Low-Temperature Arrest of the Triiodothyronine-Dependent Transcription in Rana catesbeiana Red Blood Cells
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内分泌学杂志 2005年第1期
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan
Address all correspondence and requests for reprints to: Dr. Kiyoshi Yamauchi, Department of Biology, Faculty of Science, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. E-mail: sbkyama@ipc.shizuoka.ac.jp.
Abstract
We examined possible molecular mechanisms for the low-temperature arrest of T3-induced Rana catesbeiana metamorphosis. Scatchard plots revealed that the ratios of maximum binding capacity/dissociation constant for high-affinity sites of tadpole serum proteins for T3 at 20 and 28 C was 3.3–4.6 times less than that at 4 C, due to the decrease in maximum binding capacity values. Kinetic studies of T3 uptake into tadpole red blood cells demonstrated that the ratio of maximum uptake rate/Michaelis constant at 23 C was approximately 13 times greater than that at 4 C. The process of intracellular transport of T3 into the nucleus was not arrested at 4 C. The ratio of T3 incorporated into nuclei to that taken up into red blood cells was not significantly different at 4, 20, and 28 C, indicating the absence of temperature-sensitive sites in this process. T3 binding to the T3 receptors and ? were not temperature sensitive at least at 4 and 20 C. Transcription of the tr genes, early primary T3 response genes, was activated by 10 nM T3 at 20 and 28 C but was barely detected at 4 C. These results indicate that the major molecular event causing the low-temperature arrest of amphibian metamorphosis occurs after T3 entry into the nucleus but before or during the transcriptional activation of the tr genes. Plasma proteins binding T3 and the cellular thyroid hormone uptake system on the plasma membrane may contribute to the slowing of the incorporation of T3 into nucleus at 4 C by decreasing the uptake velocity of T3.
Introduction
TEMPERATURE IS AN important environmental factor that can affect the development, metabolism, and physical activity of cold-blooded animals. In homeotherms, thyroid hormones (THs) play an important role in calorigenesis: maintaining body temperature. Oxygen consumption, an indicator of the basal metabolic rate, increases with TH treatment. However, oxygen consumption does not increase during spontaneous amphibian metamorphosis that is obligatorily dependent on THs (1). This evidence suggests that THs do not influence the basal metabolic rate of amphibians (1). Rana catesbeiana tadpoles have an extended larval life that sometimes includes overwintering before metamorphosis. Overwintering occurs for 1 yr or more at high latitudes where tadpole development does not progress at air temperatures lower than 12.8 C (2). Huxley (3) demonstrated experimentally that the metamorphosis of R. temporaria tadpoles exposed to temperatures less than 5 C was suspended even if they were bred in water containing powder of grounded thyroid gland. It was later demonstrated using T4-treated hypophysectomized tadpoles that the metamorphic response at different temperatures was largely dependent on the sensitivity of peripheral tissues to TH (4). Tail regression, increased urea-N excretion and increased activities of the urea cycle enzymes, typical TH-induced responses during spontaneous and T3-induced amphibian metamorphosis, were absent in TH-treated tadpoles at 5 C (5, 6, 7, 8). When tadpoles that had been maintained for many weeks at 5 C after T3 injection were transferred to water at 25 C, a rapid metamorphic response occurred within a few days (6, 8). In contrast to the dramatic temperature sensitivity of the above responses, general metabolic pathways, such as whole protein and RNA synthesis, were not temperature dependent (9) with only some alteration in the amount and intracellular distribution of RNA in liver cells (10). These observations suggest that one or more processes responsible for low-temperature arrest of tadpole metamorphosis may exist in peripheral tissues.
At least eight possible aspects of TH homeostasis can be considered when determining the temperature-sensitive TH response described above: 1) transport of TH by specific binding protein(s) in blood plasma; 2) TH uptake into cells; 3) intracellular transport of TH into nucleus; 4) TH metabolism by cytoplasmic enzymes; 5) TH incorporation into nucleus; 6) TH binding to TH nuclear receptors (TRs); 7) transcription of TH-responsive target genes; and 8) translation of TH-responsive target gene transcripts. Three of the above processes, cellular uptake (11), incorporation into the nucleus (11, 12), and binding to TRs (13), were proposed to be temperature-sensitive. Griswold et al. (11) found that most of the T4 taken up into liver cells at 5 C was not distributed in nuclei. However, it was reported that a significant amount of T3 taken up by Xenopus (12) and Rana cells (14, 15) was distributed in the nuclei when the tadpoles were treated with T3 at 4–5 C. Although there is the possibility that the intracellular T4 transport and T3 transport systems are affected at different temperatures, the low-temperature arrest of metamorphosis occurs in both T4-treated and T3-treated tadpoles (5, 6).
