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Comparison of the Effects of Thyroxine and Triiodothyronine on Protein Turnover and Apoptosis in Primary Chick Muscle Cell Cultures
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
251, 442– 448 (1998)
RC989483
Comparison of the Effects of Thyroxine and Triiodothyronine on Protein Turnover and Apoptosis in Primary Chick Muscle Cell Cultures Kazuki Nakashima, Akira Ohtsuka, and Kunioki Hayashi1 Department of Biochemical Science and Technology, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890, Japan
Received August 29, 1998
Primary chick muscle cells were treated with physiological level of thyroxine (T4) or triiodothyronine (T3) to examine the effects of the hormones on growth, protein turnover, and apoptosis of the cells. Creatine kinase activity, as an index of differentiation, was increased by both T4 and T3. Even when the conversion from T4 to T3 was blocked by iopanoic acid, T4 increased creatine kinase activity. The rate of protein degradation estimated from [3H] tyrosine release was increased by T3 but not by T4. DNA cleavage and fragmentation, as indices of apoptosis, were induced by T3 but not by T4. These results show that T4 stimulates cell differentiation but not protein degradation and apoptosis in primary chick muscle cells, while all events are stimulated by T3. © 1998 Academic Press
It is well known that thyroid hormones play important roles in growth, development, cellular differentiation and metabolism of tissues. The most important effects of thyroid hormone are on basal metabolic rate and protein synthesis and breakdown to maintain homeostasis. As a part of this homeostatic role and the necessity for a response to demand, it is teleologically plausible that thyroid hormones strongly affect skeletal muscle protein metabolism. It is generally thought that pharmacological dose of thyroid hormone produces a catabolic response in skeletal muscle although the effects and modes of action are still largely unknown. Either thyroxine (T4) or triiodothyronine (T3) administered at a replacement dose in hypophysectomized animals stimulates both rates of protein synthesis and, to a lesser extent, protein breakdown; it causes further stimulation of protein breakdown at a high level (1, 2). 1 To whom correspondence should be addressed. Fax: 181-99-2858652. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Recent advances in understanding of the mechanism of hormone action at the cellular level have obtained through the employment of myoblast primary cultures which retain many muscle functions and provide a useful experimental model for the study on hormonal regulation. The present cultured muscle cell consists of mononucleated cells that undergo a period’s proliferation, followed by fusion, to form postmitotic multinucleated myotubes. This differentiation process is accompanied by an increase in the level of muscle specific proteins such as creatine kinase, myosin, and actin (3). In contrast to whole animals, where general anabolic thyroid hormone activity is well established, results from in vitro experiments with cultured myoblasts are controversial. No changes in the growth related processes of cultured myoblasts were reported after thyroid hormone treatment (4). On the other hand, stimulation of myotube formation but not cell proliferation of chick embryo myoblasts was observed after treatment with T4 (5) while physiological amounts of T3 reduces the proliferation of myoblasts (6). There is general agreement that T3 is responsible for the most physiological actions of thyroid hormones in mammals (7), although T4 is normally the main secretary product of the thyroid and the extracellular pool of T4 is much greater than that of T3. Most T3 derives through its conversion from T4 in peripheral tissues (7–10). The existence of iodothyronine 59-deiodinase, which catalyzes the monodeiodination of prohormone T4 to produce T3 has been reported in the thyroid gland (11) as well as in the liver and kidney (12, 13). However, it might be an error to assume that T4 is the prohormone of T3 in various tissues, especially in skeletal muscle because little deiodination of T4 occurs in the muscle (14, 15). Therefore, in the present investigation, two experiments were conducted to show the direct effect of T4 on growth and protein turnover in primary chick muscle cell cultures.
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Furthermore, in the present study, apoptotic effect of T3 was clarified because it was shown in the preceding experiment that cell proliferation was inhibited by T3 but not by T4. Apoptosis has been recognized as a distinct form of cell death that has an essential function in the regulation of cell turnover during development, tissue homeostasis, and cancer. Apoptosis entails characteristic morphological and biochemical changes such as cell shrinkage, chromatin condensation, cell fragmentation called apoptotic body and chromosome DNA cleavage at internecleosomal sites (16, 17). Apoptosis is believed to play an important role in embryonic development. However, the molecular mechanism of apoptosis remains unknown. Recent studies have further indicated that apoptosis may be tightly associated with cell cycle regulation in proliferating cells and may represent a form of “abortive mitosis” in certain cell system (18). Thus, cell death, along with cell cycle progression and differentiation, plays a critical role in tissue homeostasis. Therefore, in the present study, the effects of T4 and T3 on apoptosis in primary cultured muscle cells were also investigated. MATERIALS AND METHODS Cell culture. Myoblasts were isolated from thigh muscle of 13day-old chick embryos. The muscle tissue obtained from the embryo was digested with dispase (2000 U/ml) for 10 min at 37°C. The cell suspension was then passed through a net and centrifuged 700 rpm for 3 min. The supernatant was aspirated and the cell pellet was dispersed into 3 ml basal growth medium (M-199 containing 15% calf serum and 2.5% chicken embryo extract). The cell suspension was transferred to a 35-mm uncoating culture dish to allow fibroblast attachment. After 40 min, unattached cells were recovered and transferred to a next dish. This protocol was repeated three times, and cell number was counted and plated onto gelatin-coated 6-wells plates at a density 2.5 3 105 cells/well. The cells were grown at 37°C in a 5% CO2-enriched atmosphere of humidified air. After 24 h, the medium was replaced by the media containing physiological levels of thyroxine sodium salt (15, 30, 60 ng/ml) or 3,39,5-triiodothyronine sodium salt (3, 6, 12 ng/ml). Thyroid hormones were dissolved in ethanol and added in the medium (The resulted ethanol concentration in the culture medium was 0.1%). The media were replaced every other days for 6 day incubation periods. The medium was collected after the final incubation and stored at 220°C until analyzed for Nt-methylhistidine. In experiments 3 and 4, cell culture was conducted similarly as experiment 1 except thyroxine sodium salt or triiodothyronine sodium salt were added at the levels of 60 and 12 ng/ml, respectively and iopanoic acid (IOP) were used to inhibit 59-deiodinase. IOP was added to the medium at a level of 1.75 mM according to the report of Galton et al. (19). Measurement of thyroxine and triiodothyronine. After the medium was collected, the cell monolayer was washed three times with ice-cold PBS and the cells were detached by scraping with a rubber policeman using 500 ml phosphate buffer (pH 7.4) containing EDTA (1 mM) and dithiothreitol (20 mM). The cells were then homogenized by a micro homogenizer and centrifuged at 2500 rpm for 10 min at 4°C. The supernatants were rapidly frozen and stored at 280°C until analysis. T4 and T3 were measured using commercial kits, EnzymunTest-T4, (Boehringer-Mannheim, Mannheim) and T3 Enzymun-Test
