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Am J Physiol Heart Circ Physiol 290: H381-H389, 2006. First published September 9, 2005; doi:10.1152/ajpheart.00576.2005
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Nuclear localization of protein kinase C-{alpha} induces thyroid hormone receptor-{alpha}1 expression in the cardiomyocyte

Agnes Kenessey,1 Elizabeth Ann Sullivan,1 and Kaie Ojamaa1,2

1Institute for Medical Research, North Shore-Long Island Jewish Health System, Manhasset; and 2Departments of Cell Biology and Medicine, New York University School of Medicine, New York, New York

Submitted 1 June 2005 ; accepted in final form 2 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Maladaptive cardiac hypertrophy results in phenotypic changes in several genes that are thyroid hormone responsive, suggesting that thyroid hormone receptor (TR) function may be altered by cellular kinases, including protein kinase C (PKC) isozymes that are activated in pathological hypertrophy. To investigate the role of PKC signaling in regulating TR function, cultured neonatal rat ventricular myocytes were transduced with adenovirus (Ad) expressing wild-type (wt) or kinase-inactive (dn) PKC{alpha} or constitutively active (ca) PKC{delta} and PKC{epsilon}. Overexpression of wtPKC{alpha}, but not caPKC{delta} or caPKC{epsilon}, induced a 28-fold increase (P < 0.001) in TR{alpha}1 protein in the nuclear compartment and a smaller increase in the cytosol. Furthermore, TR{alpha}1 mRNA was increased 55-fold (P < 0.001). This effect of PKC{alpha} was dependent on its kinase activity because dnPKC{alpha} was without effect. Phorbol 12-myristate 13-acetate (PMA) induced nuclear translocation of endogenous PKC{alpha} and Ad-wtPKC{alpha} concomitantly with an increase in nuclear TR{alpha}1 protein. In contrast, PMA-induced nuclear translocation of dnPKC{alpha} resulted in a decrease of TR{alpha}1. The increase in TR{alpha}1 protein in Ad-wtPKC{alpha}-transduced cardiomyocytes was not the result of a reduced rate of protein degradation, nor was the half-life of TR{alpha}1 mRNA prolonged, suggesting a PKC{alpha}-mediated effect on TR{alpha} transcription. Although phosphorylation of ERK1/2 was increased in Ad-wtPKC{alpha}-transduced cells, inhibition of phospho-ERK did not change TR{alpha}1 expression. PKC{alpha} overexpression in cardiomyocytes caused marked repression of triiodothyronine (T3)-responsive genes, {alpha}-myosin heavy chain, and the sarcoplasmic reticulum calcium-activated adenosinetriphosphatase SERCA2. Treatment with T3 for 4 h resulted in significant reductions of PKC{alpha} in nuclear and cytosolic compartments, and decreased TR{alpha}1 mRNA and protein, with normalization of phenotype. These results implicate PKC{alpha} as a regulator of TR function and suggest that nuclear localization of PKC{alpha} may control transcription of the TR{alpha} gene, and consequently, affect cardiac phenotype.

adenovirus; heart; triiodothyronine; extracellular signal-regulated kinase; sarcoplasmic reticulum calcium-activated adenosinetriphosphatase; myosin heavy chain

MALADAPTIVE CARDIAC HYPERTROPHY results in phenotypic changes that include genes that are known to be thyroid hormone responsive, such as {alpha}- and {beta}-myosin heavy chain (MHC), sarcoplasmic reticulum calcium-activated ATPase (SERCA2), phospholamban, and {beta}-adrenergic receptors (reviewed in Refs. 13, 25). This observation has provided support of the hypothesis that thyroid hormone effects on the myocardium may be impaired in the failing heart, which may include alterations in nuclear thyroid hormone receptors (TRs) and their transcriptional coregulators, or alternatively, a decrease in serum or tissue levels of thyroid hormones. Studies of patients with ischemic and dilated cardiomyopathy have reported contradictory results of both increases and decreases in the TR mRNA isoforms, and TR protein data are lacking (7, 23, 39). An experimental animal model of pathological cardiac hypertrophy has shown that mRNA levels of three TR isoforms were downregulated, and it was concluded that this resulted in the hypertrophic phenotype (24). Recent identification of specific thyroid hormone transporters in the myocardium raises the possibility that alterations in these transporters under pathological conditions may affect hormone uptake and thus cardiac-specific thyroid hormone function (14). Alternatively, tissue content of thyroid hormones may be decreased by induction of thyroid hormone-degrading deiodinases as has been shown in a rodent model of cardiac hypertrophy and failure (43). Thus the role of thyroid hormones in the setting of heart disease has received considerable attention (20, 33).

Recent studies have provided evidence that thyroid hormones activate intracellular signaling pathways, including the ERK (9) and phosphatidylinositol 3-kinase (PI3K)-Akt pathways (5). Additionally, a role for TR{alpha}1 in the cytoplasmic compartment has been recently reported, supporting extranuclear functions for TRs (8, 22). Therefore, it was of interest to consider an interactive role for thyroid hormones, TRs, and the protein kinase C (PKC) family of serine-threonine kinases that have been implicated in most intracellular signaling pathways regulating cardiomyocyte hypertrophy, contractile function, and gene expression (12, 29, 36). Studies of cardiac pathological hypertrophy and failure in humans and animal models have reported increased activity of calcium-dependent isotypes, PKC{alpha} and PKC{beta} (2, 42). Although PKC{alpha} has recently been associated with myocyte hypertrophic growth (3, 41), in vivo studies using transgenic models have shown that PKC{alpha} may play a potentially more important role in modifying systolic and diastolic contractile function than inducing hypertrophy (4, 16). Calcium-independent PKC{epsilon} and PKC{delta} have been identified as downstream mediators of Gq protein-coupled receptor-initiated signals; however, each isotype activates distinct intracellular responses (6). PKC{epsilon} appears to selectively activate the ERK cascade that is implicated in myocyte growth responses and cell survival, whereas PKC{delta} preferentially activates JNK and p38MAPK pathways, resulting in apoptosis and contractile dysfunction (17, 19, 31, 38). Thyroid hormone has been reported to cause subcellular redistribution and activation of PKC{alpha} (1), whereas another study reported repression of PKC{alpha} and PKC{epsilon} expression in response to thyroid hormone treatment in cultured cardiomyocytes (35). Thus, on the basis of a potentially significant interaction of PKCs and thyroid hormone, we proposed to examine the effects of PKC{alpha}, -{delta}, and -{epsilon} isozymes on TR function.

