©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
M-CAT, CArG, and Sp1 Elements Are Required for -Adrenergic Induction of the Skeletal -Actin Promoter during Cardiac Myocyte Hypertrophy
TRANSCRIPTIONAL ENHANCER FACTOR-1 AND PROTEIN KINASE C AS CONSERVED TRANSDUCERS OF THE FETAL PROGRAM IN CARDIAC GROWTH (*)

(Received for publication, June 30, 1994; and in revised form, October 21, 1994)

Larry R. Karns Ken-ichi Kariya Paul C. Simpson (§)

From the Division of Cardiology and Research Service, Veterans Affairs Medical Center, San Francisco, California 94121 and the Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Induction of the fetal isogenes skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) is characteristic of cardiac growth in many models, suggesting a conserved signaling pathway. However, divergent regulation has also been observed. beta-Protein kinase C (PKC) and transcriptional enhancer factor-1 (TEF-1) are involved in induction of beta-MHC in alpha(1)-adrenergic-stimulated hypertrophy of cultured cardiac myocytes (Kariya, K., Farrance, I. K. G., and Simpson, P. C.(1993) J. Biol. Chem. 268, 26658-26662; Kariya, K., Karns, L. R., and Simpson, P. C.(1994) J. Biol. Chem. 269, 3775-3782). In the present study, we asked whether the skACT promoter used the same mechanism. A mouse skACT promoter fragment (-113/-46) was induced by both alpha(1)-adrenergic stimulation and co-transfection of activated beta-PKC, and contained three required DNA sequence elements: M-CAT, CArG, and Sp1. The skACT M-CAT element bound TEF-1 in cardiac myocytes. Thus the skACT and beta-MHC promoters both require a TEF-1 binding site for activation by alpha(1)-adrenergic stimulation, but differ in that skACT also requires a CArG box. These results provide a potential molecular basis for divergent regulation of the fetal program, and also imply that PKC and TEF-1 are conserved transducers for this program during cardiac growth.


INTRODUCTION

A characteristic feature of hypertrophic growth in adult cardiac muscle is qualitative changes in gene expression, changes that have come to be known as the ``fetal program'' (for reviews, see (1, 2, 3, 4) ). The fetal program, most evident in the rat heart subjected to pressure overload, but seen also in man(1) , consists of the recapitulation of a pattern of cardiac-specific gene expression that characterizes the pre-natal developing heart. For example, among the contractile protein genes, the skeletal isoform of alpha-actin (skACT) (^1)and the beta-isoform of myosin heavy chain (beta-MHC) are expressed in the fetal ventricle and are replaced by the respective adult isoforms, cardiac alpha-actin and alpha-MHC, during rat post-natal development. Induction of pressure overload, as by aortic banding, up-regulates skACT and beta-MHC, and this up-regulation is clearly pre-translational and probably transcriptional(5) . The fetal program includes up-regulation of other gene products, including atrial natriuretic factor (ANF), smooth muscle actin, ventricular myosin light chain 1, beta-tropomyosin, the alpha3 subunit of the Na,K-ATPase, B creatine kinase, and the T-type calcium current, as well as down-regulation of others, such as the sarco(endo)plasmic reticulum calcium ATPase, which is expressed at very low levels in the fetal heart. The similarity of gene expression during these major cardiac growth transitions, the fetal period, and pressure overload, has raised the possibility of a conserved signaling pathway for transcriptional regulation, one that is particularly important in cardiac growth.

Additional evidence for a conserved pathway is provided by studies with cultured cardiac myocytes. Cultured myocytes from both the neonatal and the adult rat heart recapitulate the fetal program when challenged with a variety of growth stimuli, including alpha(1)-adrenergic agonists, endothelin 1, angiotensin II, TGF-beta and bFGF, myotrophin, and mechanical stretch (for references, see (6) ). Thus if there is a conserved signaling pathway for the fetal program, this pathway might be activated by a variety of different extracellular stimuli.

Despite this evidence for a conserved pathway, it is also clear that the cardiac fetal genes are not regulated identically. Divergent regulation within the fetal program is well exemplified by beta-MHC and skACT (for a review, see (3) ). With pressure overload of the rat heart, skACT mRNA is detected in the ventricle much earlier than is beta-MHC mRNA(7) ; and the skACT mRNA elevation is transient despite continued load, whereas that of beta-MHC is sustained(8, 9) . Furthermore, skACT mRNA is found throughout the ventricle after pressure overload, whereas beta-MHC mRNA tends to be localized to the inner wall of the ventricle and around large coronary arteries(7) . In the aging heart, beta-MHC mRNA reaccumulates, whereas skACT mRNA remains at a low level(10, 11) . When the aged heart is subjected to pressure overload, beta-MHC mRNA is further up-regulated, but skACT mRNA is not induced(10) . Thus, it is evident that the signals for induction of skACT and beta-MHC during cardiac growth in vivo must diverge in some way, despite the fact that they also might share some conserved pathway for transcriptional regulation.

We have been using a model of cultured neonatal rat cardiac myocytes to define conserved and divergent pathways for transcriptional signaling during cardiac growth(12) . In this model, both skACT and beta-MHC are up-regulated at the transcriptional level during hypertrophic growth stimulated by an alpha(1)-adrenergic agonist(13, 14, 15) . Therefore, this system provides the opportunity to compare the induction of skACT and beta-MHC by the same growth stimulus in the same population of cardiac myocytes, asking whether they share a common, conserved pathway for induction or whether they are activated by divergent pathways.

