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Am J Physiol Heart Circ Physiol 278: H1153-H1162, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 4, H1153-H1162, April 2000

Regulation of DHP receptor expression by elements in the 5'-flanking sequence

Lei Liu1,*, Q. Ivy Fan1,*, Mohamad R. El-Zaru1, Kathleen Vanderpool1, Ronald N. Hines2, and James D. Marsh1

1 Program in Molecular and Cellular Cardiology and 2 Departments of Medicine and Pharmacology, Harper Hospital, Wayne State University School of Medicine, and Department of Veterans Affairs Medical Center, Detroit, Michigan 48201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The alpha 1-subunit of the cardiac/vascular Ca2+ channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca2+ entry. It contains the binding sites for multiple classes of drugs collectively known as Ca2+ antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac alpha 1C-subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking sequences and identified the alpha 1C-subunit gene transcription start site. The rat alpha 1C-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of alpha 1C-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5'-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca2+ channel alpha 1C-subunit gene. Electrophoretic mobility shift assays identified a region of the alpha 1C-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.

calcium channel; promoter; transcription; myocyte; heart; dihydropyridine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

L-TYPE CALCIUM CHANNELS play a central role in cardiac and smooth muscle excitation-contraction coupling. Abundance and function of the L-type Ca2+ channel is a major determinant of Ca2+ transients and contractile performance of myocytes (7, 34). When the abundance of functional Ca2+ channels is increased or decreased in physiological or pathological conditions, there are associated changes in excitation-contraction coupling (7, 13, 19, 28, 34, 41).

At least five types of Ca2+ channels have been defined electrophysiologically: L, T, N, P/Q, and R (11, 38). The L-type channel is the predominant type in heart and vascular tissue. There are four subunits of the L-type Ca2+ channel proven to be present in heart: alpha 1, alpha 2, delta , and beta . The alpha 1-subunit alone exhibits limited function as a voltage-dependent Ca2+ channel. Distinct isoforms of the alpha 1-subunit, each a unique gene product, are present in various tissues. Thus alpha 1S is the isoform expressed in skeletal muscle, alpha 1D in neuroendocrine tissues, and alpha 1C in cardiac and vascular smooth muscle as well as in brain. Neuronal tissue expresses alpha 1A-, alpha 1B-, and alpha 1E-subunits, which are insensitive to dihydropyridine (DHP) (36). The alpha 1C-subunit is known to undergo alternative splicing, leading to functionally and molecularly distinct isoforms: alpha 1C-A appears to be the predominant isoform expressed in heart, alpha 1C-B in smooth muscle, and alpha 1C-C in neuronal tissue. The alpha 1C-subunit gene is the subject of the present investigation. The product of the gene is often termed the DHP receptor because of its ability to bind organic Ca2+ channel antagonists.

The abundance of DHP receptors may be regulated by several processes; there are data suggesting that regulation of mRNA abundance (mRNA synthesis and degradation rates) plays an important role. However, despite the evidence suggesting that transcriptional control is critical for regulation of Ca2+ channel expression, little direct information is available regarding mechanisms regulating transcription of the alpha 1C-subunit gene, and little is known about control of tissue-specific and cell type-specific expression, despite progress in these areas for other ion channel genes. We previously reported that when myocytes are stimulated with norepinephrine or isoproterenol, the increase in DHP receptor mRNA abundance is followed by increases in Ca2+ current and DHP-binding sites (26). Also, when adult cultured myocytes are grown in the presence of high extracellular Ca2+, the increase in DHP receptor mRNA levels is followed by increases in Ca2+ current and DHP-binding sites (7). Exposure of myocytes to agents activating protein kinase C decreases Ca2+ channel expression (25). During development, there appears to be an important role for translational or posttranslational control of alpha 1C-subunit expression (25). However, it appears very likely that pretranslational control is important in immature and adult cardiac myocytes. The precise mechanism of pretranslational control of the DHP receptor has not been rigorously demonstrated, and there is only limited knowledge regarding molecular mechanisms controlling expression of other cation channels in heart or vascular smooth muscle; more data are available for neuronal cation channels. For the rat brain type II Na+ channel, there is a silencer region in the 5'-flanking region of the gene (21). The expression of the rat alpha 1D-subunit gene is increased during differentiation in the presence of retinoic acid or prostaglandin E1 (16). The rat alpha 1D-subunit gene has a novel enhancer comprised of an (ATG)7 trinucleotide repeat (16). Finally, it has recently been reported that the alpha 1B-subunit gene of rat contains a TATA-less promoter and a repressor in the distal upstream region (18).