T3 (Elsia-Auto T3, International Reagents Corporation, Kobe, Japan). Creatine kinase (CK) activity assay. After the medium was collected, the cell monolayer was washed three times with ice-cold PBS and the cells were detached by scraping with a rubber policeman using 2 ml Tris buffer (pH 6.8). The cells were then homogenized by a micro homogenizer and centrifuged at 2500 rpm for 10 min at 4°C. The supernatants were rapidly frozen and stored at 280°C until analysis. CK activity was analyzed by the method of Rosalki (20). ATP formed by the action of the enzyme on ADP and CP is linked to the reduction of nicotinamide-adenine dinucleotide phosphate (NADP) with glucose, hexokinase, and glucose-6-phosphate dehydrogenase. The increase in optical density at 340 nm which depends upon NADP reduction is followed by spectrophotometry and provided a measure of CK activity. One enzyme unit is the amount of CK that catalyses formation of 1 mmol/min at 30°C, pH 6.8. DNA analysis. DNA content was determined according to the modified method described by Erwin et al. (21). Cell homogenate was treated with 10% TCA, and the precipitate was removed by centrifugation and suspended by vortexing in 500 ml 5% TCA, followed by the hydrolysis at 90°C for 30 min. The samples were then cooled to 4°C and centrifuged at 3000 rpm, 4°C for 15 min. A 300 ml portion of the supernatant was added to 100 ml diaminobenzoic acid dihydrochloride (DABA 0.2 g/ml) and kept at 60°C for 1 h. Two milliliters of 1 N HCl was added to terminate the reaction and DNA was quantified by a fluorescence spectrophotometer (Hitachi F-200, Hitachi Electric Ltd., Japan) using excitation wavelength of 415 nm and emission at wavelength of 520 nm. Nt-Methylhistidine analysis. Nt-Methylhistidine was analyzed by a modification of the method of Hayashi et al. (22). The media were hydrolyzed with 12 N HCl (final concentration 6 N) at 110°C for 20 h. After the hydrolysate was cooled and passed through filter paper, the hydrochloric acid was removed subsequently by evaporation. The residue was dissolved in 5 ml of 0.2 M pyridine; 4.5 ml of this was applied to an anion-exchange resin column (7 3 60 mm, Dowex 50 x 8, 200 to 400 mesh, pyridine form). After eluting most of the acidic and neutral amino acids with 20 ml of 0.2 M pyridine, Nt-methylhistidine was eluted with 20 ml of 1 M pyridine and collected. The eluent was then dried, and the residue was dissolved in 1 ml of mobile phase (15 mM sodium octane sulfonate in 20 mM KH2PO4). Fifty ml of this was used for HPLC analysis. The HPLC system incorporated the reverse-phase separation with ion-pairing, using Shim-pack ODS column (6.0 3 150 mm) and the post-column fluorescence derivatization with orthophthalaldehyde. Measurement of protein degradation rate. Preparation of muscle cells for measurement of long-lived protein degradation involved prelabeling protein by incubation in complete medium containing T4 or T3 and 1 mCi of [3H] tyrosine/ml for 2 days. After radiolabeling, cells were rinsed and placed in nonradioactive chase medium for 2 h to allow degradation of very-short-lived proteins. The cells were subsequently incubated in isotope free-medium supplemented with excess unlabeled 2 mM tyrosine. Excess unlabeled amino acid was included in the medium to prevent radiolabeled amino acid from being reutilized. The muscle cells were harvested at 6 h after incubation. Degradation of short lived proteins was determined after labeling with 5 mCi of [3H] tyrosine for 1 h. Cells were rinsed, chased for 10 min, and placed in isotope free-medium supplemented with excess unlabeled 2 mM tyrosine. The muscle cells were harvested after 1 h incubation. At the end of experiment, culture medium was transferred to a microcentrifuge tube containing 100 ml of BSA (10 mg/ml) and TCA was added to a final concentration of 10% (w/v). After incubation at 4°C for at least 1 h, sample were centrifuged for 5 min. The precipitates were then dissolved with tissue solubilizer. The cell
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monolayers were washed with ice-cold phosphate buffered saline and solubilized 0.5 M NaOH containing 0.1% Triton X-100. Radioactivities in the cell monolayer and TCA soluble and insoluble fractions were measured using scintillation counter. Rates of protein degradation were expressed as the percentage degraded per day. Measurement of growth rates (Kg), protein degradation rates (Kd), and protein synthesis rates (Ks). Growth rates (Kg) were measured as the net accumulation of protein in muscle cells between 1 day and 7 day of the experiment. Proteins were measured by Lowry method by using bovine serum albumin as a standard (23). Growth rates were then expressed as the percentage increment of the protein content per day. Because growth arises from the difference between in the rates of degradation (Kd) and synthesis, protein synthesis (Ks) rate was calculated from the two measured parameters, i.e., Ks 5 Kg 1 Kd. DNA extraction and electrophoresis. Myoblasts were harvested and lysed in 50 mM Tris–HCl, pH 7.8 containing 0.5% sodium lauroyl sarcosinate and 10 mM EDTA. The samples were treated with RNase A (10 mg/ml) at 50°C for 30 min, then with proteinase K (10 mg/ml) at 50°C for 60 min. DNA fragments were separated on a 1.5% agarose gel and visualized with ethidium bromide. Measurement of DNA fragmentation. After incubation, myoblasts were harvested and lysed in 0.5% SDS containing 25 mM EDTA and 75 mM NaCl for over 3 h on ice. The fragmented DNA and intact DNA were separated by centrifugation at 100,000g for 30 min at 0°C. The rotor was cooled to 0°C before use to remove SDS completely. The pellet was dissolved in the same amount of the solution containing 25 mM EDTA and 75 mM NaCl. DNA in the lysate and that in the pellet solution were again precipitated by cold ethanol treatment and then dissolved in 10 mM Tris–HCl (pH 7.4) containing 1 mM EDTA. The DNA content was measured by a fluorometric methods using DABA. Statistical analysis. Data were analyzed by analysis of variance (ANOVA), and means were further tested by Duncan’s multiple range test. A p value ,0.05 was considered to be statistically significant. Each result is the mean 6 standard deviation of the values obtained from six replicates.
