Introduction

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1-Adrenergic receptors (1-ARs), one of three families of receptors for the endogenous catecholamines norepinephrine and epinephrine, are transcribed at different levels in different tissues. For example, 1-AR transcription is robust in rat heart muscle but low or nondetectable in skeletal muscle (Rokosh et al., 1994). Differential transcription is further evident within the rat heart, where 1-ARs are expressed only in cardiac myocytes, and not in cardiac parenchymal nonmyocytes or fibroblasts (Stewart et al., 1994). In adult mouse heart also, 1-ARs are functional in myocytes but not fibroblasts (X. F. Deng, D. G. Rokosh, T. D. O'Connell, S. Cotecchia, and P. Simpson, in preparation). The myocyte-specific transcription of 1-ARs in heart contrasts distinctly with -ARs, which are expressed by both cardiac myocytes and nonmyocytes (Lau et al., 1980). Thus, catecholamine signaling in the myocardium activates 1-ARs only on myocytes, but -ARs on both myocytes and nonmyocytes, which has important implications for catecholamine biology in the heart, and for cardiac hypertrophy and failure.

Suprisingly little is known about the mechanisms that control cell-specific transcription of ARs. Previous studies defined cell-specific promoters for the 1B, the 1C (also called the 1A-AR²), and the 2C (Saulnier-Blache et al., 1996; Razik et al., 1997; Gao and Kunos, 1998; Zuscik et al., 1999). However, only for the 1B in hepatocytes have further studies documented specific functional DNA sequence elements and their corresponding transcription factors (Gao and Kunos, 1998).

Here we investigated mechanisms for transcription of the 1C-AR in cardiac myocytes. We focused on the 1C, rather than the other two subtypes, the 1B and 1D, because the 1C subtype mediates cardiac myocyte hypertrophy and hypertrophic gene induction in cultured rat myocytes (Knowlton et al., 1993; Autelitano and Woodcock, 1998). Furthermore, the 1C is itself induced by 1-AR and other hypertrophic agonists in cultured rat myocytes and the intact rat (Rokosh et al., 1996; Autelitano and Woodcock, 1998).

To study myocyte-specific expression of the 1C-AR, we first cloned and characterized the mouse gene and its transcription in mouse heart. We then defined a mouse 1C promoter in cultured neonatal rat cardiac myocytes, and examined the role of MCAT elements in activation of this promoter. MCAT DNA sequences share a consensus 5'-CATNC(C/T)(T/A) and bind members of the family of transcription factors called transcriptional enhancer factor-1 (TEF-1) (Larkin and Ordahl, 1998). We focused on MCAT elements because they are required for transcription of several other cardiac myocyte genes in culture and in the intact heart (- and -myosin heavy chain, skeletal -actin, cardiac troponin T, and B-type natriuretic peptide) (Larkin and Ordahl, 1998). In addition, MCATs are required for increased transcription during cardiac hypertrophy induced by 1C-AR and other hypertrophic agonists (Kariya et al., 1994; MacLellan et al., 1994; Karns et al., 1995; Gupta et al., 1997; Thuerauf and Glembotski, 1997; He and LaPointe, 1999). However, no receptor gene in any tissue has so far been shown to require an MCAT for activity. Therefore, we tested the idea that MCATs might be a common transcriptional regulatory element shared by the 1C-AR and the other cardiac genes regulated by this receptor.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cloning of the Mouse 1C-AR Gene. Initially, a FIX II 129SV mouse spleen genomic library (Stratagene, La Jolla, CA) was screened using rat 1C-AR cDNA probes to the first exon (PstI probe, 450 to 732 relative to ATG) or the second exon (SmaI probe from 1267 to 1827) (Stewart et al., 1994). Restriction fragments from positive clones were subcloned into pBluescript (Stratagene) and sequenced using Thermo-sequenase PCR-based DNA sequencing (Amersham Pharmacia Biotech, Cleveland, OH).

