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Am J Physiol Endocrinol Metab 286: E968-E974, 2004. First published February 17, 2004; doi:10.1152/ajpendo.00478.2003
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Tissue-specific regulation of cytochrome c oxidase subunit expression by thyroid hormone

Treacey E. Sheehan,1 Ponni A. Kumar,1 and David A. Hood1,2

1School of Kinesiology and Health Science, and 2Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada

Submitted 21 October 2003 ; accepted in final form 10 February 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The influence of thyroid hormone (T3) on respiration is partly mediated via its effect on the cytochrome c oxidase (COX) enzyme, a multi-subunit complex within the mitochondrial respiratory chain. We compared the expression of COX subunits I, III, Vb, and VIc and thyroid receptors (TR){alpha}1 and TR{beta}1 with functional changes in COX activity in tissues that possess high oxidative capacities. In response to 5 days of T3 treatment, TR{beta}1 increased 1.6-fold in liver, whereas TR{alpha}1 remained unchanged. T3 also induced concomitant increases in the protein and mRNA expression of nuclear-encoded subunit COX Vb in liver, matched by a 1.3-fold increase in binding to a putative thyroid response element (TRE) within the COX Vb promoter in liver, suggesting transcriptional regulation. In contrast, T3 had no effect on COX Vb expression in heart. T3 produced a significant increase in COX III mRNA in liver but decreased COX III mRNA in heart. These changes were matched by parallel alterations in mitochondrial transcription factor A expression in both tissues. In contrast, COX I protein increased in both liver and heart 1.7- and 1.5-fold (P < 0.05), respectively. These changes in COX I closely paralleled the T3-induced increases in COX activity observed in both of these tissues. In liver, T3 induced a coordinated increase in the expression of the nuclear (COX Vb) and mitochondrial (COX I) genomes at the protein level. However, in heart, the main effect of T3 was restricted to the expression of mitochondrial DNA subunits. Thus our data suggest that T3 regulates the expression of COX subunits by both transcriptional and posttranscriptional mechanisms. The nature of this regulation differs between tissues possessing a high mitochondrial content, like liver and heart.

mitochondrial biogenesis; mitochondrial transcription factor A; 3,3',5-triiodo-L-thyronine; thyroid receptors; gene transcription

MITOCHONDRIAL BIOGENESIS is a complex process that results in an enhanced ability of the cell to generate the energy required for cellular activities. Physiological stimuli that induce this process include endurance training (9, 11), chronic stimulation (18), and thyroid hormone (3,3',5-triiodo-L-thyronine, T3) treatment (19, 46). The mitochondrion is unique in that it has its own DNA and protein synthesis apparatus that are physically distinct from those found in the nucleus and the cytoplasm. Mitochondrial DNA (mtDNA) encodes 13 proteins of the respiratory chain, 2 rRNAs, and 22 tRNAs (18, 48). However, this represents only a minor fraction of the proteins required for mitochondrial function. Because of this limited coding capacity of mtDNA, the expression of nuclear-encoded genes is essential for the proper assembly of the organelle. Therefore, mitochondrial biogenesis requires the cooperation of both the mitochondrial and the nuclear genomes.

In the nucleus, the action of T3 is mediated via thyroid receptors (TRs). TRs are members of a family of hormone receptor transcription factors that regulate the expression of numerous genes (34). TRs associate with chromatin and bind T3 with high affinity and specificity (23, 30). Two genes encoding nuclear TRs have been characterized: the c-erb A{alpha} gene encodes the TR{alpha}1 and TR{alpha}2 isoforms (48), whereas the c-erb A{beta} gene encodes three isoforms, TR{beta}1, TR{beta}2, and TR{beta}3 (45). Only the TR{alpha}1, -{beta}1, -{beta}2, and -{beta}3 isoforms bind T3 and transactivate genes that contain T3 response elements (TREs) within their promoter regions (39).

