Transcription Factor RTEF-1 Mediates α1-Adrenergic Reactivation of the Fetal Gene Program in Cardiac Myocytes
Abstract—α1-Adrenergic receptor stimulation induces cardiac myocytes to hypertrophy and reactivates many fetal genes, including β-myosin heavy chain (βMyHC) and skeletal α-actin (SKA), by signaling through myocyte-specific CAT (M-CAT) cis elements, binding sites of the transcriptional enhancer factor-1 (TEF-1) family of transcription factors. To examine functional differences between TEF-1 and related to TEF-1 (RTEF-1) in α1-adrenergic reactivation of the fetal program, expression constructs were cotransfected with βMyHC and SKA promoter/reporter constructs in neonatal rat cardiac myocytes. TEF-1 overexpression tended to transactivate a minimal βMyHC promoter but significantly interfered with a minimal SKA promoter. In contrast, RTEF-1 transactivated both the minimal βMyHC and SKA promoters. TEF-1 and RTEF-1 also affected the α1-adrenergic response of the βMyHC and SKA promoters differently. TEF-1 had no effect. In contrast, RTEF-1 potentiated the α1-adrenergic responses of the SKA promoter and of a −3.3-kb βMyHC promoter. To determine why the promoters responded differently to TEF-1 and RTEF-1, promoters with mutated M-CAT elements were tested in the same way. The βMyHC promoter required an intact M-CAT element to respond to TEF-1 and RTEF-1, whereas the SKA promoter M-CAT was required for the TEF-1 response but not for the RTEF-1 response, suggesting that SKA promoter–specific cofactors may be involved. By competition gel shift assay, the M-CAT of the minimal βMyHC promoter had a lower affinity than that of the SKA promoter, which partly explains the different responses of these promoters to TEF-1. These results highlight functional differences between TEF-1 and RTEF-1 and suggest a novel function of RTEF-1 in mediating the α1-adrenergic response in hypertrophic cardiac myocytes.
Cardiac myocytes respond to α1-adrenergic receptor stimulation by a progressive hypertrophy1 accompanied by a characteristic reactivation of many fetal genes, including βMyHC2 and SKA.3 We and others have studied the transcriptional mechanisms that control these fetal genes in order to better understand the maladaptive process of cardiac hypertrophy. The minimal sequences required for α1-adrenergic stimulation of the βMyHC (−215 bp from the start of transcription) and SKA (−113 bp) promoters have been determined4,5; both promoters are activated via slightly different M-CAT elements (CATNT/CT/C).6 TEF-1 binds to M-CAT elements in the promoters of many genes expressed in cardiac and skeletal muscle cells (for review, see Reference 77 ). Thus, a role for TEF-1 in mediating the α1-adrenergic response of the βMyHC and SKA genes has been proposed.4 5 8
TEF-1 was originally described as a transcription factor required for SV40 enhancer function in HeLa cells,9 10 and a human TEF-1 cDNA was cloned from HeLa cells.11 The cis elements to which TEF-1 binds in the SV40 enhancer, the GTIIC motif, CATTCCA, and the SPH motifs, CATACTT and CATGCTT,10 are now recognized as variants of the M-CAT motif.6 TEF-1 is the prototype of a family of transcription factors that share sequence homology in the TEA (or ATTS) DNA binding domain.12 13 The TEA domain family of transcription factors includes human TEF-1, TEC1 from yeast,14 and ABAA from Aspergillus.15 In Aspergillus, ABAA regulates the development of the conidiophore, the asexual reproductive apparatus.15 In yeast, TEC1 regulates the development of pseudohyphal growth.16 A TEF-1–related gene from Drosophila called scalloped17 and another in Caenorhabditis18 have also been identified. Whereas the function for TEF-1 in the nematode is not yet known, in Drosophila, scalloped is required for the development of sensory organs on the wing blade17 and normal taste behavior.19 Remarkably, human TEF-1 can substitute for Drosophila scalloped during wing blade development, demonstrating the high degree of evolutionary and functional conservation among TEF-1–related TEA domain transcription factors.20
Ablation of TEF-1 expression in the mouse causes abnormal cardiac development and embryonic lethality.21 However, it does not prevent the expression of M-CAT–dependent cardiac genes, suggesting that other M-CAT binding factors are present. Recently, we cloned a cDNA encoding a TEF-1–related protein, RTEF-1, from a human heart cDNA library.22 Thus, the existence of at least 2 functional M-CAT binding factors in cardiac myocytes suggests that both play a part in transcriptional regulation.
