion of the DNA-binding domain (JunDeltaRK), deletion of the dimerization domain (JunDeltaLZ), or partial deletion of the trans-activation domain (TAM67). Each of the three Jun mutants also failed to transactivate, by themselves, the full-length SkA reporter gene.^5 Conversely, in cells cotransfected with Jun, the synergistic effect of wild-type SRF was abolished with SRFpm1, a point mutation of SRF which cannot bind DNA, and was markedly impaired with a mutation that deletes a trans-activation domain, SRF(1-338); the remaining cooperative effect is consistent with the residual activation domain of this mutation (43) . As the high levels of endogenous Jun and SRF in myocardium(6, 44) , together with the limited efficiency for transfection of ventricular myocytes, would confound efforts to compare the levels of each protein in vivo, we synthesized each Jun and SRF mutation in vitro in the presence of [S]methionine and verified that the translational efficiency and inherent stability of each protein were equivalent to that of wild-type Jun and SRF.^5 Thus, functional DNA-binding, dimerization, and trans-activation domains were each necessary for up-regulation of the SkA promoter by Jun. The necessity for full-length Jun contrasts with effects shown for the isolated Jun trans-activation domain in repression of myogenin activity(22) .

Activation of SRF Transcription by Jun

A requirement for full-length Jun would be anticipated by either of two contrasting models: activation through direct binding of Jun, suggested previously (18) , or indirect activation by a Jun-induced protein. Given the requirement for SRF binding to SRE1 (Fig. 2), Jun might thus up-regulate the SkA promoter indirectly, by augmenting SRF expression or activity. Although the small proportion of cardiac myocytes that take up foreign genes during transient transfection precludes a direct test of whether Jun induces SRF in this background, we co-transfected ventricular muscle cells with Jun together with a murine SRF luciferase reporter gene. Forced expression of Jun increased transcription of the SRF promoter (2.29 ± 0.19; p leq 0.01). However, it is implausible that changes in SRF abundance alone could explain induction of the SkA promoter by Fos/Jun. First, the magnitude of SRF induction was modest. More importantly, overexpression of SRF at up to 100 ng/culture did not increase transcription of the isolated SREs (Fig. 3), but causes squelching at several higher concentrations(^6); thus, SRF is not limiting in ventricular muscle cells. Therefore, it is necessary to consider that AP-1 factors might modulate activity of SRF, or promote transcription via SREs, by other mechanisms.

To test whether Jun could augment gene expression via the trans-activation domain of SRF, we employed a GAL4 fusion protein comprising the DNA-binding domain of yeast GAL4 fused to SRF amino acids 266-508, GAL4SRF(266-508). Five µg of GAL4SRF(266-508) were co-transfected with or without the Jun expression vector, using a GAL4-dependent luciferase reporter (Fig. 4). In the absence of exogenous Jun, the GAL4/SRF fusion protein increased transcription of the reporter 8.39 ± 0.42 (p < 0.01); transcription via the SRF activation domain was not augmented significantly by the addition of exogenous Jun. Thus, Jun did not increase transcription, when the SRF activation domain was tethered to DNA by a heterologous DNA-binding domain. This also argues against a generalized activation of transcription by Jun.


Figure 4: Jun does not potentiate the SRF trans-activation domain. Cardiac myocytes transfected with the GAL4-dependent luciferase reporter gene, pUAS(GAL4)EpLuc3, were co-transfected as shown with 5 µg of GAL4-SRF(266-508), 5 µg of the Jun expression vector, or both. Luciferase activity (mean ± S.E.) is expressed relative to that in vehicle-treated, vector-transfected cells. n = 6 for each condition tested. *p < 0.01, versus the control cells.



To address two further possibilities, that Jun might activate the SRE by physical association with native SRF, or facilitate DNA binding by SRF, gel mobility-shift assays were performed using SRF, Fos, and Jun, produced by in vitro transcription and translation (Fig. 5A). A HindIII-PvuII DNA fragment of the SkA SRE1-Delta56Fos luciferase reporter gene was used as probe, encompassing the SkA SRE1 plus all sequences of the core c-fos promoter including the TATA box. A double-stranded oligonucleotide containing the consensus AP-1 binding site was used, for comparison. Recombinant SRF bound the SRE1-Delta56 Fos probe, was displaced by the competitor that binds SRF but not YY1 (M-81/-79), and was displaced poorly by the reciprocal mutation, which disrupts SRF binding (M-94/-89). Jun, Fos, and co-translated Fos/Jun showed no direct binding to the probe, did not form a higher order complex with DNA-bound SRF, and did not alter binding of SRF to DNA in any fashion. To ensure that the lack of protein-protein interaction was not due to inadequate expression of Jun or Fos, parallel experiments were performed using the AP-1 consensus probe. Jun and co-translated Fos/Jun bound the TRE, were displaced by excess unlabeled TRE, and were unaffected by the mutated TRE. Thus, both AP-1 factors were expressed as stable proteins with the expected DNA-binding and dimerization properties. To exclude direct association between Fos/Jun proteins and SRF, we demonstrated no binding of recombinant SRF to Jun homodimers or Fos/Jun heterodimers, and no effect of SRF on the binding of AP-1 factors to the TRE probe (Fig. 5B).