Our recent studies have outlined a pathway that includes transcriptional enhancer factor-1 (TEF-1) and beta-protein kinase C (PKC) for alpha(1)-adrenergic induction of the beta-MHC promoter in the cultured cardiac myocytes. An alpha(1)-adrenergic agonist and activated beta-PKC both stimulate the beta-MHC promoter through an enhancer core/muscle CAT (M-CAT) element(6, 16) . This alpha(1)-adrenergic and beta-PKC response element binds cardiac myocyte TEF-1 in vitro(17) , and a mutation that disrupts TEF-1 binding abolishes both alpha(1)-adrenergic and beta-PKC induction of the beta-MHC promoter(6) . Thus TEF-1 is involved somehow in beta-MHC promoter induction by alpha(1)-adrenergic agonists in cardiac myocytes, and beta-PKC appears to be a transducer for alpha(1)-adrenergic signaling at TEF-1.

TEF-1 was cloned as a HeLa cell transcription factor for a Simian virus 40 (SV40) enhancer core motif (18) (for reviews, see (19) and (20) ). Recently, TEF-1 has been found to have a role in cardiac-specific transcription, even though TEF-1 mRNA (20, 21) and binding activity (M-CAT binding factor, MCBF) (6, 22, 23) are not restricted to myocardium. An M-CAT element, 5`-CATNC(C/T)(T/A)-3`(22, 24) , is present in the promoters of several cardiac- and skeletal muscle-specific genes(25) ; mediates activity in cultured cardiac myocytes of the promoters of cardiac troponin T(26) , beta-MHC(6, 27, 28) , and skACT(29) ; and binds TEF-1 in cardiac muscle (20, 23, 24) and cardiac myocytes(17, 29) . As assayed by promoter-reporter injection in the adult heart in vivo, TEF-1 binding sites are required for expression of beta-MHC (30) (see sequence in (6) ) and alpha-MHC (23) . In transgenic mice, beta-MHC promoter fragments of 600 bp (31) and 354 bp(32) , which contain at least two TEF-1 binding sites(6, 17) , are sufficient for developmentally regulated cardiac-specific expression. Finally, disruption of the TEF-1 gene in mice causes impaired cardiac growth with embryonic lethality(33) . Thus a target for alpha(1)-adrenergic stimulation of the beta-MHC promoter is critical for cardiac growth and gene transcription.

In the present study, we asked whether skACT induction during alpha(1)-adrenergic-stimulated cardiac myocyte hypertrophy also involved beta-PKC and TEF-1. We present three main findings. First, activation of the skACT promoter requires at least three DNA sequence elements: M-CAT, CArG, and Sp1. Second, the skACT promoter fragment activated by alpha(1)-adrenergic stimulation is also activated by beta-PKC. Third, the skACT M-CAT element binds TEF-1 in cardiac myocytes. We interpret these data to support two main conclusions. First, there is divergence in transcriptional activation of skACT and beta-MHC, even by the same agonist in the same population of cardiac myocytes. Induction of the skACT promoter requires a CArG box, and, by inference, the serum response factor (SRF), whereas this is not the case for beta-MHC. These results therefore provide a potential molecular basis for differential regulation within the fetal program, as exemplified by skACT and beta-MHC. Second, TEF-1 is a common factor in activation of both the skACT and beta-MHC promoters during cardiac myocyte growth stimulated through an alpha(1)-adrenergic receptor, and PKC might transduce a signal from the receptor to TEF-1. Since TGF-beta induces a skACT promoter in cardiac myocytes through the same response elements as an alpha(1)-adrenergic agonist(29) , PKC and TEF-1 might be conserved elements for activation of the fetal program by diverse stimuli for cardiac growth.


MATERIALS AND METHODS

Reporter Plasmids

The reporter plasmids RSV-CAT and RSV-LUX contained the chloramphenicol acetyltransferase (CAT) or the luciferase (LUX) coding sequences, respectively, expressed from the Rous sarcoma virus (RSV) long terminal repeat. The chicken beta-actin-CAT plasmid, beta-ACT-275-CAT(34) , was obtained from J. Mar and C. Ordahl (University of California, San Francisco). To make SP64-CAT, the promoterless CAT vector used throughout this work, the CAT-SV40 BamHI fragment from beta-ACT-275-CAT was subcloned into pGEM-3Z (Promega), then the CAT-SV40 fragment was cut out with HinDIII and XbaI, and inserted into the HinDIII-NheI sites of pSP64.

The mouse skACT gene (pMACT-alpha) was provided by M. Hu and N. Davidson (35) . The pMACT SacI-HinfI fragment (-46 to +47) was inserted into SP64-CAT cut with SacI and SmaI, to generate -46-skACT-CAT. The plasmid -46-skACT-CAT contained 47 of the 57 bp of the skACT first (untranslated) exon fused to the CAT gene (see Fig. 1). The deletions between -1400 and -113 were constructed by using convenient restriction sites, as shown in Fig. 1A, and converting the sites at the 5`-deletion end point to a SacI site by linker addition. These SacI fragments, from the new SacI site at the 5`-deletion end point to the SacI site at -46, were inserted into the SacI site of -46-skACT-CAT. SkACT-CAT plasmids containing the first intron were made by fusing the skACT promoter to CAT within the untranslated portion of exon 2, utilizing the DraI site at +1031 (1 bp 5` of the initiator ATG) (see Fig. 1A). Site-directed mutagenesis was performed on the -113/-46 skACT promoter fragment subcloned in pGEM-3Z, using the polymerase chain reaction(6) . The resulting polymerase chain reaction-generated fragments were inserted into the SacI site of -46 skACT-CAT. The thymidine kinase-LUX plasmids were constructed by inserting the -113/-46 and the -155/-76 skACT fragments into the SacI site of pT81-LUX(36) , containing the -81/+52 herpes simplex virus thymidine kinase promoter 5` of the LUX gene. Mutant sequences and the end points and orientation of the promoter fragments in the reporter plasmids were verified by DNA sequencing.