The structure of the human alpha 1C-subunit gene was reported by Soldatov (40) and Yamada et al. (47). However, the sequence of the human alpha 1C-subunit exons near the 5'-end of the gene is substantially different from the cDNA sequence of the rat aortic voltage-dependent channel alpha 1-subunit reported by Koch et al. (20) and the rabbit heart alpha 1C-subunit cDNA described by Mikami et al. (30). Furthermore, the structure of the 5'-flanking region and promoter of the cardiac DHP receptor has not been reported.

As a first step toward understanding the mechanisms controlling transcription of the rat cardiac alpha 1C-subunit gene, we cloned a 2.3-kb fragment of the 5'-flanking sequences and identified the alpha 1C-subunit gene transcription start site. Here we report that the rat alpha 1C-subunit gene contains a complex TATA-less promoter. In addition, using deletion analysis of the alpha 1C-subunit promoter-luciferase reporter gene constructs and transfections and transient expression in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells, we have characterized the transcriptional modulating activity of the 5'-flanking region. Regulatory regions in the 5'-flanking region are identified that likely contribute to tissue-specific expression and may contribute to hormone responsiveness of the gene.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Genome Walking

Genomic clones containing the first exon and 5'-flanking sequences were obtained from a modified rat genomic library (PromoterFinder, Clontech). Briefly, we examined five samples of uncloned rat genomic DNA fragments, which had been digested with five different restriction enzymes and subsequently ligated to a common adapter. A primary PCR was carried out using a primer complementary to the adapter (AP1) and a gene-specific primer (GSP1). The amplification was accomplished using Taq polymerase, 1.5 mM Mg2+, for 30 cycles at 62°C and a thermal cycler (model 2400, Perkin-Elmer). The primary PCR mixture was diluted and used as a template for a secondary PCR with use of a nested adapter primer (AP2) and a nested gene-specific primer (GSP2). PCR conditions were the same as for the initial and secondary PCR. The PCR products were examined on a 1% agarose gel, and DNA fragments of interest were then cloned into the plasmid pCRII (Invitrogen) for further sequencing and functional analysis. The gene-specific primers were designed on the basis of the sequence of the rat aortic voltage-dependent Ca2+ channel alpha 1-subunit gene cDNA (20): 5'-CCC TTG AAC TTC CTC TCT GTG TC-3' (GSP1) and 5'-CCA GGA GAA ATG TAG AAA CAG TTC A-3' (GSP2).

DNA Sequencing and Computer Analysis

Subclone identity and orientation were verified by restriction endonuclease analysis and DNA sequencing. Sequencing was performed using the dideoxy-mediated chain-termination method of Sanger et al. (37) on alkaline-denatured, double-stranded plasmid DNA (6). Programs used for the analysis of DNA sequence included TFSEARCH (46) (University of Kyoto, Kyoto, Japan), ALIGN, and CLONE Manager (Scientific and Educational Software, Durham, NC).

Primer Extension Analysis and Rapid Amplification of cDNA Ends

The transcription start site for the rat alpha 1C-subunit mRNA was determined by primer extension and by 5'-rapid amplification of cDNA ends (RACE). Primer extension analysis was performed with a 25-bp antisense oligonucleotide primer corresponding to the 5'-region of the rat aortic voltage-dependent Ca2+ channel alpha 1-subunit gene cDNA(20). The primer was end labeled with [gamma -32P]ATP (3,000 Ci/mmol) by use of T4 polynucleotide kinase. Primer extension was carried out using avian myeloblastosis virus reverse transcriptase (Promega). The sequence of the primer for primer extension analysis was 5'-TAT CCC ACT TGT ATT TTT CCT TGT A-3'. All the primers were selected from the cDNA sequence of rat aortic voltage-dependent Ca2+ channel alpha 1-subunit (20). On completion of the extension reaction, the sample was fractionated by electrophoresis in a 9% polyacrylamide-7 M urea gel along with products of a sequencing reaction performed with the same primer. 5'-RACE analysis (35) was performed using a 5'-RACE system kit (GIBCO/BRL) following the manufacturer's protocol. The primers for 5'-RACE were 5'-CCC TTG AAC TTC CTC TCT GTC TC-3' and the anchor primer supplied by the manufacturer. PCR was conducted for 35 cycles. Products of the primer extension studies and 5'-RACE experiments were analyzed by 1% agarose electrophoresis; products were isolated from the gel and subcloned into pCR2.1 (Invitrogen) and sequenced.

Isolation and Culture of Neonatal Rat Myocytes and Vascular Smooth Muscle Cells

Neonatal myocytes were isolated and cultured using standard techniques, as previously described (26), and used on day 2. Cells were maintained in chemically defined medium plus 3% fetal bovine serum (34, 39). PAC-1 cells, a cell line derived from pulmonary artery vascular smooth muscle, were a generous gift of Li Li (Wayne State University). Cells were maintained in DMEM plus 10% fetal bovine serum and used during passages 2-3.