T3 can have both direct and indirect effects on mitochondria. For instance, T3 induces the expression of mitochondrial transcription factor A (Tfam), a nuclear-encoded protein that binds mtDNA to regulate its transcription (31). Another transcription factor, p43, is a truncated form of the nuclear TR{alpha}1 located in the mitochondrial matrix (4). Its binding to mtDNA is regulated by T3, suggesting that it also plays an important role in the regulation of mtDNA transcription. However, whether the expression of this protein is responsive to T3 is currently unknown.

A well-known effect of T3 is its profound influence on mitochondrial respiration, mediated, in part, by altering the expression and activity of various components of the mitochondrial electron transport chain (31). For instance, the mRNAs encoding several nuclear-encoded respiratory genes are upregulated in response to T3 treatment. These include {beta}-F1-ATPase and several subunits of the cytochrome c oxidase (COX) enzyme (22, 44). In mammals, COX is composed of 13 polypeptides. Ten subunits are transcribed within the nucleus, and the remaining three subunits are products of the mitochondrial genome (19). COX regulation is therefore a useful indicator of mitochondrial biogenesis, since a functional holoenzyme requires the coordination of both of these genomes. Although the coordinate induction of COX subunit mRNA derived from the nuclear and mitochondrial genomes is well established with contractile activity (20, 33), this does not appear to be the case in response to altered T3 status in which nuclear-encoded mRNAs appear to remain unchanged (40) or change with different kinetics (44) from mtDNA-encoded transcripts. Therefore, the purposes of our study were 1) to determine the extent of coordination between the nuclear and mitochondrial genomes at the protein level of expression, 2) to compare this to functional measures of holoenzyme activity, 3) to examine the role of transcriptional activation in the T3-induced response, and 4) to compare our measure of transcriptional activation with the expression of TR isoforms in tissues that are known to be responsive to T3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The COX Vb cDNA was a generous gift from Dr. N. G. Avadhani (University of Pennsylvania, Philadelphia, PA). T3 and the DR2 oligonucleotide were purchased from Sigma-Aldrich (St. Louis, MO). The TR antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The COX Vb, COX VIc, and COX I antibodies were purchased from Molecular Probes (Eugene, OR). The rat Tfam antibody was used as previously described (14).

Animal care and treatments. Adult male Sprague-Dawley rats (250–300 g) were injected with T3 (0.4 mg/kg body wt) dissolved in vehicle (100% propylene glycol-0.9% NaCl [1.5:1 vol/vol)] for a period of 5 consecutive days. This dosage produces significant alterations in the mitochondrial import machinery and phenotype (7, 8, 36), and a similar time course has been used by others (29). Concentrations of T3 were increased 50-fold at 2 h and declined to four- to sixfold by 24 h after injection. This was repeated for each of the 5 days of T3 injection (Sheehan TE and Hood DA, unpublished observations).

Twenty-four hours after the last injection, the animals were anesthetized with an intraperitoneal injection of pentobarbital sodium, and the liver and heart were removed and quick-frozen in liquid nitrogen. The mass of the left ventricle was measured and compared with body mass to assess the extent of cardiac hypertrophy. Each tissue was subsequently pulverized in liquid N2 for RNA and protein extractions.

RNA isolation and analysis. Total RNA was isolated from frozen tissue powders (150 mg) with a modified Tri reagent protocol using TRIzol reagent (Invitrogen, Burlington, ON, Canada). The RNA concentration was obtained by measuring the absorbance at 260 nm. RNA (10–30 µg) was then electrophoresed on 1% agarose gels containing 0.02% formaldehyde, transferred, and fixed to nylon membranes (Hybond N; Amersham Pharmacia Biotech, Mississauga, ON, Canada). The membranes were then prehybridized and hybridized overnight at 42°C with a 32P-labeled cDNA probe encoding either COX III (20) or COX Vb. The cDNA probes were radiolabeled with [{alpha}-32P]dCTP and a random primer labeling kit (New England Biolabs, Beverly, MA). The membrane was also hybridized with radiolabeled 18S rRNA to correct for any loading differences between samples. The membranes were washed three times at room temperature using 2x SSC (0.15 M NaCl, 0.03 M sodium citrate) containing 0.1% SDS and then twice at 60°C with 0.1x SSC containing 0.1% SDS. The membranes were then exposed to film and quantified using Sigma Gel software (Jandel Scientific).