Figure 1: The -113/-46 sequence of the skACT promoter is required for activation by alpha(1)-adrenergic stimulation. A, the upper diagram represents the -1400-skACT-CAT reporter plasmid. Shown are the restriction sites used to generate the series of 5` promoter deletions, and the locations of the CArG, M-CAT, Sp1, and TATA elements. The lower diagram illustrates the structure of the skACT-CAT plasmids containing the first intron. Arrows indicate the start site of transcription at +1; and dark boxes, untranslated skACT exons. B, cardiac myocytes were transfected with the series of skACT-CAT plasmids illustrated in A. Cells were treated for 24 h with 20 µM phenylephrine (alpha1) or its vehicle (Basal). All dishes were co-tranfected with a constant amount of RSV-LUX as a control for transfection efficiency. CAT activity was assayed and calculated relative to the LUX activity measured in the same sample. The basal activity of -1400-skACT-CAT was set at 100. Values are the mean ± S.E. from at least three experiments, with duplicate dishes for each plasmid in each experiment.



Expression Plasmids

Expression plasmids for mutant beta- and alpha-PKCs, constitutively activated by deletion of the regulatory domain (see (16) ), and the expression vector without a PKC cDNA insert, pcDSRalpha, were obtained from M. Muramatsu and K. Arai (DNAX Research Institute, Palo Alto, CA, and Tokyo University, Tokyo, Japan). These PKC mutants are expressed at equivalent levels after transfection into the cardiac myocytes(16) .

Cell Culture and Transfection

Low density (150 cells/mm^2) primary cultures of neonatal rat cardiac myocytes were established and maintained in 60-mm dishes containing minimal essential medium with 5% calf serum (HyClone, Logan, UT), exactly as described previously(37) . Contaminating fibroblasts were leq10% of total cell number. NIH 3T3 cells were obtained from the UCSF Cell Culture Facility and were grown to 50% confluence in the same medium used for the cardiac myocytes.

Twenty h after plating, neonatal myocytes or 3T3 cells were refed with minimal essential medium containing 5% calf serum and 30 mM HEPES, pH 7.5. Equimolar amounts of the CAT plasmids (1.8 pmol, 5.0-6.8 µg) were transfected into duplicate dishes using the CaPO(4) method(16) . RSV-LUX (0.04 pmol, 0.2 µg) was included in all plates as an internal control for variability of transfection efficiency. In transfections with pT81-LUX plasmids, RSV-CAT (0.06 pmol, 0.2 µg) was included as the internal control. This amount of co-transfected RSV-LUX or RSV-CAT has no inhibitory effect on expression from reporter plasmids(16) . For the PKC co-transfection experiments, 0.2 or 0.8 pmol (0.5 or 2.0 µg) of the expression plasmids were included per plate. Total DNA per plate was maintained at 25 µg by the addition of variable amounts of pSP64 or pUC18. The CaPO(4) precipitate was removed from the cells after 2 h, and the medium was changed to serum-free minimal essential medium with porcine insulin (10 µg/ml) (Lilly), bovine transferrin (10 µg/ml) (HyClone), and bovine serum albumin (1 mg/ml) (Intergen no. 3130-00, Armour). After 16 h, the cells were refed with the same medium and treated for 24 h with various agents or their vehicle (100 µM ascorbic acid): 20 µML-phenylephrine HCl (Sigma); 0.2 µM WB4101 (Research Biochemicals, Natick, MA); 0.2 µML-isoproterenol HCl (Sigma); or 5% calf serum. Harvesting of transfected cells, preparation of cell extracts, and CAT and LUX assays were performed as described elsewhere(16) . Data are expressed as the relative activity, determined by normalizing the reporter plasmid CAT or LUX activity to the activity of the co-transfected RSV control plasmid in the same cell extract.

Gel Mobility Shift Assay (GMSA)

Nuclear extracts were prepared from the cultured neonatal rat cardiac myocytes, and GMSA was performed using a P-labeled double-stranded oligonucleotide probe representing the -215/-196 sequence of the rat beta-MHC promoter, both as described previously(6) . Competition for binding to the -215/-196 oligonucleotide probe was tested using a 100-fold molar excess of the -113/-46 skACT promoter fragment, containing the wild-type sequence or a 3-base change in the M-CAT consensus sequence (see Fig. 4).