Construction of alpha 1C-Subunit Promoter-Luciferase Fusion Plasmid

The alpha 1C-subunit promoter-luciferase fusion plasmid (pJDM10) containing the full-length alpha 1C-subunit gene 5'-flanking sequence (fragment DHPRP, 2,055 bp) was constructed by subcloning DHPRP into the polylinker region of the pXp1 plasmid (a gift from Li Li) between restriction enzyme sites for BamH I and Xho I; pXp1 contains the firefly luciferase gene and is derived from a pSV40 vector (9, 33). Sequential deletion constructs were generated from the pJDM10 plasmid by use of the Erase-a-Base System (Promega, Madison, WI), which utilizes exonuclease III (14). Four deletion constructs (D1-D4) were generated with different alpha 1C-subunit 5'-flanking sequence lengths ranging up to 1,984 bp. All plasmid constructs were sequenced to determine the deletion end points.

Transfection of Mammalian Cells (Neonatal Cardiac Myocytes, PAC-1 Cells, and HEK-293 Cells)

All cells were plated onto 35-mm six-well dishes, as previously described (26). Freshly plated neonatal myocytes, 40-60% confluent PAC-1 cells, and HEK-293 cells were subjected to transfection. Transient transfections were carried out in regular culture medium with Fugene 6 (Boehringer). Briefly, 2 µg of promoter construct and 0.1 µg of beta -gal DNA vector were diluted into 10-20 µl of serum-free medium. This mixture was added to 100 µl of serum-free medium with 5 µl of Fugene 6. After gentle mixing, the mixture was set at room temperature for 15 min and then applied to the cultured cells; 72 h later the cells were harvested, and luciferase and beta -galactosidase activity were measured. Ninety percent of the myocytes were contracting at the time of harvest. Cells were lysed, and luciferase activity was assayed (9). The luminescence was measured over a 5-s period on a plate luminometer (Berthold Instruments) and expressed as arbitrary units. beta -Galactosidase activity was measured on the same lysate. The luciferase activity of each sample was normalized to beta -galactosidase activity for the same sample and expressed as percentage of the activity of the full-length alpha 1C-subunit promoter-luciferase fusion construct. Transient transfections for each construct were conducted on at least three separate cell cultures; data for each construct are presented as means ± SE. In preliminary experiments the amount of plasmid added for deletion mutants was adjusted; similar results, after normalization, were obtained, so a fixed amount of plasmid was used for subsequent experiments. For some experiments, cells were transfected with the full-length flanking sequence (pJDM10), and cells were treated 72 h later with isoproterenol, phenylalanine plus propranolol, 8-bromocAMP (8-BrcAMP), or testosterone. For analysis of deletions, data were analyzed by the nonparametric Kruskil-Wallis one-way ANOVA with the Systat program (Systat, Evanston, Il). Values are means ± SE.

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays (EMSAs) were carried out using nuclear extract from adult rat ventricular myocytes obtained as described by Dignam et al. (10). The DNA probe used in the assay was a PCR product from the full-length alpha 1C-subunit 5'-flanking sequence. It was labeled by T4 polynucleotide kinase and was subsequently gel purified. The binding assay contained 50,000 counts/min of probe and 6.25 µg of nuclear extract protein in binding buffer (40 mM KCl, 15 mM HEPES, pH 7.9, 1 mM EDTA, 0.5 mM dithiothreitol, 50% glycerol). After preincubation of the nuclear extract with 1 µg of poly[d(I-C)] for 10 min with or without a specific competitor, the binding reaction was conducted for 30 min at 22°C and then subjected to electrophoresis on a 4% polyacrylamide gel. DNA-protein complexes were detected by autoradiography.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Structure of the 5'-Flanking Sequence of the alpha 1C-Subunit Gene

On the basis of the cDNA sequence published by Koch et al. (20), specific primers for the alpha 1C-subunit of the L-type Ca2+ channel were designed and used to screen the genomic library by using PCR-based genome walking techniques. The secondary PCR gave rise to four positive fragments from the five predigested genomic DNA samples. The longest fragment, from the Ssp I library, was cloned into the pCRII plasmid and sequenced (Fig. 1). The fragment is 2,297 bp long, the most-3' 274 bp exhibiting 98% sequence identity with position +3 to +277 of the rat voltage-dependent Ca2+ channel alpha 1-subunit cDNA (20) (Fig. 2). Thus the last 274 bp we cloned likely comprise the first exon of the rat heart alpha 1C-subunit. The major transcription start site we identified (see below) is an additional 38 bp upstream, suggesting a total of 314 bp of the first exon in our genomic clone. Of interest, there is only 71% sequence identity between the last 314 bp of this cloned rat sequence and the cDNA sequence for the rabbit heart L-type Ca2+ channel alpha 1-subunit (30). No significant homology was seen between the rat and the reported human DNA sequences for 314 bp of coding sequence of the L-type Ca2+ channel alpha 1-subunit (40), indicating that the NH2-terminal amino acid sequence for the human and rat alpha 1C-subunits may differ significantly.


