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Endocrinology Vol. 138, No. 1 114-120
Copyright © 1997 by The Endocrine Society

ARTICLES

{alpha}1-Adrenergic Stimulation Inhibits 3,5,3'-Triiodothyronine-Induced Expression of the Rat Heart Sarcoplasmic Reticulum Ca2+ Adenosine Triphosphatase Gene1

Patricia S.-C. Wu, Anselmo S. Moriscot, Kirk U. Knowlton, Randa Hilal-Dandan, Huaping He and Wolfgang H. Dillmann

Department of Medicine, Divisions of Endocrinology (P.S.W., A.S.M., H.H., W.H.D.), Cardiology (K.U.K.), and Pharmacology (R.H.-D.), University of California-San Diego, La Jolla, California 92093-0618

Address all correspondence and requests for reprints to: Wolfgang H. Dillmann, M.D., Department of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0618.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The interactions between the ß-adrenergic system and thyroid hormone (T3) on cardiac function have been investigated in detail. In addition to ß-adrenoceptors, {alpha}1-adrenergic receptors are present in the mammalian heart. The interactions between T3 and the {alpha}1-adrenergic system remain, however, poorly understood. T3 stimulates the expression and transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2) gene, a protein vital in the control of cardiac calcium transients and contractility. We show that in rat cardiac myocytes, the stimulatory effect of T3 on SERCA2 messenger RNA expression and gene transcription is inhibited by an {alpha}1-adrenergic agonist. We demonstrate that direct activation of the {alpha}1-adrenergic signaling pathway, using a mutant constitutively active G protein (Gq) similarly down-regulated the T3 effect on SERCA2 transcription. The combined effect of thyroid hormone receptor and retinoid X receptors on T3-stimulated SERCA2 gene transcription was also markedly attenuated by {alpha}1-adrenergic stimulation. These results suggested that activation of the {alpha}1-adrenergic signaling pathway has an inhibitory effect on T3-dependent SERCA2 gene transcription. As this inhibitory effect of {alpha}1-adrenergic stimulation occurs when only one thyroid hormone response element (TRE) drives reporter expression, it is most likely mediated by an alteration of the nuclear factors binding to the TRE or by influencing the interaction of the TRE complex with the basal transcriptional machinery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIAC FUNCTION is markedly influenced by {alpha}- and ß-adrenergic effects and thyroid hormone action. The similarities in the cardiac manifestations of thyrotoxicosis, increased ß sympathetic stimulation, and the therapeutic benefit of ß-adrenergic blockade in hyperthyroidism are well established. In contrast, the interaction between thyroid hormone and the {alpha}1-adrenergic system in modifying cardiac action remains poorly understood. Currently there is evidence supporting two distinct mechanisms through which thyroid hormone may interact with the adrenergic system. Firstly, thyroid hormone has been shown to alter tissue sensitivity to catecholamines by affecting the quantity of ß-adrenergic receptors (1). Secondly, thyroid hormone may serve to augment the responsiveness to catecholamines by amplifying sympathetic effects at a postreceptor level (2, 3, 4). Thyroid hormone may also enhance ß-adrenergic receptor responsiveness by mediating changes in the quantity of the guanine nucleotide regulatory protein (Gs) in myocardial membranes. An increased atrial membrane level of Gs has been reported in hyperthyroid pigs (5). However, in addition to ß-adrenoceptors, mammalian cardiac myocytes also possess {alpha}1-adrenergic receptors. Changes in the quantity of cardiac {alpha}1-adrenergic receptors with different thyroid status have not been consistent (6, 7, 8, 9, 10) and are generally not the converse of accompanying changes in the ß-adrenergic receptors. Few attempts have been made to investigate the influence of thyroid hormone on specific {alpha}-mediated postreceptor biochemical responses.