Figure 4: Three elements in the -113/-46 sequence, M-CAT, CArG, and Sp1, are required for activation. The lower part of the figure gives the sequence of the -113/-46 wild-type (wt) mouse skACT promoter, with the CArG, M-CAT, and Sp1 elements underlined. The -108/-43 chicken skACT promoter (58) is shown also, indicating the conservation of these motifs across species. Dots indicate identity with mouse wild-type sequence. Region I and Region II of the chicken skACT promoter are protected in DNase I footprinting by embryonic muscle nuclear extract(22) , and the footprint over Region II is competed completely by an oligonucleotide that binds TEF-1(22, 24) . Mutations (mut) of the mouse -113/-46 sequence were generated by polymerase chain reaction. As shown in the upper part of the figure, the wild-type and mutant mouse -113/-46 fragments, inserted into -46-skACT-CAT, were transfected into duplicate dishes of cardiac myocytes, along with the internal control RSV-LUX; and the cells were treated with vehicle (Basal), 20 µM phenylephrine (alpha1), or 5% calf serum (Serum) for 24 h. The basal CAT activity of -46-skACT-CAT was set at 1. The basal and serum values are the mean ± S.E. of at least three experiments, and the alpha(1) values are the mean ± variation of two experiments. The 5` (-111/-108) and 3` (-54/-51) mutations had no effect on promoter activity (data not shown). Also, a different mutation of the chicken skACT promoter just 5` to the CArG/SRE1 (M -100/-95) has no effect on basal or TGF-beta-induced activity in rat cardiac myocytes(29) .



Statistics

Results are presented as the mean ± S.E. from the number of experiments indicated, with duplicate plates used in each experiment. Treated/control ratios were tested for deviation from unity by calculation of confidence limits.


RESULTS

The skACT Promoter Is Activated by alpha(1)-Adrenergic Stimulation in Cardiac Myocytes

In the low density neonatal rat cardiac myocyte cultures used for this work, alpha(1)-adrenergic stimulation induces accumulation of skeletal alpha-actin mRNA (13) and transcription of the skeletal alpha-actin isogene(14) . We first tested if alpha(1)-adrenergic stimulation activated a skACT promoter in the cardiac myocytes. A mouse skACT-CAT reporter plasmid, -1400-skACT-CAT, which extended from -1400 bp to within the untranslated first exon of the skACT gene (Fig. 1A), was transfected into cardiac myocytes and NIH 3T3 cells. CAT activity from -1400-skACT-CAT was detected only in the cardiac myocytes, whereas beta-ACT-CAT and RSV-CAT were expressed efficiently in both cell types (data not shown). Thus, -1400-skACT-CAT was expressed in cardiac myocytes but not in fibroblasts.

Treatment of transfected cardiac myocytes with the alpha(1)-adrenergic agonist phenylephrine (20 µM, 24 h) increased the activity of -1400 skACT-CAT by 2-fold (phenylephrine/vehicle = 2.00 ± 0.09-fold, n = 21, p < 0.01) (and see Fig. 1B). The alpha(1)-adrenergic antagonist, WB4101 (0.2 µM), blocked the phenylephrine-stimulated increase in CAT activity (0.94 ± 0.05-fold, n = 6, p = NS); and the beta-adrenergic agonist, isoproterenol (0.2 µM), did not increase CAT activity (isoproterenol/vehicle = 1.08 ± 0.06-fold, n = 13, p = NS). The promoterless vector, SP64-CAT (0-skACT-CAT) was not activated by phenylephrine (Fig. 1B) or isoproterenol (data not shown). These data confirmed that the mouse skACT promoter was activated through an alpha(1)-adrenergic receptor, and a similar finding has been reported previously for the human skACT promoter(38) .

The magnitude of skACT promoter activation by alpha(1)-adrenergic stimulation (2-fold) was less than that found previously for alpha(1)-adrenergic induction of skACT mRNA (11-fold) (13) or skACT gene transcription (6-fold)(14) . We attribute this lesser degree of promoter activation to differences in experimental protocols. In the prior studies of endogenous skACT mRNA abundance and transcription, myocytes were maintained in serum-free culture for at least 72 h prior to treatment with an alpha(1)-adrenergic agonist, and basal skACT transcripts and transcription were absent or very low(13, 14) . In the present studies, myocytes were serum-depleted for only 16 h prior to alpha(1)-adrenergic stimulation, and basal skACT promoter activity was detectable easily. Further, serum (5% v/v) activated -1400-skACT-CAT potently (3.86 ± 0.36-fold, n = 12, p < 0.01; and see Fig. 4). Thus it seemed likely that the ``basal'' activity of -1400-skACT-CAT was higher than might have been expected from our prior studies of endogenous skACT transcription, due to the residual effects of serum on the promoter, and that this elevation of ``basal'' activity reduced apparent induction by alpha(1)-adrenergic stimulation. An additional factor reducing apparent induction of the skACT promoter was that alpha(1)-adrenergic stimulation increased expression from the co-transfected control plasmid, RSV-LUX, slightly but significantly (1.28 ± 0.04-fold, n = 87, p < 0.01). Since alpha(1)-adrenergic activation of skACT-CAT was calculated relative to LUX activity, phenylephrine stimulation of the skACT promoter was relatively underestimated. We cannot exclude that basal activity was spuriously high due to the absence of negative regulatory elements outside the 1400-bp skACT promoter fragment, or that induction was less due to absence of positive elements. However, 730 bp of rat skACT 5`-flanking sequence is sufficient for appropriate expression during development in transgenic mice(39) . The magnitude of promoter induction with alpha(1)-adrenergic stimulation was not a function of the particular reporter gene, since 2-fold induction was found also with a LUX reporter (see below and Fig. 2).