Immunoblotting. Total protein was isolated from tissue powders (20 mg/sample) as done previously (38). Protein extracts (100–150 µg/sample) were electrophoresed on 12–18% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Hybond C, Amersham Pharmacia Biotech). Antibodies directed toward TR{alpha}1 (1:500), TR{beta}1 (1:1,000), COX I (1:250), COX Vb (1:1,000), COX VIc (1:500), and Tfam (1:1,000) were incubated with the membrane overnight at 4°C. Signals were detected with anti-mouse (for TR{beta}1, COX Vb, COX VIc, COX I) or anti-rabbit (for TR{alpha}1 and Tfam) IgG coupled to horseradish peroxidase. The membrane was then subjected to enhanced chemiluminescence (Amersham Pharmacia Biotech) and exposed to film. Signals were quantified using Sigma Gel software.

Electromobility shift assay. Frozen whole tissue powders were used for the preparation of cell extracts. Powders (25 mg) were suspended in 15-fold volumes of buffer C [25% glycerol, 20 mM HEPES (pH 7.9), 0.42 M NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF] and sonicated for 10 s on ice. Samples were centrifuged, and the supernatant, representing the cellular extract used for the assay, was stored at –20°C. Equal amounts (50 µg) of protein were incubated with binding buffer [20 mN Tris (pH 8.0), 1 mM EDTA, 50 mM NaCl, 10% glycerol, 0.03% BSA, 1 mM DTT], containing 2% poly(dI-dC), 50 µM sodium pyrophosphate, and a 32P-labeled, double-stranded oligonucleotide representing the DR2 sequence of mtDNA (sense: 5'-CCGTCAAGGCATGAAGGTCAGCAC-3'), which has been shown to bind p43 (4), or the TRE within the nuclear-encoded COX Vb promoter (sense: 5'-ACGCGGACAGGTCATGAACCCGAAGC-3') (2). The incubation was carried out for 30 min at room temperature. After the binding reaction, the mixture was separated on 5% polyacrylamide gels (acrylamide-bisacrylamide, 29:1). Gels were fixed with acetic acid-methanol-H2O (10:30:60) and dried for 1 h, and the signals were quantified using electronic autoradiography.

COX activity. The activity of the COX holoenzyme was determined as described previously (6). Enzyme activity in liver and heart samples was determined by the maximal rate of oxidation of fully reduced cytochrome c (Sigma-Aldrich) measured by the change in absorbance at 550 nm in a Beckman DU-64 spectrophotometer.

Statistical analysis. Data are expressed as means ± SE. Differences between values obtained from vehicle-treated animals and T3-treated animals were determined using Student's t-tests. Tissue differences were analyzed using Student's t-tests. Data were reported as significant if P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Effect of T3 on cardiac and body mass. To assess the effectiveness of our T3 treatment, both heart mass and body mass were measured. T3 treatment attenuated the normal gain in body mass observed in the vehicle-treated animals (P < 0.05) and increased heart mass (Table 1). The heart mass-to-body mass ratio was significantly greater in the T3-treated animals and indicated a 31% cardiac hypertrophy in the T3-treated group (P < 0.001).


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  		Table 1. Effect of T3 treatment on body mass, heart mass, and hypertrophy

 
TR isoform protein expression and response to T3 treatment. To assess whether T3 could regulate the expression of its own receptor, we measured the protein expression of TR{beta}1 and TR{alpha}1 in liver and heart. A 1.6-fold induction of TR{beta}1 was observed in liver (P < 0.05; Fig. 1A). In contrast, T3 treatment did not alter TR{alpha}1 levels in either of the two tissues (Fig. 1B).