RESULTS Experiment 1. Concentrations of T4 and T3 in the medium were increased dose-dependently by additions of T4 and T3, respectively. T3 concentration was not increased by T4 supplementation, indicating that little deiodination of T4 was occurred (Figs. 1A and 1B). DNA content was significantly (p , 0.05) decreased when T3 was added at the levels of 6 and 12 ng/ml. However, T4 did not affect DNA content (Figs. 1A–1C). This indicates that T3 but not T4 inhibits cell proliferation. CK activity was significantly (p , 0.05) increased by T4 at the levels of 30 and 60 ng/ml and by T3 at all the levels tested (Figs. 1A–1D). These results indicate that both T4 and T3 induce differentiation. Nt-Methylhistidine release was significantly (p , 0.05) increased by T3 when the concentrations were 6 and 12 ng/ml. However, T4 did not affect Nt-methylhistidine release (Figs. 1A–1E). These results show that T3 but not T4 stimulates muscle protein breakdown. Experiment 2. The results are summarized in Table 1. There were little difference of intracellular concentration of T3 between control and T4 group, and IOP did
not influence the T3 concentration. In the T3 group, intracellular T3 concentration was about 4- fold higher than those of the control. DNA content of the cell was significantly (p , 0.05) decreased by T3 but not by T4. IOP did not affect the DNA content. This indicate that T3 but not T4 inhibits cell proliferation. CK activity was significantly (p , 0.05) increased by both T4 and T3 despite the addition of IOP. These results indicate that both T4 and T3 stimulate differentiation in cultured chick muscle cells. Nt-Methylhistidine release was increased by T3 but not by T4 and there was no influence of IOP, showing that T3 but not T4 induces myofibrillar proteolysis. Experiment 3. The effects of T4 and T3 on the turnover rates of short and long lived protein estimated by [3H] tyrosine release are shown in Table 2. Both Rates of short and long lived protein degradation were significantly (p , 0.05) increased by T3, but not by T4. Rate of muscle protein synthesis (Ks) calculated as a sum of the rates of protein accumulation (Kg) and breakdown (Kd) was increased by both T4 and T3. These results strongly indicate that both T4 and T3 are active in stimulating protein synthesis, while only T3 is active in stimulating proteolysis. Experiment 4. As it was shown in the previous experiments that cell proliferation was inhibited by T3 but not by T4, the effects of T4 and T3 on apoptosis were observed using DNA cleavage and fragmentation as indices of apoptosis (Fig. 2). DNA cleavage occurred in T3 group after 2 days of incubation, but not in T4 group. On Day 4 and 6, DNA cleavage was not observed. DNA fragmentation (% of total DNA) was also significantly increased by T3, but not by T4 on day 2 (control, 5.09 6 1.52; T4, 5.82 6 1.93; T3, 17.88 6 3.44) and day 4 (control, 4.94 6 0.70; T4, 5.36 6 1.02; T3, 7.87 6 1.48). However, on day 6, DNA fragmentation was not observed because myotube formation completed. It has been reported that differentiated myotubes are resistant to cell death (24). These results show that T3 but not T4 induces apoptosis of chick myoblasts. DISCUSSION The present experiments were conducted to compare the effects of T4 and T3 on growth and protein turnover in primary cultured muscle cells because we have previously observed in chicks that skeletal muscle protein synthesis is accelerated by exogenous T4, while plasma T3 concentration is not changed(unpublished data). It is well known that T4 is converted to the biologically potent T3 in many tissues by 59-deiodinase. However, activity of 59-deiodinase in muscle is much lower than those of liver and kidney (14) and little or no monodeiodination of T4 or T3 occurs in skeletal muscle (15).
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FIG. 1. Concentration of thyroxine (T4, A) and triiodothyronine (T3, B) in the medium at the final day of incubation and effects of T4 and T3 on DNA contents (C), Creatine kinase activity (D) and Nt-methylhistidine release (E) in cultured chick muscle cells. T4 was added at the levels of 15, 30, or 60 ng/ml in the medium. T3 was added at the levels of 3, 6 or 12 ng/ml in the medium. Values not sharing a common superscript are significantly different (p , 0.05). Values given are means 6 SD.
Thus, it is plausible that T4 stimulates muscle growth without conversion to T3. Two enzymes, type I and type II 59-deiodinase catalyze deiodination of T4. Both type I (25) and type II (26) deiodinases are present but small amount in skeletal muscle. Iopanoic acid (IOP) is a specific inhibitor of these enzymes (11, 27–30) and thus used in the present experiment. Both extracellular and intracellular concentrations of T3 were not increased in both T4 group and T4 plus IOP group. However, creatine kinase activity was increased by T4 even when IOP was included in the medium. This shows that conversion of T4 to T3 is not necessary to stimulate the cell differentiation.
