- Email: [email protected]
α1-Adrenergic activation of the cardiac ankyrin repeat protein gene in cardiac myocytes
Gene 297 (2002) 1–9 www.elsevier.com/locate/gene
a1-Adrenergic activation of the cardiac ankyrin repeat protein gene in cardiac myocytes Tomoji Maeda a, Jorge Sepulveda b, Hsiao-Huei Chen c, Alexandre F.R. Stewart a,* a
Cardiovascular Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA c Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15213, USA b
Received 19 June 2002; received in revised form 30 July 2002; accepted 20 August 2002 Received by A.J. van Wijnen
Abstract Cardiac ankyrin repeat protein (CARP) is a nuclear transcription cofactor that is activated by multiple signaling pathways in hypertrophic cardiac myocytes. Since CARP has been reported to be a transcriptional co-repressor, its activation during hypertrophy might contribute to the deregulation of gene expression leading to heart failure. Here, we found that a1-adrenergic signaling activates CARP mRNA expression in rat cardiac myocytes. To examine how a1-adrenergic signaling activates the CARP gene, a 660 bp fragment of the mouse CARP promoter was cloned. Previous reports suggested that the mouse CARP promoter was dependent on the GATA4 transcription factor whereas the human CARP promoter was dependent on transcriptional enhancer factor-1 (TEF-1). TEF-1 and GATA4 transcription factors, known mediators of a1-adrenergic signaling, bound to the mouse CARP promoter at several sites as determined by gel mobility shift assays. These sites are highly conserved between the mouse and human promoters, suggesting that they are functionally important in both. Mutation analysis showed that binding of TEF-1 factors is required for basal activity of the CARP promoter in cardiac myocytes. However, over-expression of TEF-1 factors could not potentiate the response of the CARP promoter to a1-adrenergic stimulation. On the other hand, the a1-adrenergic response was potentiated by GATA4 over-expression. Taken together, our results demonstrate that a1-adrenergic signaling regulates CARP expression in cardiac myocytes, in part through the transcription factor GATA4. q 2002 Elsevier Science B.V. All rights reserved. Keywords: TEF-1; RTEF-1; GATA4; Cardiac myocytes; DNA binding; Transcription factors
1. Introduction The cardiac ankyrin repeat protein (CARP) is an early marker of cardiac myogenic differentiation, expressed at high levels in the heart during embryonic and fetal development and at progressively lower levels in the neonate and adult (Jeyaseelan et al., 1997; Zou et al., 1997). CARP contains four ankyrin repeats thought to play a role in protein/protein interaction. CARP is not known to bind to DNA, but rather is thought to modulate transcription as a cofactor. CARP was isolated from the heart as a cofactor of YB-1, a transcription factor that is required for the activation of the myosin light chain 2v gene during cardiac
Abbreviations: CARP, cardiac ankyrin repeat protein; MCAT, muscle CAT element; RTEF-1, related to transcriptional enhancer factor-1; TEF-1, transcriptional enhancer factor-1 * Corresponding author. Cardiovascular Institute, School of Medicine, University of Pittsburgh, BST 1704.3, 200 Lothrop Street, Pittsburgh, PA 15213, USA. Tel.: 11-412-383-9761; fax: 11-412-383-8997. E-mail address: [email protected] (A.F.R. Stewart).
myogenesis (Zou et al., 1997). Although it is not clear how CARP regulates YB-1 function during cardiac fetal development, CARP over-expression in postnatal cardiac myocytes generally inhibits the expression of cardiac genes and is thought to act as a transcriptional co-repressor (Jeyaseelan et al., 1997; Zou et al., 1997). In addition to its function as a transcription regulator in the nucleus, CARP is known to interact with cytoplasmic proteins. CARP interacts with myopalladin and participates in the maintenance of sarcomere integrity (Bang et al., 2001). Mice with a null mutation in the muscle LIM protein (MLP) that disrupts myocyte sarcomere integrity develop a dilated cardiomyopathy and express elevated levels of CARP in the heart (Arber et al., 1997). When skeletal muscle is passively stretched due to denervation, CARP expression is also activated at the ends of myocytes near the myotendinous junction (Baumeister et al., 1997), where new muscle proteins are known to be synthesized in response to stretch (Dix and Eisenberg, 1990). CARP is known to shuttle from the cytoplasm to the nucleus (Zou et al., 1997). Through its interaction with myopalladin, CARP
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00924-1
2
T. Maeda et al. / Gene 297 (2002) 1–9
has been suggested to form a functional bridge between the sarcomere and transcription regulation (Bang et al., 2001). CARP expression is activated during cardiac hypertrophy in vivo (Kuo et al., 1999; Aihara et al., 2000a; Redfern et al., 2000). In cultured cardiac myocytes, the CARP promoter is activated by p38 kinase (Aihara et al., 2000a). p38 kinase is a known mediator of several hypertrophic signaling pathways, including a1-adrenergic signaling. Since a1-adrenergic stimulation of cultured cardiac myocytes is a well-characterized in vitro model of hypertrophy associated with the transcriptional activation of several genes, it is likely that a1-adrenergic signaling regulates CARP promoter activity. The CARP gene is a downstream target of the cardiacrestricted homeobox gene Nkx2.5 (Zou et al., 1997; Takimoto et al., 2000). Nkx2.5 interacts with the zinc finger transcription factor GATA4 and activates the mouse CARP promoter through a proximal GATA cis-element (Kuo et al., 1999). GATA4 is known to activate the endothelin-1 (Morimoto et al., 2000) and brain natriuretic peptide (Liang et al., 2001) promoters in response to a1-adrenergic stimulation in cardiac myocytes. Thus, the presence of at least one GATA element in the mouse CARP promoter suggests that GATA factors might participate in CARP activation during myocyte hypertrophy induced by a1-adrenergic stimulation. The activity of the human CARP promoter depends on an MCAT cis-element (Aihara et al., 2000a). It is not known whether MCAT elements play a role in regulating the mouse CARP promoter. MCAT elements, 5 0 -CATDYY-3 0 , are the cognate binding site of the transcriptional enhancer factor-1 (TEF-1) family of transcription factors and are required for transcriptional activation of many cardiac and skeletal muscle specific genes (Larkin et al., 1996). TEF-1 factors have also been implicated in the activation of fetal genes during cardiac myocyte hypertrophy. One member of the Table 1 Oligonucleotides used in gel mobility shift assays and for site-directed mutagenesis Oligonucleotide
Sequence a
CARP MCAT1 CARP MCAT2 CARP MCAT3 CARP MCAT4 SKA MCAT
5 0 -GAATTTTCATTCCAGACTTAG-3 0 5 0 -AGAGGGTCATTCCTTTGGCAG-3 0 5 0 -TGACTCGCATTGCTGAGCGGT-3 0 5 0 -CCTGTGACATGCCATCGTACC-3 0 5 0 -GCAGCAACATTCTTCGGGGC-3 0
MCAT2mut
5 0 -GAAATGTGAGAGGGTCCTTCCTTTGGCAGTGACC-3 0
CARP GATA1 CARP GATA2 CARP GATA3 CARP GATA4 rBNP GATA
5 0 -CTGGGTGAGATAATCTTCCA-3 0 5 0 -CACTTACAGCCATATCTTTCTGATAACTGG-3 0 5 0 -GGAGTGCTGATAAGTCCAGT-3 0 5 0 -TCCAAACAGATAGAGACAAG-3 0 5 0 -CACTTGATAACAGAAAGTGATAACTCT-3 0
GATA1mut
5 0 -CCAGGCTGGAAGATTACTTCACCCAGCCCTAGC-3 0
a To facilitate visualization, the strand containing the consensus transcription factor binding site (underlined) is shown for all oligonucleotide pairs.
TEF-1 family, related to TEF-1 (RTEF-1), mediates the a1-adrenergic induction of fetal genes in neonatal rat cardiac myocytes in culture (Ueyama et al., 2000). Thus, if MCAT elements are involved in the activation of the CARP promoter during hypertrophy induced by a1-adrenergic signaling, RTEF-1 is a potential mediator of this effect. The mechanisms of transcriptional regulation of the mouse CARP promoter remain poorly characterized, despite the use of this promoter to drive cardiac fetal specific transgene expression (Kuo et al., 1999) and to selectively activate therapeutic transgenes in hypertrophic hearts (Manning et al., 2000). To better understand the regulation of the CARP promoter in response to hypertrophic signals, we examined its modulation by GATA4 and TEF-1 transcription factors in response to a1-adrenergic stimulation in cardiac myocytes. 2. Materials and methods 2.1. Culture and transfection of neonatal rat cardiac myocytes Neonatal rat cardiac myocytes were isolated from 1-dayold Sprague–Dawley rats, cultured, and transfected as described previously (Stewart et al., 1998). The Institutional Animal Care and Use Committee of the University of Pittsburgh approved the procedures followed for the culture of neonatal rat cardiac myocytes. 2.2. Northern blot analysis Total RNA was prepared from freshly isolated cardiac myocytes (T0) or myocytes plated at a density of 4.5 £ 10 6 cells per 100 mm dish in medium supplemented with 5% bovine calf serum. Myocytes were either left untreated, or treated with 12.5 mmol/l prazosin (an a1-adrenergic antagonist), 12.5 mmol/l timolol (a b-adrenergic antagonist) or cotreated with 12.5 mmol/l each of prazosin and timolol in separate dishes. Medium was changed once and myocytes were harvested after 48 h in culture. Northern blot analysis was carried out using a 40-mer oligonucleotide complementary to sequence in the 3 0 untranslated region of the rat CARP cDNA, 5 0 -CCCATTGCCTACTCCCTCCATCCCCCTCCACACACGCTCC-3 0 . 2.3. Cloning of the mouse CARP promoter The mouse CARP promoter was amplified from published sequences in two fragments using polymerase chain reaction (PCR) and the 5 0 oligonucleotides: CARP 660, 5 0 -CGACGCGTGAACACGGTGAGCCTCTGGTG3 0 and CARP 295, 5 0 -CGACGCGTGTGTGCAATATTAACAGGC-3 0 each containing an MluI restriction site and the 3 0 oligonucleotide CARP3, 5 0 -GAAGATCTCATGTTGGCAGCCGTGAGTC-3 0 containing a BglII restriction site. The CARP promoter fragments were subcloned into the pGL2-Basic vector (Promega, Madison, WI) upstream of
T. Maeda et al. / Gene 297 (2002) 1–9
the firefly luciferase reporter. The mouse CARP promoter was sequenced on both strands and the sequence was submitted to GenBank (Accession number: AF478692). 2.4. Site-directed mutagenesis Site-directed mutagenesis of the proximal GATA element (GATA1) and an MCAT element (MCAT2) shown to mediate p38 MAPK signaling in the human CARP promoter (Aihara et al., 2000a) was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the oligonucleotides shown in Table 1.