Northern Blot. Total RNA was isolated from adult mouse heart, brain, and liver using guanidinium thiocyanate and extraction with phenol:chloroform:isoamyl alcohol. Thirty micrograms of total RNA from each tissue was separated on a 2.2% agarose/formaldehyde gel, transferred to a nylon membrane (Nytran plus; Schleicher & Schuell, Keene, NH), and cross-linked with UV irradiation. The blot was probed with an RNA probe to the first 494 bp of the first exon, and then reprobed with a 250 bp -actin RNA probe. Template DNA for the 1C-AR probe was amplified from the BAC clone containing the first exon. RNA probes were transcribed (Maxiscript T7 kit; Ambion, Austin, TX) using [-³²P]UTP 3000 Ci/mmol (PerkinElmer Life Science Products, Boston, MA) and then gel-purified. Blots were hybridized in Church buffer (0.1 M Na₂HPO₄ pH 7.2, 1% bovine serum albumin, 1 mM EDTA, 7% SDS, and 35% formamide) for 20 h at 65°C, washed (twice with 0.1 M Na₂HPO₄/1% SDS for 15 min at 65°C, twice with 33 mM Na₂HPO₄/0.33% SDS for 15 min at 65°C, and finally twice with 3.3 mM Na₂HPO₄/0.03% SDS for 15 min at 65°C), and exposed to film (Biomax-MR; Eastman Kodak, Rochester, NY) at 70°C with an intensifying screen.

RNase Protection Assays (RPAs). To locate the transcription initiation site of the 1C gene, total RNA from adult mouse heart was analyzed by RPA (RPA II kit; Ambion) using antisense RNA probes to the 5'-flanking sequence (probes 1 and 2: 650/+1 and 801/302, where ATG is +1). Template DNA for the 1C-AR probes was amplified from the BAC clone containing the first exon. RNA probes were transcribed (Maxiscript T7 kit; Ambion) using [-³²P]UTP 800 Ci/mmol (PerkinElmer Life Science Products) and then gel-purified. Purified probes (1 × 10⁶ cpm) were hybridized with 50 µg of total RNA (or tRNA as a control) at 45°C overnight in 20 µl of 80% deionized formamide, 100 mM sodium citrate pH 6.4, 300 mM sodium acetate pH 6.4, and 1 mM EDTA. Nonhybridized RNA was digested with RNase A (1 U/ml) and RNase T1 (40 U/ml). RNA duplexes were precipitated and separated on an 8 M urea/6% acrylamide gel. Gels were then exposed to film (Biomax-MR; Eastman Kodak) at 70°C with an intensifying screen.

Primer Extension Assays. A 20-bp antisense oligonucleotide primer was designed complimentary to the 5'-flanking sequence from 520 to 540 (relative to ATG) of the mouse 1C gene. The primer was end-labeled with [-³²P]ATP (PerkinElmer Life Science Products) using T4 polynucleotide kinase (Life Technologies, Gaithersburg, MD). Labeled primer was annealed for 10 min at 65°C to 2 µg of mouse heart poly(A) RNA (purified from total RNA using the Poly(A) Pure kit; Ambion). Annealed RNA was then reverse transcribed with avian myeloblastosis virus reverse transcriptase for 30 min at 41°C according to the Primer Extension kit (Promega, Madison, WI), with the exception that the reverse transcriptase reaction was in the presence of 25 µCi of [-³²P]dCTP. The same 20 bp primer was used to sequence DNA from the mouse 1C-AR gene (Thermo-sequenase, Amersham). Primer extension and sequencing reactions were run on an 8 M urea/6% acrylamide gel, and gels were dried and exposed to film (Biomax-MR, Eastman Kodak) at 70°C with an intensifying screen.

Mouse 1C-AR Gene Promoter Constructs. Promoter constructs were cloned into a pUC9-CAT reporter plasmid (Kariya et al., 1994), containing the multicloning site from pBluescript (KS). 5'-Flanking sequences were amplified from the HindIII BAC DNA clone by PCR using primers with unique restriction sites on each primer (SalI on the 5' primers and XbaI on the 3' primer). The 5' primers containing a SalI restriction site started at 4417, 3009, and 1307 bp relative to the transcription start site. A common 3' primer containing an XbaI restriction site started at +14 relative to the transcription start site. Amplified products were digested sequentially with SalI and XbaI and cloned directionally into p0CAT. The resulting plasmids were named p4417-1CAR-CAT, p3009-1CAR-CAT, and p1307-1CAR-CAT. A 79-bp reporter construct was cloned in a similar manner. In this case, the 5'-flanking sequences were amplified from the HindIII BAC DNA clone by PCR using primers (5' primer 79 bp and 3' primer +586 bp relative to the transcriptional start site) to amplify a blunt-ended product that was cloned into the SacI site in p0CAT.