Using adenoviral gene transfer to overexpress PKC{alpha} in cultured cardiomyocytes, we show for the first time that nuclear localization of PKC{alpha} induced the expression of TR{alpha}1 in both nuclear and cytosolic compartments. This effect was not observed with overexpression of constitutively active PKC{delta} or PKC{epsilon}. The potential physiological effects of high levels of unliganded TR{alpha}1 within the pathological myocardium of patients with heart disease, which is often associated with low circulating levels of thyroid hormones (33), may induce the "failing" phenotype and also alter cytoplasmic nongenomic activities of TRs, including activation of the ERK, p38MAPK, and PI3K/AKT pathways (5, 9, 22). Thus therapeutic approaches directed at cardiac TR{alpha}1 function or activity of the PKC{alpha} signaling pathway may prove efficacious in attenuating the dysfunction associated with pathological cardiac hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Cell culture reagents were obtained from GIBCO-BRL (Grand Island, NY). L-Triiodothyronine (T3), phorbol 12-myristate 13-acetate (PMA), cyclohexamide (CHX), and actinomycin D were obtained from Sigma (St. Louis, MO). MEK1 inhibitor PD-98059 was from Calbiochem (San Diego, CA). Anti-TR{alpha}1 (PA1–211A) antibodies were from Affinity BioReagents (Golden, CO). Antibodies to TR{beta}1 (J52), PKC{alpha}, PKC{delta}, and PKC{epsilon}, and TFIID (TATA box binding protein, TBP) were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, CA). Chemiluminescent reagents for Western blot analysis and radioactive chemicals were from PerkinElmer (Boston, MA). Real-time quantitative PCR reagents were from Eurogentec (Belgium). All other reagents, including protease and phosphatase inhibitors, were of the highest available quality from Sigma, Calbiochem, or Pierce (Rockford, IL).

Isolation, culture, and viral transduction of neonatal rat ventricular myocytes. Animals were treated in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHHS Publication No. 85–23), and study protocols were approved by the Institutional Animal Care and Use Committee. Ventricular myocytes were isolated from hearts of 2-day-old rats by collagenase digestion as we have previously described (21). Myocytes were plated at ~1.5 x 104/cm2 on collagen-coated six-well plates (RNA analysis) or 60-mm dishes (protein analysis) and cultured for the first 20 h in DMEM/F12 medium containing 10% FBS, L-glutamine, cytosine {beta}-D-arabinofuranoside (10 µM), and antibiotics. After the first 20 h, the neonatal rat ventricular myocyte (NRVM) cultures were washed with HBSS, and in select experiments, cells were exposed to adenovirus (at a multiplicity of infection as indicated in RESULTS) in DMEM/F12 for 1 h in the cell culture incubator (5% CO2, 37°C). Subsequently, the cells were washed and maintained in serum-free medium containing transferrin (5 mg/l), selenium (5 µg/l), L-glutamine, and antibiotics for 24 or 40 h before experimentation as indicated. Reagents used in cell culture were added to the final concentrations as follows: T3 (10–8 M), PMA (2 x 10–7 M), PD-98059 (10–5 M), actinomycin D (5 µg/ml), and CHX (10 µg/ml).

Adenoviral constructs. Replication-defective adenoviruses containing coding sequences of wild-type PKC{alpha} (Ad-wtPKC{alpha}), kinase-inactive mutant of PKC{alpha} (Ad-dnPKC{alpha}), constitutively active PKC{delta} (Ad-caPKC{delta}) and PKC{epsilon} (Ad-caPKC{epsilon}) were kindly provided by Dr. Allen Samarel (Loyola Univ., Maywood, IL) (17, 41). Adenovirus for nuclear-encoded {beta}-galactosidase (Ad-ne{beta}gal) was used to control for nonspecific effects of adenoviral transduction of cultured cells. The recombinant adenoviruses were amplified by sequential infection of HEK-293 cells and purified by CsCl gradient ultracentrifugation. The viral titer of each preparation of replication-defective adenovirus was determined by dilution assay in HEK-293 cells.

Cell fractionation and immunoblot analysis. For cytosolic and nuclear protein analysis, cells were washed and then scraped into homogenization buffer containing 20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 10% glycerol, 0.2% NP-40 plus protease and phosphatase inhibitors. Cells were lysed by Dounce homogenization and fractionated by centrifugation at 12,000 g for 1 min at 4°C. The resulting supernatant was used as the cytosolic fraction. The nuclear fraction was prepared by resuspension of the pellet in buffer containing 20 mM HEPES, pH 7.9, 0.42 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 25% glycerol, and protease inhibitor cocktail, and incubated with agitation for 30 min at 4°C before centrifugation for 10 min at 12,000 g. The resulting supernatants were collected and used for nuclear protein analysis.

For studies of PKC translocation in response to PMA or T3 treatment, a commercially available extraction kit (Pierce) was used to isolate cytosolic, membrane, and nuclear fractions. Cells were prepared essentially as described by Vijayan et al. (41) in which cells were initially lysed by freeze-thaw in a homogenization buffer containing 20 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA plus protease and phosphatase inhibitors. The cell lysate was fractionated by centrifugation for 2 min at 500 g, 4°C, and the resulting supernatant was used as the cytosolic fraction. The pellet was resuspended in buffers provided by the manufacturer and further fractionated to obtain membrane and nuclear fractions. Protein concentrations in all fractions were determined by Micro-BCA assay (Pierce). After normalization of sample protein content, proteins were resolved by electrophoresis on 5% stacking/10% polyacrylamide-SDS gels, and the resolved proteins were transferred to nitrocellulose membrane (Bio-Rad) and probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies as previously described (21). Primary antibodies used were polyclonal anti-TR{alpha}1, and monoclonal antibodies against TBP, TR{beta}1, PKC{alpha}, PKC{delta}, and PKC{epsilon}. Secondary antibodies used were either goat anti-rabbit or goat anti-mouse IgG conjugated with horseradish peroxidase. Protein bands were detected using chemiluminescence reagent and visualized by exposure to X-ray film. Protein band intensity was quantified by laser scanning densitometry (GS-800 Calibrated Densitometer; Bio-Rad). Results are expressed as arbitrary densitometric units (du).

Real-time quantitative RT-PCR. RNA samples were prepared from ~106 cells plated in six-well plates using a commercially available kit (RNeasy Mini Kit; Qiagen, Valencia, CA) and were treated with deoxyribonuclease during purification. The integrity of the RNA was verified before analysis. Real-time quantitative reverse-transcriptase-PCR utilized TaqMan chemistry and an ABI Prism 7700 Sequence Detector (Perkin Elmer-Applied Biosystems, Foster City, CA). Primers and TaqMan probes for TR{alpha}1, TR{beta}1, and GAPDH mRNAs were synthesized using Applied Biosystems 394 DNA Synthesizer in the Institutional Core Facility as we have previously published (21). GAPDH mRNA was used for normalization of each sample, and analysis of each specific mRNA was conducted in duplicate. Relative expression of the mRNAs was calculated as previously described (21), and results are expressed as fold change with respect to the corresponding experimental control.