3
mRNA expression (Fig. 1B). Low levels of CARP mRNA were detected in freshly isolated cardiac myocytes (T0), but CARP expression was highly elevated after 48 h in 5% bovine calf serum. When serum was withdrawn at 24 h and replaced by serum-free medium (TIBSA), CARP mRNA levels fell approximately 30% over the next 48 h (n ¼ 2). Catecholamines in bovine serum could explain part of the serum response. CARP activation by serum was blunted by the a1-adrenergic receptor antagonist prazosin, but not by the b-adrenergic receptor antagonist timolol (Fig.
2.5. Gel mobility shift assay Oligonucleotides to the sense and antisense strands containing putative TEF-1 and GATA4 binding sites were synthesized by Operon Technologies Inc. (Alameda, CA) and purified by high-pressure liquid chromatography. Sequences are shown in Table 1 and mapped on the CARP promoter sequence in Fig. 2. Gel mobility shift assays were carried out as described previously (Stewart et al., 1998). The TEF-1 specific antibody (BD Transduction Laboratories, San Diego, CA) and the GATA4 specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were obtained commercially. 2.6. Luciferase assay After transfection with various expression vectors and CARP promoter/luciferase constructs (see figure legends), cardiac myocytes were treated for 24 h with either a vitamin C vehicle (20 mmol/l) or phenylephrine (20 mmol/l). Myocytes were harvested as described previously (Stewart et al., 1998) and the luciferase activity in myocyte extracts was measured using 0.3 mg/ml d-luciferin (sodium salt, Analytical Luminescence Laboratory, Ann Arbor, MI) on a luminometer (LB501, Berthold Systems, Inc., Aliquippa, PA). 2.7. Statistical analysis An unpaired two-tailed t-test was used for statistical analysis, with P , 0:05 considered significant. Data are presented as mean ^ standard error. 3. Results 3.1. a 1-Adrenergic signaling activates CARP expression CARP mRNA expression is activated three fold relative to vehicle control by treatment of serum-free cultures with the a1-adrenergic receptor agonist phenylephrine in the presence of the b-adrenergic receptor antagonist timolol (n ¼ 3, normalized to G3PDH, P , 0:05, Fig. 1A). Neonatal rat cardiac myocytes are typically cultured in serum on the first day, a treatment that markedly induces CARP
Fig. 1. CARP mRNA is induced in cultured neonatal rat cardiac myocytes. (A) Northern blot of RNA from cardiac myocytes cultured in serum-free medium treated with vehicle control (VC) or with the a1-adrenergic agonist phenylephrine (PE, 20 mmol/l) in the presence of the b-adrenergic-selective antagonist timolol (10 mmol/l). CARP expression was activated three fold relative to control (n ¼ 3). (B) Northern blot of RNA isolated from neonatal rat cardiac myocytes at the time of plating (T0) or after 48 h of culture in medium supplemented with 5% bovine calf serum (BCS) or serum-free medium supplemented with transferrin, insulin and bovine serum albumin (TIBSA). (C) Northern blot of RNA isolated from cardiac myocytes cultured in 5% BCS treated with the a1-adrenergic-selective antagonist prazosin (Pra, 12.5 mmol/l), the b-adrenergic-selective antagonist timolol (Tim, 12.5 mmol/l), or a combination of adrenergic antagonists (Pra/Tim) for 24 h. Prazosin partially blocked the expression of CARP mRNA whereas timolol did not (n ¼ 3). All blots were probed sequentially for CARP and where applicable, for b-actin (which cross-hybridizes preferentially with a-skeletal actin, a-sk actin), mouse atrial natriuretic factor (ANF), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) to control for loading.
4
T. Maeda et al. / Gene 297 (2002) 1–9
Fig. 2. The mouse and human proximal CARP promoters are highly conserved. A 660 bp fragment of the mouse CARP (mCARP) promoter was amplified by PCR from published sequences and aligned to the sequence of the human CARP (hCARP) promoter (lower sequence, GenBank 16156721). Putative regulatory elements are identified by open boxes. Gray-filled boxes identify MCAT and GATA elements that were functional in a gel shift assay, whereas dashed-line boxes identify putative MCAT and GATA sites that did not produce a shift. Sequence identity is indicated by dots and gaps are shown as dashes. Only the mouse sequence is numbered and the mouse transcription start site is indicated by an arrow.
1C). A 50% reduction in CARP expression was seen with prazosin. ANF and skeletal a-actin are two other a1-adrenergic-responsive transcripts that were also downregulated by prazosin treatment of cardiac myocyte cultures in serum, whereas glyceraldehyde 3-phosphate dehydrogenase (G3PDH) levels remained unchanged (n ¼ 3, Fig. 1C). Taken together, these results support a role for a1-adrenergic signaling in CARP activation. 3.2. Functional TEF-1 and GATA4 sites in the CARP promoter To characterize the mechanism of a1-adrenergic receptor mediated activation of CARP at the transcriptional level, mouse CARP promoter constructs driving firefly luciferase were synthesized by PCR from published sequences (Baumeister et al., 1997; Zou et al., 1997). The CARP promoter sequence is highly conserved between the mouse and human (78% identity) and contains several consensus TEF-1 (MCAT) and GATA4 binding sites (Fig. 2). Because TEF-1 and GATA factors have been implicated in mediating a1-adrenergic induced gene activation in cardiac myocytes, we determined whether the putative sites are functional using a gel mobility shift assay. Oligonucleotides corresponding to TEF-1 and GATA4 sequences from the
mouse CARP promoter (Table 1) were tested in competitive gel shift assays and specific complexes were supershifted with TEF-1 and GATA4 specific antibodies (Fig. 3). Thus, four of seven putative TEF-1 binding sites and four of five putative GATA binding sites were found to bind to TEF-1 and GATA factors, respectively. Two of the four MCAT sites, MCAT1 and MCAT3, bound to TEF-1 factors with lower affinity than MCAT2 or MCAT4 (Fig. 3A). Binding to all of the CARP MCAT sites could be specifically competed by an oligonucleotide containing an MCAT element from the skeletal a-actin promoter (SKA), and a TEF-1 specific antibody supershifted the MCAT-binding factor (MCBF) in each case. The TEF-1 antibody supershift provides the first direct evidence that TEF-1 regulates the CARP promoter. The MCAT sites are conserved between the mouse and human CARP promoters (Fig. 2). Three of the GATA elements produced a robust shift, whereas the most distal GATA element (GATA4) gave only a modest shift (Fig. 3B). This GATA element is not conserved between mice and humans, where the terminal purine of the consensus 5 0 -WGATAR-3 0 is a pyrimidine in the human sequence (Fig. 2). Binding to all of the GATA elements was specific and was selectively competed by an oligonucleotide containing two GATA elements from the
T. Maeda et al. / Gene 297 (2002) 1–9
5
adrenergic signaling was tested in transient transfection assays. 3.4. RTEF-1 squelches the mouse CARP promoter
Fig. 3. The CARP promoter contains multiple functional MCAT and GATA elements. Gel mobility shift assays using the radiolabeled oligonucleotides shown in Table 1 were carried out to test for specific binding to putative MCAT and GATA sequences. (A) To test for MCAT binding, the MCAT element of the a-skeletal actin promoter was used as a specific competitor. The TEF-1 specific antibody was used to supershift (SS) the MCAT-binding factor (MCBF). An oligonucleotide with a single point mutation in MCAT2 (M2m) does not bind to TEF-1. (B) To test for GATA binding, a known GATA binding oligonucleotide corresponding to sequences in the rat b-type natriuretic peptide (BNP) promoter was used as a specific competitor. The shifted complex (S) was further supershifted with a GATA4 specific antibody. An oligonucleotide containing a dual mutation in the proximal GATA element (G1M) does not bind GATA4.