{alpha}1-Adrenergic stimulation of the myocardium results in changes in chronotropic, contractile, hypertrophic, and metabolic responses (11). {alpha}1-Adrenergic stimulation has been shown to transcriptionally regulate the expression of certain cardiac genes, including immediate early genes, such as c-fos and c-jun, and constitutive contractile protein genes, such as myosin light chain-2 and cardiac {alpha}-actin (12, 13), but it has no effect on the sodium channel gene (14). Previous studies have suggested that {alpha}1-adrenergic receptors signal through coupling to a pertussis toxin-insensitive GTP-binding protein (Gq) which then activates phospholipase C, which catalyzes the hydrolysis of phosphatidylinositol 4,5-biphosphate to inositol 1,4,5-triphosphate and 1,2-diacylglycerol. The mediators ultimately activate mitogen-activated protein kinases and protein kinase C cascades (11, 15). In addition, the ras protooncogene has been implicated in the signaling pathway mediating the {alpha}1-adrenergic stimulatory effect on gene regulation (16, 17), but the precise details remain largely unknown.

Alterations in thyroid status exert profound effects on the electrical and contractile functions of the heart. Influences on diastolic relaxation (lusitropic activity) represents one of the major changes altered by thyroid hormone (T3) in the mammalian heart (18, 19, 20). In hyperthyroidism, the speed of diastolic relaxation is shortened, whereas in hypothyroidism, a prolonged relaxation occurs (20). These alterations can be attributed to changes in the enzyme activity of the sarcoplasmic reticulum (SR) Ca2+ adenosine triphosphatase (ATPase) pump (SERCA) that transports calcium from the cytosol to the SR after muscle contraction. The gene expression of the cardiac isoform SERCA2 is induced by T3 in the hearts of intact animals (21) and in cultured cardiac myocytes (22). However, it is not known whether {alpha}1-adrenergic stimulation may affect the T3-regulated expression of SERCA2. Previous experiments have shown that the use of isolated cardiac myocytes in the study of hormonal regulation of SERCA2 in vitro faithfully mimics in vivo observations. Using this model, we investigated the effect of the interaction between T3 and the {alpha}1-adrenergic system on the SERCA2 gene.

In this study, we examined the effect of the interaction of T3 and {alpha}1-adrenergic stimulation on SERCA2 messenger RNA (mRNA) expression and gene transcription using transient transfection assays. Our results indicate that the T3-stimulated SERCA2 gene expression and transcription are down-regulated by {alpha}1-adrenergic stimulation. We investigated whether direct stimulation of the {alpha}1-adrenergic signaling pathway using a constitutively active mutant Gq or an activated H-Ras protein would result in a similar effect on T3-dependent SERCA2 gene transcription. The effect of {alpha}1-adrenergic stimulation on the T3 response element (TRE) and thyroid hormone receptor (TR) complex was explored using coregulators, such as retinoid X receptors (RXR), and a specific TRE sequence in transient transfection assays.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Cultured neonatal cardiac myocytes (NCM) were prepared from 1- to 3-day-old Sprague-Dawley rats as described previously (22). Minced ventricles were subjected to a series of 20-min digestion with collagenase II (Worthington Biochemical Corp., Freehold, NJ) and pancreatin (Life Technologies, Grand Island, NY). Pooled digests were kept in newborn calf serum before purification of NCM by discontinuous Percoll (Pharmacia LKB Biotechnology, Piscataway, NJ) gradient centrifugation. The myocyte-enriched fraction that sedimented between the two layers of Percoll was saved and washed twice with a balanced salt solution. The final cell pellet was resuspended in a 4:1 mixture of DMEM-medium 199 supplemented with antibiotics (penicillin/streptomycin/fungizone, Life Technologies), 10% (vol/vol) horse serum, and 5% (vol/vol) FCS stripped of thyroid hormone using Bio-Rad AG1-X8 resin (Bio-Rad, Richmond, CA) (23). The cells were plated to a density of 2 x 106 cells/10-cm tissue culture plate precoated with 1% (wt/vol) gelatin. The NCM were allowed to adhere to the plates for 24 h before switching to serum-free medium or medium containing 3.4% (vol/vol) horse serum and 1.6% (vol/vol) FCS stripped of thyroid hormones. For RNA analysis, all cells were maintained in serum-free medium for 48 h before the addition of drugs. For transfection assays, the NCM were maintained in medium composed of 5% (vol/vol) stripped serum throughout and for 24 h after transfection. The cells were then washed twice, and the media were replaced by a 4:1 mixture of DMEM-medium 199 supplemented with transferrin (10 µg/ml), insulin (10 µg/ml), vitamin B12 (1.5 µM), and BSA (1 mg/ml) for at least 8 h before drug treatment.