The cultured muscle cell undergoes a period’s proliferation followed by fusion to form postmitotic multinucleated myotubes. This differentiation process accompanies with an increase in the level of muscle specific proteins such as creatine kinase, myosin, and actin (3). Since most creatine kinase activity is derived from myotube, the activity is often used as an index of myotube growth (31). Increases in creatine kinase activities have been shown to accompany with differentiation of myogenic cells in the muscle cell culture systems (32). On the other hand, DNA content has been measured as an index of proliferation of cultured muscle
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Effects of Thyroxine (T4) or Triiodothyronine (T3) with or without Iopanoic Acid (IOP) on Intracellular T3 Concentration, DNA Content, Creatine Kinase (CK) Activity, and Nt-Methylhistidine Release in Cultured Chick Muscle Cells Parameter
Control
IOP
Intracellular T3 concentration (pmol/mg protein) DNA content (mg/well) Creatine kinase activity (IU/mg protein) Nt-Methylhistidine release (nmol/h/mg protein)
148 6 20 12.5 6 1.1a 1.67 6 0.22c 0.38 6 0.05c
140 6 23 13.6 6 1.3a 1.79 6 0.24c 0.44 6 0.08c
b
T4 b
157 6 22 12.5 6 0.7a 2.55 6 0.20b 0.42 6 0.03c b
T4 1 IOP
T3
145 6 10 14.7 6 0.6a 2.62 6 0.20b 0.47 6 0.08c
421 6 56a 8.08 6 0.6b 2.42 6 0.41b 0.91 6 0.16b
b
Note. T4, T3, and IOP were added at 60 ng/ml, 12 ng/ml, and 1.75 mM in the medium, respectively. Values not sharing a common superscript are significantly different (p , 0.05). Values given are means 6 SD.
cells (21).The present results of DNA content show that cell proliferation was inhibited by T3 but not by T4. This finding is consistent with the results of Kumegawa et al. (5)indicating that T4 stimulates myotube formation (differentiation) but not cell proliferation of chick embryo myoblasts. On the other hand, Marchal et al. (6) has reported that physiological amounts of T3 reduces the proliferation of myoblasts while T3 accelerates their fusion rate and thus the appearance of myosin heavy chain. This is consistent with our results. In the present study, DNA fragmentation and cleavage were increased by T3 showing that T3 but not T4 stimulated apoptosis. Recently, Wang et al. (24) have reported that proliferating C2C12 myoblasts can undergo either differentiation or programmed cell death under conditions of mitogen deprivation, and they concluded that a portion of myoblasts undergo apoptosis during in vitro myogenesis whereas other continue differentiation and form myotubes. Yaoita et al. (33) have also reported that T3 induces apoptosis in a myoblast prepared from tadpole tail. However, there is no paper reporting that thyroid hormone induces apoptosis in avian or mammalian myoblasts. Marchal et al. (6) have reported that T3 inhibits DNA synthesis and stimulates differentiation in quail myo-
blasts. This finding suggests that T3 may play a role in the induction of apoptosis. Indeed, our study show that myoblasts become committed suicide by apoptosis by T3 treatment whereas a small proportion of cells can survive and undergo myogenic differentiation at a later stage. Since both apoptosis and differentiation are terminal cellular process, concurrent activation of the two processes can result in adverse depletion of mitotic cell populations. It is relevant to mention that cell cycle arrest is normally required for muscle cell differentiation (34). It is likely that growth factors such as insulin and insulin-like growth factors may exert their myogenic effect by suppressing the expression of apoptotic genes. T3 may stimulate expression of apoptotic gene. The balance between apoptosis and differentiation may play a crucial role in controlling the number of cells capable in maintaining muscle growth and regeneration capacity. Nt-Methylhistidine release was increased by T3, but not by T4. Rates of breakdown of short and long lived proteins measured by tyrosine release were consistent with the result of Nt-methylhistidine release. Thus, it is clear that T3 but not T4 increases protein breakdown. Rate of protein synthesis calculated as the sum of the rates of growth and breakdown of long lived protein was increased by both T4 and T3 as expected.
TABLE 2
Effects of Thyroxine (T4) and Triiodothyronine (T3) on Rates of Short- and Long-Lived Protein Degradation, Growth (Kg), and Synthesis (Ks) of Long-Lived Protein in Cultured Chick Muscle Cells (%/day) Parameter Rate Rate Rate Rate
of of of of
short-lived protein degradation long-lived protein degradation growth (Kg) protein synthesis (Ks)
Control
T4
T3
231.8 6 9.68b 29.40 6 1.93b 15.42 6 0.22b 44.82 6 1.97c
218.6 6 10.5b 31.75 6 0.96b 22.84 6 0.42a 54.68 6 1.11a
249.6 6 6.48a 34.11 6 0.88a 15.39 6 0.10b 49.50 6 0.91b
Note. T4 and T3 were added at 60 and 12 ng/ml in the medium, respectively. Kd was calculated from long-lived protein degradation. Growth rate was expressed as the percentage increment of the protein per day. Protein was measured by Lowry method. Ks was calculated from the following equation: Ks 5 Kg 1 Kd. Values not sharing a common superscript are significantly different (p , 0.05). Values given are means 6 SD.
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REFERENCES
FIG. 2. Effects of thyroxine (T4) and triiodothyronine (T3) on DNA cleavage in chick myoblasts. Chick myoblasts were incubated for 2, 4, and 6 days with T4 or T3. T4 and T3 were added to the medium at the levels of 60 and 12 ng/ml, respectively. DNA was extracted from myoblasts and separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. l-DNA/HindIII was used as a maker.
During fusion and differentiation, protease activity and proteolysis increase (35, 36). Apoptosis pathway is thought to be correlated with the activation of a proteolytic cascade mediated by proteases such as caspases(37), lysosomal proteases (38), calpain (39), and proteasome (40, 41). These observations suggest that enhanced proteases activity play a key mechanistic role in initiating apoptosis. Thus, proteolytic systems may be associated with differentiation or apoptosis at a later stage of myogenesis. We have observed that T3 stimulates non-lysosomal protease and lysosomal protease such as calpain, proteasome and cathepsin D (unpublished data). It is known that nonlysosomal proteolysis is stimulated during apoptosis and translocation of cathepsin from the cytoplasm to the nucleus occurs during apoptosis of hepatocytes (38, 42). Degradation of nuclear structural proteins such as lamins and histones occurs in apoptosis (43). T3 may induce nuclear structural proteins degradation and promote nuclear disruption and DNA fragmentation leading to apoptosis. In conclusion, the present study shows that T4 stimulates protein synthesis and differentiation but not protein degradation, while T3 stimulates protein synthesis, protein degradation, cell differentiation and apoptosis and inhibits cell proliferation. ACKNOWLEDGMENTS The authors thank Dr. A. L. Goldberg for helpful comments and suggestions on this study. This study was supported by a Grant-inAid for Scientific Research (10660273) from the Ministry of Education, Science, Sports, and Culture, Japan.