Northern blot analysis was used for quantitation of expression of cardiac {alpha}- and {beta}-MHC and SERCA2 mRNAs. Five micrograms of total RNA were fractionated by denaturing agarose gels and blotted onto synthetic membrane as we have previously published (34). Detection of {alpha}- and {beta}-MHC mRNAs was accomplished using radiolabeled oligodeoxynucleotide probes, whereas a cloned SERCA2 cDNA fragment was radiolabeled by random-priming methodology to detect rat SERCA2 mRNA as described (34). Ribosomal RNA (18S rRNA), measured by radiolabeled oligodeoxynucleotide probe, was used for sample normalization. Specific mRNAs were detected by exposure to X-ray film.

Statistical analysis. All data are presented as means ± SE derived from three to six individual samples per experiment derived from a minimum of two separate cell preparations. One-way ANOVA was used for statistical analysis of mean values among experimental groups, with pairwise multiple comparisons conducted using the Holm-Sidak method. Differences between means were considered significant at P < 0.05. Data were analyzed using SigmaStat 3.1 (Systat Software, Richmond, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PKC{alpha} overexpression significantly increases TR{alpha}1 protein. In light of recent studies showing nuclear translocation of PKC{alpha} in response to PMA and insulin-like growth factor-1 (41), we explored the possibility that intracellular signaling through the PKC{alpha} pathway may alter gene expression by modulating expression or activity of thyroid hormone receptors TR{alpha}1 and TR{beta}1. As shown in Fig. 1A, transduction of cultured cardiomyocytes with replication-deficient Ad-wtPKC{alpha} resulted in a significant increase in both nuclear and cytosolic TR{alpha}1 protein compared with myocyte cultures transduced with control Ad-ne{beta}gal. Furthermore, by increasing the multiplicity of infection (moi) of Ad-wtPKC{alpha} from 5 to 25, immunoreactive PKC{alpha} was increased from 1.7- to 4.5-fold compared with Ad-ne{beta}gal (Fig. 1, B and C), concomitantly with an increase in TR{alpha}1 protein from 28-fold to 63-fold compared with Ad-ne{beta}gal (5 moi) (Fig. 1, B and D). In the present studies, we routinely used 5 moi of Ad-wtPKC{alpha}, which provided an increase of ~2-fold in total immunoreactive PKC{alpha} compared with control cultures (Fig. 1C). We have previously published that expression of the TR{beta}1 isoform is not detectable in cardiomyocytes cultured in the absence of thyroid hormone (21). In the present studies, overexpression of wtPKC{alpha} in cardiomyocytes cultured in serum-free medium had no effect on TR{beta}1 expression (data not shown).



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  		Fig. 1. Adenoviral-mediated transduction of wild-type protein kinase C{alpha} (Ad-wtPKC{alpha}) into cultured neonatal rat ventricular myocytes increases thyroid hormone receptor (TR)-{alpha}1 expression. A: TR{alpha}1 protein in both nuclear and cytosolic compartments was increased in cardiomyocytes transduced with Ad-wtPKC{alpha} at a multiplicity of infection of 5 (5 moi) compared with myocytes transduced with adenovirus for nuclear-encoded {beta}-galactosidase (Ad-ne{beta}gal, 5 moi). Myocytes were harvested 48 h after viral transduction, and cell lysates were fractionated and analyzed for PKC{alpha} and TR{alpha}1 expression by SDS-PAGE and Western blotting. B: total cell PKC{alpha} was increased from 1.7- to 4.5-fold with viral-mediated transduction of Ad-wtPKC{alpha} from 5 to 25 moi. C: quantitation of total PKC{alpha} in Ad-wtPKC{alpha}-transduced cells compared with Ad-ne{beta}gal-transduced cells at 5 moi. D: increasing Ad-wtPKC{alpha} from 5 to 25 moi progressively increased total TR{alpha}1 protein from 28- to 63-fold compared with Ad-ne{beta}gal (5 moi).

 
PKC{alpha} effect on TR{alpha}1 is dependent on its kinase activity and is isozyme specific. To determine whether the effect of PKC{alpha} on TR{alpha}1 was dependent on its kinase activity, we overexpressed a kinase-inactive form of PKC{alpha} (Ad-dnPKC{alpha}) in which the ATP binding site had been mutated (41). At similar levels of expression as the wtPKC{alpha}, the kinase-defective mutant dnPKC{alpha} had no effect on expression of TR{alpha}1 protein (Fig. 2A). Shown for comparison is the level of expression of nuclear TR{alpha}1 in cardiomyocytes transduced with control Ad-ne{beta}gal. Immunoreactive TATA box binding protein (TBP) shows that equivalent amounts of protein were analyzed per sample.



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  		Fig. 2. Effects of PKCs on TR{alpha}1 are isozyme specific and dependent on PKC{alpha} kinase activity. A: neonatal rat ventricular myocytes (NRVM) were virally transduced with Ad-ne{beta}gal, Ad-wtPKC{alpha}, or kinase-defective mutant PKC{alpha} (Ad-dnPKC{alpha}) at 5 moi as described in MATERIALS AND METHODS and cultured in defined medium for 48 h before harvest. Western blot analysis shows that adenovirally expressed PKC{alpha} distributed to both nuclear and cytosolic compartments, whereas endogenous PKC{alpha} in control Ad-ne{beta}gal-transduced cells appears to reside primarily in the cytoplasmic fraction. Only cells transduced with Ad-wtPKC{alpha}, but not kinase-inactive PKC{alpha} (Ad-dnPKC{alpha}), showed increased expression of TR{alpha}1 protein compared with control Ad-ne{beta}gal-transduced myocytes. Immunoreactive TATA box binding protein (TBP) shows equivalent protein content per lane. B: adenoviral-mediated expression of constitutively active PKC{delta} and PKC{epsilon} (Ad-caPKC{delta}, Ad-caPKC{epsilon}, 5 moi) in NRVM had no effect on expression of TR{alpha}1 compared with Ad-wtPKC{alpha}. Western blot analyses with the respective PKC isozyme antibodies showed nuclear and cytosolic localization of PKC{delta} and PKC{alpha}, whereas PKC{epsilon} was confined to the cytosolic compartment. Nuclear TBP shows equivalent protein content per lane.

 
To address the PKC isoenzyme-specificity of this effect, constitutively active PKC{delta} and PKC{epsilon}, were overexpressed in the cardiomyocytes by adenovirus gene transfer. As shown in Fig. 2B, neither PKC{delta} nor PKC{epsilon} had an effect on TR{alpha}1 protein content, suggesting that this effect was specific to the PKC{alpha} isozyme. Furthermore, constitutively active PKC{delta}, but not PKC{epsilon}, localized to the nuclear fraction. Thus, despite the similarity in nuclear localization of PKC{delta} and PKC{alpha}, only PKC{alpha} increased TR{alpha}1 protein expression.