The present study was designed to examine the functional differences between TEF-1 and RTEF-1 in the activity of the M-CAT–dependent βMyHC and SKA promoters, which are known to be activated by α1-adrenergic stimulation in hypertrophic cardiac myocytes. The results demonstrate that RTEF-1, but not TEF-1, can potentiate the α1-adrenergic response of these promoters. Therefore, RTEF-1 might be important in mediating the process of α1-adrenergic–induced hypertrophy.
Materials and Methods
Plasmids and Expression Constructs
The CMV enhancer–driven human TEF-1 expression plasmid pXJ40-TEF-1A and the parental plasmid pXJ40 have been described.11 The human RTEF-1 expression plasmid was constructed by releasing the BamHI/XhoI insert containing the full-length cDNA encoding human RTEF-122 from pBluescript and cloning this fragment into pXJ40. The rat −3.3-kb βMyHC/CAT, the minimal −215-bp βMyHC/CAT, and the mouse −113-bp skeletal α-actin/CAT reporter constructs, as well as the M-CAT mutants of these minimal constructs, have been described.4 5
Cell Culture and Transfections
Neonatal rat cardiac myocytes were isolated and cultured at low density (106 viable cells plated per 60-mm dish) in minimal essential medium supplemented with 5% calf serum (HyClone) and bromodeoxyuridine (100 μmol/L) to prevent nonmyocyte proliferation as described previously.1 Twenty-four hours after plating, the medium was replaced with serum-free minimal essential medium supplemented with human insulin (10 μg/mL, Sigma), human transferrin (10 μg/mL, Sigma), and bovine serum albumin (1 mg/mL, Sigma). The following day, cardiac myocytes were transfected using the calcium phosphate precipitation method for 2 hours with serum-containing medium. A total of 20 μg DNA was added per plate in duplicate: 5 μg of reporter plasmid (−3.3-kb βMyHC, −215-bp βMyHC, −215-bp mM-CAT βMyHC, −113-bp SKA, and −113-bp mM-CAT SKA) and 10, 50, or 100 ng of CMV-driven expression plasmid (pXJ40, pXJ40-TEF-1 A, and pXJ40-RTEF-1) and adjusted to 20 μg with pBluescript plasmid. The transfections were carried out using several different preparations of the expression and reporter plasmids in a minimum of 6 experiments (independent cardiac myocyte preparations).
To control for promoter competition, increasing doses of the empty CMV expression vector were tested for their effects on the basal and phenylephrine-induced activities of the reporter plasmids. Since no significant effects of CMV were noted by ANOVA, fold activities at each dose of empty CMV vector were grouped. An internal control reporter plasmid, such as RSV luciferase, was not used because TEF-1 and RTEF-1 overexpression strongly interfered with the viral promoter (data not shown). Two hours after transfection, the plates were rinsed several times in fresh medium and allowed to recover overnight in serum. The next day, medium was replaced with serum-free medium containing either the 100 μmol/L vitamin C vehicle or 100 μmol/L vitamin C and 100 μmol/L phenylephrine. Cells were maintained 2 additional days in serum-free medium and then harvested for CAT assay.
CAT activity was assessed using half of the cell lysate, prepared by sequential freeze/thaw cycles of the cells in 100 μL of assay buffer (100 mmol/L potassium phosphate [pH 7.4], 1 mmol/L dithiothreitol, and 1 mmol/L EDTA). The reaction was carried out for 2 hours at 37°C in 100 μL of assay buffer containing 0.05 μCi [14C]chloramphenicol (Moravek) and 3 mmol/L acetyl coenzyme A (Pharmacia). Samples were extracted with ethyl acetate, concentrated in a SpeedVac (Savant), resuspended in 20 μL of ethyl acetate, and spotted onto thin-layer chromatographic plates developed in 10% methanol/90% chloroform in a chromatography tank. Plates were air-dried and exposed to x-ray film overnight. After autoradiography, spots were cut out and counted in Optifluor (Packard) using a scintillation counter. The percent conversion from unacetylated to acetylated chloramphenicol for each promoter/reporter plasmid, normalized to their basal activities in the presence of empty CMV expression plasmid, was presented as fold activities. Maximal activation seldom exceeded 80% conversion, and assays were performed within the linear range.
