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-mutant 
337T alters PPAR
signaling in heart
1Division of Cardiology, Department of Pediatrics, Children's Hospital and Regional Medical Center, and University of Washington School of Medicine, Seattle, Washington; 2United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Baylor College of Medicine, Department of Pediatrics; 3Department of Biostatistics and Applied Mathematics, University of Texas M. D.Anderson Cancer Center, Houston, Texas; and 4Center for Expression Arrays, Department of Microbiology, University of Washington, School of Medicine, Seattle, Washington
Submitted 5 June 2006 ; accepted in final form 12 September 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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and TR independently regulate cardiac metabolism. Although ligands for both these receptors are currently under evaluation for treatment of congestive heart failure, their interactions or signaling cooperation have not been investigated in heart. We tested the hypothesis that cardiac TRs interact with PPAR regulation of target genes and used mice exhibiting a cardioselective 
337T TR
1 mutation (MUT) to reveal cross-talk between these nuclear receptors. This dominant negative transgene potently inhibits DNA binding for both wild-type (WT) TR and TR. We used UCP3 and MTE-1 as principal reporters and analyzed gene expression from hearts of transgenic (MUT) and nontransgenic (WT) littermates 6 h after receiving either specific PPAR ligand (WY-14643) or vehicle. Interactions were determined through qRT-PCR analyses, and the extent of these interactions across multiple genes was determined using expression arrays. In the basal state, we detected no differences between groups for protein content for UCP3, PPAR, TR2, RXR, or PGC-1. However, protein content for TR1 and the PPAR heterodimeric partner RXR was diminished in MUT, whereas PPAR increased. We demonstrated cross-talk between PPAR and TR for multiple genes, including the reporters UCP3 and MTE1. WY-14643 induced a twofold increase in UCP3 gene expression that was totally abrogated in MUT. We demonstrated variable cross-talk patterns, indicating that multiple mechanisms operate according to individual target genes. The non-ligand-binding TR1 mutation alters expression for multiple nuclear receptors, providing a novel mechanism for interaction that has not been previously demonstrated. These results indicate that therapeutic response to PPAR ligands may be determined by thyroid hormone state and TR function. cardiac metabolism; nuclear receptors; microarrays
Similarly, mild reduction in circulating thyroid hormone levels occurs in aging rats. This triggers shifts in myosin isoform profile that are typically observed with more overt hypothyroidism (10). These elderly rats also show myocardial dysfunction and left ventricular dilation. In addition to disruptions in circulating thyroid hormone homeostasis, these rats exhibit reduced cardiac expression for the principal thyroid hormone receptor (TR) isoform TR1. Failing human hearts also show reduced TR1 relative to TR2, compared with control. (12) The TR2 isoform does not bind ligand and strongly inhibits DNA binding by other TRs, including TR1 and TR1. Thus the shift toward TR2 prominence provides a plausible signaling mechanism for eliciting the myosin isoform shift and the accompanying left ventricular hypertrophy during heart failure. Maladaptive myocardial remodeling may then result from thyroid receptor imbalance, caused initially by mild decreases in thyroid hormone levels (10).
The thyroid hormone receptors are members of the nuclear steroid receptor family, which includes the peroxisome proliferator-activated receptor (PPAR) family. These receptors have recently received considerable attention as putative targets for treatment of congestive heart failure. Thyroid receptors interact with PPARs in part by sharing binding sites and heterodimeric partners such as the retinoid X receptors (RXR) (2, 5, 16). PPARs and TRs also share coactivation by the PPAR
coactivator-1 (PGC-1). In particular, the PPARs regulate multiple genes involved in myocardial substrate oxidation, as well as other cellular processes, that respond to thyroid hormone. Agonists for isoforms PPAR and PPAR ameliorate maladaptive hypertrophy in cardiac myocytes (14) and in the heart in vivo (18). Reduction in myocyte lipid content plays a role in the pharmacological response to these drugs, including the specific PPAR agonist WY-14643 (18).