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  		Fig. 1.   Nucleotide sequence of 5'-flanking region of rat dihydropyridine (DHP) receptor alpha 1C-subunit gene. Clone DHPRP was sequenced as described in MATERIALS AND METHODS. Consensus sequences for selected transcription factors are indicated by single bold underline. Bold brackets indicate sites of progressive deletions of 5'-flanking sequence. Major and minor transcription initiation sites identified by primer extension analysis are indicated by hearts.



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  		Fig. 2.   Comparison of rat genomic clone DHPRP and rat aorta cDNA for DHP receptor. A: alignment of rat DHPRP genomic clone and sequence for rat aorta cDNA (20). Base pair numbering for genomic clone and cDNA is shown after optimal alignment. Rat genomic clone was sequenced 3 times in this region to confirm sequence, with identical results from each sequence analysis. Translation start site is shown in boldface (ATG). B: alignment of deduced amino acid sequence from rat genomic DNA and rat aorta cDNA.

Primer extension analysis was employed to confirm tentative identification of the first exon and to identify the transcription start site(s) of the alpha 1C-subunit gene (35). Six start sites for transcription were identified over a 100-bp range (Fig. 3). The most intense band likely represents the major transcription start site and is designated +1. There was no canonical TATA box found in ~1,984 bp of the 5'-flanking region of the gene. Primer extension analysis with full sequencing was also performed and identified two additional transcription initiation sites, for a total of eight possible sites, within the 100-bp region where other transcription initiation sites were identified. The cDNA sequence we obtained from 5'-RACE, by use of the same primer used for primer extension experiments, was identical to that obtained by sequencing the genomic clone.


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  		Fig. 3.   Determination of transcription initiation sites of rat DHP receptor alpha 1C-subunit gene. Transcription start site was mapped using a primer extension assay. Arrow, major transcription start site. Minor transcription start sites are also in lane labeled PE. Corresponding DNA sequence is shown at right.

For genes expressed at low levels, as occurs in myocytes for the DHP receptor, TATA-less promoters are not uncommon (39). Transcription initiation sites may occur at initiator (Inr) sequences with the sequence of 5'-Py-Py-A-N-A/T-Py-Py, where Py = C or T (3)'(5). Such an Inr sequence spans the +1 base identified as the major transcription initiation site by primer extension analysis. Other minor transcription start sites are indicated in Fig. 1. These minor transcription start sites do not display characteristics of an Inr. The 5'-flanking sequence of the 1,984 bp is shown in Figs. 1 and 2, as well as the sequence from +1 to +314. To confirm the primary transcription initiation site, we isolated and sequenced the products of two primer extension experiments. The sequences were identical to those immediately 3' to the major transcription initiation site identified in Fig. 1.

A putative translation start site with an appropriate open reading frame is present at bp +226. When translation is initiated at this site, the deduced amino acid sequence from the rat genomic clone is identical to that for the sequence deduced by Koch et al. (20) for the rat aorta voltage-dependent Ca2+ channel, except for one amino acid substitution at position 25 (Fig. 2).

Analysis of the Promoter and Upstream Putative Cis-Acting Elements

Because no Ca2+ channel alpha 1C-subunit promoter has been analyzed and reported in detail, a transcription factor database was searched to identify potentially important cis-acting regulatory elements (46). Analysis of the 1,984 nucleotides upstream of the major 5'-cap site revealed several putative cis-acting elements that exhibited >85% sequence identity to reported consensus recognition sites. Potentially important cis-acting sequences are shown in Fig. 1. Multiple consensus sequences for the cardiac-specific homeobox transcription factor Nkx2.5 are present (3, 5). Also present are consensus sequences for CRE-BP, HRE, C/EBPb, and AP-1 (4, 32, 44, 45).

Analysis of Rat Ca2+ Channel alpha 1C-Subunit Promoter Construct In Vitro

As an initial approach to localizing DNA sequences that may play a role in controlling transcription of the DHP receptor and confirming or eliminating a role for the putative sites identified above, a chimeric gene was created by cloning the DHPRP fragment (the 5'-flanking sequence and a portion of exon 1 of the DHP receptor, bp -1984 to +71) upstream of a luciferase reporter gene. Deletions were made starting at the 5'-end of the chimera and progressing in the 3'-direction.