Nuclear Protein Extracts
Nuclear protein extracts from cultured neonatal rat cardiac myocytes were prepared essentially as described by Farrance and Ordahl.23 For nuclear extracts from transfected cells, 5×106 cardiac myocytes were plated on 100-mm culture dishes and transfected as described above, except that 1 μg of TEF-1 or RTEF-1 expression plasmid was used. Cells were harvested 24 hours after transfection. Nuclear protein concentration was determined by the bicinchoninic acid method (Sigma) against a bovine serum albumin standard curve.
Gel Mobility Shift Assay
The gel mobility shift assays were carried out as described,4 except that 100 μg of bovine serum albumin (Sigma) was added as a nonspecific protein competitor, using the following oligonucleotides: the βMyHC promoter proximal M-CAT element,8 βMyHC M-CAT, 5′-CATGCCATACCACAACAATG-3′; the albumin promoter D element,24 AlbD, 5′-TCCTACCCCATTACAAAATCATACCA-3′; the tandemly duplicated SV40 GTIIC element,11 2XGTIIC, 5′-CCGAGAGACACATTCCACACATTCCACTGC-3′; the two SPH motifs of the SV40 enhancer,11 2XSPH, 5′-CCGAGAGATGCATGCTTTGCATACTTCTGC-3′; the mouse SKA promoter M-CAT element,5 SKA M-CAT, 5′-GCAGCAACATTCTTCGGGGC-3′; and the mutant SKA M-CAT element5 (mutation in italics), 5′-GCAGCAAGGTACTTCGGGGC-3′. All oligonucleotides were synthesized and HPLC-purified by Operon Technologies, Ltd. For the competitive gel shifts, equal amounts of the SKA and βMyHC M-CAT oligonucleotides were simultaneously labeled to the same specific activities on the sense strand (shown) with polynucleotide kinase using [γ-32P]ATP, purified of unincorporated isotope on a NucTrap column (Stratagene), and annealed to a 2-fold molar excess of the unlabeled strand.
Values are presented as mean±SE. All statistical analyses were performed using the SPSS program (SPSS Inc). Univariate multifactorial ANOVA was used to determine whether TEF-1 or RTEF-1 expression vectors affected fold activity of promoter/reporter plasmids in vehicle and phenylephrine-treated cardiac myocytes for each reporter construct. The dependent variable was fold activity, and the 3 factors tested were PHE, TEF, and RTEF. PHE was coded as 0 when phenylephrine was absent and coded as 1 when present. TEF was coded as 0 when TEF-1 was absent and as 1 when 10 ng, 2 when 50 ng, and 3 when 100 ng of TEF-1 expression vector was used. RTEF was coded in the same way. The control CMV-treated group was identified when both TEF and RTEF were 0. Interaction between TEF-1 or RTEF-1 and phenylephrine (TEF*PHE and RTEF*PHE) was included in the ANOVA model. However, interaction between TEF-1 and RTEF-1 (TEF*RTEF) or second-order interaction (TEF*RTEF*PHE) was not included in the ANOVA model, since TEF-1 and RTEF-1 were never cotransfected simultaneously. The Dunnett test, where CMV in the absence of phenylephrine was considered as the control group, was used for post hoc comparison where necessary. Effects were considered significant at P<0.05.