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  		Fig. 1. Effect of 3,3',5-triiodo-L-thyronine (T3) treatment on thyroid receptor (TR){alpha}1 and TR{beta}1 protein expression in liver (L) and heart (H). Protein extracts (100–150 µg) from liver and heart of vehicle (V)- and T3-treated animals were subjected to immunoblotting using antibodies directed toward TR{alpha}1 and TR{beta}1. AU, arbitrary units. A: representative Western blots and graphic representation of the effect of T3 treatment on TR{beta}1 expression (n = 4). *P < 0.05 vs. V-treated animals; {ddagger}P < 0.001 vs. V-treated liver samples. B: representative Western blots and graphic representation of the effect of T3 treatment on TR{alpha}1 expression (n = 3).

 
Binding to the TRE in COX Vb promoter and COX Vb mRNA expression. Heart tissue had significantly greater constitutive binding to the TRE compared with liver (P < 0.001; Fig. 2B). In response to 5 days of T3 treatment, binding to the TRE in liver was increased 1.3-fold (P < 0.01). However, there was no effect of T3 on TRE binding in heart. The specificity of binding was demonstrated by 1) the strong inhibition of binding with the addition of a 100-fold excess of unlabeled probe (Fig. 2A, lane 2), and 2) the ~50% interference in TRE binding observed when liver extracts were preincubated with TR{alpha}1 or TR{beta}1 antibodies (results not shown). Thus binding to the TRE within the authentic COX Vb promoter is most likely binding by nuclear TRs, and it appears to be regulated by T3 in a tissue-specific manner.



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  		Fig. 2. Effect of T3 on thyroid response element (TRE) binding within the cytochrome c oxidase (COX) Vb promoter and on COX Vb mRNA. A: tissues from V- and T3-treated animals were excised, and cell extracts were prepared. Electromobility shift assays (EMSAs) were performed using an oligonucleotide representing the TRE in the COX Vb promoter. Free probe (FP) and preincubation with 100-fold molar excess of nonlabeled oligonucleotide (C) are shown along with tissue samples. Specificity of binding was demonstrated by 1) strong inhibition of binding in liver extracts with the addition of a 100-fold excess of unlabeled probe (lane 2), and 2) the ~50% interference in TRE binding observed when liver extracts were preincubated with TR{alpha}1 or TR{beta}1 antibodies (results not shown). The second band, apparent only with liver extracts from T3- and V-treated animals, was included in the quantification of the liver samples. B: quantification of TR/TRE-binding experiments (n = 5). *P < 0.05 vs. V-treated animals; {ddagger}P < 0.001 vs. V-treated liver samples. C: representative Northern blot and graphic analysis of COX Vb mRNA from tissues of V- and T3-treated animals (n = 3–4). *P < 0.05 vs. V-treated animals; {ddagger}P < 0.001 vs. V-treated liver samples.

 
To determine whether the changes observed in TRE binding within the COX Vb promoter resulted in concomitant alterations in mRNA expression, Northern blots were performed (Fig. 2C). Constitutive levels of COX Vb mRNA approximately paralleled differences in TRE binding. In response to T3, the twofold increase in mRNA observed in liver (P < 0.01) was matched by the T3-induced increase in TRE binding. No changes in binding or COX Vb mRNA were observed in heart. Thus tissue-specific differences in transcriptional and posttranscriptional mechanisms exist that regulate COX Vb mRNA.

Effect of T3 on protein expression of nuclear and mitochondrial subunits of the COX enzyme. We investigated the protein expression of several subunits of the COX enzyme that represent transcripts from the nuclear and mitochondrial genomes. Mitochondrially encoded COX subunit I (COX I) protein expression was increased 1.7- (P < 0.01) and 1.5-fold (P < 0.05) in liver and heart, respectively, in response to T3 treatment (Fig. 3A). We also investigated the protein expression of two nuclear-encoded subunits, COX Vb, and COX VIc (Fig. 3, B and C). T3 treatment resulted in an increase in the protein expression of COX Vb in liver (P < 0.05; Fig. 3B), a finding that paralleled the T3-induced increase in COX Vb mRNA. Unlike COX Vb, COX VIc protein did not change in response to T3 treatment. Furthermore, although the expression of COX Vb and COX VIc was unchanged with T3 treatment in heart, the constitutive expression of these subunits was significantly greater than levels found in liver (P < 0.001; Fig. 3, B and C).