PMA stimulates nuclear translocation of PKC{alpha} and increases nuclear TR{alpha}1. We sought to determine whether the increase in TR{alpha}1 protein seen in response to overexpression of wtPKC{alpha} occurred with the activation of endogenous PKC{alpha}. After treatment with PMA to activate PKC{alpha}, we used subcellular fractionation to show that endogenous PKC{alpha} translocated to the nucleus as had been previously reported (41). As shown by Western blot analysis in Fig. 3, both endogenous PKC{alpha} (cells transduced with Ad-ne{beta}gal) and adenoviral-expressed wtPKC{alpha} and dnPKC{alpha} translocated to the nucleus and the membrane fractions, concomitant with a marked decrease in the cytosolic fraction, when treated with PMA for 30 min. Four hours after PMA-induced PKC{alpha} nuclear translocation, TR{alpha}1 protein in the nucleus was increased in both Ad-wtPKC{alpha}- and control Ad-ne{beta}gal-transduced cells (Fig. 3). In contrast, TR{alpha}1 protein decreased in Ad-dnPKC{alpha}-transduced myocytes treated with PMA, suggesting that nuclear translocation of the kinase-inactive dnPKC{alpha} attenuated the effect of PMA-activated endogenous PKC{alpha} on TR{alpha}1 protein.



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  		Fig. 3. PMA induces PKC{alpha} translocation to the nucleus and increases nuclear TR{alpha}1. NRVM were transduced with Ad-ne{beta}gal, Ad-wtPKC{alpha}, or Ad-dnPKC{alpha} (5 moi) and then cultured in serum-free medium for 48 h before treatment with PMA (200 nM) for 30 min. Cells were fractionated into soluble (sol), membrane (mb), and nuclear (nuc) fractions as described in MATERIALS AND METHODS and analyzed by Western blotting. Treatment with PMA induced translocation of endogenous PKC{alpha} (Ad-ne{beta}gal) and adenovirally expressed wt- and dnPKC{alpha} to the membrane and nuclear fractions (top). Four hours after PMA treatment, immunoreactive TR{alpha}1 protein increased in the nuclear fraction of control and Ad-wtPKC{alpha}-transduced cardiomyocytes (bottom), whereas TR{alpha}1 decreased in Ad-dnPKC{alpha}-transduced cells. TBP shows equivalent protein content per lane.

 
PKC{alpha}-induced TR{alpha}1 expression is mediated at the transcriptional level. Several potential mechanisms could account for the PKC{alpha}-induced increase of TR{alpha}1 protein, including prolonged TR{alpha}1 protein half-life, increased TR{alpha}1 mRNA stability, or increased transcription and mRNA production. To address these possibilities we initially tested the hypothesis that the half-life of TR{alpha}1 protein was prolonged. Using CHX, an inhibitor of translation, we showed that the rate of degradation of TR{alpha}1 protein was not significantly different in cardiomyocytes transduced with Ad-wtPKC{alpha} compared with Ad-ne{beta}gal (Fig. 4). The estimated half-life of TR{alpha}1 was ~4 h. As seen in Fig. 4, the decrease in TBP after 6 h of treatment with CHX reflects the indiscriminate effect of CHX on inhibition of translation of all proteins. Because TR{alpha}1 expression was ~30-fold higher in Ad-wtPKC{alpha}-transduced cells compared with Ad-ne{beta}gal, TBP was not detectable under optimum conditions for TR{alpha}1 measurement and thus was not used for normalization.



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  		Fig. 4. Ad-wtPKC{alpha}-induced increase of TR{alpha}1 is not mediated by increasing TR{alpha}1 protein stability. NRVM were transduced with Ad-ne{beta}gal or Ad-wtPKC{alpha} (5 moi) and cultured in serum-free medium for 48 h before cyclohexamide (CHX) treatment. Cells were harvested after 0, 2, 4, and 6 h exposure to CHX (10 µg/ml), and the nuclear fractions were used for analysis of TR{alpha}1 by immunoblotting. Laser-scanning analyses are shown as the densitometric units (du) of the amount of TR{alpha}1 protein normalized to TBP (bottom, Ad-ne{beta}gal) relative to untreated 0-h control. TR{alpha}1 protein measurements in Ad-wtPKC{alpha}-transduced cardiomyocytes were not normalized to TBP, as discussed in RESULTS.

 
Since we had previously reported that TR{alpha}1 protein content was regulated at the level of mRNA (21), we quantified TR{alpha}1 mRNA in response to wtPKC{alpha} overexpression using real-time PCR and found that it was increased by 55-fold (P < 0.001) (Fig. 5A). Furthermore, PMA stimulation for 4 h increased TR{alpha}1 mRNA by an additional 60 ± 2%. TR{beta}1 mRNA content was unaffected by PKC{alpha} overexpression (data not shown).



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  		Fig. 5. Ad-wtPKC{alpha}-mediated increase of TR{alpha}1 mRNA is not the result of increased mRNA half-life. A: quantitative real-time PCR (Q-PCR) measurements of TR{alpha}1 mRNA showed 55-fold higher levels of TR{alpha}1 expression in NRVM transduced with Ad-wtPKC{alpha} compared with cardiomyocytes transduced with control Ad-ne{beta}gal. Mean ± SE; difference between groups, P < 0.0001. n = 6 per group, 2 separate experiments. B: uninfected (UI) ({circ}) or Ad-wtPKC{alpha} ({bullet})-transduced NRVM were cultured for 48 h in serum-free medium. After actinomycin D (5 µg/ml) was added, selected cultures were harvested at 15, 30, 50, 60, 90, 120, 150, and 240 min as indicated, and TR{alpha}1 mRNA was quantified by Q-PCR. Data are expressed relative to the value at 15 min within each group (UI or Ad-wtPKC{alpha}). Each data point is mean ± SE; n = 3–6 of 3 separate experiments.

 
To determine whether PKC{alpha} increased the stability of TR{alpha}1 mRNA, we measured mRNA half-life by inhibiting transcription using the RNA polymerase II inhibitor, actinomycin D, as we have previously reported (21). As shown in Fig. 5B, the rate of decay of TR{alpha}1 mRNA in cardiomyocytes transduced with Ad-wtPKC{alpha} was not prolonged compared with that in control uninfected cells; therefore, increased stability of TR{alpha}1 mRNA could not explain the PKC{alpha}-induced increase in the mRNA. Itshould be noted that total TR{alpha}1 mRNA in Ad-wtPKC{alpha}-transduced myocytes was 55-fold higher than uninfected cells; therefore, each decay curve is compared with its own control value after 15-min pretreatment with actinomycin D. These data support a potential role of PKC{alpha} on TR{alpha}1 transcription either by directly targeting specific transcription factors or by activating other downstream kinases.