Transcriptional activity in cardiac myocytes. To localize DNA elements that may play a role in controlling transcription of the DHP receptor gene in cardiac myocytes, cultured neonatal cardiac myocytes were transiently transfected with the plasmid pJDM10 containing the full-length flanking sequence or with sequential deletion constructs (D1-D4). Normalized activities of the reporter gene constructs in myocytes are shown in Fig. 4. Deletion of the 933 bp of the 5'-flanking region (deletion D1, bp -1984 to -1051) produced a modest but significant increase in reporter gene activity (P < 0.01), suggesting the presence of a repressor element in the segment that was deleted. With deletion of an additional 325 bp (D2), reporter gene activity was indistinguishable from that produced by the full-length 5'-flanking sequence. When an additional 492 bp were deleted (D3), there was marked attenuation of reporter gene activity (P < 0.01), with activity declining to ~20% of that for the full-length 5'-flanking sequence. This deletion removes consensus sequences for transcription factors NF-E2, c-Ets, C/EBP, AP-1, and two Nkx2.5 sites. Thus, in the sequence of bp -726 to -234, there appear to be elements critical for basal promoter activity. When the remaining bases of the 5'-flanking sequence upstream from the apparent major transcription initiation site were deleted (D4), all reporter gene activity was abolished (Fig. 4). Taken together, these data indicate that, within this putative proximal-promoter element from -726 to -234 bp, there are critical enhancer elements that act autonomously or, more likely, in concert to control basal expression of the rat cardiac DHP receptor gene.


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  		Fig. 4.   Transient expression analysis of rat Ca2+ channel alpha 1C-subunit gene promoter in cardiac myocytes. Results of DHP receptor-luciferase fusion gene activity are shown after transient transfection into cardiac myocytes. Luciferase activity has been corrected for transfection efficiency by normalization to beta -galactosidase activity exhibited by cotransfected pbeta Gal plasmid. Full-length DHP receptor-luciferase construct activity is expressed as 100%. Right: normalized luciferase activity; means ± SE. Left: length of 5'-flanking sequence. Luciferase activity after deletions (D1-D4) is shown. Plasmid pXp1 demonstrates basal luciferase activity of assay when control plasmid is used for transient transfection. Deletions D1, D3, and D4 each produced a significant change in reporter activity compared with full-length 5'-flanking sequence. P < 0.01; n = 8-17.

Transcriptional activity in vascular smooth muscle cells. To localize DNA elements that may play a role in controlling transcription of the DHP receptor gene in vascular smooth muscle cells, a series of experiments were conducted in PAC-1 cells that were analogous to those in cardiac myocytes. The vascular smooth muscle cells were transiently transfected with the plasmid pJDM10 containing the full-length DHPRP or with sequential deletion mutants (D1-D4). As was the case for myocytes, assays of reporter gene activity were conducted. For vascular smooth muscle cells, there is marked enhancement of reporter gene expression with deletion D1 (P < 0.01), suggesting the interaction of factor(s) from PAC-1 cells with repressor element(s) in this domain. Deletion D3 had a profound effect on reporter gene activity; this deletion almost abolished activity (Fig. 5), indicating the presence of basal promoter elements in this domain.


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  		Fig. 5.   Transient expression analysis of rat Ca2+ channel alpha 1C-subunit gene promoter in PAC-1 cells. Format is identical to that of Fig. 4. Deletions D1, D3, and D4 produced significant alterations in reporter gene activity. P < 0.01; n = 8-15.

To determine whether there is some degree of tissue specificity of the DHP receptor 5'-flanking sequence/luciferase reporter gene expression in vitro, we compared expression in cardiac myocytes, vascular smooth muscle cells (PAC-1), and a renal cell line (HEK-293). When equal amounts of the full-length chimeric gene were transfected (and normalized for transfection efficiency), there were marked differences in expression among the tissues (Fig. 6). There was robust expression in myocytes, ~50% less in vascular smooth muscle cells, and only ~6% relative expression in HEK-293 cells. The cloned full-length promoter region was also cloned into a beta -galactosidase expression vector and was transfected into myocytes and PAC-1 cells. Subsequently, X-gal staining was performed. This putative promoter was able to drive beta -galactosidase expression in these two types of cells, although it showed less strong promoter activity than that of the positive control vector with an SV40 promoter (data not shown).


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  		Fig. 6.   Tissue specificity of expression of rat Ca2+ channel alpha 1C-subunit gene. Relative reporter gene (luciferase) activity is shown. Control experiment was transient transfection of control plasmid (pXp1) into respective cell types. Relative activity in HEK-293 cells, myocytes, and PAC-1 cells is shown. Values are means ± SE of 3 experiments in triplicate.