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Comparison of the Effects of Thyroxine and Triiodothyronine on Protein Turnover and Apoptosis in Primary Chick Muscle Cell Cultures Kazuki Nakashima, Akira Ohtsuka, and Kunioki Hayashi1 Department of Biochemical Science and Technology, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890, Japan
Received August 29, 1998
Primary chick muscle cells were treated with physiological level of thyroxine (T4) or triiodothyronine (T3) to examine the effects of the hormones on growth, protein turnover, and apoptosis of the cells. Creatine kinase activity, as an index of differentiation, was increased by both T4 and T3. Even when the conversion from T4 to T3 was blocked by iopanoic acid, T4 increased creatine kinase activity. The rate of protein degradation estimated from [3H] tyrosine release was increased by T3 but not by T4. DNA cleavage and fragmentation, as indices of apoptosis, were induced by T3 but not by T4. These results show that T4 stimulates cell differentiation but not protein degradation and apoptosis in primary chick muscle cells, while all events are stimulated by T3. © 1998 Academic Press
It is well known that thyroid hormones play important roles in growth, development, cellular differentiation and metabolism of tissues. The most important effects of thyroid hormone are on basal metabolic rate and protein synthesis and breakdown to maintain homeostasis. As a part of this homeostatic role and the necessity for a response to demand, it is teleologically plausible that thyroid hormones strongly affect skeletal muscle protein metabolism. It is generally thought that pharmacological dose of thyroid hormone produces a catabolic response in skeletal muscle although the effects and modes of action are still largely unknown. Either thyroxine (T4) or triiodothyronine (T3) administered at a replacement dose in hypophysectomized animals stimulates both rates of protein synthesis and, to a lesser extent, protein breakdown; it causes further stimulation of protein breakdown at a high level (1, 2). 1 To whom correspondence should be addressed. Fax: 181-99-2858652. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Recent advances in understanding of the mechanism of hormone action at the cellular level have obtained through the employment of myoblast primary cultures which retain many muscle functions and provide a useful experimental model for the study on hormonal regulation. The present cultured muscle cell consists of mononucleated cells that undergo a period’s proliferation, followed by fusion, to form postmitotic multinucleated myotubes. This differentiation process is accompanied by an increase in the level of muscle specific proteins such as creatine kinase, myosin, and actin (3). In contrast to whole animals, where general anabolic thyroid hormone activity is well established, results from in vitro experiments with cultured myoblasts are controversial. No changes in the growth related processes of cultured myoblasts were reported after thyroid hormone treatment (4). On the other hand, stimulation of myotube formation but not cell proliferation of chick embryo myoblasts was observed after treatment with T4 (5) while physiological amounts of T3 reduces the proliferation of myoblasts (6). There is general agreement that T3 is responsible for the most physiological actions of thyroid hormones in mammals (7), although T4 is normally the main secretary product of the thyroid and the extracellular pool of T4 is much greater than that of T3. Most T3 derives through its conversion from T4 in peripheral tissues (7–10). The existence of iodothyronine 59-deiodinase, which catalyzes the monodeiodination of prohormone T4 to produce T3 has been reported in the thyroid gland (11) as well as in the liver and kidney (12, 13). However, it might be an error to assume that T4 is the prohormone of T3 in various tissues, especially in skeletal muscle because little deiodination of T4 occurs in the muscle (14, 15). Therefore, in the present investigation, two experiments were conducted to show the direct effect of T4 on growth and protein turnover in primary chick muscle cell cultures.
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Furthermore, in the present study, apoptotic effect of T3 was clarified because it was shown in the preceding experiment that cell proliferation was inhibited by T3 but not by T4. Apoptosis has been recognized as a distinct form of cell death that has an essential function in the regulation of cell turnover during development, tissue homeostasis, and cancer. Apoptosis entails characteristic morphological and biochemical changes such as cell shrinkage, chromatin condensation, cell fragmentation called apoptotic body and chromosome DNA cleavage at internecleosomal sites (16, 17). Apoptosis is believed to play an important role in embryonic development. However, the molecular mechanism of apoptosis remains unknown. Recent studies have further indicated that apoptosis may be tightly associated with cell cycle regulation in proliferating cells and may represent a form of “abortive mitosis” in certain cell system (18). Thus, cell death, along with cell cycle progression and differentiation, plays a critical role in tissue homeostasis. Therefore, in the present study, the effects of T4 and T3 on apoptosis in primary cultured muscle cells were also investigated. MATERIALS AND METHODS Cell culture. Myoblasts were isolated from thigh muscle of 13day-old chick embryos. The muscle tissue obtained from the embryo was digested with dispase (2000 U/ml) for 10 min at 37°C. The cell suspension was then passed through a net and centrifuged 700 rpm for 3 min. The supernatant was aspirated and the cell pellet was dispersed into 3 ml basal growth medium (M-199 containing 15% calf serum and 2.5% chicken embryo extract). The cell suspension was transferred to a 35-mm uncoating culture dish to allow fibroblast attachment. After 40 min, unattached cells were recovered and transferred to a next dish. This protocol was repeated three times, and cell number was counted and plated onto gelatin-coated 6-wells plates at a density 2.5 3 105 cells/well. The cells were grown at 37°C in a 5% CO2-enriched atmosphere of humidified air. After 24 h, the medium was replaced by the media containing physiological levels of thyroxine sodium salt (15, 30, 60 ng/ml) or 3,39,5-triiodothyronine sodium salt (3, 6, 12 ng/ml). Thyroid hormones were dissolved in ethanol and added in the medium (The resulted ethanol concentration in the culture medium was 0.1%). The media were replaced every other days for 6 day incubation periods. The medium was collected after the final incubation and stored at 220°C until analyzed for Nt-methylhistidine. In experiments 3 and 4, cell culture was conducted similarly as experiment 1 except thyroxine sodium salt or triiodothyronine sodium salt were added at the levels of 60 and 12 ng/ml, respectively and iopanoic acid (IOP) were used to inhibit 59-deiodinase. IOP was added to the medium at a level of 1.75 mM according to the report of Galton et al. (19). Measurement of thyroxine and triiodothyronine. After the medium was collected, the cell monolayer was washed three times with ice-cold PBS and the cells were detached by scraping with a rubber policeman using 500 ml phosphate buffer (pH 7.4) containing EDTA (1 mM) and dithiothreitol (20 mM). The cells were then homogenized by a micro homogenizer and centrifuged at 2500 rpm for 10 min at 4°C. The supernatants were rapidly frozen and stored at 280°C until analysis. T4 and T3 were measured using commercial kits, EnzymunTest-T4, (Boehringer-Mannheim, Mannheim) and T3 Enzymun-Test