Despite the above-detailed studies, evidence elucidating the molecular processes involved in the low-temperature arrest of TH-induced amphibian metamorphosis has not yet been obtained. In the present study, we investigated the possible temperature-sensitive processes in R. catesbeiana existing in the TH-signaling pathway after THs are secreted into the bloodstream including T3 binding to plasma proteins, T3 uptake into red blood cells (RBCs), T3 incorporation into nuclei, T3 binding to TRs and the induction of early primary TH response genes, the tr genes, and a delayed secondary TH-responsive gene, ornithine transcarbamylase (otc). A process in the T3-signaling pathway within the nucleus that occurs before or during the transcription of the tr gene, increasing the transcription of the tr gene, may be affected by temperature and contribute predominantly to the low-temperature arrest of T3-induced metamorphosis.
Materials and Methods
Preparation of serum and RBCs
Bullfrog R. catesbeiana tadpoles at stages X–XV (16) were collected from ponds in Saitama Prefecture and in Yaizu, Shizuoka Prefecture, Japan, from February 2000 to July 2004. The tadpoles were maintained in aerated, dechlorinated tap water at 20–25 C, unless otherwise noted, and fed boiled spinach three times a week. They were anesthetized by immersion in 0.2% ethyl 3-aminobenzoate methanesulfonic acid (Sigma-Aldrich, St. Louis, MO). The truncus arteries of the tadpoles were cut with scissors and blood samples were collected. After leaving the blood samples for several hours at room temperature, the serum was separated from the blood clot by centrifugation at 4000 x g for 15 min at 4 C. Before incubation at room temperature, a part of the blood sample was collected in heparinized tubes or tubes containing sodium citrate (final concentration 0.3%) to obtain RBCs. The RBCs were separated from the plasma by centrifugation at 1500 x g for 15 min at 4 C. After removal of the plasma and the buffy coat, the RBC fraction was washed six times with 70% Leibovitz’s L-15 medium (Sigma-Aldrich). The serum was used immediately or stored in aliquots at –20 C for later use, and the RBC suspension was kept on ice and used within a day.
Expression and purification of recombinant proteins
cDNAs encoding the ligand binding domain (LBD) of bullfrog TR (bTR), corresponding to amino acids 129–418 (17), and bTR?, corresponding to amino acids 87–373 (18), were amplified from a mixture of hepatic cDNAs, synthesized using RNA extracted from stage XIX tadpole livers, by PCR. The sense and antisense primers for amplifying the bTR LBD and bTR? LBD, designed with specific enzyme restriction sites, were 5'-AAGGATCCGCAATGGACCTTGTCCTGGATGATTC-3' and 5'-AAGTCGACTCACTGCCTTTTAGACTTCCTGGTCC-3' for bTR LBD, and 5'-AAGGATCCGCAACAGATTTGGTTTTGGACGACAG-3' and 5'-AACTCGAGCTGAAGCACAGTCTCTTCTAATCCTC-3' for bTR? LBD. The amplified cDNAs were ligated into the BamHI/XhoI or BamHI/SalI site of pGEX-6p-3 (Amersham Biosciences Corp., Piscataway, NY) to express glutathione-S-transferease (GST) bTR LBD or GST-bTR? LBD fusion proteins, respectively. cDNA encoding bTTR [bullfrog (b) transthyretin] (19) was ligated into the NdeI/BamHI site of pET3a (Novagen, Madison, WI). The resultant plasmids were introduced into Escherichia coli BL21. Bacteria were grown at 37 C until late-logarithmic phase, after which the recombinant proteins were expressed using 0.1 mM isopropyl-1-thio-?-galactopyranoside for 3 h at 28 C. The soluble fraction containing the recombinant proteins was obtained as described previously (20). bTTR was purified by affinity column chromatography using human retinal-binding protein coupled to Sepharose 4B as described by Larsson et al. (21). The GST-bTR LBD fusion proteins were purified by affinity column chromatography using glutathione coupled to Sepharose 4B (Amersham Biosciences) according to the manufacturer’s directions. Once purified, the proteins were stored in 10% glycerol at –85 C until required.
[125I]T3 binding assay
Serum proteins (8.2 μg/tube) or purified recombinant bTTR (10 ng/tube) were incubated in 250 μl of 20 mM Tris-HCl (pH 7.4), 93 mM NaCl, and 1 mM CaCl2 (TNC buffer), and GST-bTR LBD fusion proteins (50 ng/tube for GST-bTR LBD and 150 ng/tube for GST-bTR? LBD) were incubated in 250 μl of 10 mM Tris-HCl (pH 7.5), 1.5 mM EDTA, 1 mM dithiothreitol, and 10% (vol/vol) glycerol, with 0.1 nM [125I]T3, for 1 h and 1.5 h, respectively, unless otherwise noted, at 4, 20, or 28 C. Protein-bound [125I]T3 was separated from free [125I]T3 using the polyethyleneglycol method (22) for the TTR assay and the Dowex method (23, 24) for the TR assay. Radioactivity was measured in a counter (Auto Well System ARC-2000, Aloka, Japan). The amount of [125I]T3 bound nonspecifically was determined from the radioactivity of samples incubated with 5 μM unlabeled T3 for the TTR assay and with 1 μM unlabeled T3 for the TR assay. The amount of specifically bound [125I]T3 was calculated by subtracting the nonspecifically bound value from the total bound value. The dissociation constant (Kd) and maximum binding capacity (MBC) for T3 binding were determined from Scatchard plots for a single class (25) and for two classes of binding sites (26), as previously reported (20, 27).