brain natriuretic peptide promoter and could be supershifted with a GATA4 specific antibody. Given the high degree of sequence conservation between the mouse and human CARP promoters and the demonstration that the MCAT and GATA sites are bound by TEF-1 and GATA transcription factors, our results suggest that these sites are functionally important in both promoters. 3.3. The mouse CARP promoter is activated by a 1adrenergic stimulation Both the 660 bp (Fig. 4A) and the 295 bp (Fig. 4B) CARP promoter fragments were activated by phenylephrine. This response to a1-adrenergic stimulation was greater for the 660 bp promoter fragment than for the 295 bp fragment. Nonetheless, the sequences contained within the 295 bp fragment are sufficient to respond to a1-adrenergic stimulation. Since the CARP promoter contains functional TEF-1 and GATA4 binding sites, the effect of these transcription factors on CARP promoter activity and induction by a1-
The transcription factor RTEF-1 was previously shown to augment the a1-adrenergic response of the fetal a-skeletal actin and b-myosin heavy chain promoters (Stewart et al., 1998). Thus, we tested the dose response of the CARP promoter to increasing amounts of the RTEF-1 expression vector and compared the response to equal amounts of the empty expression vector pXJ40. When compared to pXJ40, RTEF-1 produced a dose-dependent inactivation of the CARP promoter. At the lowest dose of the expression vector tested (1 ng), RTEF-1 over-expression slightly increased the a1-adrenergic response of the CARP promoter but at higher doses RTEF-1 interfered with the a1-adrenergic response (Fig. 4A). A similar dose-dependent inactivation was also seen with TEF-1 and DTEF-1 (divergent from transcriptional enhancer factor-1) over-expression (data not shown). Squelching by TEF-1 over-expression has been observed for a number of MCAT-dependent promoters (Stewart et al., 1994, 1998; Gupta et al., 1996) and is believed to result from the tritration of a limiting coactivator. When the MCAT2 element of the mouse CARP promoter was mutated so that it no longer bound to TEF-1 factors (Fig. 3B), the basal promoter activity was significantly lowered (Fig. 4C) but this site does not appear to be essential for the a1-adrenergic response. We chose to mutate this site because it was previously shown to be required for the p38 kinase-dependent activation of the human CARP promoter (Aihara et al., 2000a). Although p38 kinase is a known mediator of a1-adrenergic signaling, our results suggest that this MCAT site is not the major target of the a1-adrenergic response. Therefore, additional MCAT elements of the proximal promoter (MCAT1 and MCAT3), or other ciselements, are likely to contribute to the a1-adrenergic response. Given that TEF-1 factors did not significantly augment the response to a1-adrenergic stimulation, it is not clear whether additional MCAT element mutations would have had an effect. 3.5. GATA4 augments the a 1-adrenergic response of the mouse CARP promoter In contrast to the effect of RTEF-1, GATA4 significantly increased the a1-adrenergic response of the 660 bp CARP promoter when compared to the CMV expression vector pCG (Fig. 4A). Interestingly, a similar effect of GATA4 was not observed for the 295 bp CARP promoter (Fig. 4B), suggesting that distal GATA elements present in the 660 bp promoter are required for the a1-adrenergic response and that the proximal GATA element is not sufficient. To test whether the proximal GATA element contributes to the a1-adrenergic response, the proximal GATA element was mutated so that it no longer binds GATA4 (Fig. 3B). Muta-
6
T. Maeda et al. / Gene 297 (2002) 1–9
Fig. 4. GATA4 augments the a1-adrenergic response of the CARP promoter. Schematic representations of the CARP promoter constructs and the location of GATA and MCAT elements are shown above each data set. Mutated elements in the proximal promoter are crossed out. (A) A CARP promoter construct with 660 bp of the proximal promoter responds to the a1-adrenergic-selective agonist phenylephrine (PE, 20 mmol/l). PE does not activate the promoter-less luciferase vector (data not shown). Fold activities are expressed relative to the activity of the CARP promoter in the presence of the pXJ40 CMV vector under vehicle control conditions. An asterisk indicates all values significantly different from one fold. RTEF-1 squelched the CARP promoter basal activity and also the a1-adrenergic response in a dose-dependent manner when compared to the pXJ40 vector at similar doses. In contrast, GATA4 increased the a1-adrenergic response of the 660 bp CARP promoter when compared to the pCG expression vector (n ¼ 4, P , 0:05). (B) A 295 bp fragment of the CARP promoter was PE-responsive but was not activated by GATA4, suggesting that the proximal GATA element (G1) alone is not sufficient to mediate the effect of GATA4. (C) Mutation of MCAT2 (M2) lowers basal CARP promoter activity (n ¼ 4, P , 0:01), whereas mutation of GATA1 (G1) has no effect. Neither mutation affected the PE response.
T. Maeda et al. / Gene 297 (2002) 1–9
tion of the proximal GATA element had no effect on the basal activity or the a1-adrenergic response of the 295 bp promoter (Fig. 4B) or the 660 bp promoter (data not shown). Thus, the proximal GATA element does not appear to be required. Taken together, our results demonstrate that a1-adrenergic signaling activates the CARP promoter in cardiac myocytes and that part of this response is conferred by GATA4. In addition to TEF-1 and GATA cis-elements, other sequences and their cognate transcription factors are likely to participate in the a1-adrenergic activation of the CARP promoter.
4. Discussion Activation of fetal genes in cardiac myocytes by a1-adrenergic stimulation is a well-characterized in vitro paradigm of cardiac hypertrophy. Many of the fetal genes activated during hypertrophy have been shown to require an MCAT element for their activation, including skeletal a-actin, bmyosin heavy chain, and brain natriuretic peptide (Kariya et al., 1994; Karns et al., 1995; Thuerauf and Glembotski, 1997). CARP was recently identified as another fetal gene activated during cardiac myocyte hypertrophy (Kuo et al., 1999; Aihara et al., 2000a; Redfern et al., 2000; Takimoto et al., 2000). Activation of CARP expression occurs largely at the level of transcription. A CARP promoter-dependent transgene is actively transcribed in the fetal heart (Kuo et al., 1999) and during hypertrophy (Kuo et al., 1999; Manning et al., 2000). Since CARP is activated during hypertrophy, we wondered whether a1-adrenergic signaling might activate CARP. Indeed, the a1-adrenergic-selective agonist phenylephrine elevated endogenous CARP expression in our cultures and the antagonist prazosin lowered CARP expression. In addition, the two CARP promoter fragments we tested were also activated by a1-adrenergic stimulation. Thus, we conclude that CARP is a bona fide a1adrenergic-responsive gene. The mouse CARP promoter is TEF-1-dependent. Mutation of the MCAT2 element, previously shown to reduce basal activity of the human CARP promoter (Aihara et al., 2000b), also lowered basal activity of the mouse CARP promoter. Thus, TEF-1 factors are required for CARP expression. In the human CARP promoter, mutation of the MCAT2 element reduced activation by p38 kinase and Rac1, suggesting that TEF-1 factors might mediate CARP gene activation in response to a1-adrenergic and other signaling pathways (Aihara et al., 2000a). However, mutation of the MCAT2 element did not block the a1-adrenergic activation of the mouse CARP promoter in our study, suggesting that the use of activated mutants of p38 kinase and Rac1 by Aihara et al. (2000a) might not reflect the physiological response to a1-adrenergic signaling. In addition, other MCAT sites might be involved. The MCAT2 site has a higher affinity than either the MCAT1 or MCAT3 sites
7
(Fig. 3). Low affinity MCAT sites have been implicated in the a1-adrenergic activation of the b-myosin heavy chain promoter (Kariya et al., 1994; Stewart et al., 1998) and might also be contributing to the a1-adrenergic activation of the CARP promoter. In addition, not all cardiac muscle promoters with functional MCAT sites are responsive to a1adrenergic stimulation. For example, the cardiac troponin T promoter contains two high affinity sites (Mar and Ordahl, 1988) yet is not further upregulated by a1-adrenergic stimulation (data not shown). Moreover, RTEF-1 over-expression did not further activate the CARP promoter in response to phenylephrine. Endogenous TEF-1 factors might be stimulating the CARP promoter at nearly maximal levels, so that over-expression of TEF-1 factors would titrate away limiting cofactors. In support of this squelching hypothesis, increasing doses of the RTEF-1 expression vector (1, 10 and 100 ng) progressively interfered with the a1-adrenergic activation of CARP promoter. Thus, TEF-1 factors may still contribute to a1-adrenergic activation of the mouse CARP promoter. GATA4 mediates part of the a1-adrenergic activation of CARP in cardiac myocytes. GATA4 over-expression increased the a1-adrenergic response of the 660 bp CARP promoter fragment. However, mutation of the proximal GATA element in the 295 bp CARP promoter fragment had no effect on its basal activity or its a1-adrenergic activation. Thus, the three additional GATA elements in the 660 bp promoter are likely to participate in the a1-adrenergic activation of the CARP promoter. In the case of the minimal 295 bp promoter, MCAT cis-elements and their transcription factors are likely to contribute to both the basal activity of the promoter and the a1-adrenergic response, since the proximal GATA element appears to be expendable. Interestingly, mutation of this same GATA element in the context of the 295 bp CARP promoter was shown previously to abrogate Nkx2.5 and GATA4 cooperative activation in CV1 cells (Kuo et al., 1999). This GATA element was suggested to account for the cardiac specific expression of a 295 bp CARP promoter-driven transgene (Kuo et al., 1999). However, given that we did not observe a reduction in basal activity of the 295 bp GATA1-mutant CARP promoter in cardiac myocytes, cardiac specific cofactors of TEF-1 might also account for CARP expression in the heart. Our results suggest that not all GATA sites are identical. Many GATA-dependent cardiac muscle gene promoters are not upregulated by a1-adrenergic signaling. The a-myosin heavy chain promoter is a good example of such a GATAdependent promoter (Lu et al., 1999). In addition, since GATA4 can augment the a1-adrenergic response, this would suggest that GATA4 levels in cardiac myocytes are limiting for the activation of the CARP promoter, since we did not observe a squelching effect by GATA4 over-expression. Other signaling pathways have also been implicated in the regulation of CARP expression. For example, CARP is
8
T. Maeda et al. / Gene 297 (2002) 1–9
activated by the cytokine IL1b in endothelial cells (Chu et al., 1995). In cardiac myocytes, IL1b activates the brain natriuretic peptide gene (He and LaPointe, 2000), but represses the skeletal a-actin promoter through the transcription factor YY1 (Patten et al., 1996). Following myocardial infarction, IL1b production is elevated in the heart (Yue et al., 1998). Although the ability of IL1b to activate CARP expression in cardiac myocytes has not been tested, concomitant activation of a co-repressor like CARP and down-regulation of skeletal a-actin expression by IL1b might contribute to the progression to heart failure. Transforming growth factor b (TGFb) is also known to activate the human CARP promoter in vascular smooth muscle cells. TGFb is a growth factor highly induced in myocardium during hypertrophy. TGFb activates the human CARP promoter at a 5 0 -CAGA-3 0 cis-element, presumably by signaling through the smad transcription factors (Kanai et al., 2001). However, in the mouse CARP promoter at position 2285, the sequence is 5 0 -CAGG-3 0 . Whether this mutation would impede a TGFb response of the mouse promoter is not known. TGFb can also signal through a serum response element and an MCAT site to activate the skeletal a-actin promoter in cardiac myocytes (MacLellan et al., 1994). Therefore, activation of the human CARP promoter by TGFb might also be mediated by TEF-1 factors. The high degree of sequence identity between the mouse and human CARP promoters points to an important conservation of functional cis-regulatory elements. Thus, studies that characterize the transcription regulation of the mouse CARP promoter are likely to shed light on the regulation of the human CARP promoter. It will be important to understand why CARP expression is maintained at relatively low levels in vivo. Given that multiple signals synergize to regulate the CARP promoter in transfected cells, further characterization of the signaling pathways that modulate CARP promoter function through GATA4, TEF-1 and other transcription factors is warranted. Acknowledgements A.F.R. Stewart was supported by a grant-in-aid from the American Heart Association (0050282N) and by a grant from the National Institutes of Health (R29 HL57211). References Aihara, Y., Kurabayashi, M., Saito, Y., Ohyama, Y., Tanaka, T., Takeda, S., Tomaru, K., Sekiguchi, K., Arai, M., Nakamura, T., Nagai, R., 2000a. Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter. Hypertension 36, 48–53. Aihara, Y., Kurabayashi, M., Tanaka, T., Takeda, S.I., Tomaru, K., Sekiguchi, K.I., Ohyama, Y., Nagai, R., 2000b. Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinasedependent pathways. J. Mol. Cell. Cardiol. 32, 1401–1414.