PKC{alpha}-induced TR{alpha}1 expression is not mediated by activated ERK1/2. Published studies have reported that PKC{alpha} activates ERK1/2 in cardiomyocytes (3, 41), suggesting that ERK may have a potential effect on nuclear factor activation. Therefore, we questioned whether the observed PKC{alpha}-induced TR{alpha}1 expression could be the result of ERK activation. As shown in Fig. 6, significant activation of ERK1/2 and increased TR{alpha}1 expression were observed in Ad-wtPKC{alpha}- compared with Ad-ne{beta}gal-transduced cardiomyocytes. Treatment with a specific MAPK kinase (MEK1) inhibitor, PD-98059, for 4 h significantly reduced ERK1/2 phosphorylation but had no significant effect on nuclear TR{alpha}1 content, suggesting that activation of the ERK pathway was unlikely to be involved in PKC{alpha}-mediated induction of TR{alpha}1.



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  		Fig. 6. Inhibition of phospho-ERK1/2 (p-ERK1/2) has no effect on TR{alpha}1 expression. NRVM were transduced with Ad-ne{beta}gal or Ad-wtPKC{alpha} and cultured in serum-free medium for 48 h, after which Ad-wtPKC{alpha}-transduced NRVM were treated with MEK1 inhibitor, PD-98059 (10–5 M), for 4 h before harvest. A: Western blot analysis of cytosolic PKC{alpha}, p-ERK1/2, total ERK1/2, and nuclear TR{alpha}1 and TBP. B: quantitation showed significant ERK1/2 activation and increased TR{alpha}1 in Ad-wtPKC{alpha}- compared with Ad-ne{beta}gal-transduced (Ad{beta}gal) myocytes. PD-98059 caused significant inhibition of ERK1/2 phosphorylation with no significant effect on nuclear TR{alpha}1 content. Data are means ± SE. *P < 0.01 vs. Ad-{beta}gal; **P < 0.01 vs. Ad-{beta}gal and Ad-wtPKC{alpha} – PD-98059.

 
Effect of T3 treatment on subcellular content and distribution of PKC{alpha}. Previous studies have reported that thyroid hormone treatment of cultured chick embryo hepatocytes caused translocation of PKC{alpha} to the membrane fraction and induced PKC{alpha} activity (1) and that T3 treatment caused a reduction in PKC{alpha} expression in cardiomyocytes (35). Therefore, it was of interest to determine whether T3 had a similar effect on PKC{alpha} in the Ad-wtPKC{alpha}-transduced cardiomyocytes. The Western blot in Fig. 7A illustrates the subcellular distribution of PKC{alpha} after T3 treatment for 30 min, 4 h, and 24 h. Quantitation of these data showed no significant redistribution of PKC{alpha} between the soluble and membrane or nuclear fractions; however, PKC{alpha} protein decreased significantly by 25% in the cytosolic fraction and 50% in the nuclear fraction after 4 and 24 h of T3 treatment (Fig. 7B). No significant differences in PKC{alpha} were observed in any fraction after 30 min of T3 exposure.



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  		Fig. 7. Effect of triiodothyronine (T3) on subcellular distribution of PKC{alpha}. NRVM transduced with Ad-wtPKC{alpha} were treated with T3 (10 nM) for 0.5, 4, 24 h and then extracted to isolate cytosolic, membrane, and nuclear fractions. A: Western blot analysis of PKC{alpha} showing subcellular distribution with T3 treatment. B: quantitation from 3 separate experiments shows significant reduction of PKC{alpha} in cytosolic and nuclear fractions at 4 and 24 h of T3 treatment. PKC{alpha} protein content was normalized to TBP content (not shown). Data are means ± SE. *P < 0.01 vs. control; {dagger}P < 0.01 vs. control and 30 min.

 
Effect of T3 treatment on PKC{alpha}-induced TR{alpha}1 expression. Since we had previously published that T3 rapidly decreased TR{alpha}1 content in the cardiomyocyte (21), we sought to determine whether the elevated level of expression of TR{alpha}1 in Ad-wtPKC{alpha}-transduced cells was similarly regulated. As shown in Fig. 8A, T3 treatment for 30 min showed no effect on nuclear TR{alpha}1 protein content, but treatment for 4 and 24 h resulted in a 45% and 55% decrease of TR{alpha}1 protein, respectively. This decrease in TR{alpha}1 protein was paralleled by a significant decrease of TR{alpha}1 mRNA (56 ± 4%) after 4 h of T3 treatment (Fig. 8B). Shown for comparison are data from control cells transduced with Ad-ne{beta}gal in which T3 treatment for 4 h significantly reduced TR{alpha}1 mRNA, as we have previously published (21). In contrast, TR{beta}1 mRNA was increased by 40–60% after 4 h of T3 treatment in both Ad-wtPKC{alpha}- and Ad-ne{beta}gal-transduced cardiomyocytes (data not shown).



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  		Fig. 8. T3 decreases TR{alpha}1 protein and mRNA. A: Ad-wtPKC{alpha}-transduced NRVM were treated with T3 (10 nM) for 0.5, 4, and 24 h. Nuclear TR{alpha}1 and TBP were analyzed by Western blotting. B: TR{alpha}1 mRNA was quantified by real-time PCR and expressed as fold change of untreated Ad-ne{beta}gal. NRVM were transduced with Ad-ne{beta}gal or Ad-wtPKC{alpha} and cultured for 48 h in serum-free medium and then supplemented with T3 (10 nM) for 4 h before harvest and RNA extraction. Data are means ± SE; n = 6–8 from 2 separate experiments. *P < 0.0001, # P < 0.05 vs. untreated of same group.

 
Cardiac phenotype in response to PKC{alpha} overexpression and T3 treatment. To understand the potential biological consequences of PKC{alpha}-induced increase in TR{alpha}1 protein in the cardiac myocyte, we examined the expression of several known thyroid hormone-responsive genes, including {alpha}- and {beta}-MHCs and SERCA2. As shown in Fig. 9, cardiomyocytes transduced with Ad-wtPKC{alpha} showed significant reductions in expression of the positively regulated T3-responsive genes {alpha}-MHC and SERCA2 compared with control Ad-ne{beta}gal-transduced cells, indicative of an increased repressive role of TR{alpha}1 in PKC{alpha}-activated myocytes. In contrast, the negatively regulated T3-responsive {beta}-MHC gene was expressed to a similar extent as control. Furthermore, the cardiomyocytes exhibited a robust response to treatment with T3 (10 nM) for 24 h with significant increases in {alpha}-MHC and SERCA2 expression and a decrease in {beta}-MHC mRNA expression (Fig. 9).