Mutations to the p4417-1CAR-CAT Plasmid. Mutations to MCAT910 and MCAT1041 were made in the p4417-1CAR-CAT plasmid using a PCR-based mutagenesis protocol. Two primers around the site to be mutated, both 5' to 3', with one primer complementary to each strand, incorporated the mutated base pairs. One primer of each pair was phosphorylated on the 5' end to facilitate ligation after amplification. The p4417-1CAR-CAT plasmid was used as the template. After amplification, the PCR products were treated with Klenow Fragment (Life Technologies) to create a blunt-ended product, digested with Dpn I (Roche Molecular Biochemicals, Indianapolis, IN) to remove the supercoiled template DNA, and ligated with T4 DNA Ligase (Roche Molecular Biochemicals) overnight at 16°C. DH5 competent cells (Life Technologies) were transformed with the ligation products. Mutations were confirmed using Thermo-sequenase PCR-based DNA sequencing (Amersham Pharmacia Biotech).

Cell Culture and Transfection. Neonatal cardiac myocytes were obtained from the hearts of day-old rats by serial trypsinization and cultured at low density in 60-mm dishes using defined medium as described previously (Long et al., 1991). Myocytes were transfected in duplicate by the calcium phosphate precipitation method with 5 pmol (10-20 µg) of one of the 1C-AR promoter constructs or p0CAT, 0.01 pmol (50 ng) of RSV-luciferase (RSV-LUX) as an internal control and differing amounts of pUC19 to adjust total DNA to 25 µg/dish (Kariya et al., 1994). In experiments with hypertrophic agonists, cells were treated with the following (from Sigma Chemical, St. Louis, MO, except as noted): norepinephrine bitartrate (RBI/Sigma, Natick, MA), timolol maleate, prazosin HCl, phenylephrine HCl, endothelin-1, phorbol-12-myristate-13-acetate (PMA), prostaglandin F-2 (R&D Systems, Minneapolis, MN), or vehicle (100 µM ascorbic acid; Sigma Chemical, or 0.05% dimethyl sulfoxide for PMA and PGF2). CAT and luciferase activities were assayed 24 h later as described previously (Kariya et al., 1994). In myocytes, luciferase activity was increased by cotransfection with the 1C-AR reporter plasmids (data not shown), and thus CAT activity was not normalized to luciferase in any experiment.

Gel Mobility Shift Assays. Rat TEF-1 for the binding reactions was produced in vitro using a reticulocyte lysate expression system (TnT Quick Coupled Transcription/Translation System; Promega). Nuclear extracts from cardiac myocytes were prepared as described previously (Kariya et al., 1993). The radiolabeled probe for the gel shift assays was the 22-bp enhancer core/MCAT (EC/MCAT) sequence (215/194) from the rat -myosin heavy chain promoter (Kariya et al., 1993, 1994). Cold competitor probes from the 1C-AR promoter were designed encoding MCAT910, a mutant MCAT910, and MCAT1041 (Fig. 4A). Binding reactions and analysis were as described previously (Kariya et al., 1993, 1994), except that the gel contained 6% acrylamide, not 4%. Binding affinities for each competing oligonucleotide were compared by IC₅₀ values estimated from a logarithmic equation used to describe the competition curve.