EMSA. We performed EMSA as an initial approach to identify potentially important regions regulating transcription. Four distinct segments of the 2-kb 5'-flanking sequence were screened; segments were selected that contained potential cis-acting sequences that were likely candidates for regulating gene expression in heart and vascular smooth muscle. In initial experiments, three of the selected sequences did not produce changes in electrophoretic mobility (data not shown). Specifically, the DNA segment bp -1701 to -1445, which contains a putative MyoD transcription factor element, did not produce a shift in mobility. A 413-bp fragment (-846 to -434 bp; Fig. 7) demonstrated specific binding with nuclear proteins of nuclear extracts from four different preparations of purified adult rat myocytes. This fragment contains putative cis-acting sequences for well-described transcription factors, including Nkx2.5, NF-E3, c-Ets, AP-1, and C/EBPb. Binding of the radiolabeled DNA-protein complex was inhibited in a graded manner by addition of nonradioactive 413-bp probe (Fig. 7). We were able to entirely abolish radiolabeled DNA-protein binding by adding 200-fold molar excess of nonradioactive probe (data not shown). We repeated the experiments four times with similar results. Interestingly, consensus sequences for Nkx2.5 and AP-1 are present within this 413-bp DNA fragment; they are candidates for regulators of basal DHP receptor expression.


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  		Fig. 7.   Electrophoretic mobility shift assay. Nuclear extract from purified adult rat myocytes was incubated with a radiolabeled DNA fragment corresponding to bp -846 to -434. Autoradiogram of resultant electrophoretic mobility shift assay is shown. Same amount of nuclear extract was used in each experiment; a montage from 1 gel is shown. Lane 1, nuclear extract and radiolabeled DNA probe. Bottom band, free DNA; top band, shift in mobility due to protein-DNA interaction. Lanes 2-4, same as lane 1, but with increasing amount (25-, 50-, and 75-fold molar excess) of unlabeled DNA, demonstrating specific competitive interaction with nuclear protein(s). Lane 5, radiolabeled DNA probe with no nuclear extract.

Hormone responsiveness. We previously reported that, in the neonatal myocyte, abundance of mRNA and protein for the DHP receptor is regulated by catecholamines over the time course of hours (26). There is indirect evidence for regulation of DHP receptor expression by catecholamines in human heart as well (41). As an initial approach to identifying functional cis-acting elements of importance in cardiac muscle, we examined the response of the intact alpha 1C-subunit promoter-luciferase chimera to catecholamine analogs in neonatal myocytes. Cells were transfected with the promoter-luciferase construct, and luciferase assays were performed. Seventy-two hours after transfection of myocytes with pJDM10, myocytes were treated with the pure beta -adrenergic agonist isoproterenol, the alpha -adrenergic agonist phenylephrine plus the beta -adrenergic antagonist propranolol (26), or the membrane-permeant 8-BrcAMP (1 mM). Cells were treated for 1 or 4 h and then assayed for luciferase activity and for beta -galactosidase activity.

The data shown in Fig. 8 demonstrate that treatment with isoproterenol stimulates reporter gene activity about twofold, whether cells were treated for 1 or 4 h. Similar results were obtained with 8-BrcAMP. We previously found that treatment of neonatal cardiac myocytes with isoproterenol increases steady-state mRNA levels for the endogenous alpha 1C-subunit twofold (26). At 1 h, phenylephrine had no effect on reporter gene activity, but by 4 h it produced strong stimulation. Taken together, these data demonstrate that 1.8 kb of 5'-flanking sequence of the DHP receptor gene contains cis-acting elements sufficient to strongly enhance transcription in response to beta - and alpha -adrenergic signaling in myocytes.


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  		Fig. 8.   DHP receptor promoter-reporter gene response to adrenergic effectors. Luciferase activity normalized for beta -galactosidase activity is plotted on x-axis and expressed as percentage of control. Control, no added effector. Response of reporter gene activity to isoproterenol, 8-bromocAMP (8-BRcAMP), phenylephrine, and testosterone is shown. This representative experiment was replicated in 2 myocyte cultures, and each assay was conducted with 4-6 replicates. * P < 0.05; ** P < 0.01 compared with control at same time point (by ANOVA followed by Newman-Keuls test).