Uptake of [125I]T3 into bRBCs
RBCs used in this experiment were prepared from tadpoles collected during June and July, 2000–2004. All solutions were maintained at the temperature required before and during the experiment. Uptake was initiated by mixing the RBC suspension (2.5 x 106 cells) with [125I]T3 solution and adjusting the final concentration of [125I]T3 to 0.1 nM in the presence or absence of 5 μM T3. At the time point indicated, the cell-associated [125I]T3 and free [125I]T3 were immediately separated using oil centrifugation at 9100 x g for 2 min (28). The tip of the tube containing the cell pellet was cut off. The amount of [125I]T3 associated with cells and [125I]T3 remaining in the extracellular fraction was determined using a -counter.
Kinetic studies were carried out at either 4 or 23 C for 2 min. Kinetic parameters were determined by fitting the plot of initial velocity (Vi) vs. T3 concentration (S) to the Michaelis-Menten equation: Vi = Vmax/(1 + Km/S), where Vmax is the maximum uptake rate and Km is the Michaelis constant.
Incorporation of [125I]T3 into the nuclei of bRBCs
RBCs used in this experiment were prepared from tadpoles collected during June and July, 2000–2004. Initially, 2.5% tadpole serum (stage XV) was preincubated with 0.5 nM [125I]T3 in 70% Leibovitz’s L-15 medium for 1 h at 4, 20, or 28 C. RBCs (4.5 x 106 cells, stage XV), at 4, 20, or 28 C, were then added to 70% Leibovitz’s L-15 preincubation medium, at the equivalent temperature, adjusting the final volume to 450 μl/tube with the final concentration of tadpole serum and [125I]T3 1% and 0.2 nM, respectively. The cell suspensions were incubated for the various time periods at 4, 20, or 28 C. At the indicated time point, 200 μl of the cell suspension (2.0 x 106 cells) were removed to determine the amount of cell-associated [125I]T3 using the oil centrifugation described above. For determining the amount of nucleus-incorporated [125I]T3, after washing the remaining cells (2.5 x 106 cells) twice with TNC buffer, they were lysed with 2 ml of buffer containing 60 mM KCl, 15 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, 2 mM EDTA, 0.5 mM EGTA, 15 mM 2-mercaptoethanol, and 0.5% Triton X-100 (29). The isolated nuclei were collected by centrifugation at 1500 x g for 5 min and the amount of [125I]T3 incorporated into the nuclei was determined using a -counter.
RNA analyses
Tadpoles (stage X–XV), collected from June to July and from December to January, 2000–2004, were divided into two groups, one of which was maintained in aerated, dechlorinated tap water at 4 C and the other at either 20 or 28 C for 1 d before starting the experiments. For the in vivo study, tadpoles from each group were transferred into tap water with or without 10 nM T3 and were maintained at their respective temperatures for indicated time periods. After anesthetizing the animals, RBCs were prepared as described earlier. Livers were removed from the tadpoles, immediately deposited in liquid nitrogen and then stored at –85 C until used. For the in vitro studies, blood was collected from tadpoles maintained in aerated, dechlorinated tap water at 20–25 C within a horizontal laminar flow hood, and RBCs were prepared under sterile conditions. The cells (1.0 x 106 cells/35 mm dish) were cultured in 70% Leibovitz’s L-15 medium in the presence or absence of 10 nM T3 for either 2 or 5 d at 4, 20, or 28 C.
Total RNA was extracted from liver using the acid guanidinium thiocyanate-phenol-chloroform extraction procedure (30) and from RBCs using a QIAamp RNA Blood Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s directions. RNA (5–10 μg per lane) was size fractionated using a 1% agarose gel containing 2.6 M formaldehyde. After visualizing the 28S and 18S rRNAs by ethidium bromide staining and confirming the integrity and equal loading of the RNA samples, RNA was transferred onto a nylon membrane. Hybridization was performed with 0.1 μg/ml of digoxigenin-labeled RNA probe overnight at 68 C in a mixture of 0.75 M NaCl, 75 mM sodium citrate, 50% formamide, 0.02% sodium dodecyl sulfate (SDS), 0.1% N-lauroylsarcosine and 2% blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany). The antisense RNA probes were transcribed from cDNAs encoding bTTR (nucleotides 1–653) (19) and bOTC (nucleotides 1–1371) (31) using either T7 or T3 RNA polymerase (Roche Diagnostics) after linearizing the plasmids with the appropriate restriction enzymes. After hybridization, membranes were washed three times in a solution consisting of 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS at 68 C. The hybridized probe was detected colorimetrically using antidigoxigenin-alkaline phosphatase-conjugated antibody followed by the reaction with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, according to the manufacturer’s instructions (Roche Diagnostics). The band intensities were quantified using the NIH Image program, version 1.62 (National Institutes of Health, Bethesda, MD).