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  		Fig. 9. Effects of PKC{alpha} overexpression on cardiomyocyte phenotype and effects of T3 treatment. Northern blot analysis of RNA isolated from NRVM transduced with Ad-ne{beta}gal or Ad-wtPKC{alpha} (5 moi) and cultured in serum-free medium for 48 h or supplemented with T3 (10 nM) for the final 24 h of culture. Radiolabeled oligodeoxynucleotide probes were used to quantify {alpha}- and {beta}-myosin heavy chain (MHC) mRNAs and 18S ribosomal RNA and a radiolabeled cDNA hybridized to sarcoplasmic reticulum calcium-ATPase (SERCA2). As illustrated, each specific mRNA was detected by exposure to X-ray film. 18S rRNA shows equivalent RNA content per lane.

 

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Genetic studies of mouse models harboring deletions of either TR{alpha} or TR{beta} isoform have provided insight into the specific effects of each receptor isoform on cardiocirculatory function and phenotype. The results largely support a predominant role of TR{alpha} in the contractile and electrophysiological functions in the heart (reviewed in Ref. 20). The prominent role of thyroid hormone and its receptors in regulating cardiac physiology is exemplified by both nuclear and extranuclear mechanisms that regulate ion channel activities, calcium transients, contractile function, and transcriptional regulation of genes encoding proteins regulating these functions (reviewed in Refs. 8, 25). Furthermore, because the phenotype associated with pathological hypertrophy and heart failure includes many thyroid hormone-associated functions, substantial interest has been generated in developing TR isoform-specific therapeutic strategies in these disease settings (32).

In the present study, we proposed that TR function in the myocardium could be altered by intracellular protein kinase pathways known to be activated in the diseased heart. PKC-{alpha} and PKC-{beta} activity and expression have been shown to be increased in failing human hearts (2). In experimental animal models of cardiac infarction, PKC{alpha}, -{beta},- {epsilon}, and -{zeta} content and calcium-dependent and calcium-independent PKC activities have been reported to be significantly increased in the remodeled left ventricle (42). Furthermore, transgenic models utilizing gain- and loss-of-function approaches of specific PKC isotypes support critical roles for PKCs in intracellular signaling pathways regulating cardiac contractile function, hypertrophy, and phenotype (4, 19, 31). We focused on PKC{alpha}, -{delta} and -{epsilon} isoenzymes, reported to lie downstream of G protein-coupled receptors and activated in the pathological hypertrophic cardiomyocyte (11, 12), to interrogate the effects of each isotype on TR activity and cardiomyocyte phenotype.

Our initial observation that a twofold increase in cellular PKC{alpha} content resulted in an increase of TR{alpha}1 protein by 28-fold was unexpected. No effect was observed on the TR{beta}1 isoform. Additionally, by progressively increasing the expression of virally transduced PKC{alpha}, TR{alpha}1 content in both nuclear and cytosolic compartments was further increased. Although we and others have shown that TR{alpha}1 distributes in the cytoplasmic fraction when virally transduced into the cardiomyocyte (21, 22), the present data showing significant cytosolic compartmentalization of endogenous TR{alpha}1 support a potential role of TR{alpha}1 outside the nuclear domain. This effect on TR{alpha}1 was specific to the PKC{alpha} isotype, as supported by the lack of effect when virally transduced constitutively active PKC{delta} and PKC{epsilon} were overexpressed in cardiomyocytes. Furthermore, the kinase activity of PKC{alpha} was necessary for the observed increase of TR{alpha}1 protein because overexpression of a kinase-inactive mutant of PKC{alpha} (Ad-dnPKC{alpha}) was ineffective.

We initially investigated several cytoplasmic mechanisms that could result in these observations, including a potential effect of PKC{alpha} on prolongation of the half-lives of TR{alpha}1 protein and mRNA. RNA analysis revealed a ~50-fold increase in TR{alpha}1 mRNA equivalent to the increase in TR{alpha}1 protein, suggesting a potential effect of PKC{alpha} on mRNA stability. However, experiments with the RNA polymerase II inhibitor actinomycin D showed that the half-life of TR{alpha}1 mRNA in Ad-wtPKC{alpha}-transduced myocytes was not increased. Similarly, CHX experiments showed that the half-life of TR{alpha}1 protein in Ad-wtPKC{alpha}-transduced cells was not sufficiently different from control cultures to explain the increase in protein. Taken together, these data support a mechanism whereby transcription of the TR{alpha} gene locus is induced by overexpression of PKC{alpha}, suggesting an important role of nuclear localization of PKC{alpha}. A recent report has shown that PKC{alpha} translocates to the nucleus in response to the phorbol ester PMA (41). Our data support this observation of nuclear translocation of endogenous PKC{alpha} as well as adenovirus-expressed PKC{alpha} in response to PMA. The PMA-induced nuclear translocation of PKC{alpha}, whether endogenous or virally expressed PKC{alpha}, resulted in a significant increase of TR{alpha}1 protein. In contrast, PMA-induced translocation of virally transduced dnPKC{alpha} repressed expression of TR{alpha}1 protein to a level below that observed in unstimulated myocytes. Whether endogenous PKC{alpha} is present in the nucleus under basal, unstimulated conditions is not clear; however, overexpression of PKC{alpha} resulted in detectable amounts in the nuclear compartment, which we hypothesize induced TR{alpha}1 expression even in the absence of activation by PMA.

Alternatively, PKC{alpha} may indirectly induce TR{alpha}1 transcription by activating other downstream kinases, including ERK1/2, as published by others (3, 41). Activation and nuclear translocation of ERKs has been reported in response to thyroxine, supporting a potential role of the ERK pathway in thyroid hormone-mediated nuclear events (9). Alternatively, ERK-mediated phosphorylation of 90-kDa ribosomal S6 kinases (p90 RSK), for example, could potentially activate nuclear transcription factors, including cAMP response element-binding protein, activating transcription factor 1, and c-Fos as has been reported in other cell systems (reviewed in Ref. 15). However, the promoter region of the TR{alpha} gene locus has not been extensively characterized (18, 27, 28), and thus the identities of plausible transcription factors as targets of PKC{alpha} will require further investigation. To address the possibility that the ERK pathway may be involved in the current observations, we studied the effects of PKC{alpha} overexpression on ERK phosphorylation and the effect of ERK inhibition on TR{alpha}1 expression. Similar to published reports, phosphorylation of ERK1/2 was increased with PKC{alpha} overexpression (3, 41); however, inhibition of the MEK1-ERK1/2 signaling pathway had no effect on expression of TR{alpha}1, suggesting that the PKC{alpha} effects on TR{alpha}1 were unlikely to be mediated by this pathway. Thus identification of potential nuclear substrates of PKC{alpha} may shed light on the mechanisms by which transcription of TR{alpha}1 is induced and determine how activation of this important intracellular kinase, as occurs in the pathological myocardium, can affect cardiac phenotype and function.