It has been suggested that expression of some ion channels may be responsive to gender determination hormones, and by this mechanism, these hormones may alter susceptibility to clinically important arrhythmias (17, 24, 27). Androgenic hormones also mediate cardiac hypertrophy (29). Accordingly, to determine whether DHP receptor expression is responsive to an androgenic hormone, we repeated the experiment, stimulating cells for 1 or 4 h with 1 µM testosterone. Figure 8 demonstrates that reporter gene activity is strongly stimulated by this hormone at 1 and 4 h. There is a hormone response element (HRE) in the 5'-flanking sequence that we have identified.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present investigation reports the cloning and partial characterization of the rat DHP receptor alpha 1C-subunit gene 5'-flanking sequence. Attention was focused on the promoter region and sequences that are necessary for basal transcription and that may direct tissue-specific expression. Sequence analysis revealed that the 5'-flanking sequence lacks a canonical TATA sequence but does contain a consensus Inr element, which corresponds to the major transcription start site determined by primer extension analysis. There appear to be minor transcription initiation sites as well. The Inr element typically contains within itself the transcription start site, and that is the case for the rat DHP receptor gene (39). The 5'-flanking sequence for the alpha 1C-subunit gene appears to contain all requisite elements for binding of general transcription factors (22). The 2-kb 5'-flanking sequence of the alpha 1C-subunit gene contains a promoter capable of directing its expression in neonatal ventricular myocytes and in vascular smooth muscle cells. Deletion analysis of the 5'-flanking region in the DHPRP-luciferase fusion gene indicates the presence of cis-acting regulatory elements in the proximal 726 bp that appear to be essential for myocyte- and smooth muscle cell-specific expression of the alpha 1C-subunit gene.

There has been considerable progress in understanding of molecular mechanisms responsible for tissue- and cell-specific expression of genes in noncardiac tissue, such as lymphocytes and hepatocytes. Although it has been somewhat slower, there has been recent progress in understanding regulation of expression of several ion channels in neurons and some progress for cardiac ion channels as well (16, 31, 42, 43). Trans-acting factors determining cardiac specificity of gene expression are beginning to be identified, notably including the mammalian analog of the Drosophila tinman gene, Nkx2.5, also called Csx (5,23). Nkx2.5 is a murine homeobox-containing gene specifically expressed in the developing heart; its expression is restricted to cardiac myocytes from day 8.5 postcoitus through adulthood (23). The 2-kb 5'-flanking sequence for the alpha 1C-subunit gene includes five consensus sequences for Nkx2.5. Nkx2.5 appears to play an important role in developmental events in the cardiac muscle lineage and in cardiac-specific gene expression. It is a modest transcriptional activator (5).

Deletion analysis of the 5'-flanking sequence of the alpha 1C-subunit promoter in myocytes and PAC-1 cells demonstrates that deletion of DNA, including the proximal two Nkx2.5 consensus sequences (deletion from position -726 to -234, D3) is associated with a substantial decrease in gene expression. EMSA demonstrates specific binding of nuclear proteins to a portion of this segment containing a consensus sequence for Nkx2.5. These data raise the possibility that these sequences may possibly play an important permissive role in alpha 1C-subunit gene expression in myocytes. The present studies of transcriptional regulation have been conducted in model systems: cultured neonatal myocytes and a vascular smooth muscle cell line. Whether similar mechanisms occur in vivo in adult myocytes and smooth muscle cells remains to be determined.

Previous work has demonstrated that two splice variants of the alpha 1C-subunit gene exist. The alpha 1C-A-isoform (also called Cach2a) can be identified by PCR and Northern analysis in heart exclusively, whereas the alpha 1C-B-isoform (also called Cach2b) can be identified in brain, heart, aorta, trachea, and lung (2, 36). The signals that dictate alternative splicing leading to expression of the alpha 1C-A-isoform exclusively in heart are unknown. It is possible that elements in the 5'-flanking region may play a role.

Kamp et al. (16) studied transcriptional regulation of a neuronal Ca2+ channel alpha 1D-subunit gene. Nkx2.5 consensus sequences are not present in the 5'-flanking sequence for this neuronal ion channel. However, the neuronal channel gene has a distinctive (ATG)7 trinucleotide repeat sequence that functions as an enhancer (16). No such sequences were observed here. A cardiac K+ channel gene, Kv1.5, demonstrates an increased transcription rate in rat ventricle and in GH3 pituitary cells in response to glucocorticoids (42, 43). Thus there is precedent for transcription of a cardiac ion channel being under control of a hormone. The consensus sequence for an HRE (15) is present in the 5'-flanking sequence for the alpha 1C-subunit gene; its definitive function remains to be determined. HREs, in the proper context, provide responsiveness to a variety of steroid hormones, and this HRE may account for the increase in transcription of the reporter gene in response to treatment of myocytes with testosterone.