Figure 2: The -113/-46 skACT promoter sequence is sufficient for activation by alpha(1)-adrenergic stimulation. Overlapping fragments of the skACT promoter were inserted into the enhancer test plasmid, pT81-LUX, 5` of the 81-bp herpes simplex virus thymidine kinase promoter, as illustrated in the lower portion of the figure. Cardiac myocytes were transfected with the skACT-thymidine kinase-LUX plasmids or pT81-LUX, as in Fig. 1B except that RSV-CAT was the internal control, and treated for 24 h with 20 µM phenylephrine (alpha1) or its vehicle (Basal). The relative LUX activity of the vector with no skeletal actin promoter fragment (pTK81-LUX) was set at 1. Values are the mean ± S.E. from three experiments.



The -113/-46 Sequence of the skACT Promoter Is Required for Activation by alpha(1)-Adrenergic Stimulation

To map the DNA sequences required for activation of the skACT promoter by alpha(1)-adrenergic stimulation, 5`-deletion mutants of the 1400-bp skACT promoter, illustrated in Fig. 1A, were transfected into the cardiac myocytes. The deletions progressively removed the four CArG box elements, which are necessary for expression of the sarcomeric actin genes in cardiac muscle cells(29, 40) . As shown in Fig. 1B, basal promoter activity was reduced by about 25% with the deletion at -360 bp, which removed the 5`-most CArG box; and by another 15% with the deletion at -155 bp, which removed 2 additional CArG boxes. The deletion at -113 bp maintained about 60% of the basal activity of the full-length promoter, whereas the deletion at -46 bp, just 5` of the skACT TATA box, reduced basal activity to the background level of the CAT vector plasmid (0-skACT-CAT in Fig. 1B). Activation of the promoter by alpha(1)-adrenergic stimulation was maintained at 2-fold with all deletions up to 113 bp, but was lost with the deletion at -46 bp (Fig. 1B). Thus, the -113/-46 skACT sequence was required for basal promoter activity and for activation by alpha(1)-adrenergic stimulation. This conclusion was supported further by deletion of the sequences between -155 and -41 (see Fig. 1A), producing a mutant, -1400Delta155-41-skACT-CAT, which was inactive both in the basal state and after alpha(1)-adrenergic stimulation (Fig. 1B).

The -113/-46 skACT Promoter Sequence Is Sufficient for Activation by alpha(1)-Adrenergic Stimulation

To determine if the -113/-46 promoter sequence was sufficient to confer activation by alpha(1)-adrenergic stimulation, skACT promoter fragments were inserted 5` of the thymidine kinase promoter in the heterologous enhancer test plasmid, pT81-LUX (Fig. 2). As shown in Fig. 2, pT81-LUX was not activated by alpha(1)-adrenergic stimulation. However, insertion of the skACT -113/-46 sequence, in -113/-46-pT81-LUX, increased basal activity markedly, and conferred a 2-fold activation by alpha(1)-adrenergic stimulation (1.92 ± 0.09-fold, n = 3, p < 0.05). In contrast, insertion of the skACT -155/-76 sequence, in -155/-76-pT81-LUX, did not produce a significant increase in basal or induced activity. As diagrammed in Fig. 2, the -155/-76 sequence shares the CArG box found in the -113/-46 sequence, and contains an Sp1 site; but the -155/-76 sequence lacks the M-CAT element found in the -113/-46 sequence. Therefore, the -113/-46 promoter sequence was sufficient to confer activation by alpha(1)-adrenergic stimulation, and an M-CAT element was present in this sequence, but not in the inactive -155/-76 sequence.

Activated beta-PKC Stimulates the skACT Promoter

We have found previously that co-transfection of a constitutively activated mutant of beta-PKC stimulates the beta-MHC promoter (16) and that stimulation by both an alpha(1)-adrenergic agonist and activated beta-PKC maps to a 9-bp element that binds TEF-1(6, 17) . To test if PKC stimulated the skACT promoter, cardiac myocytes were co-transfected with -1400-skACT-CAT or -113-skACT-CAT, and activated mutants of beta-PKC or alpha-PKC (Fig. 3). Promoter stimulation was calculated relative to myocytes co-transfected with the expression vector, pcDSRalpha, without the PKC cDNA insert. As shown in Fig. 3, both the 1400-bp skACT promoter and the 113-bp promoter were stimulated by activated beta-PKC (-1400-skACT-CAT/vector = 1.64 ± 0.17-fold, n = 5, p < 0.05; -113-skACT-CAT/vector = 1.85 ± 0.19-fold, n = 6, p < 0.01). Neither skACT promoter was stimulated by activated alpha-PKC (Fig. 3). Thus activated beta-PKC, but not activated alpha-PKC, stimulated the skACT promoter; and the response element or elements were contained within the 113-bp promoter.


Figure 3: Activated beta-PKC stimulates the skACT promoter. The -1400-skACT-CAT and -113-skACT-CAT plasmids were co-transfected with activated mutants of beta-PKC (act beta-PKC) or alpha-PKC (act alpha-PKC), or with the expression vector without the PKC cDNA (vector). RSV-LUX, which is not affected by the PKC mutants(16) , was also co-transfected as an internal control. CAT activity was measured after 24 h and normalized to the LUX activity in each sample. The relative CAT activity for each skACT plasmid co-transfected with the vector was set at 1. Values are the mean ± S.E. of five to six experiments. The autoradiograph is a representative assay showing the increase in CAT activity from the -113-skACT-CAT plasmid co-transfected with activated beta-PKC.