The response to these PPAR agonists may depend on the interactions between PPARs and TRs. A recent study (2) showed complex regulation of PPAR activity by liganded and unliganded WT TRs and a nonligand binding TR1 mutant, TRPV. The dominant negative action by TRPV represses troglitazone (ligand)-dependent PPAR-mediated transcriptional activity in cultured thyroid cells and CV-1 cells (2). These studies in isolated cells suggest the hypothesis that TRPV and dominant negative TR modifies PPAR mediation of transcriptional activity in intact organs.
As stated, cooperation between PPARs and TRs is well recognized, although, to our knowledge, such interaction between these receptors has not been investigated in heart. Therefore, in this study, we tested our hypothesis in heart using a mouse expressing the 337T TR1 human mutation, selectively targeted to cardiomyocytes by linking to the -myosin promoter (20). These mice show decreased cardiac function and bradycardia at rest and develop left ventricular hypertrophy with aging. This mutation expressed in multiple tissues in some humans with resistance to thyroid hormone syndrome (RTH) fulfills the requirements as a dominant negative nonligand binding TR. RTH is characterized by reduced biochemical and clinical manifestations of thyroid hormone action relative to the circulating thyroid hormone levels (1, 12, 20). We determined whether presence of this mutation in mouse heart altered the immediate gene response to the PPAR agonist WY-14643. Uncoupling protein-3 (UCP3), a gene highly responsive to PPAR ligand (17), served as the primary reporter for these experiments, whereas extent of interactions between the receptors was confirmed by gene expression array.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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337T is a naturally occurring mutation in humans exhibiting resistance to RTH (1, 20). A threonine deletion at amino acid position 337 of TR1 abrogates ligand binding and transforms the TR1 receptor into a constitutive and dominant negative repressor. The murine -myosin heavy chain promoter was used to direct transgene expression selectively to cardiac myocytes. Despite thyroid regulation of this promoter, these mice adequately express the TR1 mutation and created a cardiac hypothyroid phenotype. Mice expressing this mutation were obtained from Dr. Fred Wondisford, University of Chicago (1, 20) and bred in the vivarium at Children's Hospital and Regional Medical Center. Previous studies have confirmed selective transgene expression in heart. The transgene was confirmed in mice (MUT) by tail PCR or Southern blot analyses. Littermates, negative for the transgene (WT), served as controls. Fed male mice, aged 4–6 mo, were used in these experiments. Gross examination of hearts showed no evidence of abnormal myocardial hypertrophy. All procedures were performed in accordance with the National Institutes of Health (NIH Publication No. 85-23, revised 1996) Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee at the University of Washington.
Specific PPAR activation.
To specifically activate the PPAR system, WY-14643 (50 mg/kg) was administered by a single injection (ip). The nontreatment groups were injected with vehicle only (1:1 vol/vol DMSO-saline). Four groups of mice were studied, with 7 mice in each group: WT with or without WY-14643 and MUT with or without WY-14643. The mice were killed 6 h after injection. Heart tissue was rapidly extracted from the left ventricle, quickly blotted dry, frozen in liquid nitrogen, and stored at –80°C.
RNA extraction, labeling, and quality control. Total mRNA was extracted from the frozen heart tissue using the RNeasy kit from Qiagen (Chatsworth, CA). Contaminating DNA was removed by deoxyribonuclease I digestion of the RNA preparation when still bound to the column. An average of 27 µg of total RNA was obtained from 20 mg of frozen mouse heart tissue. The total RNA and cRNA was analyzed using the Agilent 2100 bioanalyzer for RNA quality. Quantity and OD260/280 of total RNA and cRNA was assessed by UV spectrophotometer.
RT-PCR analyses. Quantitative real-time PCR (qRT-PCR) analyses of samples were performed using previously described methods (15, 27). Specific quantitative assays were designed from mouse sequences available in GenBank (Table 1). Standard RNA was made for all assays by the T7 polymerase method (Ambion, Austin, Texas) with the use of total RNA isolated from mouse hearts. The correlation between the number of PCR cycles required for the fluorescent signal to reach a detection threshold and the amount of standard was linear over at least a five-log range of RNA for all assays. The level of transcripts for the constitutive housekeeping gene product 18S rRNA was quantitatively measured in each sample to control for sample-to-sample differences in RNA concentration. PCR data are reported as the number of transcripts per number of 18S rRNA molecules. Some samples were repeated using cyclophilin, which showed no general difference from normalization with 18S rRNA. For initial surveys of mutation, WY-14643, and interaction effects, expression patterns for nine genes were examined. The choice of these genes was based on previous studies performed in rodent heart (17, 27). Since a robust response to WY-14643 has been shown to occur for UCP3 and mitochondrial thioesterase 1 (MTE1), these were used as the primary reporter genes.