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  		Fig. 3. Effect of T3 on protein levels of nuclear- and mitochondrially encoded subunits of the COX holoenzyme and COX enzyme activity. Representative immunoblots and graphic quantification of the results are shown. A: COX I protein expression (n = 3–4). *P < 0.05 vs. V-treated animals. B: COX Vb protein expression (n = 4–7). *P < 0.05 vs. V-treated animals; {ddagger}P < 0.0001 vs. V-treated liver samples. C: COX VIc protein expression (n = 3–5). {ddagger}P < 0.001 vs. V-treated liver samples. D: COX enzyme activity, used as an indicator of mitochondrial biogenesis (n = 6–8). *P < 0.05 vs. V-treated animals; {ddagger}P < 0.001 vs. V-treated liver samples.

 
We also assessed the effect of T3 on the activity of the COX holoenzyme (Fig. 3D). COX activity was increased in response to T3 ~1.7-fold in liver (P < 0.001) and 1.2-fold in heart(P < 0.05). This increase observed in heart in response to T3 occurred in parallel with COX I subunit changes, despite a lack of increase in the levels of COX Vb or COX VIc subunits.

Effect of T3 on DR2 binding within mtDNA. Transcriptional regulation of mtDNA in response to T3 involves the matrix-localized T3 receptor termed p43. This protein binds to four putative TRE sequences within the mitochondrial genome (4, 47). One such TRE is located within the D-loop of mtDNA, the site of transcriptional initiation called the DR2. In response to T3, the binding affinity to this sequence was unaltered in both liver and heart (Fig. 4, A and B). In addition, there was no difference in DR2 binding between heart and liver.



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  		Fig. 4. Effect of T3 on DR2 binding. A: Tissues from V- and T3-treated animals were excised, and cell extracts were prepared. EMSAs were performed using an oligonucleotide representing the DR2 sequence within the D-loop of mitochondrial (mt)DNA. FP and preincubation with 100-fold molar excess of nonlabeled oligonucleotide (C) are shown along with tissue samples. B: Summary of DR2 binding (n = 5–8).

 
COX III mRNA and Tfam protein expression. We compared our findings of DR2 binding with potential effects on the levels of mtDNA transcript levels such as COX III. T3 increased transcript levels of COX III in liver (P < 0.05; Fig. 5A). In contrast, COX III mRNA expression in heart was decreased by 26% in response to T3 treatment (P < 0.01). These changes occurred despite unaltered DR2 binding but paralleled alterations in Tfam expression. Similar to p43, Tfam can bind mtDNA and regulate transcription of mitochondrially encoded genes. A 1.3-fold increase in Tfam expression was observed in liver (P < 0.05; Fig. 5B), whereas the heart displayed a 30% decrease in this transcription factor (P < 0.05) in response to T3.



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  		Fig. 5. Effect of T3 on COX III mRNA and mitochondrial transcription factor A (Tfam) protein expression. A: quantification of COX III mRNA (n = 4–6). Representative autoradiogram of COX III mRNA experiments is also shown. *P < 0.05 vs. V-treated animals; {ddagger}P < 0.001 vs. V-treated liver samples. B: Protein extracts (50–75 µg) were subjected to immunoblotting using an antibody directed toward Tfam. Representative Western blots and graphic representation of the effect of T3 treatment on Tfam expression are shown (n = 5–6). *P < 0.05 vs. V-treated animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The regulation of mitochondrial activity by T3 is well documented. The best known action of T3 is its effect on cellular respiration and energy metabolism, the magnitude of which is most pronounced in hypothyroid individuals. Animals treated with T3 exhibit increases in metabolic rate (15, 26), and patients with hyper- and hypothyroidism exhibit high and low metabolic rates, respectively (3). The cellular adaptations to increased T3 levels are partly mediated by alterations in the expression of components of the mitochondrial respiratory chain, a principal component of which is COX. COX is composed of 13 subunits, 10 that are nuclear encoded and 3 that are derived from mtDNA (19). Thus COX represents an excellent model for the study of holoenzyme assembly and mitochondrial biogenesis in response to physiological stimuli. Previous studies have indicated that the expression of nuclear and mitochondrial COX subunits is coordinated among tissues possessing a wide range of COX enzyme activities (12, 17). However, the current evidence suggests that this may not be the case in response to T3 at the mRNA level (40, 44). Therefore, we evaluated the mechanisms involved in T3-induced COX subunit expression at the level of transcription, as well as at the level of mRNA and protein expression, with the aim of further understanding the mechanisms of COX assembly.