Three Elements in the -113/-46 Sequence, M-CAT, CArG, and Sp1, Are Required for Activation

The results above indicated that the -113/-46 skACT promoter sequence was necessary and sufficient for activation by alpha(1)-adrenergic stimulation. Within the -113/-46 sequence were three known recognition sites for DNA-binding proteins, M-CAT(41) , CArG(40) , and Sp1 (42) (Fig. 4). To assess the importance of these motifs for skACT promoter activation by alpha(1)-adrenergic stimulation, substitution mutations were introduced into each site in the context of the active 113-bp skACT promoter, as shown in Fig. 4. Wild-type and mutant promoters were transfected into the cardiac myocytes; and the cells were treated with an alpha(1)-adrenergic agonist. Mutation of either the CArG box or the M-CAT element eliminated basal expression of the 113-bp skACT promoter, and no induction by alpha(1)-adrenergic stimulation was detectable (Fig. 4). With the Sp1 mutation, basal promoter activity was reduced significantly (25% of wild-type), and induction was detectable but was not statistically significant (Fig. 4). Serum, a more potent stimulus for promoter activation, produced the same result as an alpha(1)-adrenergic agonist (Fig. 4). Control mutations at the 5` and 3` ends of the -113/-46 fragment, outside the known recognition motifs (Fig. 4), had no effect on promoter activity (data not shown). Therefore, mutation of the M-CAT or CArG motifs eliminated basal and induced expression from the active skACT promoter, and mutation of the Sp1 site attenuated expression.

The skACT M-CAT Motif Binds Myocyte TEF-1

The M-CAT motif in the skACT -113/-46 promoter fragment (CATTCTT, see Fig. 4) is similar to the enhancer core/M-CAT motif in the 20-bp alpha(1)-adrenergic- and PKC-inducible beta-MHC element that binds TEF-1 (CATACCA)(6, 17) . To determine if the skACT M-CAT sequence bound TEF-1, GMSA was done with cardiac myocyte nuclear extract and the -215/-196 beta-MHC promoter sequence as probe (Fig. 5). The characteristic band pattern is shown in Fig. 5, lane 1. Complex C2 is produced by the interaction of TEF-1 with the beta-MHC enhancer core/M-CAT(17) , whereas complex C1 is of questionable specificity and complex C3 is nonspecific(6, 17) . Complex C2 was reduced by competition with the -113/-46 skACT promoter fragment (Fig. 5, lane 2), whereas the -113/-46 fragment with the M-CAT mutation (sequence shown in Fig. 4) did not compete (Fig. 5, lane 3). Thus the M-CAT motif in the skACT promoter bound TEF-1 in cardiac myocytes.


Figure 5: The skACT M-CAT motif binds myocyte TEF-1. A double-stranded oligonucleotide of the rat -215/-196 beta-MHC promoter sequence was end-labeled with P and incubated with 10 µg of cardiac myocyte nuclear extract. Free and complexed probe were separated on a 4% native gel. Arrows indicate the position of protein-DNA complexes (C1, C2, and C3), identified after autoradiography. GMSA was done with no competitor (none, lane 1), with 100-fold molar excess of unlabeled wild-type -113/-46 skACT promoter fragment (-113/-46 skACT, lane 2), or with the -113/-46 fragment with the M-CAT mutations shown in Fig. 4(-113/-46 M-CAT mutant, lane 3). Complex C2 is specific (6) and is produced by binding of TEF-1(17) .



Intron 1 of the skACT Gene Does Not Contain an Enhancer for Expression in Cardiac Myocytes

Many genes for contractile proteins have a large (geq1 kilobase) intron within the 5`-untranslated region. In several cases, this intron contains an enhancer element for the expression of the gene in skeletal muscle cells, including muscle creatine kinase (43) and slow/cardiac troponin C(44) . To test for the presence of an intronic enhancer in the skACT gene, pairs of CAT reporter plasmids were constructed which contained the same length 5`-flanking sequence, without or with the first intron. The -360 and -46-skACT promoter fragments were fused to the CAT gene in exon 1, as in the experiments described above; or within the untranslated portion of exon 2, such that the entire 1-kilobase first intron was present in the normal position (see Fig. 1A). As shown on the right side of Fig. 1B, the plasmids containing the 360- or 46-bp promoter fragments plus intron 1 were no more active than the same plasmids without this intron. Thus, in cardiac myocytes the first intron of the skACT gene did not contain inherent promoter activity (-46-skACT-CAT and intron 1), nor did it enhance the activity of the 5` skACT promoter (-360-skACT-CAT and intron 1). These results agree with prior studies in cardiac myocytes on regulation of muscle creatine kinase (43) and slow/cardiac troponin C(44) .


DISCUSSION

There are three main results of the present study. First, activation of the skACT promoter during alpha(1)-adrenergic-stimulated cardiac myocyte hypertrophy requires at least three DNA sequence elements: M-CAT, CArG, and Sp1; second, the skACT promoter fragment activated by alpha(1)-adrenergic-stimulation is also activated by beta-PKC; and third, the skACT M-CAT element binds TEF-1 in cardiac myocytes. Because we have examined skACT promoter activation by the same agonist and in the same population of myocytes used for recent studies of the beta-MHC promoter(6, 16, 17) , we can draw inferences about conserved and divergent signaling pathways for alpha(1)-adrenergic-stimulated transcription during cardiac myocyte growth. Taken with those recent studies of the beta-MHC promoter, the present results have two main implications. First, they suggest that TEF-1 is a common factor in activation of the skACT and beta-MHC promoters during cardiac myocyte growth stimulated through an alpha(1)-adrenergic receptor, and that PKC might transduce the signal from the receptor to TEF-1. Since TGF-beta induces a skACT promoter in cardiac myocytes through the same response elements as an alpha(1)-adrenergic agonist(29) , PKC and TEF-1 might be conserved elements for activation of the fetal program by diverse stimuli for cardiac growth. Second, the results show that there is divergence in transcriptional activation of skACT and beta-MHC, even by the same agonist in the same population of myocytes. Induction of the skACT promoter requires a CArG box, and, by inference, the SRF, whereas this is not the case for beta-MHC. Thus, these results provide a potential molecular basis for differential regulation of the fetal program. It would now be interesting to test whether differences in the amount or activity of TEF-1 or the SRF contribute to the spatial and temporal differences in skACT and beta-MHC expression known to occur during cardiac hypertrophy in vivo (see Introduction).