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Image analysis. Image processing and initial extraction of single-chip expression data were performed using the Affymetrix Gene Chip Operating Software (GCOS). Each gene chip genome array hybridization that was performed initially underwent MAS 5.0 absolute expression analysis. The quality of hybridizations and overall chip performance were determined by visual inspection of the raw scanned data. Data were complied with the minimum information about a microarray experiment standard and deposited at http://www.ncbi.nlm.gov/projects/geo/ with accession no. GSE3067.
Immunoblotting.
Content for these proteins was analyzed by Western blots. Fifty micrograms of total protein extracts from mouse heart tissue were electrophoresed along with two lanes of molecular weight size markers (precision plus protein standards, Bio-Rad and chemichrome western control; Sigma) in 4.5% stacking and a 7.5, 10, or 12% running SDS-polyacrylamide gels, depending on the molecular weight of the protein of interest. The gels were then electroblotted onto polyvinylidene fluoride plus membranes. The Western blots were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline plus Tween-20 (TBST; 10 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20), followed by overnight incubation at 4°C with each primary antibody diluted in the appropriate blocking solution as recommended by the supplier. The primary antibodies used in the study are PPAR (PPAR, sc-9000; PPAR, sc-7197), PGC-1 (sc-5816), RXR (RXR, sc-553; RXR, sc-831), and UCP3 (sc-31387) obtained from Santa Cruz Biotechnology. The thyroid hormone receptor primary antibodies (TR1, PA1–211A; TR2, PA1–216A; TR1, PA1-213A) were obtained from Affinity BioReagents. After two 5-min washes with TBST and one 5-min wash with Tris-buffered saline (TBS), membranes were incubated at room temperature for 1 h with the appropriated secondary antibody conjugated to horseradish peroxidase (HRP). The membranes were washed twice for 10 min with TBST and visualized with enhanced chemiluminescence after exposure to Kodak biomax light ML-1 film. The membranes were stripped by washing them two times for 30 min with 200 mM glycine, 0.1% SDS, and 1% Tween-20, (pH adjusted to 2.2), followed by three 10-min washes with TBS. Then the membranes were again blocked for 1 h as described above, followed by overnight incubation at 4°C with a GAPDH antibody diluted 1:200 in blocking solution. The next day, the membranes were washed (as above), the appropriate secondary HRP antibody was applied, and the remaining procedures as described above were followed. GAPDH (sc-25778; Santa Cruz Biotechnology) was used as an internal reference to verify protein lane loadings.
Data and statistical analysis. Data were analyzed using the Bioconductor software. Background correction and normalization were performed before statistical analysis using robust multiarray average (28). In addition, all background adjusted and normalized intensities were log base 2 transformed.
A two-factor ANOVA was used to statistically analyze the expression data. To derive the significance levels for the ANOVA, permutation tests were performed to empirically determine the significance of the computed F statistic. For calculating the main effect of a factor, the levels of only that factor were randomized, keeping the others constant.
The transgene effect was determined from the two-factor ANOVA of the control with and without drug vs. the transgenic with and without drug. The drug effect was obtained from the two-factor ANOVA of the control and transgenic without the drug and with the drug. An interaction effect exists when differences on one factor depend on the level of another factor (Fig. 1). For calculating the interaction effect, the residuals were permutated after the main effects for the interaction terms were removed. Two-thousand permutations were chosen to provide a reasonably small P value for the most significant genes. The qRT-PCR data were similarly analyzed using the two-factor ANOVA. Student's t-test was also used for intergroup comparisons when appropriate after significance was determined by ANOVA.