In some cases, RNA, after being treated with reverse transcriptase (TaqMan Reverse Transcription Reagents, Applied Biosystems), was analyzed by real-time PCR using SYBR Green Master Mix and ABI Prism 7000 (Applied Biosystems, Foster City, CA). Each PCR was run in triplicate to control for PCR variation. The endpoint used in real-time PCR quantification, Ct, is defined as the PCR cycle number that crosses an arbitrarily placed signal threshold and is a function of the amount of target cDNA present in the starting material. Quantification was determined by applying the 2-Ct formula and calculating the average of the three values obtained for each sample. To standardize each experiment, the amount of TR transcript was divided by the amount of 18S rRNA present in the same sample. Primer sequences used were as follows: sense 5'-GCGTCGGAAGGAGGAGATG-3' (nucleotide numbers 476–494) and antisense 5'-TCCCACTCCTCGCTGCTT-3' (524–541) for bTR (17); sense 5'-AAGTCACTGGAAACAGAAACGAAAA-3' (444–468) and antisense 5'-CCCTCCGGTGCATTAACTATAGG-3' (496–518) for bTR? (18); and sense 5'-TGTGCCGCTAGAGGTGAAATT-3' (908–928) and antisense 5'-TGGCAAATGCTTTCGCTTT-3' (952–970) for bullfrog 18S rRNA, which were designed using the corresponding Xenopus laevis and human 18S rRNA sequences (32).
Western blotting and protein determination
Serum for Western blotting and protein determination was collected from the tadpoles used for RNA analysis. After boiling for 5 min under reducing conditions, 2 μl of serum for each tadpole was electrophoresed in a SDS 15% polyacrylamide gel (33). Proteins in the gel were then transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) at 1.75 mA/cm2 for 1 h. After blocking with 10% (wt/vol) skimmed milk in 50 mM Tris-buffered saline overnight at 4 C, the nitrocellulose membrane was immunoblotted for 1 h at room temperature with a rabbit polyclonal antibody against the purified bTTR [1:500 diluted with 50 mM Tris-buffered saline (pH 7.6), containing 1% skimmed milk] as described previously (28). Binding was visualized using a detection kit for alkaline phosphatase according to the manufacturer’s directions (Promega, Madison, WI). The band intensities were quantified using NIH Image, version 1.62. Protein concentration was measured by the Bradford dye-binding method using bovine -globulin as the standard (34).
Statistical analysis
The data are presented as mean ± SEM. Differences between groups were analyzed using either the Student’s t test or the Cochran-Cox test to evaluate the significance of the differences. P < 0.05 was considered statistically significant.
Results
Effects of temperature on T3 binding to plasma TH-binding proteins and the expression of TTR mRNA in liver
The TH-binding protein responsible for binding the majority of T3 in tadpole plasma is TTR (22). To examine whether the incubation temperature influenced T3 binding to tadpole plasma proteins, Scatchard analysis of T3 was performed using tadpole serum at 4, 20, or 28 C (Fig. 1A). Two classes of T3 binding sites were detected at these temperatures. The Kd values for high-affinity sites at 20 C, 1.13 nM, and at 28 C, 0.97 nM, were similar to the value determined at 4 C, 0.98 nM. However, the MBC values for high-affinity sites at 20 C, 1.8 pmol/mg protein, and at 28 C, 2.1 pmol/mg protein, were 4.0 times less (P < 0.05) and 3.4 times less (P < 0.01), respectively, than that determined at 4 C, 7.2 pmol/mg protein (Table 1). There were no significant temperature effects on both Kd and MBC values for low-affinity sites. To exclude the possibility that the plasma TH-binding proteins partially and/or irreversibly lost their activity during incubation at 20 or 28 C, all serum samples for the 4 C experiments were used after preincubation at 20 or 28 C for 1 h. This pretreatment did not change the T3 binding activity.
FIG. 1. Effect of temperature on T3 binding to serum proteins or purified TTR. Proteins were incubated with 0.1 nM [125I]T3 in the presence of various concentrations of unlabeled T3 for 1 h at 4 (closed circles), 20 (closed triangles), and 28 C (closed squares). Data are presented as Scatchard plots. Broken lines represent the Scatchard plots resolved into two lines showing the high-affinity and low-affinity sites. A, Serum proteins obtained from the stage X–XV tadpoles; B, purified recombinant TTR. Each point represents the mean of triplicate determinations. These experiments were repeated at least three times.
TABLE 1. Effects of temperature on T3 binding to tadpole plasma proteins and on T3 uptake into tadpole RBCs
Similar results were obtained from the Scatchard analysis of T3 binding to the purified recombinant bTTR (Fig. 1B). The Kd values of bTTR at 20 and 28 C, which were four times greater than those for the high-affinity sites of serum proteins, were not significantly different from that determined at 4 C, whereas the MBC values at 20 and 28 C were 1.7 times less (P < 0.05) and 2.0 times less (P < 0.01) than that determined at 4 C, respectively (Table 1).