Data Analysis. Results are given as mean ± S.E.M. for the number of experiments indicated in each figure. Treated versus control values were tested for deviation from unity by calculation of confidence limits. Mean values were compared by the paired, two-tailed Student's t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Mouse 1C-AR Gene Cloning. We previously cloned the rat 1C-AR cDNA (Stewart et al., 1994). To obtain the mouse 1C-AR gene, we screened a mouse spleen genomic library with rat 1C-AR cDNA probes to exons 1 and 2. We isolated two genomic clones (Fig. 1A, i and ii). The first (Fig. 1A, i) contained 1.2 kb of 5'-flanking sequence, the entire 0.88 kb of coding sequence for the first exon of the mouse 1C-AR, and 11.9 kb of intron sequence. The second clone (Fig. 1A, ii) contained approximately 6 kb of intron sequence, the entire 0.52 kb of coding sequence for the second exon and 8.5 kb of 3'-flanking sequence. To obtain additional 5'-flanking sequence, we screened a mouse BAC library with the first exon PstI probe. We isolated a 7.7 kb HindIII fragment that contained 5 kb of 5'-flanking sequence, the entire 0.88 kb of coding sequence for the first exon and 1.8 kb of intron sequence (Fig. 1A, iii). Analysis of the mouse 1C-AR primary sequence showed that the gene contained two exons separated by a large intron of at least 18 kb, inserted between the coding sequences for the sixth transmembrane domain and the third extracellular loop. This gene structure was identical to that of the 1B-AR (Ramarao et al., 1992; Gao and Kunos, 1993).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Cloning of the mouse 1C-AR gene. A, structure of the mouse 1C-AR gene. The genomic organization of the mouse 1C-AR gene is represented in the upper portion of the figure, where the striped boxes represent the two exons, which are separated by an intron at least 18 kb long. The transcriptional initiation site, 588 bp upstream of the ATG (Fig. 2, below), is indicated by an arrow, and four potential polyadenylation signals are marked 3' to the second exon. The three genomic clones used to characterize the gene are labeled i, ii, and iii. The PstI (450 to 732 of exon 1) and SmaI (1267-1827 of exon 2) probes used in the cloning are shown beneath clones ii and iii. Enzyme abbreviations are given in the inset. B, alignment of 1C-AR deduced amino acid sequences from mouse, rat, and human. Dots indicate homology in all species. Nonconserved changes in amino acid sequence that could have structural significance are in boldface. Amino acids conserved with the 1B-AR and implicated in intracellular signaling are underlined, including residues important for agonist binding (C109 and S188/192) (Hwa and Perez, 1996; Perez et al., 1996) and G protein coupling (V209 to A243 and K268/A271) (Cotecchia et al., 1992; Wu et al., 1995; Perez et al., 1996). N13 is a potential site for N-linked glycosylation, and is also conserved among 1 subtypes and across species in the 1C. The location of the intron is noted by double arrows. The seven transmembrane (TM) spanning regions and three intracellular loops are indicated. Asterisks are spaced every 10 amino acids. Rat sequences are from Stewart et al. (1994), and human are from Schwinn et al. (1990).

Mouse 1C-AR Amino Acid Sequence. Figure 1B compares the mouse 1C-AR deduced amino acid sequence with the rat and human 1C-AR sequences. There was a 97% homology between the mouse and rat sequences and a 92% homology between the human and mouse. Most of the amino acids important for agonist binding or G protein coupling were conserved in the mouse 1C-AR (Fig. 1B legend).

1C-AR Gene Transcription Initiation Site in Mouse Heart. We located the transcription initiation site of the 1C in adult mouse heart, using RPAs with probes from the cloned gene, followed by primer extension. As shown in Fig. 2A, RPAs were done using 50 µg of total RNA from adult mouse heart and two different probes. Probe 1 (650 to 1 relative to the ATG) gave a specific (absent in tRNA lane) 600-bp fragment (Fig. 2A). Based on these results, probe 2 was designed further upstream (801 to 302 relative to the ATG) and gave a specific 275 bp fragment (Fig. 2A). Together, these results suggested the presence of a single major transcription initiation site, located between ~577 and ~600 bp upstream from the ATG.

View larger version (64K): [in this window] [in a new window]   Fig. 2.   Transcription of the 1C-AR in mouse heart. A, identification of the mouse 1C-AR transcriptional initiation site in heart by RPA. Antisense RNA probes from 650 to +1 and 801 to 302 (ATG +1) of the 5'-flanking sequence of the mouse 1C-AR (shown on left) were labeled with [-32P]dUTP and used in a RPA with 50 µg of total RNA from adult mouse heart or 50 µg of control tRNA. The gel lanes show the probe alone (Pr), tRNA control (tR), and heart RNA (H). Protected fragments in the heart RNA lane are labeled by approximate size, 600 bp with probe 1 and 275 bp with probe 2. These results indicate a single major transcriptional start site located approximately 577 to 600 bp upstream of the ATG. B, identification of the mouse 1C-AR transcriptional initiation site in heart by primer extension. Primer extension was used to localize the transcription initiation site precisely. Poly(A) RNA (2 µg) from mouse heart was used in a primer extension assay with a 20 bp antisense oligonucleotide primer against the sequence from 520 to 540 (ATG +1). The same primer was used to sequence DNA from the 5'-flanking sequence of the mouse 1C gene. The sequence is shown in the figure aligned with the primer extension reaction. The transcription initiation site is 588 bp upstream of the ATG and encompasses an Inr sequence TCAG(588)ATA (sequence shown on the left). C, identification of mouse 1C-AR by Northern blot. Total RNA (30 µg) from adult mouse heart (H), brain (B), and liver (L) was probed with an [-32P]dUTP-labeled antisense RNA probe encompassing nucleotides 3 to 497 of the first exon of the 1C-AR gene (top). The same blot was stripped and reprobed with a 250 bp -actin probe (bottom); sarcomeric (seen only in heart) and cytoskeletal actin are labeled. The molecular weights of the 1C-AR mRNAs are shown on the left. Molecular weight size markers and ribosomal RNA mobility are shown on the right. D, 1-AR subtype mRNAs in mouse heart development by RPA. Antisense RNA probes to the rat 1-AR subtypes 1B, 1C, and 1D (Rokosh et al., 1994), and to -actin, were labeled with [-32P]dUTP and used in RPA with 25 µg of total RNA from a newborn rat heart (day 1), newborn mouse heart (day 1; one litter pooled), and weanling mouse heart (day 21). RNA was made from hearts of three different mouse strains: C57BL/6J (BL/6), CD-1, and FVB/NJ (FVB). The 1C was not detected in the newborn heart in any mouse strain.