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RESULTS |
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, its cofactors, and other receptors that form heterodimers with PPAR or compete with PPAR for heterodimeric partners could determine response to ligand. Accordingly, we performed immunoblotting analysis to determine protein expression for these factors (Fig. 2). The protein level for GAPDH did not vary between the two groups. Expression for the important cofactor PGC-1 is not altered by cardioselective expression of 337T TR. Furthermore, no differences in expression were found for PPAR itself. However, TR1 is mildly, although significantly, depressed in MUT, as is the heterodimeric binding partner RXR. Although available antibodies could not detect PPAR in these hearts, PPAR was significantly elevated in MUT. TR2 and TR1 showed no difference between groups. Expression for UCP3 was not altered in MUT compared with WT. No changes in protein content for these receptors occurred over the 6-h time period of this protocol (data not shown).
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and the coactivator PGC-1. Expression patterns for metabolic related genes were surveyed by PCR and then by differential microarrays. Genes surveyed by PCR showed no differences in expression for MUT compared with WT using multiple statistical methods. The first two columns in Fig. 3 show group comparisons between MUT and WT.
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PPAR activation by WY-14643.
The PCR analyses detected significant differences in expression for three of nine genes caused by WY-14643. UCP3, known previously to exhibit extreme sensitivity to WY-14643 in rat heart (17, 26), showed an approximately twofold increase by this ligand (Fig. 3). Stavinoha et al. (26) previously showed that rat heart MTE1 mRNA increased significantly 6 h after WY-14643 intraperitoneal injection, although the magnitude of the response was less than for UCP3. Our WT mice followed a similar pattern, where UCP3 increased substantially, and MTE1 (also known as acyl-CoA thioesterase) increased significantly, but to a lesser degree.
Expression array assay detected multiple responses to PPAR activation that have not been previously reported in heart (Supplemental Table S1). Interestingly, a recent report (28) suggested that the G0/G1 switch gene is a PPAR target gene and is upregulated by WY-14643 in liver. However, the response is directly opposite in heart, implying tissue specificity. Although data from our previous study (15) indicated that PPAR is under autoregulation at the posttranscriptional level, we noted that WY-14643 decreased PPAR mRNA expresssion over the brief time course of these experiments.
Modification of PPAR activation by 337T TR.
The PCR analysis demonstrated multiple and significant modifications to the WY-14643 response in MUT (Fig. 3 and Supplemental Table S1). For instance, 337T abrogated the robust UCP3 and the lesser MTE1 response seen in WT. In some cases, WY-14643 induced a response that was not apparent in WT. For instance, WY-14643 reduces PPAR expression in MUT, but not in WT. Isocitrate dehydrogenase 3, acetyl-CoA carboxylase-, cytochrome c oxidase subunit VIIIa, and AMP deaminase 3 are among others noted in Supplemental Table S1. Differences in the directions of these interactions indicate that multiple mechanisms were operative and resulted from insertion of 337T. For instance, pyruvate dehydrogenase kinase-4 (PDK4) was not affected by WY-14643 in WT but was downregulated by this ligand in MUT.
The array studies indentified novel interactions at genes unsuspected for either TR or PPAR regulation in heart. Consistent with the theme that insulin signaling is altered in MUT, we noted by gene chip a strong interaction for adiponectin (adipocyte complement-related protein). This protein has previously been considered as selectively produced adipocytes (24a). However, a recent study (21) shows that this protein, known to modulate insulin signaling, is synthesized and secreted by murine cardiomyocytes.
PCR and gene chip consistency. As noted, the PCR experiments were peformed initially as a survey for drug, mutation, and interaction effects in this model. The followup study by array was performed to identify other genes that might demonstrate similar responses. Sequences for all nine genes were represented on the Affymetrix Gene Chip. The relative magnitude of expression differences among groups varied for multiple genes between PCR and gene chip analyses. However, for the most part, these methods were consistent in validating the existence or absence of response. The two major conflicts involved the UCP3 and PDK4 genes. For UCP3, the gene chip indicated a WY-14643-induced decrease in expression and no interaction, whereas PCR detected the robust increase in expression by this ligand that was abrogated in MUT. PCR detected downregulation in MUT after WY-14643, which was not detected by array analyses. Unlike the sequences used for PCR analyses, the mRNA sequences for these two genes on the Affymetrix array differ from mature mRNA sequences isolated in mouse heart tissue. We believe the differences between the mature mouse sequences and the sequences reported as mouse by Affymetrix are caused by organ-specific alternate splicing of mouse mRNAs. Therefore, the more specific analyses conducted by PCR should carry precedence.