Effects of TEF-1 and RTEF-1 Overexpression on the Basal and α1-Adrenergic–Stimulated Activity of the −215-bp βMyHC Promoter
Figure 1A⇓ shows the activity of the −215-bp βMyHC promoter in the absence or presence of α1-adrenergic stimulation and in response to cotransfection with the empty CMV, the CMV-TEF-1, or the CMV-RTEF-1 expression vectors, each at 3 different doses. The basal activity of the −215-bp βMyHC promoter in neonatal rat cardiac myocytes cotransfected with the empty CMV expression vector and treated with 100 μmol/L of the vitamin C vehicle was set at 1-fold. Fold activities at the 3 doses of empty CMV vector were grouped, since increasing doses had no significant effect on the basal and phenylephrine-induced activities of the reporter plasmids.
As shown by the stippled bars, TEF-1 overexpression tended to transactivate the −215-bp βMyHC promoter, although the effect did not reach significance by ANOVA (P=0.06). In contrast, RTEF-1 significantly transactivated the −215-bp βMyHC promoter in a dose-dependent manner (P<0.001). The α1-adrenergic agonist phenylephrine (100 μmol/L) increased the −215-bp βMyHC promoter activity (P<0.001), as reported previously.4 There were no interactions between TEF-1 or RTEF-1 overexpression and the presence of phenylephrine, suggesting that these transcription factors do not potentiate the α1-adrenergic response of the minimal −215-bp βMyHC promoter.
Response of the −215-bp βMyHC Promoter With a Mutated M-CAT Element
To test whether transactivation of the −215-bp βMyHC promoter by TEF-1 or RTEF-1 overexpression was dependent on an intact M-CAT element, a −215-bp βMyHC promoter with a mutated M-CAT element (−215-bp mM-CAT βMyHC) was analyzed in the same way. This reporter was previously called mutation D.4
As shown in Figure 1B⇑, TEF-1 or RTEF-1 overexpression did not transactivate the −215-bp mM-CAT βMyHC promoter but, instead, significantly interfered with promoter activity (P<0.001 and P<0.02, respectively). Thus, transactivation of the wild-type −215-bp βMyHC promoter by TEF-1 and RTEF-1 requires binding of TEF-1 and RTEF-1 to an intact M-CAT element. However, treatment with 100 μmol/L phenylephrine significantly increased the −215-bp mM-CAT βMyHC promoter activity (P<0.001), suggesting that other transcription factors also participate in the α1-adrenergic response. TEF-1 or RTEF-1 overexpression did not potentiate the α1-adrenergic response to phenylephrine.
Effects of TEF-1 and RTEF-1 Overexpression on the Basal and α1-Adrenergic–Stimulated Activity of the −113-bp SKA Promoter
Unlike the effect on the −215-bp βMyHC promoter, TEF-1 overexpression interfered with the activity of the −113-bp SKA promoter (Figure 2A⇓, P<0.002). In contrast, RTEF-1 overexpression produced a dose-dependent transactivation of the −113-bp SKA promoter (P<0.001). No squelching was observed even at the highest dose of RTEF-1. The −113-bp SKA promoter activity was increased by phenylephrine stimulation (P<0.001). There was no interaction between TEF-1 overexpression and the phenylephrine response of the −113-bp SKA promoter. In contrast, there was a significant interaction between RTEF-1 and phenylephrine (P<0.02), suggesting that RTEF-1 potentiated the effects of phenylephrine.
Response of the −113-bp SKA Promoter With a Mutated M-CAT
An M-CAT mutation in the −113-bp SKA promoter was tested in the same way. TEF-1 overexpression did not significantly affect the −113-bp mM-CAT SKA promoter (Figure 2B⇑). In contrast, RTEF-1 overexpression transactivated the −113-bp mM-CAT SKA promoter in a dose-dependent manner (P<0.001). Thus, unlike the βMyHC promoter, transactivation of the −113-bp SKA promoter by RTEF-1 was independent of the mutated M-CAT. The −113-bp mM-CAT SKA promoter was significantly activated by phenylephrine stimulation (P<0.001). There was no interaction between TEF-1 overexpression and the phenylephrine response. In contrast, RTEF-1 and phenylephrine had a synergistic effect in activating the −113-bp mM-CAT SKA promoter (P<0.001).