T3 (Elsia-Auto T3, International Reagents Corporation, Kobe, Japan). Creatine kinase (CK) activity assay. After the medium was collected, the cell monolayer was washed three times with ice-cold PBS and the cells were detached by scraping with a rubber policeman using 2 ml Tris buffer (pH 6.8). The cells were then homogenized by a micro homogenizer and centrifuged at 2500 rpm for 10 min at 4°C. The supernatants were rapidly frozen and stored at 280°C until analysis. CK activity was analyzed by the method of Rosalki (20). ATP formed by the action of the enzyme on ADP and CP is linked to the reduction of nicotinamide-adenine dinucleotide phosphate (NADP) with glucose, hexokinase, and glucose-6-phosphate dehydrogenase. The increase in optical density at 340 nm which depends upon NADP reduction is followed by spectrophotometry and provided a measure of CK activity. One enzyme unit is the amount of CK that catalyses formation of 1 mmol/min at 30°C, pH 6.8. DNA analysis. DNA content was determined according to the modified method described by Erwin et al. (21). Cell homogenate was treated with 10% TCA, and the precipitate was removed by centrifugation and suspended by vortexing in 500 ml 5% TCA, followed by the hydrolysis at 90°C for 30 min. The samples were then cooled to 4°C and centrifuged at 3000 rpm, 4°C for 15 min. A 300 ml portion of the supernatant was added to 100 ml diaminobenzoic acid dihydrochloride (DABA 0.2 g/ml) and kept at 60°C for 1 h. Two milliliters of 1 N HCl was added to terminate the reaction and DNA was quantified by a fluorescence spectrophotometer (Hitachi F-200, Hitachi Electric Ltd., Japan) using excitation wavelength of 415 nm and emission at wavelength of 520 nm. Nt-Methylhistidine analysis. Nt-Methylhistidine was analyzed by a modification of the method of Hayashi et al. (22). The media were hydrolyzed with 12 N HCl (final concentration 6 N) at 110°C for 20 h. After the hydrolysate was cooled and passed through filter paper, the hydrochloric acid was removed subsequently by evaporation. The residue was dissolved in 5 ml of 0.2 M pyridine; 4.5 ml of this was applied to an anion-exchange resin column (7 3 60 mm, Dowex 50 x 8, 200 to 400 mesh, pyridine form). After eluting most of the acidic and neutral amino acids with 20 ml of 0.2 M pyridine, Nt-methylhistidine was eluted with 20 ml of 1 M pyridine and collected. The eluent was then dried, and the residue was dissolved in 1 ml of mobile phase (15 mM sodium octane sulfonate in 20 mM KH2PO4). Fifty ml of this was used for HPLC analysis. The HPLC system incorporated the reverse-phase separation with ion-pairing, using Shim-pack ODS column (6.0 3 150 mm) and the post-column fluorescence derivatization with orthophthalaldehyde. Measurement of protein degradation rate. Preparation of muscle cells for measurement of long-lived protein degradation involved prelabeling protein by incubation in complete medium containing T4 or T3 and 1 mCi of [3H] tyrosine/ml for 2 days. After radiolabeling, cells were rinsed and placed in nonradioactive chase medium for 2 h to allow degradation of very-short-lived proteins. The cells were subsequently incubated in isotope free-medium supplemented with excess unlabeled 2 mM tyrosine. Excess unlabeled amino acid was included in the medium to prevent radiolabeled amino acid from being reutilized. The muscle cells were harvested at 6 h after incubation. Degradation of short lived proteins was determined after labeling with 5 mCi of [3H] tyrosine for 1 h. Cells were rinsed, chased for 10 min, and placed in isotope free-medium supplemented with excess unlabeled 2 mM tyrosine. The muscle cells were harvested after 1 h incubation. At the end of experiment, culture medium was transferred to a microcentrifuge tube containing 100 ml of BSA (10 mg/ml) and TCA was added to a final concentration of 10% (w/v). After incubation at 4°C for at least 1 h, sample were centrifuged for 5 min. The precipitates were then dissolved with tissue solubilizer. The cell
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monolayers were washed with ice-cold phosphate buffered saline and solubilized 0.5 M NaOH containing 0.1% Triton X-100. Radioactivities in the cell monolayer and TCA soluble and insoluble fractions were measured using scintillation counter. Rates of protein degradation were expressed as the percentage degraded per day. Measurement of growth rates (Kg), protein degradation rates (Kd), and protein synthesis rates (Ks). Growth rates (Kg) were measured as the net accumulation of protein in muscle cells between 1 day and 7 day of the experiment. Proteins were measured by Lowry method by using bovine serum albumin as a standard (23). Growth rates were then expressed as the percentage increment of the protein content per day. Because growth arises from the difference between in the rates of degradation (Kd) and synthesis, protein synthesis (Ks) rate was calculated from the two measured parameters, i.e., Ks 5 Kg 1 Kd. DNA extraction and electrophoresis. Myoblasts were harvested and lysed in 50 mM Tris–HCl, pH 7.8 containing 0.5% sodium lauroyl sarcosinate and 10 mM EDTA. The samples were treated with RNase A (10 mg/ml) at 50°C for 30 min, then with proteinase K (10 mg/ml) at 50°C for 60 min. DNA fragments were separated on a 1.5% agarose gel and visualized with ethidium bromide. Measurement of DNA fragmentation. After incubation, myoblasts were harvested and lysed in 0.5% SDS containing 25 mM EDTA and 75 mM NaCl for over 3 h on ice. The fragmented DNA and intact DNA were separated by centrifugation at 100,000g for 30 min at 0°C. The rotor was cooled to 0°C before use to remove SDS completely. The pellet was dissolved in the same amount of the solution containing 25 mM EDTA and 75 mM NaCl. DNA in the lysate and that in the pellet solution were again precipitated by cold ethanol treatment and then dissolved in 10 mM Tris–HCl (pH 7.4) containing 1 mM EDTA. The DNA content was measured by a fluorometric methods using DABA. Statistical analysis. Data were analyzed by analysis of variance (ANOVA), and means were further tested by Duncan’s multiple range test. A p value ,0.05 was considered to be statistically significant. Each result is the mean 6 standard deviation of the values obtained from six replicates.