Arber, S., Hunter, J.J., Ross Jr., J., Hongo, M., Sansig, G., Borg, J., Perriard, J.C., Chien, K.R., Caroni, P., 1997. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 88, 393–403. Bang, M.L., Mudry, R.E., McElhinny, A.S., Trombitas, K., Geach, A.J., Yamasaki, R., Sorimachi, H., Granzier, H., Gregorio, C.C., Labeit, S., 2001. Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. J. Cell Biol. 153, 413–427. Baumeister, A., Arber, S., Caroni, P., 1997. Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube end compartments during muscle morphogenesis. J. Cell Biol. 139, 1231–1242. Chu, W., Burns, D.K., Swerlick, R.A., Presky, D.H., 1995. Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells. J. Biol. Chem. 270, 10236–10245. Dix, D.J., Eisenberg, B.R., 1990. Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers. J. Cell Biol. 111, 1885–1894. Gupta, M.P., Amin, C.S., Gupta, M., Hay, N., Zak, R., 1996. Cooperative activation of cardiac muscle gene expression by transcription enhancer factor-1 (TEF-1) and a basic helix-loop-helix leucine zipper protein Max. Circulation 94, I-526. He, Q., LaPointe, M.C., 2000. Interleukin-1b regulates the human brain natriuretic peptide promoter via Ca( 21)-dependent protein kinase pathways. Hypertension 35, 292–296. Jeyaseelan, R., Poizat, C., Baker, R.K., Abdishoo, S., Isterabadi, L.B., Lyons, G.E., Kedes, L., 1997. A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes. J. Biol. Chem. 272, 22800–22808. Kanai, H., Tanaka, T., Aihara, Y., Takeda, S., Kawabata, M., Miyazono, K., Nagai, R., Kurabayashi, M., 2001. Transforming growth factor-b/ Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells. Circ. Res. 88, 30–36. Kariya, K., Karns, L.R., Simpson, P.C., 1994. An enhancer core element mediates stimulation of the rat b-myosin heavy chain promoter by an a1-adrenergic agonist and activated b-protein kinase C in hypertrophy of cardiac myocytes. J. Biol. Chem. 269, 3775–3782. Karns, L.R., Kariya, K., Simpson, P.C., 1995. M-CAT, CArG, and Sp1 elements are required for a1-adrenergic induction of the skeletal aactin promoter during cardiac myocyte hypertrophy. Transcriptional enhancer factor-1 and protein kinase C as conserved transducers of the fetal program in cardiac growth. J. Biol. Chem. 270, 410–417. Kuo, H., Chen, J., Ruiz-Lozano, P., Zou, Y., Nemer, M., Chien, K.R., 1999. Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis. Development 126, 4223–4234. Larkin, S.B., Farrance, I.K.G., Ordahl, C.P., 1996. Flanking sequences modulate the cell specificity of M-CAT elements. Mol. Cell. Biol. 16, 3742–3755. Liang, Q., De Windt, L.J., Witt, S.A., Kimball, T.R., Markham, B.E., Molkentin, J.D., 2001. The transcription factors GATA4 and GATA6 regulate cardiomyocyte hypertrophy in vitro and in vivo. J. Biol. Chem. 276, 30245–30253. Lu, J.R., McKinsey, T.A., Xu, H., Wang, D.Z., Richardson, J.A., Olson, E.N., 1999. FOG-2, a heart- and brain-enriched cofactor for GATA transcription factors. Mol. Cell. Biol. 19, 4495–4502. MacLellan, W.R., Lee, T.C., Schwartz, R.J., Schneider, M.D., 1994. Transforming growth factor-b response elements of the skeletal a-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1. J. Biol. Chem. 269, 16754–16760. Manning, B.S., Shotwell, K., Mao, L., Rockman, H.A., Koch, W.J., 2000. Physiological induction of a b-adrenergic receptor kinase inhibitor transgene preserves b-adrenergic responsiveness in pressure-overload cardiac hypertrophy. Circulation 102, 2751–2757. Mar, J.H., Ordahl, C.P., 1988. A conserved CATTCCT motif is required for
T. Maeda et al. / Gene 297 (2002) 1–9 skeletal muscle-specific activity of the cardiac troponin T gene promoter. Proc. Natl. Acad. Sci. USA 85, 6404–6408. Morimoto, T., Hasegawa, K., Kaburagi, S., Kakita, T., Wada, H., Yanazume, T., Sasayama, S., 2000. Phosphorylation of GATA-4 is involved in a1-adrenergic agonist-responsive transcription of the endothelin-1 gene in cardiac myocytes. J. Biol. Chem. 275, 13721–13726. Patten, M., Hartogensis, W.E., Long, C.S., 1996. Interleukin-1b is a negative transcriptional regulator of a1-adrenergic induced gene expression in cultured cardiac myocytes. J. Biol. Chem. 271, 21134– 21141. Redfern, C.H., Degtyarev, M.Y., Kwa, A.T., Salomonis, N., Cotte, N., Nanevicz, T., Fidelman, N., Desai, K., Vranizan, K., Lee, E.K., Coward, P., Shah, N., Warrington, J.A., Fishman, G.I., Bernstein, D., Baker, A.J., Conklin, B.R., 2000. Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy. Proc. Natl. Acad. Sci. USA 97, 4826–4831. Stewart, A.F.R., Larkin, S.B., Farrance, I.K.G., Mar, J.H., Hall, D.E., Ordahl, C.P., 1994. Muscle-enriched TEF-1 isoforms bind M-CAT elements from muscle-specific promoters and differentially activate transcription. J. Biol. Chem. 269, 3147–3150. Stewart, A.F.R., Suzow, J., Kubota, T., Ueyama, T., Chen, H.H., 1998.