RNA isolation and analysis
NCM were maintained in 15% serum for 24 h after harvest and then switched to serum-free medium. After 48 h, 10-4 M phenylephrine and 2 x 10-6 M propranolol (PP) were added to the NCM, either with or without the simultaneous addition of 10-7 M T3. Twenty-four hours after the drug treatment, total cellular RNA was isolated from NCM using the guanidinium thiocyanate phenol/chloroform method (24). The yield and purity of RNA samples were assessed by ratio optical density at 260 and 280 nm. For hybridization, total RNA was size-fractionated by denaturing agarose gel electrophoresis, visualized by ethidium bromide staining, transferred to nylon membrane in 10 x SSC (standard saline citrate) by capillary diffusion and fixed by baking at 80 C for 2 h before hybridization with complementary DNA (cDNA) probes labeled with [32]phosphorous by random priming. After hybridization, the membranes were washed with increasing stringency from 2 x SSC-0.1% SDS at room temperature to 0.1 x SSC-0.1% SDS at 55 C before exposure to Kodak XAR 5 film (Eastman Kodak, Rochester, NY) at -70 C. Quantification of autoradiograms was carried out by scanning densitometry using the NIH Image software on a Macintosh personal computer with an attached high resolution camera.

cDNA probes
The SR Ca2+-ATPase cDNA was a rat cardiac cDNA cloned and previously characterized, containing a 1.7-kilobase (kb) EcoRI fragment spanning the 5'-extent of pCC1 and a 4.1-kb cDNA was isolated from a cardiac-specific cDNA library by screening with a 3'-end internal fragment derived from a rabbit slow isoform SR Ca2+ ATPase cDNA pCA3 (22). Three other cDNA probes were used as references for equivalent RNA loading and have been previously described: 1) a 1.5-kb HindIII/SphI fragment of the cDNA encoding the constitutive form of heat shock protein 70 (cHSP70) p333 (25), 2) a 780-bp fragment of the human glyceraldehyde-3-phosphate-dehydrogenase cDNA (American Type Culture Collection, Rockville, MD) (26), and 3) the 29-bp oligomer cDNA encoding the mouse 28S ribosomal RNA.

Transient transfection assays
The NCM culture was established as previously described. After 40- to 48-h incubation in 5% serum stripped of thyroid hormones, the NCM were transiently transfected with a total of 20 µg DNA/plate using a calcium phosphate-DNA coprecipitation method (27). The NCM were transfected with 7 µg 3.2-kb SERCA2 CAT reporter plasmid DNA, 3 µg pCMVßGal (CMV, cytomegalovirus; ßGal, ß-galactosidase), 5 µg rat TR{alpha}1, and either 5 or 7 µg of the appropriate expression vectors (GqWT, Gq1, H-Ras, or TREpal), as described in the text and figure legends. The empty vectors pCDNA1 and pBLCAT2 were cotransfected as the control in the appropriate experiments. The empty vector pBS was used to compensate for a constant total DNA when necessary. After 16–20 h of incubation in 3% CO2, the precipitate was washed off, and the NCM was placed in medium containing 5% stripped serum for 24 h. Fresh serum-free medium supplemented with insulin, transferrin, and BSA was added to the cells before drug treatment. After 8 h in the serum-free medium, 10-4 M phenylephrine plus 2 x 10-6 M PP were added, with or without 10-7 M T3. For the experiments examining the effects of the {alpha}1-adrenergic antagonist prazosin (PR), PR at 10-5 M was added to the myocytes simultaneously with PP and or T3 at this time point. Twenty-four hours after treatment, the NCM were harvested in 0.25 M Tris-HCl, pH 7.5, and subjected to three cycles of freeze-thawing to lyse the cells. The cell debris was pelleted, and the supernatant was collected and aliquoted for ßGal and chloramphenicol acetyltransferase (CAT) assays. ßGal activity was measured by the ONPG method (28), and CAT assays were performed using a phase extraction technique (29). CAT activities were normalized to their corresponding ßGal activities to correct for variation in transfection efficiency. Each experiment was carried out in triplicate and repeated on at least three separate occasions, except where otherwise specified.