RESULTS Experiment 1. Concentrations of T4 and T3 in the medium were increased dose-dependently by additions of T4 and T3, respectively. T3 concentration was not increased by T4 supplementation, indicating that little deiodination of T4 was occurred (Figs. 1A and 1B). DNA content was significantly (p , 0.05) decreased when T3 was added at the levels of 6 and 12 ng/ml. However, T4 did not affect DNA content (Figs. 1A–1C). This indicates that T3 but not T4 inhibits cell proliferation. CK activity was significantly (p , 0.05) increased by T4 at the levels of 30 and 60 ng/ml and by T3 at all the levels tested (Figs. 1A–1D). These results indicate that both T4 and T3 induce differentiation. Nt-Methylhistidine release was significantly (p , 0.05) increased by T3 when the concentrations were 6 and 12 ng/ml. However, T4 did not affect Nt-methylhistidine release (Figs. 1A–1E). These results show that T3 but not T4 stimulates muscle protein breakdown. Experiment 2. The results are summarized in Table 1. There were little difference of intracellular concentration of T3 between control and T4 group, and IOP did
not influence the T3 concentration. In the T3 group, intracellular T3 concentration was about 4- fold higher than those of the control. DNA content of the cell was significantly (p , 0.05) decreased by T3 but not by T4. IOP did not affect the DNA content. This indicate that T3 but not T4 inhibits cell proliferation. CK activity was significantly (p , 0.05) increased by both T4 and T3 despite the addition of IOP. These results indicate that both T4 and T3 stimulate differentiation in cultured chick muscle cells. Nt-Methylhistidine release was increased by T3 but not by T4 and there was no influence of IOP, showing that T3 but not T4 induces myofibrillar proteolysis. Experiment 3. The effects of T4 and T3 on the turnover rates of short and long lived protein estimated by [3H] tyrosine release are shown in Table 2. Both Rates of short and long lived protein degradation were significantly (p , 0.05) increased by T3, but not by T4. Rate of muscle protein synthesis (Ks) calculated as a sum of the rates of protein accumulation (Kg) and breakdown (Kd) was increased by both T4 and T3. These results strongly indicate that both T4 and T3 are active in stimulating protein synthesis, while only T3 is active in stimulating proteolysis. Experiment 4. As it was shown in the previous experiments that cell proliferation was inhibited by T3 but not by T4, the effects of T4 and T3 on apoptosis were observed using DNA cleavage and fragmentation as indices of apoptosis (Fig. 2). DNA cleavage occurred in T3 group after 2 days of incubation, but not in T4 group. On Day 4 and 6, DNA cleavage was not observed. DNA fragmentation (% of total DNA) was also significantly increased by T3, but not by T4 on day 2 (control, 5.09 6 1.52; T4, 5.82 6 1.93; T3, 17.88 6 3.44) and day 4 (control, 4.94 6 0.70; T4, 5.36 6 1.02; T3, 7.87 6 1.48). However, on day 6, DNA fragmentation was not observed because myotube formation completed. It has been reported that differentiated myotubes are resistant to cell death (24). These results show that T3 but not T4 induces apoptosis of chick myoblasts. DISCUSSION The present experiments were conducted to compare the effects of T4 and T3 on growth and protein turnover in primary cultured muscle cells because we have previously observed in chicks that skeletal muscle protein synthesis is accelerated by exogenous T4, while plasma T3 concentration is not changed(unpublished data). It is well known that T4 is converted to the biologically potent T3 in many tissues by 59-deiodinase. However, activity of 59-deiodinase in muscle is much lower than those of liver and kidney (14) and little or no monodeiodination of T4 or T3 occurs in skeletal muscle (15).
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FIG. 1. Concentration of thyroxine (T4, A) and triiodothyronine (T3, B) in the medium at the final day of incubation and effects of T4 and T3 on DNA contents (C), Creatine kinase activity (D) and Nt-methylhistidine release (E) in cultured chick muscle cells. T4 was added at the levels of 15, 30, or 60 ng/ml in the medium. T3 was added at the levels of 3, 6 or 12 ng/ml in the medium. Values not sharing a common superscript are significantly different (p , 0.05). Values given are means 6 SD.
Thus, it is plausible that T4 stimulates muscle growth without conversion to T3. Two enzymes, type I and type II 59-deiodinase catalyze deiodination of T4. Both type I (25) and type II (26) deiodinases are present but small amount in skeletal muscle. Iopanoic acid (IOP) is a specific inhibitor of these enzymes (11, 27–30) and thus used in the present experiment. Both extracellular and intracellular concentrations of T3 were not increased in both T4 group and T4 plus IOP group. However, creatine kinase activity was increased by T4 even when IOP was included in the medium. This shows that conversion of T4 to T3 is not necessary to stimulate the cell differentiation.