9
Transcription factor RTEF-1 mediates a1-adrenergic reactivation of the fetal gene program in cardiac myocytes. Circ. Res. 83, 43–49. Takimoto, E., Mizuno, T., Terasaki, F., Shimoyama, M., Honda, H., Shiojima, I., Hiroi, Y., Oka, T., Hayashi, D., Hirai, H., Kudoh, S., Toko, H., Kawamura, K., Nagai, R., Yazaki, Y., Komuro, I., 2000. Up-regulation of natriuretic peptides in the ventricle of Csx/Nkx2-5 transgenic mice. Biochem. Biophys. Res. Commun. 270, 1074–1079. Thuerauf, D.J., Glembotski, C.C., 1997. Differential effects of protein kinase c, ras, and raf-1 kinase on the induction of the cardiac b-type natriuretic peptide gene through a critical promoter-proximal M-CAT element. J. Biol. Chem. 272, 7464–7472. Ueyama, T., Zhu, C., Valenzuela, Y.M., Suzow, J.G., Stewart, A.F.R., 2000. Identification of the functional domain in the transcription factor RTEF-1 that mediates a1-adrenergic signaling in hypertrophied cardiac myocytes. J. Biol. Chem. 275, 17476–17480. Yue, P., Massie, B.M., Simpson, P.C., Long, C.S., 1998. Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure. Am. J. Physiol. 275, H250–H258. Zou, Y., Evans, S., Chen, J., Kuo, H.C., Harvey, R.P., Chien, K.R., 1997. CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway. Development 124, 793–804.
a1-Adrenergic activation of the cardiac ankyrin repeat protein gene in cardiac myocytes Tomoji Maeda a, Jorge Sepulveda b, Hsiao-Huei Chen c, Alexandre F.R. Stewart a,* a
Cardiovascular Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA c Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15213, USA b
Received 19 June 2002; received in revised form 30 July 2002; accepted 20 August 2002 Received by A.J. van Wijnen
Abstract Cardiac ankyrin repeat protein (CARP) is a nuclear transcription cofactor that is activated by multiple signaling pathways in hypertrophic cardiac myocytes. Since CARP has been reported to be a transcriptional co-repressor, its activation during hypertrophy might contribute to the deregulation of gene expression leading to heart failure. Here, we found that a1-adrenergic signaling activates CARP mRNA expression in rat cardiac myocytes. To examine how a1-adrenergic signaling activates the CARP gene, a 660 bp fragment of the mouse CARP promoter was cloned. Previous reports suggested that the mouse CARP promoter was dependent on the GATA4 transcription factor whereas the human CARP promoter was dependent on transcriptional enhancer factor-1 (TEF-1). TEF-1 and GATA4 transcription factors, known mediators of a1-adrenergic signaling, bound to the mouse CARP promoter at several sites as determined by gel mobility shift assays. These sites are highly conserved between the mouse and human promoters, suggesting that they are functionally important in both. Mutation analysis showed that binding of TEF-1 factors is required for basal activity of the CARP promoter in cardiac myocytes. However, over-expression of TEF-1 factors could not potentiate the response of the CARP promoter to a1-adrenergic stimulation. On the other hand, the a1-adrenergic response was potentiated by GATA4 over-expression. Taken together, our results demonstrate that a1-adrenergic signaling regulates CARP expression in cardiac myocytes, in part through the transcription factor GATA4. q 2002 Elsevier Science B.V. All rights reserved. Keywords: TEF-1; RTEF-1; GATA4; Cardiac myocytes; DNA binding; Transcription factors
1. Introduction The cardiac ankyrin repeat protein (CARP) is an early marker of cardiac myogenic differentiation, expressed at high levels in the heart during embryonic and fetal development and at progressively lower levels in the neonate and adult (Jeyaseelan et al., 1997; Zou et al., 1997). CARP contains four ankyrin repeats thought to play a role in protein/protein interaction. CARP is not known to bind to DNA, but rather is thought to modulate transcription as a cofactor. CARP was isolated from the heart as a cofactor of YB-1, a transcription factor that is required for the activation of the myosin light chain 2v gene during cardiac
Abbreviations: CARP, cardiac ankyrin repeat protein; MCAT, muscle CAT element; RTEF-1, related to transcriptional enhancer factor-1; TEF-1, transcriptional enhancer factor-1 * Corresponding author. Cardiovascular Institute, School of Medicine, University of Pittsburgh, BST 1704.3, 200 Lothrop Street, Pittsburgh, PA 15213, USA. Tel.: 11-412-383-9761; fax: 11-412-383-8997. E-mail address: [email protected] (A.F.R. Stewart).
myogenesis (Zou et al., 1997). Although it is not clear how CARP regulates YB-1 function during cardiac fetal development, CARP over-expression in postnatal cardiac myocytes generally inhibits the expression of cardiac genes and is thought to act as a transcriptional co-repressor (Jeyaseelan et al., 1997; Zou et al., 1997). In addition to its function as a transcription regulator in the nucleus, CARP is known to interact with cytoplasmic proteins. CARP interacts with myopalladin and participates in the maintenance of sarcomere integrity (Bang et al., 2001). Mice with a null mutation in the muscle LIM protein (MLP) that disrupts myocyte sarcomere integrity develop a dilated cardiomyopathy and express elevated levels of CARP in the heart (Arber et al., 1997). When skeletal muscle is passively stretched due to denervation, CARP expression is also activated at the ends of myocytes near the myotendinous junction (Baumeister et al., 1997), where new muscle proteins are known to be synthesized in response to stretch (Dix and Eisenberg, 1990). CARP is known to shuttle from the cytoplasm to the nucleus (Zou et al., 1997). Through its interaction with myopalladin, CARP
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00924-1
2
T. Maeda et al. / Gene 297 (2002) 1–9
has been suggested to form a functional bridge between the sarcomere and transcription regulation (Bang et al., 2001). CARP expression is activated during cardiac hypertrophy in vivo (Kuo et al., 1999; Aihara et al., 2000a; Redfern et al., 2000). In cultured cardiac myocytes, the CARP promoter is activated by p38 kinase (Aihara et al., 2000a). p38 kinase is a known mediator of several hypertrophic signaling pathways, including a1-adrenergic signaling. Since a1-adrenergic stimulation of cultured cardiac myocytes is a well-characterized in vitro model of hypertrophy associated with the transcriptional activation of several genes, it is likely that a1-adrenergic signaling regulates CARP promoter activity. The CARP gene is a downstream target of the cardiacrestricted homeobox gene Nkx2.5 (Zou et al., 1997; Takimoto et al., 2000). Nkx2.5 interacts with the zinc finger transcription factor GATA4 and activates the mouse CARP promoter through a proximal GATA cis-element (Kuo et al., 1999). GATA4 is known to activate the endothelin-1 (Morimoto et al., 2000) and brain natriuretic peptide (Liang et al., 2001) promoters in response to a1-adrenergic stimulation in cardiac myocytes. Thus, the presence of at least one GATA element in the mouse CARP promoter suggests that GATA factors might participate in CARP activation during myocyte hypertrophy induced by a1-adrenergic stimulation. The activity of the human CARP promoter depends on an MCAT cis-element (Aihara et al., 2000a). It is not known whether MCAT elements play a role in regulating the mouse CARP promoter. MCAT elements, 5 0 -CATDYY-3 0 , are the cognate binding site of the transcriptional enhancer factor-1 (TEF-1) family of transcription factors and are required for transcriptional activation of many cardiac and skeletal muscle specific genes (Larkin et al., 1996). TEF-1 factors have also been implicated in the activation of fetal genes during cardiac myocyte hypertrophy. One member of the Table 1 Oligonucleotides used in gel mobility shift assays and for site-directed mutagenesis Oligonucleotide
Sequence a
CARP MCAT1 CARP MCAT2 CARP MCAT3 CARP MCAT4 SKA MCAT
5 0 -GAATTTTCATTCCAGACTTAG-3 0 5 0 -AGAGGGTCATTCCTTTGGCAG-3 0 5 0 -TGACTCGCATTGCTGAGCGGT-3 0 5 0 -CCTGTGACATGCCATCGTACC-3 0 5 0 -GCAGCAACATTCTTCGGGGC-3 0
MCAT2mut
5 0 -GAAATGTGAGAGGGTCCTTCCTTTGGCAGTGACC-3 0
CARP GATA1 CARP GATA2 CARP GATA3 CARP GATA4 rBNP GATA
5 0 -CTGGGTGAGATAATCTTCCA-3 0 5 0 -CACTTACAGCCATATCTTTCTGATAACTGG-3 0 5 0 -GGAGTGCTGATAAGTCCAGT-3 0 5 0 -TCCAAACAGATAGAGACAAG-3 0 5 0 -CACTTGATAACAGAAAGTGATAACTCT-3 0
GATA1mut
5 0 -CCAGGCTGGAAGATTACTTCACCCAGCCCTAGC-3 0
a To facilitate visualization, the strand containing the consensus transcription factor binding site (underlined) is shown for all oligonucleotide pairs.
TEF-1 family, related to TEF-1 (RTEF-1), mediates the a1-adrenergic induction of fetal genes in neonatal rat cardiac myocytes in culture (Ueyama et al., 2000). Thus, if MCAT elements are involved in the activation of the CARP promoter during hypertrophy induced by a1-adrenergic signaling, RTEF-1 is a potential mediator of this effect. The mechanisms of transcriptional regulation of the mouse CARP promoter remain poorly characterized, despite the use of this promoter to drive cardiac fetal specific transgene expression (Kuo et al., 1999) and to selectively activate therapeutic transgenes in hypertrophic hearts (Manning et al., 2000). To better understand the regulation of the CARP promoter in response to hypertrophic signals, we examined its modulation by GATA4 and TEF-1 transcription factors in response to a1-adrenergic stimulation in cardiac myocytes. 2. Materials and methods 2.1. Culture and transfection of neonatal rat cardiac myocytes Neonatal rat cardiac myocytes were isolated from 1-dayold Sprague–Dawley rats, cultured, and transfected as described previously (Stewart et al., 1998). The Institutional Animal Care and Use Committee of the University of Pittsburgh approved the procedures followed for the culture of neonatal rat cardiac myocytes. 2.2. Northern blot analysis Total RNA was prepared from freshly isolated cardiac myocytes (T0) or myocytes plated at a density of 4.5 £ 10 6 cells per 100 mm dish in medium supplemented with 5% bovine calf serum. Myocytes were either left untreated, or treated with 12.5 mmol/l prazosin (an a1-adrenergic antagonist), 12.5 mmol/l timolol (a b-adrenergic antagonist) or cotreated with 12.5 mmol/l each of prazosin and timolol in separate dishes. Medium was changed once and myocytes were harvested after 48 h in culture. Northern blot analysis was carried out using a 40-mer oligonucleotide complementary to sequence in the 3 0 untranslated region of the rat CARP cDNA, 5 0 -CCCATTGCCTACTCCCTCCATCCCCCTCCACACACGCTCC-3 0 . 2.3. Cloning of the mouse CARP promoter The mouse CARP promoter was amplified from published sequences in two fragments using polymerase chain reaction (PCR) and the 5 0 oligonucleotides: CARP 660, 5 0 -CGACGCGTGAACACGGTGAGCCTCTGGTG3 0 and CARP 295, 5 0 -CGACGCGTGTGTGCAATATTAACAGGC-3 0 each containing an MluI restriction site and the 3 0 oligonucleotide CARP3, 5 0 -GAAGATCTCATGTTGGCAGCCGTGAGTC-3 0 containing a BglII restriction site. The CARP promoter fragments were subcloned into the pGL2-Basic vector (Promega, Madison, WI) upstream of
T. Maeda et al. / Gene 297 (2002) 1–9
the firefly luciferase reporter. The mouse CARP promoter was sequenced on both strands and the sequence was submitted to GenBank (Accession number: AF478692). 2.4. Site-directed mutagenesis Site-directed mutagenesis of the proximal GATA element (GATA1) and an MCAT element (MCAT2) shown to mediate p38 MAPK signaling in the human CARP promoter (Aihara et al., 2000a) was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the oligonucleotides shown in Table 1.