Plasmid constructs
The rat SERCA2 promoter CAT expression vector contains the 3.2-kb upstream promoter sequence of the SERCA2 gene fused to the bacterial gene coding for CAT inserted into a promoterless vector pBLCAT3 (22); the parent vector contains translational termination signals and simian viurs 40 polyadenylation signal to enable the expression of CAT protein in eukaryotic cells. Five micrograms of rat TR{alpha}1 (rTR{alpha}1) isoform expressed in Escherichia coli BL21 (30) was used in all transfection experiments. The wild-type G{alpha}q (GqWT) and the constitutively active guanine triphosphatase-deficient mutant (R183C) of G{alpha}q (Gq1) were both cloned into a cytomegalovirus promoter expression vector pCDNA1 (Invitrogen, San Diego, CA) (16). The constitutively active H-Ras expression plasmid used was cloned into a pDCR vector (31). The RXR{gamma} plasmid construct was a gift from Dr. C. Glass and was described previously (40). The TREpal plasmid contained a synthetic derivative of an idealized TRE, a palindromic oligomer (AGGTCATGACCT) fused to a minimal viral thymidine kinase (TK) promoter in the expression vector pBLCAT2 (32). The expression vectors pCDNA1 and pBLCAT2 without insert were used as the control DNA in the experiments with G{alpha}q and TREpal, respectively.

Data analysis
Results of multiple experiments are shown as the mean ± SE. The results were analyzed using a statistics software Statsview 4.0 on a Macintosh personal computer. Statistical comparison used ANOVA (Fisher’s protected least significant difference test) for group comparison at a level of significance of P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
{alpha}1-Adrenergic stimulation markedly diminished T3-induced SERCA2 gene expression
It has previously been shown that T3 stimulates SERCA2 gene expression and increases SERCA2 mRNA levels in NCM in cell culture (22) and under in vivo conditions (21). The aim of the present study was to investigate the effects of {alpha}1-adrenergic stimulation on T3-dependent SERCA2 gene regulation in NCM, using Northern analysis of mRNA and transient transfection assays. Primary cultures of NCM were incubated for 24 h in the presence of 10-7 M T3, with or without 10-4 M phenylephrine and 2 x 10-6 M PP, which produced a pure {alpha}1-adrenergic effect. The cHSP70, which is known to be unresponsive to T3, was used as a mRNA loading standard. T3 increased SERCA2 mRNA expression by 2.45 ± 0.35-fold compared to the control. Addition of the {alpha}1-adrenergic agonist PP alone resulted in a reduction of SERCA2 mRNA levels to 53.2 ± 27.7% of the control value. When PP was added with T3, SERCA2 mRNA level was only 79.9 ± 27.4% of the control, a 3-fold reduction from the T3-stimulated levels in the absence of PP (Fig. 1Go). Similar results were obtained using glyceraldehyde-3-phosphate-dehydrogenase and 28S RNA as loading controls (results not shown). In the presence of PP, the T3-stimulated increase in SERCA2 mRNA was only 1.5-fold of the basal expression. This represented a 61% reduction compared to the 2.45-fold increase in SERCA2 mRNA induced by T3 in the absence of PP. Thus, this suppression of T3-stimulated SERCA2 mRNA expression could not be accounted for solely by a decrease in the basal SERCA2 mRNA level in the presence of PP, but was likely to be an effect of the interaction between T3 and {alpha}1-adrenergic stimulation. We have, therefore, shown that {alpha}1-adrenergic stimulation down-regulated the T3-induced SERCA2 gene expression, resulting in a reduction of SERCA2 mRNA levels.



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  		Figure 1. Effect of {alpha}1-adrenergic agonist treatment with and without T3 on SERCA2 messenger RNA expression in cultured neonatal cardiac myocytes. A, Primary cultures of neonatal cardiac myocytes were serum-starved for 24 h before treatment with 10-7 M T3, 10-4 M phenylephrine and 2 x 10-6 M PP, or both in combination. Twenty-four hours after treatment, total RNA was isolated. Five micrograms of total RNA from each treatment were used. Hybridization was carried out with the 1.7-kb SERCA2 and 1.5-kb cHSP70 cDNA probes. The cHSP70 probe was used as a loading control. Lane 1, Control; lane 2, T3; lanes 3 and 4, PP; lane 5, T3 and PP. This is a representative Northern blot. Similar results were obtained in three separate experiments. B, Fold induction of SERCA2 mRNA, expressed as ratio of SERCA2/cHSP70. The results shown represent a mean of three experiments. *, P < 0.02 compared to T3 treatment alone.