Figure 5: Fos/Jun proteins do not bind the SkA SRE1 or DNA-bound SRF in vitro. SRE1-Delta56 Fos (A) and TRE (B) probes were end-labeled with P, incubated with Fos, Jun, and SRF proteins produced in vitro, and analyzed by the electrophoretic mobility shift assay in the presence or absence of a 100-fold excess of the indicated unlabeled competitor. The amount of Fos, Jun, and SRF designates microliters of the programmed reticulocyte lysate. The total quantity of reticulocyte lysate per lane was maintained at 6 µl using lysate programmed with the empty SV40 vector. ns, nonspecific binding produced by the control reticulocyte lysate.




DISCUSSION

The present investigations show that transcription of SkA, a ``fetal'' cardiac gene associated with myocardial hypertrophy, can be augmented by AP-1 transcription factors (Jun, Fos plus Jun, Fos plus JunB, or Jun plus JunB) in ventricular myocytes from neonatal rats. Despite marked differences in the constructs and procedures used, this corroborates and extends the report of Bishopric et al.(18) , that forced expression of Jun or Fos plus Jun up-regulates the human SkA promoter in cardiac cells. Because dichotomous results, both induction and repression, were reported for the ANF gene(27, 28) , consensus regarding the functional role of Fos/Jun factors in hypertrophy has been lacking. Although JunB, like Fos and Jun, is highly induced by both mechanical load (13, 14) and trophic factors that up-regulate the endogenous SkA gene (TGF-beta, catecholamines, and angiotensin II^2(18, 45) ), even less functional evidence has been available for JunB in cardiac muscle(24) . Whereas Fos proteins do not form homodimers, Jun and JunB form both homodimers and heterodimers with Fos/Jun proteins and more distantly related factors, via the leucine zipper. Our results demonstrate functional synergy between JunB and Fos in cardiac myocytes; either alone had no effect or was inhibitory, while Fos plus JunB activate the SkA promoter. Permutations of Fos, Jun, and JunB that activate the SkA gene correspond to those that induced the human collagenase TRE, a canonical AP-1-responsive element.

Our results diverge from previous findings, however, on mechanisms to explain trans-activation of SkA by AP-1. Here, SRE1 was both necessary and sufficient. A molecular basis for activation of the SkA promoter by SRF in concert with TEF-1 has been proposed(6, 7) , with virtually identical conclusions for the avian and rat promoters. Cooperation of SRF and TEF-1 also was implicated in two distinct transduction pathways for hypertrophy, TGF-beta and alpha(1)-adrenergic agonists. By contrast, the TEF-1 site is dispensable for full augmentation of the SkA promoter by Jun. In agreement, nested deletions of the human SkA promoter suggested that nucleotides encompassing the first SRE (-153 to -87) were required for maximal trans-activation by Fos plus Jun, whereas more proximal sequences including the TEF-1 site at nucleotides -71 to -65 do not mediate AP-1 responsiveness(18) . While direct binding of Jun and Fos/Jun to a noncanonical site near SRE1 was reported for the human SkA promoter, our results point instead toward an indirect mechanism, mapped to SRE1 itself. First, Jun can induce the avian SkA promoter in the absence of TEF-1, Sp1, or YY1 binding, yet the SRF binding site and TATA box are indispensable. This is intriguing, given that SRF contacts the RAP74 subunit of transcription factor IIF(39, 41) , while Fos and Jun contact other basal factors, transcription factor IIB and TATA box-binding protein(42, 46) . Second, the isolated SkA SRE1 is sufficient for AP-1-dependent expression and is interchangeable, in this respect, with the c-fos SRE. Third, using recombinant Fos and Jun produced in reticulocyte lysates, we detected no binding to the SkA SRE1. This discrepancy with direct binding of AP-1 factors reported for the human SkA promoter (18) may be explained by technical differences: in the earlier study, proteins were produced in E. coli, truncated Fos and Jun were used, and five times more protein was used for the SkA probe versus the authentic TRE. Alternatively, sequence dissimilarities may be germane. Among the characterized vertebrate SkA genes, only the human promoter matches the TGACTCA consensus TRE at five positions that include both cytosine residues.