alpha(1)-Adrenergic Stimulation of Both the SkACT and beta-MHC Promoters Requires an M-CAT Element Binding TEF-1

In the present study, a 67-bp skACT fragment (-113/-46) was found to be both necessary and sufficient for skACT promoter activation by alpha(1)-adrenergic stimulation. This fragment contained a conserved M-CAT motif that bound TEF-1 in GMSA, and a mutation that disrupted TEF-1 binding abolished alpha(1)-adrenergic activation of the 113-bp skACT promoter. A 20-bp oligonucleotide containing a TEF-1 binding site from the beta-MHC promoter can serve in isolation as an alpha(1)-adrenergic response element, and activation of a 215-bp beta-MHC promoter by alpha(1)-adrenergic stimulation is eliminated by disruption of TEF-1 binding(6, 17) . In the case of beta-MHC, unlike skACT, disruption of TEF-1 binding does not inactivate the promoter(6) , highlighting that TEF-1 is required for induction.

Other observations are consistent with a role for TEF-1 in gene expression during cardiac growth. An M-CAT element binding TEF-1 is required for induction of the chicken skACT promoter by a different growth agonist in cultured rat cardiac myocytes, TGF-beta(29) . In vivo, TEF-1 is up-regulated in the rat heart by pressure overload hypertrophy(23) . TEF-1 knockout mice have impaired cardiac growth (33) .

Critical to testing the involvement of TEF-1 in cardiac growth is understanding the mechanism of TEF-1 regulation by alpha(1)-adrenergic agonists or other stimuli. Evidence suggests that PKC might transduce a signal from the alpha(1)-adrenergic receptor to TEF-1 (see below), and PKC could regulate TEF-1 directly or indirectly (for a review, see (17) ). The mechanism is likely to be complex, since multiple TEF-1 isoforms, produced by alternate splicing, are present in the chick (20) and in the rat(45) , and TEF-1-related MCBFs have also been proposed (21) .

A CArG Box Is Required for Expression of the SkACT Promoter, but Not the beta-MHC Promoter

The CArG sequence is similar to the c-fos serum response element (SRE), and both bind the SRF (46, 47) . The SRF is present in rat cardiac myocytes, and interacts with the CArG/SRE sequences required for basal expression in cardiac myocytes of the human cardiac alpha-actin and chicken skACT promoters (29, 40, 48) .

In the present study, a CArG mutation inactivated the 113-bp skACT promoter, suggesting that the SRF is required for basal expression of the mouse skACT promoter in rat cardiac myocytes, in agreement with prior studies of the chicken skACT promoter(29, 48) . In contrast, there is no CArG box in the active 215-bp beta-MHC promoter(6, 27) , nor has the SRF been implicated in expression of the beta-MHC promoter in cardiac myocytes(6, 27, 28) . Our prior results on skACT and beta-MHC mRNA levels in the cultured cardiac myocytes are also consistent with a differential dependence on the SRF for basal expression. Specifically, skACT mRNA (13) and transcription (14) are very low or undetectable after prolonged culture in the absence of serum, whereas beta-MHC mRNA is well expressed in serum-free cultures(15) . This difference might be explained by serum regulation of SRF abundance and activity(49) , and skACT, but not beta-MHC, dependence on the SRF. Activation by the SRF could also explain why the basal activity of the skACT promoter was higher in this study than had been expected from prior study of endogenous skACT transcription, as mentioned under ``Results.'' Thus skACT and beta-MHC appear to differ in that the SRF, or another CArG-binding factor, is required for expression of skACT but not beta-MHC.

It is not clear whether the SRF is a target for alpha(1)-adrenergic signaling at the skACT promoter, or whether the SRF is simply a part of a basal transcriptional complex required for detection of skACT induction. In this study, an skACT promoter fragment (-155/-76) containing a CArG and a putative Sp1 site did not confer basal or alpha(1)-adrenergic-stimulated expression on the 81-bp thymidine kinase promoter. In contrast, the chicken skACT SRE1/CArG confers both basal expression in cardiac myocytes and responsiveness to bFGF (48) and TGF-beta (29) on the 56-bp c-fos promoter. Although it is likely that bFGF and TGF-beta signal differently in some respects from an alpha(1)-adrenergic agonist, the different heterologous promoters might also contribute to these results(50) .

An Sp1 Site Is Required for Activation of the skACT Promoter

In this study, an intact Sp1 site was required for maximum basal and induced expression in cardiac myocytes of the mouse skACT promoter, in agreement with a recent study of the chicken skACT promoter(29) . An Sp1 site in the human cardiac alpha-actin promoter is also required for basal expression in cardiac myocytes(40) , and both the cardiac and skeletal actin Sp1 sites bind cardiac myocyte Sp1 or an Sp1-related protein(29, 40) .