3
mRNA expression (Fig. 1B). Low levels of CARP mRNA were detected in freshly isolated cardiac myocytes (T0), but CARP expression was highly elevated after 48 h in 5% bovine calf serum. When serum was withdrawn at 24 h and replaced by serum-free medium (TIBSA), CARP mRNA levels fell approximately 30% over the next 48 h (n ¼ 2). Catecholamines in bovine serum could explain part of the serum response. CARP activation by serum was blunted by the a1-adrenergic receptor antagonist prazosin, but not by the b-adrenergic receptor antagonist timolol (Fig.
2.5. Gel mobility shift assay Oligonucleotides to the sense and antisense strands containing putative TEF-1 and GATA4 binding sites were synthesized by Operon Technologies Inc. (Alameda, CA) and purified by high-pressure liquid chromatography. Sequences are shown in Table 1 and mapped on the CARP promoter sequence in Fig. 2. Gel mobility shift assays were carried out as described previously (Stewart et al., 1998). The TEF-1 specific antibody (BD Transduction Laboratories, San Diego, CA) and the GATA4 specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were obtained commercially. 2.6. Luciferase assay After transfection with various expression vectors and CARP promoter/luciferase constructs (see figure legends), cardiac myocytes were treated for 24 h with either a vitamin C vehicle (20 mmol/l) or phenylephrine (20 mmol/l). Myocytes were harvested as described previously (Stewart et al., 1998) and the luciferase activity in myocyte extracts was measured using 0.3 mg/ml d-luciferin (sodium salt, Analytical Luminescence Laboratory, Ann Arbor, MI) on a luminometer (LB501, Berthold Systems, Inc., Aliquippa, PA). 2.7. Statistical analysis An unpaired two-tailed t-test was used for statistical analysis, with P , 0:05 considered significant. Data are presented as mean ^ standard error. 3. Results 3.1. a 1-Adrenergic signaling activates CARP expression CARP mRNA expression is activated three fold relative to vehicle control by treatment of serum-free cultures with the a1-adrenergic receptor agonist phenylephrine in the presence of the b-adrenergic receptor antagonist timolol (n ¼ 3, normalized to G3PDH, P , 0:05, Fig. 1A). Neonatal rat cardiac myocytes are typically cultured in serum on the first day, a treatment that markedly induces CARP
Fig. 1. CARP mRNA is induced in cultured neonatal rat cardiac myocytes. (A) Northern blot of RNA from cardiac myocytes cultured in serum-free medium treated with vehicle control (VC) or with the a1-adrenergic agonist phenylephrine (PE, 20 mmol/l) in the presence of the b-adrenergic-selective antagonist timolol (10 mmol/l). CARP expression was activated three fold relative to control (n ¼ 3). (B) Northern blot of RNA isolated from neonatal rat cardiac myocytes at the time of plating (T0) or after 48 h of culture in medium supplemented with 5% bovine calf serum (BCS) or serum-free medium supplemented with transferrin, insulin and bovine serum albumin (TIBSA). (C) Northern blot of RNA isolated from cardiac myocytes cultured in 5% BCS treated with the a1-adrenergic-selective antagonist prazosin (Pra, 12.5 mmol/l), the b-adrenergic-selective antagonist timolol (Tim, 12.5 mmol/l), or a combination of adrenergic antagonists (Pra/Tim) for 24 h. Prazosin partially blocked the expression of CARP mRNA whereas timolol did not (n ¼ 3). All blots were probed sequentially for CARP and where applicable, for b-actin (which cross-hybridizes preferentially with a-skeletal actin, a-sk actin), mouse atrial natriuretic factor (ANF), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) to control for loading.
4
T. Maeda et al. / Gene 297 (2002) 1–9
Fig. 2. The mouse and human proximal CARP promoters are highly conserved. A 660 bp fragment of the mouse CARP (mCARP) promoter was amplified by PCR from published sequences and aligned to the sequence of the human CARP (hCARP) promoter (lower sequence, GenBank 16156721). Putative regulatory elements are identified by open boxes. Gray-filled boxes identify MCAT and GATA elements that were functional in a gel shift assay, whereas dashed-line boxes identify putative MCAT and GATA sites that did not produce a shift. Sequence identity is indicated by dots and gaps are shown as dashes. Only the mouse sequence is numbered and the mouse transcription start site is indicated by an arrow.
1C). A 50% reduction in CARP expression was seen with prazosin. ANF and skeletal a-actin are two other a1-adrenergic-responsive transcripts that were also downregulated by prazosin treatment of cardiac myocyte cultures in serum, whereas glyceraldehyde 3-phosphate dehydrogenase (G3PDH) levels remained unchanged (n ¼ 3, Fig. 1C). Taken together, these results support a role for a1-adrenergic signaling in CARP activation. 3.2. Functional TEF-1 and GATA4 sites in the CARP promoter To characterize the mechanism of a1-adrenergic receptor mediated activation of CARP at the transcriptional level, mouse CARP promoter constructs driving firefly luciferase were synthesized by PCR from published sequences (Baumeister et al., 1997; Zou et al., 1997). The CARP promoter sequence is highly conserved between the mouse and human (78% identity) and contains several consensus TEF-1 (MCAT) and GATA4 binding sites (Fig. 2). Because TEF-1 and GATA factors have been implicated in mediating a1-adrenergic induced gene activation in cardiac myocytes, we determined whether the putative sites are functional using a gel mobility shift assay. Oligonucleotides corresponding to TEF-1 and GATA4 sequences from the
mouse CARP promoter (Table 1) were tested in competitive gel shift assays and specific complexes were supershifted with TEF-1 and GATA4 specific antibodies (Fig. 3). Thus, four of seven putative TEF-1 binding sites and four of five putative GATA binding sites were found to bind to TEF-1 and GATA factors, respectively. Two of the four MCAT sites, MCAT1 and MCAT3, bound to TEF-1 factors with lower affinity than MCAT2 or MCAT4 (Fig. 3A). Binding to all of the CARP MCAT sites could be specifically competed by an oligonucleotide containing an MCAT element from the skeletal a-actin promoter (SKA), and a TEF-1 specific antibody supershifted the MCAT-binding factor (MCBF) in each case. The TEF-1 antibody supershift provides the first direct evidence that TEF-1 regulates the CARP promoter. The MCAT sites are conserved between the mouse and human CARP promoters (Fig. 2). Three of the GATA elements produced a robust shift, whereas the most distal GATA element (GATA4) gave only a modest shift (Fig. 3B). This GATA element is not conserved between mice and humans, where the terminal purine of the consensus 5 0 -WGATAR-3 0 is a pyrimidine in the human sequence (Fig. 2). Binding to all of the GATA elements was specific and was selectively competed by an oligonucleotide containing two GATA elements from the
T. Maeda et al. / Gene 297 (2002) 1–9
5
adrenergic signaling was tested in transient transfection assays. 3.4. RTEF-1 squelches the mouse CARP promoter
Fig. 3. The CARP promoter contains multiple functional MCAT and GATA elements. Gel mobility shift assays using the radiolabeled oligonucleotides shown in Table 1 were carried out to test for specific binding to putative MCAT and GATA sequences. (A) To test for MCAT binding, the MCAT element of the a-skeletal actin promoter was used as a specific competitor. The TEF-1 specific antibody was used to supershift (SS) the MCAT-binding factor (MCBF). An oligonucleotide with a single point mutation in MCAT2 (M2m) does not bind to TEF-1. (B) To test for GATA binding, a known GATA binding oligonucleotide corresponding to sequences in the rat b-type natriuretic peptide (BNP) promoter was used as a specific competitor. The shifted complex (S) was further supershifted with a GATA4 specific antibody. An oligonucleotide containing a dual mutation in the proximal GATA element (G1M) does not bind GATA4.