In previous studies (19, 28), it was demonstrated that the amount of TTR in plasma and its mRNA in liver decreased during the climax stages of spontaneous metamorphosis. However, it is not known whether the expression of the ttr gene is controlled by a developmentally programmed process or is down-regulated by TH. Premetamorphic tadpoles at stages X–XV, collected during summer (panel A) and winter (panel B), were bred in dechlorinated tap water with or without 10 nM T3 for 0–6 d at 4 or 28 C. The amount of TTR mRNA in liver did not change significantly with T3 treatment at either 4 or 28 C in both summer and winter tadpoles, suggesting that the expression of the ttr gene is not down-regulated by T3-treatment for at least 6 d (Fig. 2, A and B). The amount of TTR in sera, prepared from these same tadpoles, did not change with T3-treatment at both temperatures, although slightly higher amount of TTR was found in sera from winter tadpoles bred at 28 C (Fig. 2, C and D).
FIG. 2. Effects of temperature on the amounts of TTR mRNA in liver and of TTR in serum. Stage X–XV tadpoles were collected in summer (A and C) and winter (B and D) and then bred in dechlorinated tap water with or without 10 nM T3 for specified periods at 4 and 28 C. A and B, Total RNA was extracted from the stocked frozen livers of the two groups at the same time, and was analyzed by Northern blot hybridization (10 μg/lane) with digoxygenin-labeled cRNAs for TTR. The quantity of RNA loaded in each lane was standardized using ethidium bromide staining of rRNAs. Positions of TTR mRNA and 28S and 18S rRNAs are shown on the left. C and D, Blood was collected from the same tadpoles that were used for Northern blot analysis. Serum was prepared as described. Two μl of the serum was mixed with the same volume of 2x SDS sample buffer, and the mixture was immediately boiled for 5 min under reducing conditions. After SDS-PAGE, the immobilized proteins were immunoblotted with a polyclonal antibody against the purified bTTR as described. These experiments were repeated two times with similar results.
Temperature-sensitive T3 uptake into the tadpole RBCs
To examine whether the T3 uptake system is a temperature-dependent process, a time course of [125I]T3 uptake into tadpole RBCs was investigated. [125I]T3 uptake was linear up to 6 min at 4 C and 3 min at 23 C (Fig. 3A). Uptake was saturable. Nonspecific uptake at 4 and 23 C, measured in the presence of 5 μM unlabeled T3, was 36–43% and 17–21% of total [125I]T3 uptake, respectively. Approximately 11% of the [125I]T3 added to the incubation medium was taken up by the RBCs after 9 min at 23 C by a saturable process. However, only 2% of the [125I]T3 added was taken up by the RBCs after 9 min at 4 C.
FIG. 3. Effect of temperature on [125I]T3 uptake into tadpole RBCs. A, Time course of T3 uptake by tadpole RBCs. RBCs were incubated with 0.1 nM [125I]T3 at 4 (open circles) and 23 C (open squares) for indicated time periods. Nonsaturable cell-associated [125I]T3 was measured in the presence of 5 μM unlabeled T3 at 4 (closed circles) and 23 C (closed squares). Each point represents the mean ± SEM of triplicate determinations. Deviations less than the size of the symbols are not shown. B, Hanes plots of T3 uptake. The initial velocity (Vi) of [125I]T3 uptake was measured after 2 min incubation with increasing unlabeled T3 concentrations (S) (0–500 nM) at 4 (closed circles) and 23 C (closed squares). The graphs are drawn after subtraction of the amounts of nonsaturable cell-associated T3. Each point represents the mean of triplicate determinations. Kinetic constants were obtained by linear regression. The abscissa intercepts of the lines correspond to –Km, the ordinate intercepts to Km/Vmax and the slopes to 1/Vmax. These experiments were repeated four to six times.
In the following experiments, the Vi values of T3 uptake were measured routinely after a 2-min incubation at 4 or 23 C. The curves obtained in the presence of 5 μM unlabeled T3 did not extrapolate to zero, indicating an instantaneous and unsaturable adsorption of T3 to the RBC membrane. The Km and Vmax for T3 were determined by plotting S/Vi as a function of T3 concentration (S). Hanes plots of the data were linear, yielding a Km of 199 nM and a Vmax for transport of 207 fmol/min/106 cells at 4 C, and a Km of 169 nM and a Vmax for transport of 2310 fmol/min/106 cells at 23 C (Fig. 3B and Table 1). Low temperature decreased the Vmax value by 11-fold but did not significantly affect the Km value.
Effect of temperature on intracellular transport of T3 in tadpole RBCs
To discover whether an intracellular T3 transport system in tadpole RBCs showed temperature dependency, time courses of T3 uptake into cells and of T3 incorporation into nuclei were investigated at 4, 20, or 28 C. The amount of cell-associated T3 reached a plateau after 6 h at 20 C (Fig. 4A) and after 3 h at 28 C (Fig. 4C), and it continued to increase even after 5 d at 4 C (Fig. 4C). Similar results were obtained for T3 incorporation into nuclei at these temperatures. Figure 4, B and D, shows the ratios of T3 uptake into cells to T3 incorporation into nuclei, at 4, 20, or 28 C, as a function of the incubation time. The ratio reached a plateau by 2 d at 28 C, by 1 d at 20 C and by 2–5 d at 4 C. The value of the ratio when it reached a plateau was similar for each temperature, 13–16%. It suggests the absence of a temperature-sensitive process in the intracellular T3 transport system in tadpole RBCs, and that a significant amount of T3 is transported into the nucleus even at 4 C.