The apparent requirement of the skACT promoter for Sp1 or a related protein might not be different from the beta-MHC promoter. The 20-bp beta-MHC promoter element (-215/-196) that confers alpha(1)-adrenergic inducibility on the 109-bp thymidine kinase promoter is bound by TEF-1, but not by Sp1(17) . However, the 109-bp thymidine kinase promoter does contain a high affinity Sp1 binding site, centered at about -100 bp(51) . The 215-bp beta-MHC promoter, which requires an intact TEF-1 binding site for induction(6) , contains two GAG-like motifs, in the betae4 and betae5 footprints(27) ; and a GAG motif in an alpha(1)-adrenergic-inducible rat ANF promoter fragment binds an Sp1-related protein in cardiac myocytes(52) . Thus Sp1 or an Sp1-related protein might also contribute to expression in cardiac myocytes of the beta-MHC promoter, as appears to be the case with the skACT, cardiac actin, and ANF promoters.

beta-PKC Is a Transducer for alpha(1)-Adrenergic Activation of the skACT and beta-MHC Promoters

PKC is activated by alpha(1)-adrenergic agonists in the cardiac myocytes(53) , and beta-PKC immunoreactivity is translocated to the nucleus on activation (54, 55) . Co-transfection of a constitutively activated mutant of beta-PKC mimics the effect of an alpha(1)-adrenergic agonist, stimulating the beta-MHC promoter but not the alpha-MHC promoter(16) . beta-PKC and alpha(1)-adrenergic stimulation converge on the identical element to activate the beta-MHC promoter, a TEF-1 binding site(6, 17) . These results suggest that beta-PKC is in the pathway for alpha(1)-adrenergic regulation of TEF-1 on the beta-MHC promoter.

In the present study, the 1400-bp skACT promoter was stimulated by both an alpha(1)-adrenergic agonist and co-transfection of activated beta-PKC, and the element or elements required for these responses were contained within the -113/-46 skACT sequence. We did not attempt to map the PKC response element or elements in the -113/-46 skACT sequence, because the M-CAT and CArG mutations each had a major effect on basal and induced expression. Nevertheless, there is indirect evidence that the M-CAT motif binding TEF-1 is also important for beta-PKC activation of the skACT promoter. Specifically, in this study, activated beta-PKC was a much more potent stimulus for the 1400- and 113-bp skACT promoters than was activated alpha-PKC. The same preference for beta-PKC over alpha-PKC is found with a 3300-bp beta-MHC promoter (16) and the 20-bp PKC-inducible element that binds TEF-1(6) , but not with the AP-1 element(16) , which binds Fos and Jun. Thus, the beta-MHC and skACT promoters are both preferentially activated by beta-PKC, implying that TEF-1 might be a better substrate for beta-PKC than for alpha-PKC, a possibility that can now be tested.

The skACT CArG might participate in the response to PKC, and hence in the response to alpha(1)-adrenergic stimulation. Indeed, the c-fos SRE is stimulated by an alpha(1)-adrenergic agonist in cardiac myocytes (^2)and by co-transfection of activated alpha-PKC or beta-PKC in cell lines (see (6) ) and in cardiac myocytes. (^3)PKC stimulation of the SRE appears to require ternary complex factors (TCFs), for example SAP-1 and Elk-1, which interact with SRF bound to the SRE (for a review, see (56) ). The pressure overload response element of the c-fos promoter, assayed by DNA injection in the isolated perfused rat heart, maps to the SRE; and a mutation that disrupts TCF binding abolishes induction(57) . These results are consistent with a role for PKC in pressure overload in vivo, and suggest that TCFs are present in cardiac muscle(57) , as reported in a preliminary fashion by others(29) . However, the skACT CArG/SRE does not form a detectable complex with TCF(47) . Therefore the CArG/SRE might not be a target for PKC on the skACT promoter, in contrast with the c-fos promoter.


FOOTNOTES

*
This work was supported by the National Institutes of Health, the Department of Veterans Affairs Research Service, and a Fellowship (to K. K.) from the American Heart Association, Northern California affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: VAMC 111-C-8, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-221-4810 (ext. 3200); Fax: 415-750-6950.

(^1)
The abbreviations used are: skACT, skeletal alpha-actin; ANF, atrial natriuretic factor; bp, base pair; beta-ACT, beta-actin; beta-MHC, beta-myosin heavy chain; CAT, chloramphenicol acetyl transferase; CArG, CC(A/T)(6)GG; FGF, fibroblast growth factor; HSV, herpes simplex virus; LUX, luciferase; M-CAT, muscle CAT heptamer CATNC(C/T)(T/A); MCBF, M-CAT binding factor; PKC, protein kinase C; RSV, Rous sarcoma virus; SRE, serum response element; SRF, serum response factor; SV40, simian virus 40; TCF, ternary complex factor; TEF-1, transcriptional enhancer factor-1; TGF, transforming growth factor; NS, not significant; GMSA, gel mobility shift assay.

(^2)
L. R. Karns and P. C. Simpson, unpublished data.

(^3)
K. Kariya and P. C. Simpson, unpublished data.


ACKNOWLEDGEMENTS

We thank K. Arai, N. Davidson, M. Hu, K. Kaibuchi, J. Mar, M. Muramatsu, and C. Ordahl for plasmids; C. Long, E. McCluskey, J. Tsoporis, and W. Zierhut for valuable discussions; A. Stewart for review of the manuscript; and G. Cuenco, G. Davis, L. Moore, and M. Paningbatan for technical assistance.


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