TRs. T3 exerts its biological effects within the nucleus primarily via binding to nuclear TRs. Thus we first examined whether T3 could influence the expression of its own nuclear receptors at the protein level and whether this effect was tissue specific. It has been reported that the mRNA expression of the TR{beta}1 isoform is inducible during conditions of elevated thyroid status (16). Our analyses support this finding at the protein level, as we observed a 60% induction of TR{beta}1 in liver with T3 treatment. Recent studies have reported decreases or no effect of T3 on TR{alpha}1 mRNA expression (16). Again, our results are consistent with these findings at the protein level, as TR{alpha}1 protein expression was unchanged with T3 treatment, suggesting that the hormone does not regulate the expression of this TR isoform. Because these isoforms can typically bind DNA as homodimers, or as heterodimers with retinoid X receptors, this altered stoichiometry of TRs in response to T3 in liver suggests that TR{beta} could become a more dominant mediator of T3 action on target genes in this tissue.

Nuclear effects. The widespread effect of T3 on nuclear gene expression is evident from the observed increases in a variety of mRNAs encoding nuclear-encoded respiratory genes (22, 27, 42). To determine whether genes encoding COX subunits are regulated by T3 in this manner, we investigated the expression of nuclear-encoded COX subunits Vb and VIc. The COX Vb promoter contains one TRE half-site at position –183 in the 5'-flanking region (2), whereas the COX VIc promoter possesses sites at –217 and +76 that are similar to the TRE (44); thus these genes may be transcriptionally regulated by T3. In liver, a concomitant increase in COX Vb DNA binding and mRNA expression was observed, providing direct evidence for this possibility. This was likely facilitated by the T3-induced increase in TR{beta}1 protein expression, which could account for the increase in binding to the TRE in this tissue.

Analysis of a wide spectrum of nuclear genes that code for mitochondrial proteins has resulted in the identification of some common transcription factors that bind DNA sequences within their promoter regions (35). One of these is nuclear respiratory factor-1 (NRF-1), which binds to the promoters of several nuclear genes encoding COX subunits, including COX Vb and COX VIc (10). Because NRF-1 mRNA expression is increased in response to T3 treatment (43), it may provide a potential link between T3 stimulation and the selective upregulation of respiratory genes. However, NRF-1 sites do not appear to be universally present within COX regulatory regions (25). Thus there must be additional proteins that participate in the upregulation of nuclear genes encoding mitochondrial proteins in response to T3. The most noteworthy of these is peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}), a nuclear localized coactivator that has the ability to increase the transcriptional activity of TR{beta}1 (32) as well as that of NRF-1 (24, 43, 49). We have recently demonstrated that T3 treatment in vivo results in a significant increase in PGC-1{alpha} protein expression in liver, but not in heart (21). Thus PGC-1{alpha} is likely important in regulating NRF-1 and TR{beta}1 transcriptional activation during conditions of T3-induced mitochondrial biogenesis in liver. However, the mechanisms involved in producing an increase in COX holoenzyme activity in heart remains unresolved, given the lack of effect of T3 on PGC-1{alpha} (21), Tfam, and TR{alpha} and TR{beta} (present study).