FIG. 4. Time courses of [125I]T3 uptake into RBCs and of [125I]T3 incorporation into nuclei at 4, 20, and 28 C. RBCs (4.5 x 106 cells, stage XV) were incubated with 0.2 nM [125I]T3 in the presence of 1% tadpole plasma (stage XV) for the indicated periods (A and C). The cell-associated [125I]T3 at 4 (open circles), 20 (open triangles), and 28 C (open squares), and the nuclei-incorporated [125I]T3 at 4 (closed circles), 20 (closed triangles), and 28 C (closed squares), were determined as described. Each point represents the mean ± SEM of triplicate determinations. B and D, The ratio of the amount of [125I]T3 incorporated into the nuclei to the amount of [125I]T3 associated with RBCs is plotted against the incubation time: 4 (closed circles), 20 (closed triangles), and 28 C (closed squares).
Effect of temperature on T3 binding to LBDs of bTRs
Recombinant GST-bTR (Fig. 5A) and GST-bTR? (Fig. 5B) LBD fusion proteins were incubated with 0.1 nM [125I]T3 for the indicated time periods at 4, 20, or 28 C. T3 binding to the recombinant proteins was saturable. Nonspecific binding was less than 26% of total binding at these temperatures. The T3 binding activity of GST was negligible when purified GST, which was expressed from pGES-6p-3 in E. coli, was substituted for the GST-bTR LBD fusion proteins in the assay. T3 binding to bTR and ? LBDs increased gradually and reached a plateau within 1.5 h at 4 and 20 C. No significant difference was found in the amount of bound [125I]T3 between samples incubated at 4 and 20 C. [125I]T3 binding activity of both bTR LBDs at 28 C was lost due to severe protein denaturation especially after 15 min incubation. This suggests that T3 binding to bTRs is a temperature-independent process at least in the range of 4–20 C. Scatchard analysis revealed a single class of T3 binding sites with Kd values, at 4 C, of 0.25 ± 0.02 nM (n = 3) for bTR LBD (Fig. 5A, inset) and 0.76 ± 0.20 nM (n = 3) for bTR? LBD (Fig. 5B, inset), which were one order of magnitude greater than those for in vitro-translated bTRs (17, 18).
FIG. 5. [125I]T3 binding to GST-fused LBDs of bTRs. A and B, GST-bTR LBD (A, 50 ng/250 μl) or GST-bTR? LBD (B, 150 ng/250 μl) fusion proteins were incubated with 0.1 nM [125I]T3 in the presence or absence of unlabeled 1 μM unlabeled T3 for the indicated periods at 4 (closed circles), 20 (closed triangles), and 28 C (closed squares). Nonspecific binding was subtracted from total binding. Each point represents the mean ± SEM of triplicate determinations. Deviations less than the size of the symbols are not shown. Inset, Purified recombinant proteins were incubated with 0.03–1.0 nM [125I]T3 in the presence or absence of 1 μM unlabeled T3 for 1.5 h at 4 C. Data were represented as Scatchard plots. Each value is the mean of triplicate determinations. These experiments were repeated at least three times.
Effect of temperature on T3-activated genes
To examine whether low temperature affects the activation of gene transcription for the urea cycle enzyme OTC by T3, Northern blot analysis was performed (Fig. 6). The premetamorphic tadpoles, which were collected in summer (panel A) and winter (panel B), were bred in dechlorinated tap water with or without 10 nM T3 at 4 or 28 C for 0–6 d. The OTC mRNA was up-regulated in T3-treated tadpole livers by 2–4 d at 28 C. Its amount increased 2- to 3-fold by d 6 at 28 C; however, no change in OTC mRNA levels was detected at 4 C even in the 6-d T3-treated tadpoles. There was no significant difference in the amount of T3-induced OTC mRNA at 28 C between the summer and winter tadpoles, suggesting that the winter tadpole livers are as TH sensitive as the summer tadpoles livers. The genes encoding the urea cycle enzymes in tadpole liver are delayed secondary TH response genes (35, 36). Therefore, the major temperature-sensitive site in the tadpole TH-signaling pathway might exist before the delayed secondary TH response genes are transcribed.
FIG. 6. Effects of temperature on the delayed secondary TH-responsive gene transcript for OTC in tadpole liver. Stage X–XV tadpoles were collected in summer (A) and winter (B) and then bred in dechlorinated tap water with or without 10 nM T3 for indicated periods at 4 and 28 C. Total RNA was extracted at the same time from the stocked frozen livers of the two groups and was analyzed by Northern blot hybridization (10 μg/lane) with digoxygenin-labeled cRNAs for OTC. The quantity of RNA loaded in each lane was standardized using ethidium bromide staining of rRNAs. Positions of rRNAs (28S and 18S) and mRNA for OTC are shown on the left.