1C-AR mRNA Transcription in Adult Mouse Heart. To compare 1C mRNA size and tissue distribution with that in the rat (Rokosh et al., 1994; Stewart et al., 1994), and to confirm the cloned gene structure, we did a Northern blot with 30 µg of total RNA isolated from adult mouse heart, brain, and liver, using an RNA probe directed against the first 500 bp of exon 1 (Fig. 2C). Mouse heart had a predominant 1C mRNA of 11 kb, with minor transcripts of 7.5 and 3.5 kb. Brain had similar mRNAs, and liver had none. Actin mRNA was detected in all tissues (Fig. 2C). Thus, mouse 1C-AR mRNA size and tissue distribution were similar to that found in the rat (Rokosh et al., 1994; Stewart et al., 1994). Notable is the much larger size of 1C mRNA compared with 1B or 1D (~2 kb) (Fig. 2C; Stewart et al., 1994).

1C-AR mRNA Transcription in Mouse Heart Development. In the rat, all three 1-AR subtype mRNAs are expressed in the neonatal heart (Stewart et al., 1994). To study expression during postnatal cardiac development in the mouse, we used RPA to detect the subtype mRNAs in mouse hearts of various ages (Fig. 2D). As shown in Fig. 2D, the 1C was not detected in newborn (day 1) mouse heart of three different strains (C57BL/6, CD-1, and FVB), but was seen by weaning (day 21) in all strains. The 1B and 1D were present in the mouse heart at each time point (Fig. 2D); and all three subtypes were present in the newborn rat, confirming prior results (Stewart et al., 1994). Thus, the 1C was not an early developmental mRNA in the mouse heart, in contrast with the rat.

Identification of a Mouse 1C-AR Promoter in Cardiac Myocytes and the Role of an MCAT Element in Promoter Activity. We showed previously that the 1C-AR is transcribed in rat cardiac myocytes, and not in cardiac nonmyocytes (fibroblasts) (Stewart et al., 1994). 1-ARs are also functional in myocytes but not fibroblasts in the adult mouse heart (X. F. Deng, D. G. Rokosh, T. D. O'Connell, S. Cotecchia, and P. Simpson, in preparation). However, little is known about mechanisms that might regulate AR transcription in specific cells such as cardiac myocytes. Here we tested fragments of the cloned mouse 1C gene for promoter activity using transfection in cultured neonatal rat cardiac myocytes. We used rat myocytes rather than mouse as a test system, because the 1C was not transcribed in neonatal mouse myocytes (Fig. 2D).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Analysis of an 1C-AR promoter in rat heart cells. A, mouse 1C-AR promoter and reporter constructs. The top of the figure shows the locations of potential cis-regulatory elements (relative to the transcription initiation site), including seven consensus MCAT motifs. An asterisk indicates elements conserved with the human sequence (Razik et al., 1997). The bottom of the figure diagrams the three CAT reporter plasmids, containing ~4.4, 3.0, and 1.3 kb of sequence. B, activity of the mouse 1C-AR promoter in cardiac myocytes and nonmyocytes. The three CAT reporter plasmids containing 4417, 3009, and 1307 bp of promoter sequence were transiently transfected into primary cultures of rat neonatal cardiac myocytes and nonmyocytes. Cardiac myocytes were cultured in serum-free medium, whereas nonmyocytes were cultured in serum-supplemented medium to maximize any expression of the reporters. These were exactly the same conditions where myocytes but not fibroblasts have 1-AR mRNAs, proteins, and function (Rokosh et al., 1996, and references therein). CAT activity was measured after 24 h. The graph shows the constructs on the left and the corresponding CAT activity on the right, expressed as fold activation relative to p0CAT (empty vector). The locations of the seven MCAT elements (M1-M7) are indicated. An RSV-luciferase construct was cotransfected as a control, but CAT activities were not corrected for luciferase expression. Each experiment was performed with duplicate dishes, and data are presented as mean ± S.E.M. (n = 10-20 experiments in myocytes, n = 3 experiments in nonmyocytes). *p < 0.05 versus p0CAT. C, activity of mouse 1C-AR promoter MCAT mutants in cardiac myocytes. Point mutations were made in MCATs M1, M2, or both (double, dbl) in the context of the full-length 4.4 kb 1C promoter (M1 CATGCCA mutated to CgatCgA, M2 CATACTT to CgatCgT, mutations in lowercase; see also Fig. 4A). Plasmids were transfected as in B. Values are mean ± S.E.M. (n = 3-5 experiments for each mutant construct). *p < 0.05 versus p0CAT; p < 0.05 versus p4417-1CAR-CAT.