Mitochondrial effects. Until recently, the nucleus was considered to be the primary target for T3-induced alterations in gene expression. However, the evidence of high-affinity binding sites for a T3 receptor within mitochondria has provided a possible explanation for the direct action of this hormone on the organelle (48). This mitochondrial TR is derived from the use of an internal start codon of the c-Erb A{alpha}1 mRNA, which encodes the full-length TR{alpha}1, as well as the truncated mitochondrial TR termed p43. In our experiments, a T3-induced increase in COX III mRNA was observed in liver, and this occurred despite a lack of increase in mtDNA binding at the DR2 sequence, a location for p43 binding. Thus, although p43 binding to mtDNA is enhanced in the presence of T3 in in vitro experiments (4), the expression of p43 does not seem to be influenced by 5 days of hormone treatment. Therefore, the increases in COX III mRNA and COX I protein in response to T3 are likely mediated by alternative transcription factors that regulate the expression of mitochondrially encoded respiratory genes. Tfam is a probable candidate. Tfam is a nuclear-encoded protein that binds mtDNA within the D-loop to regulate its transcription and replication (5). NRF-1 sites are present in the Tfam promoter (41), and therefore the established induction of both NRF-1 (43) and PGC-1{alpha} (21) by T3 can lead to an increase in Tfam expression. In support of this, Garstka et al. (13) have shown that T3 treatment augments Tfam mRNA in liver. Our results are consistent with the suggestion that Tfam is the most important transcription factor mediating changes in the expression of COX subunits, since we observed an increase in the expression of Tfam in liver.

COX holoenzyme activity. T3-induced changes in COX Vb, COX VIc, and COX I protein expression closely paralleled each other. Thus T3 induced a coordinated increase in the expression of these COX subunits in liver. This coordination between the expression of proteins encoded by both the nuclear and mitochondrial genomes differs somewhat from findings previously reported at the mRNA level (40, 44). This is not unusual given the different turnover rates of mRNA and protein and the knowledge of multiple posttranscriptional mechanisms that regulate the concentration of the final protein product.

Both liver and heart responded to T3 with an enhanced expression of the COX I subunit, and this increase most closely reflected the change in COX enzyme activity produced by the hormone. The three mitochondrially encoded subunits are vital for the catalytic function of COX, since they contain the copper and heme metal centers that are responsible for the final reduction of oxygen to water (1). Therefore, unlike the nuclear-encoded subunits, which may be responsible for modulating the respiratory rate, the expression of the mitochondrially encoded subunits is more likely expected to parallel COX enzyme activity. This is consistent with suggestions that COX I may regulate assembly of the holoenzyme complex and that the nuclear-encoded subunits are not limiting for this assembly (28).

Heart. Although heart displayed increased COX activity with T3 treatment, it remained the least responsive to T3 with respect to changes in the expression of COX subunits. It is noteworthy that the constitutive expression of all of the COX subunits, at both the protein and the mRNA levels, was markedly higher in heart than in liver after normalization for loading. This is likely related to the relatively unresponsive character of this tissue to T3 treatment despite a high abundance of TR{alpha} and TR{beta} isoforms. This finding is in contrast to the idea that the responsiveness of tissues to T3 is simply mediated by differences in the distribution of TRs (37). The lack of response to T3 in heart may be related to a saturation of TR-TRE binding in nuclear DNA, since a high constitutive degree of binding was observed. In addition, the lack of response in heart suggests a role for TR-interacting proteins, such as corepressors, that could participate in a cellular feedback mechanism limiting the further induction of gene transcription in tissues in which constitutive expression is already very high. Thus future work in this area could focus on the involvement of corepressor/TR interactions in limiting further gene transcription in this tissue.

In summary, our data indicate that T3 induces a coordinate increase in the expression of nuclear and mitochondrial COX gene products at the protein level, leading to functional increases in COX enzyme activity. These changes are tissue specific (i.e., evident in liver but not in heart) and involve both transcriptional and posttranscriptional mechanisms.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Canadian Institutes of Heath Research. D. A. Hood is the holder of a Canada Research Chair in Cell Physiology.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. N. G. Avadhani (University of Pennsylvania, Philadelphia, PA) for providing the COX Vb cDNA and to Dr. H. Inagaki (National Industrial Research Institute of Nagoya, Nagoya, Japan) for the provision of the rat Tfam antibody. The technical assistance of Sari Herman for help with the COX Vb mRNA analysis is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. Hood, Dept. of Biology, York Univ., Toronto, ON M3J 1P3, Canada (E-mail: dhood{at}yorku.ca).

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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