Comparison of SKA and βMyHC Promoter M-CATs by Competition Gel Shift Assay
As previously reported,4 5 we also observed that the −215-bp βMyHC promoter did not require its M-CAT for basal activity, whereas the −113-bp SKA promoter showed a marked decrease in basal activity when it carried a mutated M-CAT (data not shown). To understand the basis for this difference, the affinity of the M-CAT elements from these 2 promoters was compared by competition gel shift assay using nuclear extracts overexpressing TEF-1 and RTEF-1 (Figure 3⇓). The shifted complex obtained for the βMyHC M-CAT was weaker than for the SKA M-CAT and was completely competed by a 50-fold molar excess of the SKA M-CAT oligonucleotide. Conversely, binding to the SKA M-CAT was not fully competed by the βMyHC M-CAT. The high-affinity GTIIC oligonucleotide11 competed both the SKA and βMyHC M-CAT elements equally, whereas the lower-affinity SPH oligonucleotide11 competed the βMyHC M-CAT completely but only partially competed for binding to the SKA M-CAT. Therefore, the relative affinities of the M-CAT containing oligonucleotides to bind to TEF-1 or RTEF-1 were as follows: βMyHC M-CAT<SPH<SKA M-CAT<GTIIC. Thus, this difference in M-CAT affinities can explain why the basal activities of the βMyHC and SKA promoters are affected differently by the M-CAT mutations. Moreover, this may also explain why TEF-1 tended to transactivate the βMyHC promoter but interfered with the SKA promoter.
Effects of TEF-1 and RTEF-1 Overexpression on the Basal and α1-Adrenergic–Stimulated Activity of the −3.3-kb βMyHC Promoter
The effects of TEF-1 and RTEF-1 overexpression on minimal βMyHC and SKA promoters might have reflected an artifact of using minimal promoters, each carrying a single M-CAT and few other cis-regulatory elements. To determine what effect TEF-1 and RTEF-1 overexpression would have in the context of additional regulatory sequences, a βMyHC promoter/reporter construct with 3300 bp of promoter and 5′ flanking sequence was used (Figure 4⇓). Phenylephrine increased the activity of the −3.3-kb βMyHC promoter (P<0.001). TEF-1 overexpression (100 ng) had no effect. In contrast, RTEF-1 significantly potentiated the α1-adrenergic response (RTEF*PHE). Although RTEF-1 had an effect overall (RTEF), post hoc comparison showed that it did not have a significant effect in the absence of phenylephrine. The synergistic effect of RTEF-1 and phenylephrine was abolished when cardiac myocytes were treated with the α1-adrenergic antagonist prazosin (10 μmol/L, n=4). This result suggests that RTEF-1 is activated by phenylephrine stimulation, either through an increase in the ability of RTEF-1 to access regulatory cis elements or by altering its interaction with other transcriptional cofactors.
The present study has examined whether human TEF-1 and RTEF-1 overexpression would influence the activity of the βMyHC and SKA promoters and alter their M-CAT–dependent α1-adrenergic response. If TEF-1 and RTEF-1 were functionally redundant, they should have had the same effect. The principal findings of the present study were as follows. First, the 2 promoters responded differently to TEF-1 and RTEF-1 overexpression: TEF-1 tended to transactivate the −215-bp βMyHC promoter with a low-affinity M-CAT but squelched the SKA promoter with a high-affinity M-CAT. In contrast, RTEF-1 transactivated both promoters. Second, RTEF-1 potentiated the effect of phenylephrine in activating the −113-bp SKA promoter and the −3.3-kb βMyHC promoter, whereas TEF-1 did not. These results demonstrate that TEF-1 and RTEF-1 are functionally different and suggest that RTEF-1 is important in mediating the α1-adrenergic activation of fetal genes in cardiac myocytes.
The Minimal Promoters Respond Differently to TEF-1 and RTEF-1 Overexpression
Until recently, attempts to demonstrate the transactivation of M-CAT–dependent promoters by TEF-1 overexpression have typically resulted in squelching, presumably because a limiting coactivator is sequestered by the overabundance of TEF-1.11 25 26 27 28 29
In the present study, TEF-1 overexpression tended to transactivate the −215-bp βMyHC promoter but interfered with the −113-bp SKA promoter in a dose-dependent manner, suggesting that TEF-1 levels exceeded the functional capacity of the SKA promoter. The competition gel shifts showed that the SKA M-CAT is a higher-affinity binding site than is the βMyHC M-CAT. Thus, squelching appears to require saturation of the M-CAT site, because the dose of TEF-1 that squelched the SKA promoter did not interfere with the βMyHC promoter.