Next, the effect of temperature on the activation of early primary TH response genes was examined. TR? is an early primary TH response gene in tadpoles (35) and its expression is up-regulated in tadpole liver during spontaneous and TH-induced metamorphosis (37, 38). The TR gene is also an early primary TH response gene in bullfrog tadpole RBCs (39, 40) and is up-regulated by TH in vivo and in vitro (39, 41). TR and ? genes were confirmed as early primary TH response genes by showing cycloheximide-insensitive T3-induction of their transcripts in tadpole RBCs (left part of Fig. 7A) and liver (left parts of Fig. 7, B and C). The TR? transcript was not detected in tadpole RBCs by real-time PCR, in agreement with a previous report (17).
FIG. 7. Effects of temperature on the early primary TH-responsive gene transcripts for TRs in tadpole RBCs and liver. In in vivo studies, tadpoles were treated with or without 10 nM T3 for 4–5 d at 4, 20, and 28 C, whereas, in the in vitro studies, the isolated RBCs were treated with and without 10 nM T3 for 2 d at 4 and 28 C. RNAs were prepared from RBCs (A) and liver (B and C), and TR (A and B) and ? (C) transcripts were analyzed by real-time PCR. Some tadpoles were treated with 1.0 μM (closed bars) cycloheximide (Chx) in the presence or absence of 10 nM T3 for 4 d at 28 C, and some RBCs were cultured with 0.5 μM (hatched bar) and 3.0 μM Chx (cross-hatched bar) in the presence or absence (open bar) of 10 nM T3 for 2 d at 28 C. Vertical axis represents the ratio of TR transcripts of T3-treated samples to those of T3-untreated samples. Each number of repeated experiments was shown in parentheses. Each value is the mean ± SEM. Statistically significant differences were: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
The amount of the TR mRNA was 7–19 times higher in T3-treated RBCs than in T3-untreated RBCs when stage X–XV tadpoles were immersed for 4–5 d in chlorinated tap water with or without 10 nM T3 at 20 or 28 C (Fig. 7A). The same result was obtained when stage X–XV tadpole RBCs were cultured for 2 d in 70% Leibovitz’s L-15 culture medium with or without 10 nM T3. Low temperature (4 C) almost completely abolished the T3 responses found at 20 and 28 C in both the in vivo and in vitro studies. When tadpole RBCs were cultured with T3 in vitro at 4 C even for 5 d, the amount of TR transcript did not increase (data not shown). Similar experiments were conducted examining the low-temperature effect on TR and ? mRNAs in tadpole livers (right parts of Fig. 7, B and C). The amount of the TR mRNA was seven to nine times higher in T3-treated tadpole livers than in T3-untreated ones, at 20 and 28 C in the in vivo study, which was similar to the response found in RBCs. The amount of the TR? mRNA increased approximately 30 and 200 times in T3-treated tadpole liver at 20 and 28 C, respectively. The induction of TR gene transcription in liver by T3 was almost completely suppressed by exposing tadpoles to 4 C for 4–5 d. The in vivo T3 response at 20 C was comparable with that found at 28 C for the amount of TR transcript, and the amount of TR? transcript at 20 C was one order of magnitude greater than that found at 4 C and one order of magnitude less than that found at 28 C. Therefore, it is likely that the most critical site for the low-temperature arrest of T3-induced metamorphosis exists in T3-signaling pathway in the nucleus, probably at the induction of the early primary TH response genes.
Discussion
In this study, we investigated the effect of temperature on the TH-signaling pathway during T3-induced tadpole metamorphosis and found that the low-temperature arrest of amphibian metamorphosis was most likely due to a TH-dependent process within the nucleus that occurred before or during the transcriptional activation of the tr genes. T3 binding to serum proteins and cellular T3 uptake were also temperature dependent and may slow the rate of T3 incorporation into the nucleus at low temperatures resulting in the delayed progress of metamorphosis. TH binding activity in R. catesebeiana plasma dramatically changes during metamorphosis. Miyauchi et al. (42) demonstrated that high-affinity sites for T3 rather than for T4 were present in plasma at the premetamorphic stage, and that their amount decreased after the beginning of metamorphic climax stage (stage XX) and disappeared at the end of metamorphic climax stage (stage XXV). These changes in the TH-binding activity of plasma were shown to be due to changes in the concentration of a major TH-binding protein, later identified as TTR (22, 28). bTTR was found to have 102 times higher affinity for T3 than for T4 (22, 43). Conversely, human TTR has 10 times higher affinity for T4 than for T3 (44). This suggested that TTR has a regulatory role in the free concentration of tadpole plasma T3.