TEF-1 Binds the Active 1C Promoter MCAT Element M1. MCAT elements bind members of the family of transcription factors known as TEF-1 (Larkin and Ordahl, 1998), and TEF-1-binding MCATs are required for transcription of several contractile protein genes and cardiac BNP (see Introduction). We have studied in detail an MCAT in a 22-bp sequence (215 to 194) in the rat -myosin heavy chain promoter, named the proximal EC/MCAT. The EC/MCAT is required for promoter activity in myocytes and binds to TEF-1 in vitro and in vivo (Kariya et al., 1993, 1994; Stewart et al., 1998). To test whether the M1 MCAT required for 1C promoter activity also bound to TEF-1, we did competition gel mobility shift assays, using the EC/MCAT as the probe and both recombinant TEF-1 and endogenous TEF-1.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Binding of TEF-1 to MCATs in the 1C-AR promoter. A, recombinant TEF-1 binds to the 1C MCAT elements. The 22 bp rat -myosin heavy chain proximal enhancer core/MCAT (EC/MCAT; 215/-194) (Kariya et al., 1993, 1994) was labeled with [-32P]dATP and incubated with in vitro translated TEF-1, in the presence or absence of increasing amounts of unlabeled competitor oligonucleotides. Sequences of the proximal enhancer core/MCAT probe and the competitor oligonucleotides are shown at the top of the figure. Bound and free probe were separated on a 6% gel and visualized on film by autoradiography. The shifted TEF-1 band is indicated by the arrow on the left, and the fold concentrations of each competitor are indicated at the top of each lane. B, TEF-1 binding affinity for MCAT elements. Densitometry was used to quantify the gel shift bands in A and in an additional identical experiment. The graph shows the percentage of labeled probe bound to TEF-1 after competitionwith the indicated concentration of cold oligonucleotides. The molar concentration of oligonucleotide giving 50% inhibition of binding to the EC/MCAT (IC50) was estimated from the competition curves as ~4 nM for MCAT M2 (MCAT1041), ~24 nM for the EC/MCAT, and ~157 nM for MCAT M1 (MCAT910). The mutated version of MCAT M1 (MCAT910 mut) did not compete 50% of TEF binding even at 100×. Points are mean ± range, n = 2. C, endogenous TEF-1 in cardiac myocyte nuclear extracts binds to the 1C MCAT elements. Gel shift was done as in A, using 10 µg of cardiac myocyte nuclear extract. The TEF-1 shifted complex (C2) is indicated on the left (see also Kariya et al., 1993), and the unlabeled competitors (100-fold) are indicated at the top of each lane.

Activation of the Mouse 1C-AR Promoter by Norepinephrine and Other Hypertrophic Agonists. In the rat, hypertrophic stimuli increase 1C-AR mRNA in cardiac myocytes in culture and in vivo (Rokosh et al., 1996). To test whether hypertrophic agonists stimulated the mouse 1C-AR promoter, rat myocytes were transfected with the full-length 4.4 kb 1C promoter, and then treated with various agonists for 24 h. Norepinephrine increased 1C promoter activity by ~4-fold versus vehicle. However, the norepinephrine effect was mediated entirely through a -AR, because induction was blocked by the -AR antagonist timolol and not by the 1-AR antagonist prazosin (Fig. 5). Phenylephrine mildly activated the promoter, but this was blocked by timolol, consistent with a slight -AR action of 20 µM phenylephrine (Fig. 5). Endothelin and PGF2 had no effect, and PMA induced the promoter only slightly, albeit significantly (Fig. 5).