In contrast, RTEF-1 transactivated both the −215-bp βMyHC and the −113-bp SKA promoters. For the −215-bp βMyHC promoter, transactivation was M-CAT site dependent, since the promoter with a mutated M-CAT was no longer activated by RTEF-1. In the case of SKA, transactivation was independent of the mutated M-CAT, since the −113-bp SKA mM-CAT promoter could still be transactivated by RTEF-1. In a gel shift assay, the mutated M-CAT element failed to compete for binding of RTEF-1 to the wild-type SKA M-CAT (data not shown). Thus, transactivation of the −113-bp SKA mM-CAT promoter by RTEF-1 was not due to residual binding at the mutated site and might reflect a difference in promoter-specific cofactors. That RTEF-1 overexpression did not squelch the SKA promoter suggests that intracellular RTEF-1 levels might not have been sufficiently elevated to become saturating. If the relative TEF-1 and RTEF-1 mRNA levels in human myocardium are an indication of the endogenous protein levels, TEF-1 should be more abundant than RTEF-1 (see Figure 2⇑ in Reference 2222 ). In addition, RTEF-1 might interact with different transcriptional cofactors that are not as limiting as the TEF-1 cofactors.
The different effects of TEF-1 and RTEF-1 overexpression on the βMyHC and SKA promoters might also be related to the different M-CAT elements. In actively transcribed SV40 late promoter, the lower-affinity SPH sites were essentially unoccupied, whereas the high-affinity GTIIC site was blocked from PvuII digestion in 36% of the complexes.30 31 Since both the low-affinity and high-affinity sites are required for high levels of transcription from the SV40 late promoter, these results suggest that there may be functionally different M-CAT binding factors at each of these sites. In the context of the SKA and βMyHC promoters, occupancy at an M-CAT site may change depending on the levels of M-CAT binding factors present or the activation state of the cardiac myocyte. Whatever the mechanism, TEF-1 and RTEF-1 are clearly different. Identifying their respective cofactors should shed light on these functional differences.
The α1-Adrenergic Response
In healthy adult rat myocardium, the αMyHC gene is the isoform normally expressed. In studies using cultured fetal rat cardiac myocytes, β-adrenergic stimulation was mimicked by elevating intracellular cAMP levels that, in turn, activated the αMyHC gene, presumably via a cAMP-dependent protein kinase (PKA) mechanism.32 A hybrid M-CAT/E-box cis element was shown to be required for the basal and cAMP-inducible expression of the αMyHC promoter.33 The recent finding that TEF-1 transactivates the αMyHC promoter in cardiac myocytes, by recruiting the basic helix-loop-helix leucine zipper protein Max as a coactivator,34 suggests that TEF-1 function might depend on promoter-specific cofactors. Although in the present study TEF-1 overexpression had a modest transactivating effect on the minimal βMyHC promoter, TEF-1 tended to lower the activity of the −3.3-kb βMyHC promoter and significantly interfered with the −113-bp SKA promoter, presumably by squelching a limiting cofactor. TEF-1, together with Max, may be important in maintaining αMyHC gene expression in healthy cardiac myocytes but not in activating the βMyHC gene.
In contrast, the βMyHC and SKA genes are activated in the fetal heart, during cardiac hypertrophy caused by aortic coarctation in the adult rat, and during α1-adrenergic–induced hypertrophy of cultured neonatal rat cardiac myocytes. The present study supports the conclusion that the M-CAT elements of the −215-bp βMyHC and the −113-bp SKA promoters are required for part of the α1-adrenergic response,4 5 since the M-CAT mutations lowered the responsiveness of both promoters. Recently, the α1-adrenergic induction of the brain natriuretic peptide gene in cardiac myocytes was also shown to require an M-CAT element.35 PKC36 and MAP kinase37 are implicated in the α1-adrenergic response in cardiac myocytes. Thus, how can M-CAT elements mediate both a PKA-dependent activation of the αMyHC promoter and a PKC/MAP kinase–dependent activation of the βMyHC and SKA promoters? The answer must lie in the different factors that bind to the M-CAT elements. Because the α1-adrenergic responses of −3.3-kb βMyHC and the −113-bp SKA promoters were potentiated by RTEF-1, but unaffected by TEF-1 overexpression, these results provide strong support to the suggestion that RTEF-1, but not TEF-1, is important in mediating the α1-adrenergic response.