 
To investigate whether this interaction occurred at the transcription level, transient transfection assays with TRE containing SERCA2 regulatory region-CAT reporter plasmids were performed in NCM maintained in hypothyroid serum. Expression plasmids coding for rTR{alpha}1 were cotransfected. After transfection, 10-7 M T3 was added, with and without PP, and reporter activity was determined after 24 h. T3 alone induced a 4.35 ± 0.4-fold rise in SERCA2-driven CAT reporter activity, confirming a significant T3-dependent effect on the SR Ca2+ ATPase gene transcription. In contrast, PP by itself had no significant effect on reporter activity. In the presence of both T3 and PP, the induction of CAT activity was reduced by 64% to 1.62 ± 0.1-fold (P < 0.0001; Fig. 2AGo). In the presence of the specific {alpha}1-adrenergic antagonist PR, the down-regulation of T3-stimulated SERCA2 transcription was abolished, resulting in a 3.6 ± 0.6-fold induction of SERCA2 CAT activity, not significantly different from that with T3 stimulation alone. PR alone has no significant effect on the T3-stimulated SERCA2 transcription (Fig. 2BGo). These results indicated that {alpha}1-adrenergic stimulation down-regulated the T3 stimulatory effect on SERCA2 gene transcription. Our results suggested that this effect was specific for {alpha}1-adrenergic stimulation, as it was reversed by a specific {alpha}1-adrenergic antagonist. This presents the first description of an inhibitory effect of {alpha}1-adrenergic stimulation on T3-induced gene transcription. To determine whether effectors of the {alpha}1-adrenergic signaling cascade that mediate postreceptor binding effects, such as the G proteins, influence the T3 effect, the experiments described below were performed.



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  		Figure 2. Effect of {alpha}1-adrenergic stimulation and blockade on SERCA2-driven CAT activity in neonatal cardiac myocytes. A, Neonatal cardiac myocytes were transfected with 7 µg SERCA2 CAT plasmid and vectors expressing rTR{alpha}1 (5 µg) and ß-galactosidase (3 µg). The cells were incubated in the absence or presence of 10-7 M T3, 10-4 M phenylephrine and 2 x 10-6 M PP, or both in combination. After 24 h, the cells were harvested, and CAT activity was determined as described in Materials and Methods. All transfections were carried out in triplicate, and the data are expressed as the mean ± SD. *, P < 0.0001 compared to T3 alone. B, This series of experiments was similar to that described above, except that 10-5 M PR was added simultaneously with T3, PP, or both agents. Results shown are representative for two independent experiments, performed in triplicate, and the data are expressed as the mean ± SD. *, P < 0.0001 compared to T3 alone, T3 plus PR, and T3, PP, and PR.

 
The activated G protein Gq and H-Ras inhibit T3-stimulated SERCA2 gene transcription
We hypothesize that the interaction between T3 and {alpha}1-adrenergic stimulation may result from a direct effect on the {alpha}1-adrenergic signaling pathway, and the direct activation of an intermediary signal-transducing protein such as the guanine nucleotide regulatory protein G{alpha}q would result in a similar down-regulation of the T3 stimulatory effect on SERCA2 transcription as using the {alpha}1-adrenergic agonist PP. To investigate this hypothesis, we cotransfected expression plasmids coding for the mutant constitutively active G{alpha}q (Gq1) and wild-type G{alpha}q (GqWT) with the SERCA2 CAT reporter plasmid in NCM with and without 10-7 M T3. The expression vector pCDNA1 without insert was transfected as a DNA control. The expression of G proteins had no effect on basal SERCA2 transcriptional activity (Fig. 3AGo). However, the addition of these G proteins resulted in a down-regulation of the T3 stimulatory effect on SERCA2 transcription. As shown in Fig. 3BGo, T3 alone induced a 4.3 ± 0.70-fold rise in SERCA2 CAT reporter activity compared to control, confirming previous results. When Gq1 was cotransfected, the addition of T3 only induced a 2.3 ± 0.2-fold increase in SERCA2 transcription, a reduction of 47% compared to T3 stimulation alone (P < 0.001). In the presence of GqWT, there was a modest reduction of T3-stimulated SERCA2 CAT activity to 3.4 ± 0.3-fold the basal transcription rate (P < 0.05 compared to T3 alone). These results suggested that direct stimulation of the {alpha}1-adrenergic signaling pathway via G proteins could attenuate the T3-stimulated SERCA2 transcription, producing a similar inhibition as an {alpha}1-adrenergic agonist. This inhibitory effect was further enhanced by the expression of an activated mutant G protein.