Because our previous study had shown that TR{alpha}1 was degraded rapidly in response to T3 treatment, it was of interest to determine how these effects of T3 interacted with the PKC{alpha}-mediated increase in TR{alpha}1 expression. Additionally, previous reports had provided evidence that thyroid hormone regulated PKC{alpha} expression as well as its subcellular localization (1). Although we found no effect of T3 on the subcellular redistribution of PKC{alpha}, T3 treatment for 4 h significantly reduced PKC{alpha} protein content in the cytosolic and nuclear fractions. Therefore, these data further support the hypothesis that PKC{alpha} activates transcription at the TR{alpha} gene locus and that the T3-mediated decrease in nuclear PKC{alpha} may in part be responsible for the decrease in TR{alpha}1 expression.

As we show in this study, PKC{alpha}-induced expression of TR{alpha}1 in the cardiomyocyte resulted in altered expression of T3-responsive genes similar to that seen in the pathological myocardium. Importantly, treating these cardiomyocytes with thyroid hormone reversed the phenotypic changes due to PKC{alpha} overexpression by decreasing TR{alpha}1 protein expression. In heart failure in which PKC{alpha} has been shown to be activated (2), expression of TR{alpha}1 may be increased as a consequence, and with decreased circulating levels of T3, as is often observed in heart failure patients (33), the unliganded TR{alpha}1 would act to repress T3-responsive genes, including SERCA2 and {alpha}-MHC. Furthermore, with the recent identification of extranuclear or cytoplasmic actions of TRs (5, 9, 22), the effects of unliganded TR{alpha}1 in the cytosolic compartment may have as yet undetermined metabolic and functional effects on the cardiomyocyte. Thus results from our studies underscore the importance of the relative concentrations of circulating thyroid hormones and the cellular content of unliganded TRs in determining cardiomyocyte phenotype and function.


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This work was supported by National Heart, Lung, and Blood Institute Grant HL-71623.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Ojamaa, Institute for Medical Research, North Shore-LIJ Health System, 350 Community Dr., Manhasset, NY 11030 (e-mail: kojamaa{at}nshs.edu)