The cultured muscle cell undergoes a period’s proliferation followed by fusion to form postmitotic multinucleated myotubes. This differentiation process accompanies with an increase in the level of muscle specific proteins such as creatine kinase, myosin, and actin (3). Since most creatine kinase activity is derived from myotube, the activity is often used as an index of myotube growth (31). Increases in creatine kinase activities have been shown to accompany with differentiation of myogenic cells in the muscle cell culture systems (32). On the other hand, DNA content has been measured as an index of proliferation of cultured muscle
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Effects of Thyroxine (T4) or Triiodothyronine (T3) with or without Iopanoic Acid (IOP) on Intracellular T3 Concentration, DNA Content, Creatine Kinase (CK) Activity, and Nt-Methylhistidine Release in Cultured Chick Muscle Cells Parameter
Control
IOP
Intracellular T3 concentration (pmol/mg protein) DNA content (mg/well) Creatine kinase activity (IU/mg protein) Nt-Methylhistidine release (nmol/h/mg protein)
148 6 20 12.5 6 1.1a 1.67 6 0.22c 0.38 6 0.05c
140 6 23 13.6 6 1.3a 1.79 6 0.24c 0.44 6 0.08c
b
T4 b
157 6 22 12.5 6 0.7a 2.55 6 0.20b 0.42 6 0.03c b
T4 1 IOP
T3
145 6 10 14.7 6 0.6a 2.62 6 0.20b 0.47 6 0.08c
421 6 56a 8.08 6 0.6b 2.42 6 0.41b 0.91 6 0.16b
b
Note. T4, T3, and IOP were added at 60 ng/ml, 12 ng/ml, and 1.75 mM in the medium, respectively. Values not sharing a common superscript are significantly different (p , 0.05). Values given are means 6 SD.
cells (21).The present results of DNA content show that cell proliferation was inhibited by T3 but not by T4. This finding is consistent with the results of Kumegawa et al. (5)indicating that T4 stimulates myotube formation (differentiation) but not cell proliferation of chick embryo myoblasts. On the other hand, Marchal et al. (6) has reported that physiological amounts of T3 reduces the proliferation of myoblasts while T3 accelerates their fusion rate and thus the appearance of myosin heavy chain. This is consistent with our results. In the present study, DNA fragmentation and cleavage were increased by T3 showing that T3 but not T4 stimulated apoptosis. Recently, Wang et al. (24) have reported that proliferating C2C12 myoblasts can undergo either differentiation or programmed cell death under conditions of mitogen deprivation, and they concluded that a portion of myoblasts undergo apoptosis during in vitro myogenesis whereas other continue differentiation and form myotubes. Yaoita et al. (33) have also reported that T3 induces apoptosis in a myoblast prepared from tadpole tail. However, there is no paper reporting that thyroid hormone induces apoptosis in avian or mammalian myoblasts. Marchal et al. (6) have reported that T3 inhibits DNA synthesis and stimulates differentiation in quail myo-
blasts. This finding suggests that T3 may play a role in the induction of apoptosis. Indeed, our study show that myoblasts become committed suicide by apoptosis by T3 treatment whereas a small proportion of cells can survive and undergo myogenic differentiation at a later stage. Since both apoptosis and differentiation are terminal cellular process, concurrent activation of the two processes can result in adverse depletion of mitotic cell populations. It is relevant to mention that cell cycle arrest is normally required for muscle cell differentiation (34). It is likely that growth factors such as insulin and insulin-like growth factors may exert their myogenic effect by suppressing the expression of apoptotic genes. T3 may stimulate expression of apoptotic gene. The balance between apoptosis and differentiation may play a crucial role in controlling the number of cells capable in maintaining muscle growth and regeneration capacity. Nt-Methylhistidine release was increased by T3, but not by T4. Rates of breakdown of short and long lived proteins measured by tyrosine release were consistent with the result of Nt-methylhistidine release. Thus, it is clear that T3 but not T4 increases protein breakdown. Rate of protein synthesis calculated as the sum of the rates of growth and breakdown of long lived protein was increased by both T4 and T3 as expected.
TABLE 2
Effects of Thyroxine (T4) and Triiodothyronine (T3) on Rates of Short- and Long-Lived Protein Degradation, Growth (Kg), and Synthesis (Ks) of Long-Lived Protein in Cultured Chick Muscle Cells (%/day) Parameter Rate Rate Rate Rate
of of of of
short-lived protein degradation long-lived protein degradation growth (Kg) protein synthesis (Ks)
Control
T4
T3
231.8 6 9.68b 29.40 6 1.93b 15.42 6 0.22b 44.82 6 1.97c
218.6 6 10.5b 31.75 6 0.96b 22.84 6 0.42a 54.68 6 1.11a
249.6 6 6.48a 34.11 6 0.88a 15.39 6 0.10b 49.50 6 0.91b
Note. T4 and T3 were added at 60 and 12 ng/ml in the medium, respectively. Kd was calculated from long-lived protein degradation. Growth rate was expressed as the percentage increment of the protein per day. Protein was measured by Lowry method. Ks was calculated from the following equation: Ks 5 Kg 1 Kd. Values not sharing a common superscript are significantly different (p , 0.05). Values given are means 6 SD.
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REFERENCES
FIG. 2. Effects of thyroxine (T4) and triiodothyronine (T3) on DNA cleavage in chick myoblasts. Chick myoblasts were incubated for 2, 4, and 6 days with T4 or T3. T4 and T3 were added to the medium at the levels of 60 and 12 ng/ml, respectively. DNA was extracted from myoblasts and separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. l-DNA/HindIII was used as a maker.
During fusion and differentiation, protease activity and proteolysis increase (35, 36). Apoptosis pathway is thought to be correlated with the activation of a proteolytic cascade mediated by proteases such as caspases(37), lysosomal proteases (38), calpain (39), and proteasome (40, 41). These observations suggest that enhanced proteases activity play a key mechanistic role in initiating apoptosis. Thus, proteolytic systems may be associated with differentiation or apoptosis at a later stage of myogenesis. We have observed that T3 stimulates non-lysosomal protease and lysosomal protease such as calpain, proteasome and cathepsin D (unpublished data). It is known that nonlysosomal proteolysis is stimulated during apoptosis and translocation of cathepsin from the cytoplasm to the nucleus occurs during apoptosis of hepatocytes (38, 42). Degradation of nuclear structural proteins such as lamins and histones occurs in apoptosis (43). T3 may induce nuclear structural proteins degradation and promote nuclear disruption and DNA fragmentation leading to apoptosis. In conclusion, the present study shows that T4 stimulates protein synthesis and differentiation but not protein degradation, while T3 stimulates protein synthesis, protein degradation, cell differentiation and apoptosis and inhibits cell proliferation. ACKNOWLEDGMENTS The authors thank Dr. A. L. Goldberg for helpful comments and suggestions on this study. This study was supported by a Grant-inAid for Scientific Research (10660273) from the Ministry of Education, Science, Sports, and Culture, Japan.
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