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DISCUSSION |
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and thyroid hormone signaling in heart. We used UCP3 as a principal reporter in vivo, although expression for multiple proteins and genes was evaluated to determine the extent of these interactions. This particular protein has previously been shown (11, 17) as highly responsive to both triiodothyronine and WY-14643 in rodent heart. However, Murray et al. (17) showed that treating WT mice with WY-14643 for 7 days substantially reduces plasma insulin and triacylglycerol levels, whereas it increases myocardial protein levels of UCP2 and UCP3. Although those authors attributed increases in UCP2 and UCP3 protein to selective PPAR activation, transcriptional action through insulin receptor might also account for these responses. To minimize these systemic and indirect influences of WY-14643 on heart, we limited the time of exposure to this PPAR agonist. The short time exposure, although it limits indirect action by WY-14643, also reduces the magnitude of the response to this drug. Therefore, only modest fold changes in expression for many genes, such as MTE1, occurred after this brief exposure. We were able to confirm in WT mice that WY-14643 induced a greater than twofold increase in expression for UCP3 (Fig. 3). Furthermore, we noted that the 337T mutation abrogated this response, thereby demonstrating the appropriate use of this gene as the principal reporter in these experiments.
The mechanisms for interaction between TRs and other nuclear receptors have been previously studied in detail using various experimental models (1a, 2a, 6a, 14a, 29a). This study presents the first evaluation for these interactions in heart. We demonstrated multiple types of interactions between TRs and PPAR, suggesting that numerous mechanisms operate in vivo and vary according to target gene. Previous studies (3, 4, 9, 16) have documented either abrogation or enhancement of PPAR response by thyroid hormone receptor. Although data identifying TR-PPAR interaction occurs in the literature, the direction of change mediated by TR on PPAR activity reporters has been inconsistent. Contradictory results are apparent in a study by Hunter et al. (9). They demonstrated in solution that TR competes with an unspecified PPAR for binding to the fatty acyl-CoA oxidase. Yet they also showed that TR stimulates PPAR/RXR transactivation in BSC40 cells.
The mechanisms proposed for interaction or cross signaling between these two transcription factors include competition for binding a response element, formation of inactive PPAR-TR heterodimers, and squelching of the RXR cofactor (3, 4, 9, 16). The 337TR mutation occurs in the ligand binding domain and represses binding of endogenous TRs. Thus our observed changes in PPAR activation likely required both TR binding to a receptor element as well as an alteration in T3 binding to the TR. This contention is supported by two prior cell transfection studies. Miyamoto et al. (16) showed in COS1 cells that introduction of a mutation into the TR1 DNA binding domain eliminated interaction with PPAR. Additionally, Chu et al. (4), using transfected rat hepatoma cells, showed T3 modulation of PPAR-TR interaction.
Limitations in data interpretation do occur when a transgenic mouse with a dominant negative mutation in a nuclear receptor is used. The 337T mutation exhibits not only loss of function for the thyroid receptor but various other properties that differ from the normally functioning TR. This mouse model may also be subject to effects caused by TR overexpression. Experimentally, this can be compensated for by using, as a control group, mice with cardioselective insertion of normal human TR1, as performed by Pazos-Moura et al. (20). Unfortunately, the mouse line with this mutation was not propagated either in the original laboratory or in our own (personal communication with Dr. F. Wondisford). However, our immunoblot data provide some evidence that no major increase in TR1 protein expression occurred in MUT.