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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  1. Alisi A, Spagnuolo S, Napoletano S, Spaziani A, and Leoni S. Thyroid hormones regulate DNA-synthesis and cell-cycle proteins by activation of PKC{alpha} and p42/44 MAPK in chick embryo hepatocytes. J Cell Physiol 201:259–265, 2004.[CrossRef][ISI][Medline]
  2. Bowling N, Walsh RA, Song G, Estridge T, Sandusky GE, Fouts RL, Mintze K, Pickard T, Roden R, Bristow MR, Sabbah HN, Mizrahi JL, Gromo G, King GL, and Vlahos CJ. Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation 99: 384–391, 1999.[Abstract/Free Full Text]
  3. Braz JC, Bueno OF, De Windt LJ, and Molkentin JD. PKC{alpha} regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase 1/2 (ERK1/2). J Cell Biol 156: 905–919, 2002.[Abstract/Free Full Text]
  4. Braz JC, Gregary K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, DePaoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, and Molkentin JD. PKC-{alpha} regulates cardiac contractility and propensity toward heart failure. Nat Med 10: 248–254, 2004.[CrossRef][ISI][Medline]
  5. Cao X, Kambe F, Moeller LC, Refetoff S, and Seo H. Thyroid hormone induces rapid activation of Akt/protein kinase B-mammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts. Mol Endocrinol 19: 102–112, 2005.[Abstract/Free Full Text]
  6. Clerk A, Bogoyevitch MA, Fuller SJ, Lazou A, Parker PJ, and Sugden PH. Expression of protein kinase C isoforms during cardiac ventricular development. Am J Physiol Heart Circ Physiol 269: H1087–H1097, 1995.[Abstract/Free Full Text]
  7. D'Amati G, Di Gioia CRT, Mentuccia D, Pistilli D, Proietti-Pannunzi L, Miraldi F, Gallo P, and Celi FS. Increased expression of thyroid hormone receptor isoforms in end-stage human congestive heart failure. J Clin Endocrinol Metab 86: 2080–2084, 2001.[Abstract/Free Full Text]
  8. Davis PJ and Davis FB. Nongenomic actions of thyroid hormone on the heart. Thyroid 12: 459–466, 2002.[CrossRef][ISI][Medline]
  9. Davis PJ, Shih A, Lin HY, Martino LJ, and Davis FB. Thyroxine promotes association of mitogen-activated protein kinase and nuclear thyroid hormone receptor (TR) and causes serine phosphorylation of TR. J Biol Chem 275: 38032–38039, 2000.[Abstract/Free Full Text]
  10. Dean JLE, Sully G, Clark AR, and Saklatvala J. The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilization. Cell Signal 16: 1113–1121, 2004.[CrossRef][ISI][Medline]
  11. Decock JBJ, Gillespie-Brown J, Parker PJ, Sugden PH, and Fuller SJ. Classical, novel and atypical isoforms of PKC stimulate ANF- and TRE/AP-1-regulated-promoter activity in ventricular cardiomyocytes. FEBS Lett 356: 275–278, 1994.[CrossRef][ISI][Medline]
  12. Dorn IIGW and Force T. Protein kinase cascades in the regulation of cardiac hypertrophy. J Clin Invest 115: 527–537, 2005.[CrossRef][ISI][Medline]
  13. Dorn IIGW, Robbins J, and Sugden PH. Phenotyping hypertrophy. Eschew obfuscation. Circ Res 92: 1171–1175, 2003.[Free Full Text]
  14. Friesema ECH, Ganguly S, Abdalla A, Fox JEM, Halestrap AP, and Visser TJ. Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J Biol Chem 278: 40128–40135, 2003.[Abstract/Free Full Text]
  15. Frodin M and Gammeltoft S. Role and regulation of 90-kDa ribosomal S6 kinase (RSK) in signal transduction. Mol Cell Endocrinol 151: 65–77, 1999.[CrossRef][ISI][Medline]
  16. Hahn HS, Marreez Y, Odley A, Sterbling A, Yussman MG, Hilty KC, Bodi I, Liggett SB, Schwartz A, and Dorn GW II. Protein kinase C-{alpha} negatively regulates systolic and diastolic function in pathological hypertrophy. Circ Res 93: 1111–1119, 2003.[Abstract/Free Full Text]
  17. Heidkamp MC, Bayer AL, Martin JL, and Samarel AM. Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase-C, -{epsilon}, and -{delta} in neonatal rat ventricular myocytes. Circ Res 89: 882–890, 2001.[Abstract/Free Full Text]
  18. Hu RM, Wu LM, Frank HJ, Pedram A, and Levin ER. Insulin stimulates thyroid hormone receptor-{alpha} gene expression in cultured bovine aortic endothelial cells. Mol Cell Endocrinol 103: 65–71, 1994.[CrossRef][Medline]
  19. Inagaki K, Chen L, Ikeno F, Lee FH, Imahashi K, Bouley DM, Rezaee M, Yock PG, Murphy E, and Mochley-Rosen D. Inhibition of {delta}-protein kinase C protects against reperfusion injury of the ischemic heart in vivo. Circulation 108: 2304–2307, 2003.[Abstract/Free Full Text]
  20. Kahaly GJ and Dillmann WH. Thyroid hormone action in the heart. Endocrinol Rev 26: 704–728, 2005.[Abstract/Free Full Text]
  21. Kenessey A and Ojamaa K. Ligand-mediated decrease of thyroid hormone receptor-{alpha}1 in cardiomyocytes by proteosome-dependent degradation and altered mRNA stability. Am J Physiol Heart Circ Physiol 288: H813–H821, 2005.[Abstract/Free Full Text]
  22. Kinugawa K, Jeong MY, Bristow MR, and Long CS. Thyroid hormone induces cardiac hypertrophy in a TR{alpha}1-specific manner that requires TAK1 and p38 MAPK. Mol Endocrinol 19: 1618–1628, 2005.[Abstract/Free Full Text]
  23. Kinugawa K, Minobe WA, Wood WM, Ridgway EC, Baxter JD, Ribeiro RCJ, Tawadrous MF, Lower BA, Long CS, and Bristow MR. Signaling pathways responsible for fetal gene induction in the failing human heart. Evidence for altered thyroid hormone receptor gene expression. Circulation 103: 1089–1094, 2001.[Abstract/Free Full Text]
  24. Kinugawa K, Yonekura K, Ribeiro RCJ, Eto Y, Aoyagi T, Baxter JD, Camacho SA, Bristow MR, Long CS, and Simpson PC. Regulation of thyroid hormone receptor isoforms in physiological and pathological cardiac hypertrophy. Circ Res 89: 591–598, 2001.[Abstract/Free Full Text]
  25. Klein I and Ojamaa K. Thyroid hormone and the cardiovascular system. N Engl J Med 344: 501–509, 2001.[Free Full Text]
  26. Kotlyarov A and Gaestel M. Is MK2 (mitogen-activated protein kinase-activated protein kinase 2) the key for understanding posttranscriptional regulation of gene expression? Biochem Soc Trans 30: 959–963, 2002.[CrossRef][ISI][Medline]
  27. Laudet A, Vanacker JM, Adelmant G, Begue A, and Stehelin D. Characterization of a functional promoter for the human thyroid hormone receptor-{alpha} (c-erbA-1) gene. Oncogene 8: 975–982, 1993.[ISI][Medline]
  28. Lazar J, Desvergne B, Zimmerman EC, Zimmer DB, Magnuson MA, and Nikodem VM. A role for intronic sequences on expression of thyroid hormone receptor-{alpha} gene. J Biol Chem 269: 20352–20359, 1994.[Abstract/Free Full Text]
  29. Malhotra A, Kang BP, Opawumi D, Belizaire W, and Meggs LG. Molecular biology of protein kinase C signaling in cardiac myocytes. Mol Cell Biochem 225: 97–107, 2001.[CrossRef][ISI][Medline]
  30. Miyata S, Minobe W, Bristow MR, and Leinwand LA. Myosin heavy chain isoform expression in the failing and nonfailing human heart. Circ Res 86: 386–390, 2000.[Abstract/Free Full Text]
  31. Mochly-Rosen D, Wu G, Hahn H, Osinska H, Liron T, Lorenz JN, Yatani A, Robbins J, and Dorn GW II. Cardiotrophic effects of protein kinase C{epsilon}. Analysis by in vivo modulation of PKC{epsilon} translocation. Circ Res 86: 1173–1179, 2000.[Abstract/Free Full Text]
  32. Morkin E, Ladenson P, Goldman S, and Adamson C. Thyroid hormone analogs for treatment of hypercholesterolemia and heart failure: past, present and future prospects. J Mol Cell Cardiol 37: 1137–1146, 2004.[ISI][Medline]
  33. Ojamaa K, Ascheim D, Hryniewicz K, and Klein I. Thyroid hormone therapy of cardiovascular disease. Cardiovasc Rev Rep 23: 20–26, 2002.
  34. Ojamaa K, Kenessey A, Shenoy R, and Klein I. Thyroid hormone metabolism and cardiac gene expression after acute myocardial infarction in the rat. Am J Physiol Endocrinol Metab 279: E1319–E1324, 2000.[Abstract/Free Full Text]
  35. Rybin V and Steinberg SF. Thyroid hormone represses protein kinase C isoform expression and activity in rat cardiac myocytes. Circ Res 79: 388–398, 1996.[Abstract/Free Full Text]
  36. Sabri A and Steinberg SF. Protein kinase C isoform-selective signals that lead to cardiac hypertrophy and the progression of heart failure. Mol Cell Biochem 251: 97–101, 2003.[CrossRef][ISI][Medline]
  37. Steinberg SF, Goldberg M, and Rybin VO. Protein kinase C isoform diversity in the heart. J Mol Cell Cardiol 27: 141–153, 1995.[ISI][Medline]
  38. Strait JB III, Martin JL, Bayer A, Mestril R, Eble DM, and Samarel AM. Role of protein kinase C-{epsilon} in hypertrophy of cultured neonatal rat ventricular myocytes. Am J Physiol Heart Circ Physiol 280: H756–H766, 2001.[Abstract/Free Full Text]
  39. Sylven C, Jansson E, Sotonyi P, Waagstein F, Barkhem T, and Bronnegard M. Cardiac nuclear hormone receptor mRNA in heart failure in man. Life Sci 59: 1917–1922, 1996.[CrossRef][ISI][Medline]
  40. Tchen CR, Brook M, Saklatvala J, and Clark AR. The stability of tristetraprolin mRNA is regulated by mitogen-activated protein kinase p38 and by tristetraprolin itself. J Biol Chem 279: 32393–32400, 2004.[Abstract/Free Full Text]
  41. Vijayan K, Szotek EL, Martin JL, and Samarel AM. Protein kinase C-{alpha}-induced hypertrophy of neonatal rat ventricular myocytes. Am J Physiol Heart Circ Physiol 287: H2777–H2789, 2004.[Abstract/Free Full Text]
  42. Wang J, Liu X, Sentex E, Takeda M, and Dhalla NS. Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction. Am J Physiol Heart Circ Physiol 284: H2277–H2287, 2003.[Abstract/Free Full Text]
  43. Wassen FWJS, Schiel AE, Kuiper GGJM, Kaptein E, Bakker O, Visser TJ, and Simonides WS. Induction of thyroid hormone-degrading deiodinase in cardiac hypertrophy and failure. Endocrinology 143: 2812–2815, 2002.[Free Full Text]