Mori et al. (31) established that, in the 5'-flanking region of the Kv1.5 gene, there is a cAMP response element (CRE) that functions as an enhancer in cardiac myocytes and as a silencer in GH3 cells. In the 5'-flanking sequence of the alpha 1C-subunit gene, the CRE consensus sequence is present (position -1823), with uncertain function. On the basis of previous work from this laboratory (26, 34), one might expect that expression of the alpha 1C-subunit gene may be enhanced by cAMP-dependent processes. We demonstrated directly that, in cardiac myocytes, cAMP-dependent signaling increased mRNA abundance for the alpha 1C-subunit gene, DHP receptors, and functional Ca2+ channels (26). Thus we hypothesized the presence of a CRE in the promoter region for the alpha 1C-subunit gene, which we now demonstrate to be present. Its functional activity remains to be confirmed. Our data demonstrating that increasing intracellular cAMP concentration via receptor-dependent and -independent processes increases reporter gene expression suggest that this CRE may be functional in cardiac muscle or that there is an additional functional CRE further 5' than we have examined. Specific mutagenesis studies are necessary to ascertain the activity of the CRE at bp -1823. There is quantitative correspondence between the increase in reporter gene expression (2-fold) induced by isoproterenol, which we observed in the present study, and the increase in steady-state mRNA levels for the alpha 1C-subunit gene in response to isoproterenol (2-fold), which we previously reported in the same myocyte culture system (26). These findings suggest that the primary process regulating alpha 1C-subunit transcript levels in response to protein kinase A-dependent signals consists of alterations in transcription initiation rate. Preliminary findings suggest that other subunits of the L-type Ca2+ channel may be under transcriptional control by isoproterenol as well (12).

An additional postulated process by which transcription of the alpha 1C-subunit gene may be modulated, on the basis of previous studies, is through an AP-1 site. A consensus AP-1 site is present at position -595 in the 5'-flanking sequence of the alpha 1C-subunit gene. A deletion from position -726 to -234 (D3) removes the AP-1 site and markedly decreases basal reporter gene activity in myocytes. We showed (7, 26) that activation of protein kinase C and elevation of cytosolic Ca2+ concentrations in myocytes alter alpha 1C-subunit gene mRNA abundance. Protein kinase C activation decreases message levels; increased cytosolic Ca2+ increases message levels (7). Both effectors activate the transcription factor AP-1 (45). We now demonstrate that, over a time course slower than that for the beta -adrenergic response, alpha -adrenergic stimulation enhances reporter gene expression. Thus the AP-1 site at position -596 may be functionally important.

In addition to consensus sequences for trans-acting factors already discussed, there are consensus sequences for factors that are less obvious candidates for regulation of alpha 1C-subunit gene expression. Consensus sequences for two muscle determination factors, MyoD and MEF2, are present, but with unknown function (8). The D1 deletion removes the MyoD and MEF2 sequences, but there is no associated repression of reporter gene expression. Indeed, in myocytes and PAC-1 cells, this deletion produced augmented basal reporter gene activity. An MEF2-like motif appears to be a requisite for cardiac-specific expression of the rat troponin T gene. An MEF2 element is present in the proximal promoter region of the feline cardiac Na+/Ca2+ exchanger (1). This exchanger is, like the DHP receptor, a sarcolemmal protein critical to transmembrane Ca2+ flux in cardiac muscle.

In summary, we report an initial characterization of the rat L-type Ca2+ channel alpha 1C-subunit gene 5'-flanking sequence, including 1984 bp upstream of the major transcription initiation site. The promoter is TATA-less but contains an Inr sequence. There are multiple transcription start sites. However, primer extension experiments show a major transcription start site. Deletion analysis of the 5'-flanking sequence in cardiac myocytes and in vascular smooth muscle cells demonstrated that the -726- to -234-bp fragment provides the majority of basal promoter activity. Regions of the 5'-flanking region containing two Nkx2.5 transcription factor consensus sequences may be critical for expression of the gene in these tissues. This putative promoter for the alpha 1C-subunit of the L-type Ca2+ channel can be regulated by neurotransmitters and hormones known to be important for cardiovascular function.


    ACKNOWLEDGEMENTS

We appreciate the technical assistance of Bin Chen.


    FOOTNOTES

*  L. Liu and Q. I. Fan contributed equally to this work.

This work was supported in part by National Heart, Lung, and Blood Institute Grant R01-HL-54086 (J. D. Marsh), a grant-in-aid from the Michigan Affiliate of the American Heart Association (J. D. Marsh), a grant from the Veterans Administration (J. D. Marsh), and a fellowship grant from the Michigan Affiliate of the American Heart Association (L. Liu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. D. Marsh, Program in Molecular and Cellular Cardiology, Wayne State University School of Medicine, 421 E. Canfield Ave., Detroit, MI 48201 (marsh{at}cardiology.harper.wayne.edu).

Received 24 June 1999; accepted in final form 27 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 278(4):H1153-H1162
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