View larger version (48K): [in this window] [in a new window]   Fig. 5.   Activation of the mouse 1C-AR promoter and promoter mutants by norepinephrine and other hypertrophic agonists. Cardiac myocytes on culture day 1 were transfected for 2 h with p4417-1CAR-CAT, and then treated the next day with various hypertrophic agonists. CAT activity was measured after 24 h, and is shown here relative to the vehicle-treated control cells. An RSV-luciferase construct was cotransfected as a control, but CAT activities were not corrected for luciferase expression. Each experiment was performed with duplicate dishes, and data are presented as mean ± S.E.M. *p < 0.05 versus vehicle. Abbreviations of agonists and numbers of experiments (in parentheses) are as follows: doses were 2 µM except as noted: VEH, vehicle (20); NE, norepinephrine (16); NE/PZN, NE plus prazosin 0.2 µM (3); NE/TML, NE plus timolol (5); PE, phenylephrine 20 µM (4); PE/TML, PE plus timolol (4); ET-1, endothelin-1 100 nM (3); PMA 100 nM (3); PGF2 1 µM (3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Here we cloned and characterized the complete structure of the 1C-AR gene and characterized transcription of the gene in adult mouse heart. We defined an 1C promoter that was active in cultured neonatal rat cardiac myocytes but not fibroblasts and showed that activity of the promoter required an MCAT element that bound TEF-1. This is the first example of a receptor gene regulated by an MCAT element, and one of the few cases (Gao and Kunos, 1998) to identify a mechanism for adrenergic receptor transcription in a specific cell.

The structure of the 1C-AR gene was similar to that of the 1B-AR (Ramarao et al., 1992; Gao and Kunos, 1993), in that the gene contained two exons separated by a large intron (at least 18 kb). Similar to the major promoter of the 1B (Gao and Kunos, 1993), the 1C promoter was TATA-less, and transcription in mouse heart was initiated from a single site that contained an Inr consensus. This Inr is possibly the same one used by the human 1C promoter for transcription initiation in neural cells (Razik et al., 1997). The predominant 1C mRNA in both mouse and rat heart, ~11 kb, is much larger than the mRNAs of the 1B or 1D in heart (both ~2 kb) (Fig. 2C; Stewart et al., 1994). Here we were able to account for this long 1C mRNA by transcription from the Inr at 588 upstream of the ATG, 1.4 kb of open reading frame, and a potential polyadenylation sequence 8.5 kb downstream.

We identified a 4.4 kb fragment of mouse 1C-AR 5'-nontranscribed sequence that was active as a promoter in cultured neonatal rat myocytes but not in nonmyocytes, and thus mimicked the myocyte-specific transcription of the endogenous 1C gene in the heart. The magnitude of 1C promoter activity, ~2.5-fold over empty vector, was congruent with the much lower abundance of endogenous 1C transcripts in comparison with the structural genes -myosin heavy chain and skeletal -actin (Bishopric et al., 1987; Waspe et al., 1990; Stewart et al., 1994; Rokosh et al., 1996); these latter promoters are activated ~10-fold over basal in the same system (Kariya et al., 1994; Karns et al., 1995).

A single MCAT element, MCAT M1 at 910 from the Inr, was required for activity of the full-length promoter in myocytes and bound recombinant and endogenous TEF-1 in gel mobility shift assay. Thus, an MCAT element that bound TEF-1 was required for 1C transcription in myocytes. This active MCAT in the 1C-AR promoter is conserved in the human gene, although its activity was not tested (Razik et al., 1997), just as an active MCAT in the rat -myosin heavy chain promoter is conserved among species (Kariya et al., 1994). It is intriguing that an identical MCAT/TEF-1 mechanism is used in myocytes for transcription of the 1C receptor gene and for transcription of several other cardiac genes (- and -myosin heavy chain, skeletal -actin, cardiac troponin T, and B-type natriuretic peptide) (see Introduction). This observation links an MCAT/TEF-1 signaling mechanism that maintains the cardiac myocyte phenotype to 1C transcription in myocytes, and suggests a possible autoregulatory system for transcription. That is, MCATs and TEF-1 promote transcription of genes that are targets for 1C signaling, such as contractile proteins and cardiac BNP (Kariya et al., 1994; MacLellan et al., 1994; Karns et al., 1995; Gupta et al., 1997; Thuerauf and Glembotski, 1997; He and LaPointe, 1999), and also transcription of the receptor gene itself. The 1B promoter similarly contains at least one MCAT (Saulnier-Blache et al., 1996; Razik et al., 1997; Gao and Kunos, 1998; Zuscik et al., 1999), although it is not known whether the 1B MCAT is functional. We are further exploring a possible connection in myocytes between the 1C receptor and TEF-1 using mice with gene knockouts (O'Connell et al., 2000a,b) and studies of 1C signaling. Consistent with a connection we find that TEF-1 phosphorylation is altered by 1C-AR stimulation (R. Q. To, C. Turck, and P. Simpson, unpublished observations).