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  		Figure 3. Activated G protein (Gq) suppressed T3-induced SERCA2 CAT activity. A, Basal SERCA2 transcription in the presence of the activated Gq (Gq*) or wild-type Gq (GqWT); the experimental design is described below. B, NCM were transfected with 7 µg SERCA2 CAT plasmid and vectors expressing rTR{alpha}1 (5 µg) and ß-galactosidase (3 µg). In appropriate groups, the cells were cotransfected with 5 µg plasmids expressing either the activated Gq (Gq*) or wild-type Gq (GqWT). The vector pCDNA1 without insert was used as control. The cells were incubated for 24 h in the absence or presence of 10-7 M T3 before harvest and assay for CAT activity. All transfections were carried out in triplicate, and the data are expressed as the mean ± SD. The results presented are typical of three separate experiments. *, P < 0.05 compared to T3 alone.

 
The ras gene family is a member of a nucleotide-binding superfamily and is involved in growth factor-stimulated proliferation in many cell types. Ras not only shares structure homology with the heterotrimeric G proteins, but is also functionally related, as both G proteins and Ras contain intrinsic GTPase activity and can alternate between active and inactive states. A functional role for Ras in {alpha}1-adrenergic stimulated hypertrophy of cardiac myocytes had been shown (17). It was found that a dominantly negative H-Ras mutant inhibited the hypertrophic effect of {alpha}1-adrenergic agonist phenylephrine, suggesting that a Ras-dependent pathway may in part mediate cardiac hypertrophy after stimulation of a classical G protein-coupled pathway. To assess whether Ras has a role as part of the {alpha}1-adrenergic signaling pathway responsible for the down-regulation of the T3 stimulatory effect on SERCA2 transcription, we cotransfected 5 µg of an expression plasmid containing a constitutively activated H-Ras in NCM with the SERCA2 CAT reporter plasmid in the presence and absence of 10-7 M T3. In this series of experiments, 10-7 M T3 alone induced a 5.9 ± 0.4-fold increase in SERCA2 CAT activity, whereas H-Ras alone had no significant effect on SERCA2 CAT reporter activity compared to that in the control. When H-Ras was cotransfected, T3-stimulated SERCA2 transactivation was significantly reduced by 39% to 3.6 ± 0.2-fold (P < 0.0001 compared to T3 alone). We have demonstrated that cotransfection of a constitutively active mutant G protein Gq1 or H-Ras resulted in a similar inhibition of T3-stimulated SERCA2 gene transcription as an {alpha}1-adrenergic agonist. These data suggested that direct activation of the {alpha}1-adrenergic pathway can result in an interference of the T3-regulated SERCA2 gene transcription.

Down-regulation of the T3 effect on SERCA2 by {alpha}1-adrenergic stimulation is TRE specific
To explore whether the interaction between T3 and {alpha}1-adrenergic stimulation was specific to particular TREs, a synthetic palindromic TRE sequence, TREpal, which is known to have significantly higher affinity for nuclear TRs than wild-type TREs (32), was used in transient transfection assays. The expression plasmid pBLCAT2 without insert was transfected as a DNA control. The T3-stimulated response of the TREpal-TKCAT construct was compared to that of SERCA2 CAT in the presence of PP with and without T3. In the presence of PP alone, there was no significant induction of either SERCA2 or TREpal. Transfection of TREpal in the presence of T3 resulted in a 9.2 ± 0.1-fold rise in CAT activity. When PP was added with T3, the CAT activity was reduced by 67% to 3.0 ± 0.04-fold (P < 0.0001 compared to T3 stimulation alone; Fig. 4Go). Using the empty reporter vector pBLCAT2 with no known TRE, there was no significant T3 stimulation nor was there an inhibitory effect of PP. This result indicated that the interactions of {alpha}1-adrenergic stimulation with T3 were TRE specific.