brain natriuretic peptide promoter and could be supershifted with a GATA4 specific antibody. Given the high degree of sequence conservation between the mouse and human CARP promoters and the demonstration that the MCAT and GATA sites are bound by TEF-1 and GATA transcription factors, our results suggest that these sites are functionally important in both promoters. 3.3. The mouse CARP promoter is activated by a 1adrenergic stimulation Both the 660 bp (Fig. 4A) and the 295 bp (Fig. 4B) CARP promoter fragments were activated by phenylephrine. This response to a1-adrenergic stimulation was greater for the 660 bp promoter fragment than for the 295 bp fragment. Nonetheless, the sequences contained within the 295 bp fragment are sufficient to respond to a1-adrenergic stimulation. Since the CARP promoter contains functional TEF-1 and GATA4 binding sites, the effect of these transcription factors on CARP promoter activity and induction by a1-
The transcription factor RTEF-1 was previously shown to augment the a1-adrenergic response of the fetal a-skeletal actin and b-myosin heavy chain promoters (Stewart et al., 1998). Thus, we tested the dose response of the CARP promoter to increasing amounts of the RTEF-1 expression vector and compared the response to equal amounts of the empty expression vector pXJ40. When compared to pXJ40, RTEF-1 produced a dose-dependent inactivation of the CARP promoter. At the lowest dose of the expression vector tested (1 ng), RTEF-1 over-expression slightly increased the a1-adrenergic response of the CARP promoter but at higher doses RTEF-1 interfered with the a1-adrenergic response (Fig. 4A). A similar dose-dependent inactivation was also seen with TEF-1 and DTEF-1 (divergent from transcriptional enhancer factor-1) over-expression (data not shown). Squelching by TEF-1 over-expression has been observed for a number of MCAT-dependent promoters (Stewart et al., 1994, 1998; Gupta et al., 1996) and is believed to result from the tritration of a limiting coactivator. When the MCAT2 element of the mouse CARP promoter was mutated so that it no longer bound to TEF-1 factors (Fig. 3B), the basal promoter activity was significantly lowered (Fig. 4C) but this site does not appear to be essential for the a1-adrenergic response. We chose to mutate this site because it was previously shown to be required for the p38 kinase-dependent activation of the human CARP promoter (Aihara et al., 2000a). Although p38 kinase is a known mediator of a1-adrenergic signaling, our results suggest that this MCAT site is not the major target of the a1-adrenergic response. Therefore, additional MCAT elements of the proximal promoter (MCAT1 and MCAT3), or other ciselements, are likely to contribute to the a1-adrenergic response. Given that TEF-1 factors did not significantly augment the response to a1-adrenergic stimulation, it is not clear whether additional MCAT element mutations would have had an effect. 3.5. GATA4 augments the a 1-adrenergic response of the mouse CARP promoter In contrast to the effect of RTEF-1, GATA4 significantly increased the a1-adrenergic response of the 660 bp CARP promoter when compared to the CMV expression vector pCG (Fig. 4A). Interestingly, a similar effect of GATA4 was not observed for the 295 bp CARP promoter (Fig. 4B), suggesting that distal GATA elements present in the 660 bp promoter are required for the a1-adrenergic response and that the proximal GATA element is not sufficient. To test whether the proximal GATA element contributes to the a1-adrenergic response, the proximal GATA element was mutated so that it no longer binds GATA4 (Fig. 3B). Muta-
6
T. Maeda et al. / Gene 297 (2002) 1–9
Fig. 4. GATA4 augments the a1-adrenergic response of the CARP promoter. Schematic representations of the CARP promoter constructs and the location of GATA and MCAT elements are shown above each data set. Mutated elements in the proximal promoter are crossed out. (A) A CARP promoter construct with 660 bp of the proximal promoter responds to the a1-adrenergic-selective agonist phenylephrine (PE, 20 mmol/l). PE does not activate the promoter-less luciferase vector (data not shown). Fold activities are expressed relative to the activity of the CARP promoter in the presence of the pXJ40 CMV vector under vehicle control conditions. An asterisk indicates all values significantly different from one fold. RTEF-1 squelched the CARP promoter basal activity and also the a1-adrenergic response in a dose-dependent manner when compared to the pXJ40 vector at similar doses. In contrast, GATA4 increased the a1-adrenergic response of the 660 bp CARP promoter when compared to the pCG expression vector (n ¼ 4, P , 0:05). (B) A 295 bp fragment of the CARP promoter was PE-responsive but was not activated by GATA4, suggesting that the proximal GATA element (G1) alone is not sufficient to mediate the effect of GATA4. (C) Mutation of MCAT2 (M2) lowers basal CARP promoter activity (n ¼ 4, P , 0:01), whereas mutation of GATA1 (G1) has no effect. Neither mutation affected the PE response.
T. Maeda et al. / Gene 297 (2002) 1–9
tion of the proximal GATA element had no effect on the basal activity or the a1-adrenergic response of the 295 bp promoter (Fig. 4B) or the 660 bp promoter (data not shown). Thus, the proximal GATA element does not appear to be required. Taken together, our results demonstrate that a1-adrenergic signaling activates the CARP promoter in cardiac myocytes and that part of this response is conferred by GATA4. In addition to TEF-1 and GATA cis-elements, other sequences and their cognate transcription factors are likely to participate in the a1-adrenergic activation of the CARP promoter.
4. Discussion Activation of fetal genes in cardiac myocytes by a1-adrenergic stimulation is a well-characterized in vitro paradigm of cardiac hypertrophy. Many of the fetal genes activated during hypertrophy have been shown to require an MCAT element for their activation, including skeletal a-actin, bmyosin heavy chain, and brain natriuretic peptide (Kariya et al., 1994; Karns et al., 1995; Thuerauf and Glembotski, 1997). CARP was recently identified as another fetal gene activated during cardiac myocyte hypertrophy (Kuo et al., 1999; Aihara et al., 2000a; Redfern et al., 2000; Takimoto et al., 2000). Activation of CARP expression occurs largely at the level of transcription. A CARP promoter-dependent transgene is actively transcribed in the fetal heart (Kuo et al., 1999) and during hypertrophy (Kuo et al., 1999; Manning et al., 2000). Since CARP is activated during hypertrophy, we wondered whether a1-adrenergic signaling might activate CARP. Indeed, the a1-adrenergic-selective agonist phenylephrine elevated endogenous CARP expression in our cultures and the antagonist prazosin lowered CARP expression. In addition, the two CARP promoter fragments we tested were also activated by a1-adrenergic stimulation. Thus, we conclude that CARP is a bona fide a1adrenergic-responsive gene. The mouse CARP promoter is TEF-1-dependent. Mutation of the MCAT2 element, previously shown to reduce basal activity of the human CARP promoter (Aihara et al., 2000b), also lowered basal activity of the mouse CARP promoter. Thus, TEF-1 factors are required for CARP expression. In the human CARP promoter, mutation of the MCAT2 element reduced activation by p38 kinase and Rac1, suggesting that TEF-1 factors might mediate CARP gene activation in response to a1-adrenergic and other signaling pathways (Aihara et al., 2000a). However, mutation of the MCAT2 element did not block the a1-adrenergic activation of the mouse CARP promoter in our study, suggesting that the use of activated mutants of p38 kinase and Rac1 by Aihara et al. (2000a) might not reflect the physiological response to a1-adrenergic signaling. In addition, other MCAT sites might be involved. The MCAT2 site has a higher affinity than either the MCAT1 or MCAT3 sites
7
(Fig. 3). Low affinity MCAT sites have been implicated in the a1-adrenergic activation of the b-myosin heavy chain promoter (Kariya et al., 1994; Stewart et al., 1998) and might also be contributing to the a1-adrenergic activation of the CARP promoter. In addition, not all cardiac muscle promoters with functional MCAT sites are responsive to a1adrenergic stimulation. For example, the cardiac troponin T promoter contains two high affinity sites (Mar and Ordahl, 1988) yet is not further upregulated by a1-adrenergic stimulation (data not shown). Moreover, RTEF-1 over-expression did not further activate the CARP promoter in response to phenylephrine. Endogenous TEF-1 factors might be stimulating the CARP promoter at nearly maximal levels, so that over-expression of TEF-1 factors would titrate away limiting cofactors. In support of this squelching hypothesis, increasing doses of the RTEF-1 expression vector (1, 10 and 100 ng) progressively interfered with the a1-adrenergic activation of CARP promoter. Thus, TEF-1 factors may still contribute to a1-adrenergic activation of the mouse CARP promoter. GATA4 mediates part of the a1-adrenergic activation of CARP in cardiac myocytes. GATA4 over-expression increased the a1-adrenergic response of the 660 bp CARP promoter fragment. However, mutation of the proximal GATA element in the 295 bp CARP promoter fragment had no effect on its basal activity or its a1-adrenergic activation. Thus, the three additional GATA elements in the 660 bp promoter are likely to participate in the a1-adrenergic activation of the CARP promoter. In the case of the minimal 295 bp promoter, MCAT cis-elements and their transcription factors are likely to contribute to both the basal activity of the promoter and the a1-adrenergic response, since the proximal GATA element appears to be expendable. Interestingly, mutation of this same GATA element in the context of the 295 bp CARP promoter was shown previously to abrogate Nkx2.5 and GATA4 cooperative activation in CV1 cells (Kuo et al., 1999). This GATA element was suggested to account for the cardiac specific expression of a 295 bp CARP promoter-driven transgene (Kuo et al., 1999). However, given that we did not observe a reduction in basal activity of the 295 bp GATA1-mutant CARP promoter in cardiac myocytes, cardiac specific cofactors of TEF-1 might also account for CARP expression in the heart. Our results suggest that not all GATA sites are identical. Many GATA-dependent cardiac muscle gene promoters are not upregulated by a1-adrenergic signaling. The a-myosin heavy chain promoter is a good example of such a GATAdependent promoter (Lu et al., 1999). In addition, since GATA4 can augment the a1-adrenergic response, this would suggest that GATA4 levels in cardiac myocytes are limiting for the activation of the CARP promoter, since we did not observe a squelching effect by GATA4 over-expression. Other signaling pathways have also been implicated in the regulation of CARP expression. For example, CARP is
8
T. Maeda et al. / Gene 297 (2002) 1–9
activated by the cytokine IL1b in endothelial cells (Chu et al., 1995). In cardiac myocytes, IL1b activates the brain natriuretic peptide gene (He and LaPointe, 2000), but represses the skeletal a-actin promoter through the transcription factor YY1 (Patten et al., 1996). Following myocardial infarction, IL1b production is elevated in the heart (Yue et al., 1998). Although the ability of IL1b to activate CARP expression in cardiac myocytes has not been tested, concomitant activation of a co-repressor like CARP and down-regulation of skeletal a-actin expression by IL1b might contribute to the progression to heart failure. Transforming growth factor b (TGFb) is also known to activate the human CARP promoter in vascular smooth muscle cells. TGFb is a growth factor highly induced in myocardium during hypertrophy. TGFb activates the human CARP promoter at a 5 0 -CAGA-3 0 cis-element, presumably by signaling through the smad transcription factors (Kanai et al., 2001). However, in the mouse CARP promoter at position 2285, the sequence is 5 0 -CAGG-3 0 . Whether this mutation would impede a TGFb response of the mouse promoter is not known. TGFb can also signal through a serum response element and an MCAT site to activate the skeletal a-actin promoter in cardiac myocytes (MacLellan et al., 1994). Therefore, activation of the human CARP promoter by TGFb might also be mediated by TEF-1 factors. The high degree of sequence identity between the mouse and human CARP promoters points to an important conservation of functional cis-regulatory elements. Thus, studies that characterize the transcription regulation of the mouse CARP promoter are likely to shed light on the regulation of the human CARP promoter. It will be important to understand why CARP expression is maintained at relatively low levels in vivo. Given that multiple signals synergize to regulate the CARP promoter in transfected cells, further characterization of the signaling pathways that modulate CARP promoter function through GATA4, TEF-1 and other transcription factors is warranted. Acknowledgements A.F.R. Stewart was supported by a grant-in-aid from the American Heart Association (0050282N) and by a grant from the National Institutes of Health (R29 HL57211). References Aihara, Y., Kurabayashi, M., Saito, Y., Ohyama, Y., Tanaka, T., Takeda, S., Tomaru, K., Sekiguchi, K., Arai, M., Nakamura, T., Nagai, R., 2000a. Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter. Hypertension 36, 48–53. Aihara, Y., Kurabayashi, M., Tanaka, T., Takeda, S.I., Tomaru, K., Sekiguchi, K.I., Ohyama, Y., Nagai, R., 2000b. Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinasedependent pathways. J. Mol. Cell. Cardiol. 32, 1401–1414.