It was initially surprising in this regard that the mouse 1C promoter was not activated by 1-AR or other Gq-coupled hypertrophic agonists (Fig. 5), whereas the rat 1C gene is activated robustly by hypertrophic stimuli in culture and in the intact rat (Rokosh et al., 1996; Autelitano and Woodcock, 1998). However, recent data confirm that the endogenous mouse 1C gene also differs from the rat. In the mouse, 1C mRNA in myocytes does not increase with hypertrophy in vivo (Snyder et al., 1999; Wang et al., 2000), in contrast with the rat where it does increase (Rokosh et al., 1996). For example, abdominal aortic banding, which increases 1C mRNA in rat heart (Rokosh et al., 1996), does not do so in the mouse (Snyder et al., 1999; D. G. Rokosh, P. C. Simpson, unpublished observations).

At least two mechanisms could account for the difference in 1C transcription with hypertrophic stimuli between rat and mouse: differences in intracellular signaling and/or differences in gene structure. Our results favor differences in gene structure, because we tested the mouse gene in rat myocytes, where signaling does activate the endogenous 1C. For example, the rat 1C gene might contain an "inducible" MCAT, or some other hypertrophic response element, not present in the mouse gene. In this regard it is interesting that inducible MCATs have an A or a T at the fourth position, rather than the G found in the "noninducible" MCAT M1 at 910 in the mouse 1C-AR promoter (Fig. 4A). A structural gene difference might also explain why -AR stimulation activates the mouse promoter (Fig. 5) but not the rat (Rokosh et al., 1996). -AR activation of the mouse 1C promoter required a CRE, rather than an MCAT/E-box as in the -myosin heavy chain promoter (Gupta et al., 1994), and the rat gene might lack the required CRE found in the mouse. The mechanism(s) require further study, but the results do indicate important differences in transcription activation between the rat and the mouse, comparable with those seen previously with atrial natriuretic factor (Seidman et al., 1991).

Another transcription difference between rat and mouse is more probably explained by differences in signaling rather than gene structure, the delayed maturation of 1C transcription during development in the mouse. In the newborn heart, the 1C is not transcribed in the mouse, but is transcribed in the rat (Fig. 2D). Because we studied the mouse promoter in neonatal rat myocytes, we know that the structure of the mouse gene supports transcription in neonatal cells. Thus absent transcription in neonatal mouse myocytes might reflect some rat-mouse difference(s) in signaling, such as a kinase that activates TEF-1, a TEF-1-interacting protein (see below), or a mechanism that unfolds chromatin over the active mouse MCAT element.

Finally, it was noteworthy that only one of the seven MCAT consensus elements in the mouse 1C promoter was required for activity in myocytes, emphasizing the need for functional studies such as those reported here. Similar observations in other promoters (Kariya et al., 1994; Larkin and Ordahl, 1998) have raised the idea that TEF-1 requires some additional cofactor for activity in myocytes. Indeed "active" MCATs in myocytes can have apparently lower binding affinity for TEF-1 than inactive MCATs (Kariya et al., 1993, 1994; I. K. G. Farrance and P. C. Simpson, unpublished observations), as we observed in this study with both recombinant and endogenous TEF-1 (Fig. 4). Lower affinity TEF-1 binding at an active MCAT might favor a protein conformation that allows for association with a required coactivator. A few TEF-1 coactivators are known, including Max in the -myosin heavy chain promoter (Gupta et al., 1997) and poly(ADP-ribose) polymerase in the cardiac troponin T promoter (Butler and Ordahl, 1999). However, the active MCAT M1 at 910 is not flanked by an E-Box (CANNTG; Max binding site) as in the -myosin heavy chain promoter (Gupta et al., 1997), or a poly(ADP-ribose) polymerase binding site (TGTTG) as in the cardiac troponin T promoter (Butler and Ordahl, 1999). In addition, MCAT element M7 (Fig. 3A) is flanked by a poly(ADP-ribose) polymerase-binding site, but was not required for 1C-AR promoter activity. Thus, it is likely that novel TEF-1-interacting proteins exist, such as the human protein TONDU, related to vestigal in Drosophila (Vaudin et al., 1999), and several labs are pursuing this idea.