The different effects of TEF-1 and RTEF-1 overexpression on the α1-adrenergic response of the βMyHC and SKA promoters must reflect differences in how TEF-1 and RTEF-1, or their associated cofactors, are modified by α1-adrenergic signaling. TEF-1 and RTEF-1 are identical in their DNA binding domains and highly conserved in their carboxyl-terminal activation domains. However, they diverge in sequences flanking the TEA DNA binding domain that differ in their content of potential consensus PKC and MAP kinase sites. To what extent these divergent sequences confer functional differences and whether direct phosphorylation of RTEF-1 (or other TEF-1–related factors) is induced by α1-adrenergic stimulation are not known, although RTEF-1 is known to be basally phosphorylated in vivo in the chicken.23 What is clear is that RTEF-1 can mediate the α1-adrenergic response in cultured cardiac myocytes. Whether RTEF-1 functions the same way in the whole heart awaits a transgenic strategy to test its function at the organismic level.
The TEF-1 Multigene Family
The Table⇓ lists the 4 vertebrate members of the TEF-1 multigene family: TEF-1, the first to be cloned11; RTEF-1, the second member cloned38; a third, named ETF39; and a fourth called DTEF-1.40 Others have cloned orthologous homologues (same genes, different species; see Reference 2222 for phylogenetic analysis) with 16 different names. Thus, to limit confusion, we propose to retain the names listed in boldface in the Table⇓.
It is now apparent that other members of the TEF-1 multigene family are also expressed in the heart. The recent observations that the mouse DTEF-143 44 and ETF41 genes are expressed in adult heart will require further investigation to determine their involvement in M-CAT–dependent gene regulation. Although the isoform of human TEF-1 tested in the present study did not augment the α1-adrenergic induction of the −3.3-kb βMyHC and SKA promoters, additional alternative splicing isoforms of TEF-1 might also participate. Recent reports have suggested that as many as 7 alternatively spliced isoforms of TEF-1, which differ in their transactivating properties, exist in neonatal rat cardiac myocytes.45 46
Taken together, the results of the present study have demonstrated functional differences between 2 members of the TEF-1 family. TEF-1 did not have an effect on the α1-adrenergic response, although TEF-1 appears to be important in maintaining gene expression in normal cardiac myocytes. In contrast, RTEF-1 potentiated the α1-adrenergic response, suggesting that it mediates α1-adrenergic receptor–induced activation of the fetal gene program in hypertrophic cardiac myocytes.
Selected Abbreviations and Acronyms
|βMyHC||=||β-myosin heavy chain|
|ETF||=||embryonic TEA domain–containing factor|
|PKA, PKC||=||protein kinase A and C|
|RSV||=||Rous sarcoma virus|
|RTEF-1||=||related to TEF-1|
|TEF-1||=||transcriptional enhancer factor-1|
This study was supported by grants to Dr Stewart from the American Heart Association, Pennsylvania Affiliate, Inc, and from the National Institutes of Health (R29-HL-57211-01). Dr Kubota is the recipient of a Japan Heart Foundation and Bayer Yakuhin Research Grant Abroad, and Dr Chen is the recipient of a National Research Service Award from the National Institutes of Health (F32-HL-09673-01). We thank Prof Pierre Chambon (IGBMC-LGME-U.184-ULP) for the human TEF-1 cDNA and the pXJ40 expression vector and Dr Paul C. Simpson for the βMyHC and SKA promoter constructs. We are also grateful to our colleagues for critical review of the manuscript.
- Received February 20, 1998.
- Accepted April 17, 1998.
- © 1998 American Heart Association, Inc.
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