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  		Figure 4. Effect of {alpha}1-adrenergic stimulation on a palindromic TRE (TREpal). NCM were transfected with 7 µg of plasmid constructs expressing SERCA2 CAT, TREpal, TKCAT, or the empty vector pBLCAT2. All cells were cotransfected with vectors expressing rTR{alpha}1 (5 µg) and ß-galactosidase (3 µg). After transfection, the cells were incubated with 10-7 M T3 alone, 10-4 M phenylephrine and 2 x 10-6 M PP, or both T3 and PP in combination. After 24 h, the cells were harvested, and CAT activity was assayed. All transfections were carried out in triplicate, and the data are expressed as the mean ± SD. The results presented are typical of three separate experiments. *, P < 0.0001 vs. T3 treatment alone.

 
To further explore whether the down-regulation of T3 effect on SERCA2 transcription by {alpha}1-adrenergic stimulation is TRE specific, we investigated the effect of {alpha}1-adrenergic stimulation on the cooperative effect of TR and nuclear TR accessory proteins (TRAPs) heterodimerization. TRs bind to DNA more stably if they form heterodimers with other nuclear proteins. Any disruption of this heterodimerization may result in a less stable TR-TRE complex, leading to reduced ligand binding and gene transcription. RXRs are a group of TRAPs that interact with TRs through heterodimerization on TREs. To investigate whether the effect of RXRs and TR on SERCA2 gene transcription was altered by {alpha}1-adrenergic stimulation, 5 µg RXR{gamma} were cotransfected with TR{alpha} and SERCA2 in the presence of PP with and without T3. The basal SERCA2 activity was not affected by the cotransfection of RXR{gamma} and/or the addition of PP alone. The cotransfection of RXR{gamma} resulted in a 4.3 ± 0.6-fold induction of the T3-stimulated SERCA2 driven reporter activity compared to the control. In the presence of PP, this T3-stimulated SERCA2 transcription was reduced to 3.3 ± 0.2-fold the control level (results not shown). Thus, in the presence of RXR{gamma}, T3 stimulated a 3.6 ± 0.5-fold increase in SERCA2 transcription compared to basal level, which was significantly reduced by the addition of PP to 2.4 ± 0.1-fold (P < 0.05). These results suggested that the interference of T3-induced SERCA2 gene transcription by {alpha}1-adrenergic stimulation also occurred when TR-RXR heterodimers form on TREs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T3 and the adrenergic system both have a profound influence on the physiological functions of the mammalian heart. Previous investigations have shown that alterations in the thyroid status affect the adrenergic receptor response to catecholamines which may or may not be accompanied by changes in receptor numbers (10). At the molecular level, T3 has been shown to regulate ß-adrenergic receptor gene transcription (34) and the expression of genes encoding G protein subunits in the rat heart (35). However, few studies have explored the interaction between {alpha}1-adrenergic stimulation and T3 on specific gene regulation in detail. In this paper, we report a novel observation showing that {alpha}1-adrenergic stimulation inhibited T3-dependent SERCA2 gene transcription in cardiac myocytes. This effect appears to be specific, as it was reversed in the presence of an {alpha}1-adrenergic antagonist.

In the myocardial {alpha}1-adrenergic signaling pathway, the coupling of the liganded receptor to the second messenger G protein Gq activates a cascade of enzymic events, including the hydrolysis of phosphatidyl inositol and the activation of protein kinase C, which catalyzes phosphorylation. We hypothesize the interaction of T3 and {alpha}1-adrenergic stimulation results from a direct effect of the signaling pathway and is not dependent on changes in the cell surface adrenoceptor numbers. We directly stimulated the {alpha}1-adrenergic pathway by cotransfecting an expression plasmid containing the constitutively activated G protein Gq1 with the SERCA2 CAT construct and rTR{alpha}1. We showed a 50% suppression in the T3-induced SERCA2 gene transcription, similar to that in the presence of an {alpha}1-adrenergic agonist. To a lesser extent, the wild-type Gq showed a comparable effect. In a similar experiment, an expression plasmid containing H-Ras was cotransfected. The T3-stimulated SERCA2 gene transcription was down-regulated in the presence of H-Ras, again suggesting that a direct effect on the {alpha}1-adrenergic cascade may be responsible for the interaction with T3. This result indirectly indicates that the interaction between the {alpha}1-adrenergic system and T3 may not depend on an alteration in the adrenergic receptor numbers, as direct stimulation of the {alpha}1-adrenergic signaling cascade produces a similar down-regulation of the T3 effect on SERCA2 gene transcription.