Control of the SkA SRE1 by Jun and synergy with SRF both required full-length Jun protein with intact DNA binding, dimerization, and trans-activation domains. As no binding of AP-1 factors was detected to SRE1, mechanisms alternative to direct association must be considered. Conceivably, AP-1 factors might increase transcriptional activity of an SRF binding site through protein-protein interactions with SRF, increasing SRF abundance, or affecting transcriptional activity of SRF. Our results provide no support for a ternary or quarternary complex of Fos/Jun with DNA-bound SRF. No physical interaction was seen between SRF and AP-1 in gel mobility-shift assays, nor did AP-1 factors augment DNA binding by SRF. However, measurements of DNA-protein interaction might overlook low affinity binding or interactions that require a co-activator. Using GAL4 fusion proteins to overcome limitations of both gel retardation assays and endogenous SRF, we found that Jun could not potentiate the C-terminal activation domain of SRF. It is a formal possibility that Jun might interact (with affinity too low to be stable in vitro) only with native SRF or the native SRFbulletSRE complex. Increased SRF abundance is unlikely to explain AP-1 dependent SkA transcription, since SRF was not limiting in cardiac myocytes. Our findings are more consistent with the alternative, that Fos/Jun transcription factors increase, instead, the transcriptional activity of SRF. In principle, this could be contingent on altered expression of an autocrine or paracrine factor(23) , a protein kinase modulating SRF activity(47) , or a co-activator. Whereas SRF accessory proteins include most obviously the ternary complex factors Elk-1/TCF and SAP-1 (48) , association and synergy with SRF both were demonstrated (49) for the cardiac-restricted homeodomain protein, Nkx-2.5, vertebrate homologue of the Drosophila tinman gene(50, 51) .

In summary, our studies reveal a novel AP-1-dependent pathway for gene induction in cardiac myocytes, via indirect activation of SREs. Hence, AP-1 factors might plausibly be involved in the up-regulation of genes containing SREs, including skeletal and smooth muscle alpha-actin and the immediate-early gene c-fos, in the setting of cardiac hypertrophy. It is unknown whether the greater induction of ``fetal'' alpha-actin transcripts relative to cardiac alpha-actin reflects inherent differences among SREs, contextual sequences, or elements elsewhere in the respective promoters. The SRE has been implicated in numerous settings as a pivotal regulatory element for cardiac gene expression during hypertrophy, for activation of the SkA promoter by TGF-beta(6) , basic FGF(52) , and alpha(1)-adrenergic agonists(7) , up-regulation of ANF by alpha(1)-adrenergic signals(53) , and induction of c-fos by load(54) , passive stretch(55) , or angiotensin II (56) . Control of SRE-dependent transcription by AP-1 factors was selective, as the neutral core promoter was unaffected by Jun, contrasting with the global increase in transcription provoked by Ras under similar conditions(36) . Conversely, TGF-beta selectively up-regulates fetal cardiac genes, with little change in RNA or protein content(57) . Hence, the overall increase in cell RNA and protein can be dissociated from the ``fetal/hypertrophic'' program. Analogously, rapamycin blocks the increase in total cell protein and p70 S6 kinase activity in angiotensin II-treated cardiac myocytes, while not affecting induction of c-fos or fetal cardiac genes(58) . The latter report highlights other data pointing to the possibility, with which the present study and our Ras results concur(36) , of distinguishable signaling cascades for these components of the hypertrophic phenotype, that the global increase of cell protein is mediated by p70 S6 kinase, while the fetal program might be mediated, at least in part, by mitogen-activated protein kinase induction of Fos, and Jun N-terminal kinase acting though Jun(58) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants R01 HL47567, P01 HL49953, and T32 HL07706 (to M. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49759[GenBank].

§
Fellow of the Fonds de la Recherche en Santé du Québec and the Heart and Stroke Foundations of Canada. Present address: Institut de Recherches Cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, Québec, Canada H2W 1R7.

Fellow of the Medical Research Council of Canada.

**
To whom correspondence should be addressed: Molecular Cardiology Unit, One Baylor Plaza, Rm. 506C, Baylor College of Medicine, Houston, TX 77030; Tel.: 713-798-6683; Fax: 713-798-7437.

(^1)
The abbreviations used are: SkA, skeletal alpha-actin; ANF, atrial natriuretic factor; SRE, serum response element; SRF, serum response factor; TGF-beta, type beta transforming growth factor; TRE, 12-O-tetradecanoylphorbol-13-acetate response element; CMV, cytomegalovirus; DF, Dulbecco's modified Eagle's medium:Ham's nutrient medium F-12.

(^2)
T. G. Parker, P. Paradis, and M. D. Schneider, unpublished results.

(^3)
T. G. Parker, personal communication.

(^4)
N. S. Belaguli and R. J. Schwartz, unpublished data.

(^5)
P. Paradis and M. D. Schneider, unpublished results.

(^6)
W. R. MacLellan and M. D. Schneider, unpublished results.


ACKNOWLEDGEMENTS

We thank T. Curran, M. Karin, R. W. Moreadith, E. Olson, R. Prywes, and I. M. Verma for plasmids cited, F. Ervin and W. Boerwinkle for assistance, M. Abdellatif for subcloning SRF and SRF(1-338), and R. Roberts for encouragement and support.


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