Hashimoto et al. (8) also used the 337T mutation to uncover cross-talk between TR and liver X receptor (LXR). For some target genes, those authors detected antagonisms between LXR and 337T that did not occur for wild-type TR1. Similarly, in our studies, we observed that WY-14643 in MUT suppressed expression for some genes displaying no response to this ligand in WT. Those authors exposed two types of mechanisms that explain the dominant negative action of 337T. First, thyroid hormone action is directly impaired by the inability to bind the mutant receptor. Second, the mutation alters functions of other nuclear receptors through various modes. These negative actions occur both in vitro and in vivo and vary according to target, such that not all genes positively regulated by thyroid hormone are repressed by this mutation in the basal state. Accordingly, MUT did not exhibit reduced mRNA or protein expression for UCP3 compared with WT. However, we showed that UCP3 response to the PPAR ligand was impaired in MUT. The PCR and array experiments confirmed that interaction between PPAR and 337T was not restricted to a single gene but involved multiple genes, regulating a full range of cellular processes, including insulin signaling pathways.
The 337T mutation in particular alters dimerization patterns for nuclear receptors. Although this phenomenon has not been studied specifically for PPAR, formation of 337T homodimers alters LXR heterodimer binding to receptor elements (8). These MUT homodimers, unlike WT TR homodimers, do not dissociate on exposure to triiodothyronine (7). Thus, in WT mice, LXR receptor elements are occupied primarily by TR/RXR heterodimers, whereas in MUT mice the same elements are occupied by LXR/RXR heterodimers. If a similar process occurs for PPAR response element, activation or repression of the target gene in MUT might occur in response to a ligand such as WY-14643, when no effect occurred in WT.
Alteration in the basal levels of expression for PPARs, TRs, and other proteins involved in their activation and binding represents another possible limitation in using the 337T transgenic model. Indeed, our study revealed that alterations in basal levels of particular nuclear receptors could account for some modifications of the PPAR response in MUT. The 337T mutation reduced basal myocardial protein content for TR1 without concomitant significant change in TR2. These findings imply that the 337T mutation enhances relative expression of another dominant negative TR isoform, possibly reducing the total capacity for TR-ligand binding further. Additionally, MUT show decreased expression for RXR, a heterodimeric binding partner for both TR1 and PPAR. The stoichiometry of these nuclear receptors has not been precisely determined; therefore, we cannot predict the overall effect of these changes on genes that are jointly regulated by PPAR-RXR and TR-RXR. However, TR regulation of expression for other nuclear receptors does represent a novel but possibly unique mode of interaction between TRs and PPAR in this model.
The physiological implications of altered signaling in this model remain to be determined. Of note is that this transgenic mouse exhibits myocardial hypertrophy (19) similar to that exhibited by cardioselective PPAR-overexpressing mouse (6). Alterations in contractile proteins are implicated in part for the hypertrophy noted in the cardioselective 337T TR mouse, although modification in PPAR signaling may also play a role. We also noted some 337T TR-mediated alterations in insulin-signaling pathways, although the implications of these disturbances require further evaluation.
In summary, we showed novel interactions between PPAR and thyroid hormone receptors in heart. We employed a strategy targeting the transgene specifically to cardiomyocytes to demonstrate these interactions, as opposed to using direct thyroid hormone supplementation. This transgenic model does not alter circulating thyroid hormone levels, ensuring that experiments were conducted under systemic euthyroid conditions. Therefore, our study design avoids confounding factors such as posttranscriptional triiodothyronine action on cardiac metabolism (13, 22) and transcriptional actions on noncardiomyocyte cells and organs that could indirectly alter cardiac response. These findings imply that these nuclear receptor pathways synergistically or cooperatively activate or repress target genes. The PPAR agonists are receiving attention as candidates for treatment of cardiac remodeling and heart failure (25). Since abnormalities in thyroid hormone metabolism frequently occur during cardiomyopathy induced by aging and other factors, these interactions may have importance in determining therapeutic response to PPAR agonists.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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and PGC-1 during mitochondrial maturation in sheep heart. Am J Physiol Heart Circ Physiol 289: H2258–H2264, 2005.-regulated gene in cardiac and skeletal muscle. Am J Physiol Endocrinol Metab 287: E888–E895, 2004.
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