Arber, S., Hunter, J.J., Ross Jr., J., Hongo, M., Sansig, G., Borg, J., Perriard, J.C., Chien, K.R., Caroni, P., 1997. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 88, 393–403. Bang, M.L., Mudry, R.E., McElhinny, A.S., Trombitas, K., Geach, A.J., Yamasaki, R., Sorimachi, H., Granzier, H., Gregorio, C.C., Labeit, S., 2001. Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. J. Cell Biol. 153, 413–427. Baumeister, A., Arber, S., Caroni, P., 1997. Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube end compartments during muscle morphogenesis. J. Cell Biol. 139, 1231–1242. Chu, W., Burns, D.K., Swerlick, R.A., Presky, D.H., 1995. Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells. J. Biol. Chem. 270, 10236–10245. Dix, D.J., Eisenberg, B.R., 1990. Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers. J. Cell Biol. 111, 1885–1894. Gupta, M.P., Amin, C.S., Gupta, M., Hay, N., Zak, R., 1996. Cooperative activation of cardiac muscle gene expression by transcription enhancer factor-1 (TEF-1) and a basic helix-loop-helix leucine zipper protein Max. Circulation 94, I-526. He, Q., LaPointe, M.C., 2000. Interleukin-1b regulates the human brain natriuretic peptide promoter via Ca( 21)-dependent protein kinase pathways. Hypertension 35, 292–296. Jeyaseelan, R., Poizat, C., Baker, R.K., Abdishoo, S., Isterabadi, L.B., Lyons, G.E., Kedes, L., 1997. A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes. J. Biol. Chem. 272, 22800–22808. Kanai, H., Tanaka, T., Aihara, Y., Takeda, S., Kawabata, M., Miyazono, K., Nagai, R., Kurabayashi, M., 2001. Transforming growth factor-b/ Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells. Circ. Res. 88, 30–36. Kariya, K., Karns, L.R., Simpson, P.C., 1994. An enhancer core element mediates stimulation of the rat b-myosin heavy chain promoter by an a1-adrenergic agonist and activated b-protein kinase C in hypertrophy of cardiac myocytes. J. Biol. Chem. 269, 3775–3782. Karns, L.R., Kariya, K., Simpson, P.C., 1995. M-CAT, CArG, and Sp1 elements are required for a1-adrenergic induction of the skeletal aactin promoter during cardiac myocyte hypertrophy. Transcriptional enhancer factor-1 and protein kinase C as conserved transducers of the fetal program in cardiac growth. J. Biol. Chem. 270, 410–417. Kuo, H., Chen, J., Ruiz-Lozano, P., Zou, Y., Nemer, M., Chien, K.R., 1999. Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis. Development 126, 4223–4234. Larkin, S.B., Farrance, I.K.G., Ordahl, C.P., 1996. Flanking sequences modulate the cell specificity of M-CAT elements. Mol. Cell. Biol. 16, 3742–3755. Liang, Q., De Windt, L.J., Witt, S.A., Kimball, T.R., Markham, B.E., Molkentin, J.D., 2001. The transcription factors GATA4 and GATA6 regulate cardiomyocyte hypertrophy in vitro and in vivo. J. Biol. Chem. 276, 30245–30253. Lu, J.R., McKinsey, T.A., Xu, H., Wang, D.Z., Richardson, J.A., Olson, E.N., 1999. FOG-2, a heart- and brain-enriched cofactor for GATA transcription factors. Mol. Cell. Biol. 19, 4495–4502. MacLellan, W.R., Lee, T.C., Schwartz, R.J., Schneider, M.D., 1994. Transforming growth factor-b response elements of the skeletal a-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1. J. Biol. Chem. 269, 16754–16760. Manning, B.S., Shotwell, K., Mao, L., Rockman, H.A., Koch, W.J., 2000. Physiological induction of a b-adrenergic receptor kinase inhibitor transgene preserves b-adrenergic responsiveness in pressure-overload cardiac hypertrophy. Circulation 102, 2751–2757. Mar, J.H., Ordahl, C.P., 1988. A conserved CATTCCT motif is required for
T. Maeda et al. / Gene 297 (2002) 1–9 skeletal muscle-specific activity of the cardiac troponin T gene promoter. Proc. Natl. Acad. Sci. USA 85, 6404–6408. Morimoto, T., Hasegawa, K., Kaburagi, S., Kakita, T., Wada, H., Yanazume, T., Sasayama, S., 2000. Phosphorylation of GATA-4 is involved in a1-adrenergic agonist-responsive transcription of the endothelin-1 gene in cardiac myocytes. J. Biol. Chem. 275, 13721–13726. Patten, M., Hartogensis, W.E., Long, C.S., 1996. Interleukin-1b is a negative transcriptional regulator of a1-adrenergic induced gene expression in cultured cardiac myocytes. J. Biol. Chem. 271, 21134– 21141. Redfern, C.H., Degtyarev, M.Y., Kwa, A.T., Salomonis, N., Cotte, N., Nanevicz, T., Fidelman, N., Desai, K., Vranizan, K., Lee, E.K., Coward, P., Shah, N., Warrington, J.A., Fishman, G.I., Bernstein, D., Baker, A.J., Conklin, B.R., 2000. Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy. Proc. Natl. Acad. Sci. USA 97, 4826–4831. Stewart, A.F.R., Larkin, S.B., Farrance, I.K.G., Mar, J.H., Hall, D.E., Ordahl, C.P., 1994. Muscle-enriched TEF-1 isoforms bind M-CAT elements from muscle-specific promoters and differentially activate transcription. J. Biol. Chem. 269, 3147–3150. Stewart, A.F.R., Suzow, J., Kubota, T., Ueyama, T., Chen, H.H., 1998.
9
Transcription factor RTEF-1 mediates a1-adrenergic reactivation of the fetal gene program in cardiac myocytes. Circ. Res. 83, 43–49. Takimoto, E., Mizuno, T., Terasaki, F., Shimoyama, M., Honda, H., Shiojima, I., Hiroi, Y., Oka, T., Hayashi, D., Hirai, H., Kudoh, S., Toko, H., Kawamura, K., Nagai, R., Yazaki, Y., Komuro, I., 2000. Up-regulation of natriuretic peptides in the ventricle of Csx/Nkx2-5 transgenic mice. Biochem. Biophys. Res. Commun. 270, 1074–1079. Thuerauf, D.J., Glembotski, C.C., 1997. Differential effects of protein kinase c, ras, and raf-1 kinase on the induction of the cardiac b-type natriuretic peptide gene through a critical promoter-proximal M-CAT element. J. Biol. Chem. 272, 7464–7472. Ueyama, T., Zhu, C., Valenzuela, Y.M., Suzow, J.G., Stewart, A.F.R., 2000. Identification of the functional domain in the transcription factor RTEF-1 that mediates a1-adrenergic signaling in hypertrophied cardiac myocytes. J. Biol. Chem. 275, 17476–17480. Yue, P., Massie, B.M., Simpson, P.C., Long, C.S., 1998. Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure. Am. J. Physiol. 275, H250–H258. Zou, Y., Evans, S., Chen, J., Kuo, H.C., Harvey, R.P., Chien, K.R., 1997. CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway. Development 124, 793–804.