Although we observed an interaction between T3 and the {alpha}1-adrenergic system on SERCA2 gene transcription, the precise mechanism of this interaction or the site at which it takes place is presently unknown. It is known that T3 regulates gene transcription through binding to nuclear TRs, which are normally bound to specific TREs in target genes either as homodimers or heterodimers with other TR auxiliary proteins, such as RXRs. The presence of three closely placed TREs upstream from the transcription start site of the SERCA2 gene in the rat heart has been identified (33). However, it is presently unclear whether {alpha}1-adrenergic stimulation may interfere with the TR-TRE interaction at one or more of the three TREs. The loss of T3-regulated transcriptional activation of the idealized TREpal provides further evidence that the interaction between T3 and {alpha}1-adrenergic stimulation is TRE specific. Neither T3 nor {alpha}1-adrenergic stimulation caused a significant change in the reporter activity of the empty expression vector pBLCAT2, which lacks TRE, further supporting this hypothesis. It is possible that the activation of the {alpha}1-adrenergic pathway results in an interference with the ligand-binding ability of the TRs at the TREs, presumably via alterations in the TR heterodimerization with TRAPs, with the resultant loss of the T3 dependent up-regulation of SERCA2 transcription.

It is known that T3 influences the TR-DNA interaction as well as TR-TRAP heterodimerization (36). We have shown that RXRs markedly enhanced the T3 effect on SERCA2 gene transcription, the binding of the TR-RXR heterodimer to the TREs being preferential to and more stable than that of TR homodimer (33). In this study, we observed that in the presence of RXR{gamma}, the T3-stimulated SERCA2 gene transcription was impaired in the presence of {alpha}1-adrenergic stimulation, suggesting that the interaction between T3 and the {alpha}1-adrenergic system may affect TR-RXR heterodimer formation on the TREs. Recently, several groups reported that phosphorylation may play an important role in the regulation of TR-mediated gene transcription. Using a protein phosphatase inhibitor, okadaic acid, to alter the phosphorylation status of the cell, Jones et al. (37) found that phosphorylation augmented the T3 stimulatory effect on transcription. The protein kinase inhibitor H7 was found to block transcriptional activity in the presence of T3 (37). Phosphorylated human TRß1 was reported to enhance homodimer binding to TREs (38) and was also found to be essential in the human TRß1-RXRß heterodimerization (39). In the heart, {alpha}1-adrenergic stimulation is known to activate protein kinase C through a cascade involving phosphatidyl inositol metabolism. In addition to its role in the phosphorylation of contractile proteins, protein kinase C may modify the phosphorylation status of the myocardial cells. A change in the phosphorylation status of the TR may result in the alteration of TR binding to TREs or heterodimerization with RXRs, possibly leading to the observed down-regulation of the T3-induced SERCA2 gene transcription in the presence of {alpha}1-adrenergic stimulation.

In this paper, we present novel results showing that {alpha}1-adrenergic stimulation inhibits T3-induced SERCA2 gene transcription in cardiac myocytes. We have shown that direct activation of the {alpha}1-adrenergic signaling cascade using a constitutively activated G protein (Gq) or H-Ras protein resulted in a similar down-regulation of the T3 effect on SERCA2 transcription. The interaction between {alpha}1-adrenergic stimulation and T3 is TRE specific and occurs in the presence of TR-RXR heterodimerization. Although the underlying mechanisms for this interaction remain unclear, it is possible that phosphorylation events may play a role.


   Footnotes
 
1 This work was supported by a grant from the Endocrine Fellows Foundation, NIH Grant HL-25022, and NIH Training Grants DK-07044 and DK-07494. Back

Received April 18, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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