Low temperature increased the MBC of high-affinity sites of plasma proteins for T3 with no significant differences in the Kd values (Fig. 1A and Table 1). Similar results were obtained when we used the purified recombinant bTTR instead of tadpole serum (Fig. 1B and Table 1), which was in agreement with a previous report (22). It is likely that low temperature affects the tertiary structure of TTR subunits, subunit-subunit interactions or a dimer-dimer interaction making a higher affinity T3-binding pocket, although we cannot fully explain how low temperature changes the apparent MBC. Quantitative analysis of temperature sensitivity using the ratio of MBC/Kd at different temperatures (see Table 1) revealed that the ratio (4 C/20 C) was 4.6 for tadpole serum and 2.2 for the purified bTTR, whereas the ratio (20 C/28 C) was 0.7 for tadpole serum and 1.2 for the purified bTTR. This result suggests that tadpole plasma creates a reserve of T3 at 4 C three to five times greater than that at 20 and 28 C, and that this reserve is predominantly created by the major T3-binding protein in tadpole plasma, TTR. Therefore, TTR might affect plasma TH homeostasis in a temperature-dependent manner, altering the equilibrium of free T3/ total T3 in plasma that determines the amount of T3 available for target cells (45, 46). Frieden et al. (6) indicated that a decrease in breeding temperature from 30 to 20 C slightly slowed the progress of tadpole metamorphosis, determined by tail length and urea-N/NH3-N excretion, and that breeding tadpoles at 15 and 10 C dramatically decreased the progress of tadpole metamorphosis. This temperature dependency may be explained in part by the thermo-sensitive characteristic of high-affinity sites for T3 present in tadpole plasma. Greater T3 binding to plasma proteins in tadpoles may decrease the free T3 concentration in plasma when they are exposed to low temperature. It is doubtful whether this decrease is compensated by a negative feedback loop of the hypothalamus/pituitary/thyroid system as found in homeotherms, because low temperature also decreases the activity of the thyroid in tadpoles (47, 48).
The T3-treated summer tadpoles maintained at low temperature had the same amount of serum TTR as those maintained at 28 C (Fig. 2C). The T3-treated winter tadpoles maintained at 28 C had a slightly higher concentration of serum TTR than those maintained at 4 C (Fig. 2D). The amounts of TTR mRNA in the liver did not change significantly during the 6 d of T3 treatment (Fig. 2, A and B). It was previously found that acclimatization (winter vs. summer) and thermal acclimation for short periods in the laboratory resulted in somewhat different hormonal responses in bullfrog tadpoles (48). However, in our study, the amount of TTR in serum and of TTR mRNA in liver did not vary between samples from acclimatization and thermal acclimation. It is therefore unlikely that an increase in the concentration of plasma TTR causes the low-temperature arrest of amphibian metamorphosis.
[125I]T3 uptake experiments in the absence of serum demonstrated that T3 uptake into tadpole RBCs was a temperature-dependent process (Fig. 3). The Vmax value at 4 C was 11 times less than that at 23 C, whereas there was no significant difference in Km values at 4 C and at 23 C. Because the time required for the cellular T3 level to reach equilibrium was longer at 4 C than at 23 C, the T3 uptake system may contribute in part to the delayed progress of T3-induced metamorphosis at low temperature. The Km values for T3 uptake, 169 nM at 23 C and 199 nM at 4 C, were in the range of those for RBCs from the same species (49) or other species (50, 51, 52, 53). The ratio of Vmax/Km at 23 C to Vmax/Km at 4 C was approximately 13 (the ratio per 10 C difference, [Vmax/Km]10 = 3.9). The [Vmax/Km]10 value for bullfrog RBCs was similar to those reported for rat RBCs, rat hepatocytes and human RBCs: 40 at 37 C/at 0 C ([Vmax/Km]10 = 2.7) (50), 50 at 25 C/at 0 C([Vmax/Km]10 = 4.7) (54) and 26 at 37 C/at 0 C ([Vmax/Km]10 = 2.4) (51), respectively. Therefore, temperature-sensitive [125I]T3 uptake into RBCs is a characteristic common to the above animal cells rather than a characteristic specific to amphibian cells.
We found that a significant proportion of [125I]T3 taken up into RBCs in vitro was transported into the nucleus at 4, 20, and 28 C and that the amount of [125I]T3 incorporated into the nucleus did not differ at these temperatures after incubation for 2–5 d. Translocation, however, was slower at 4 C than at 20 and 28 C (Fig. 4), which was in agreement with previous in vivo (12, 14, 15) and in vitro studies (55). It is unlikely that the intracellular transport of TH plays an important role in the low-temperature arrest of amphibian metamorphosis. The amounts of T3 incorporated into nuclei at 4 C did not reach a plateau even after incubation for 5 d; however, the ratio of the amount of T3 incorporated into the nucleus to the amount of T3 taken up into RBCs reached a plateau at 1–5 d. This suggests that a limiting process responsible for the slow T3 incorporation into nucleus at 4 C would be a cellular T3 uptake system but not an intracellular T3 transport system into nucleus.
We found no temperature dependency during a time course study of in vitro [125I]T3 binding to purified bTRs at 4 or 20 C (Fig. 5), although their T3 binding activity at 28 C was inhibited during incubations longer than 15 min. It may be concluded that T3 binding to bTRs is not critical in the low-temperature arrest of amphibian metamorphosis. However, our result does not exclude the possibility of other temperature-dependent factors that may interact with the TH-signaling pathway in the nucleus, such as the cold-inducible coactivator for peroxisome proliferator-activated receptor (PGC-1), which is dramatically induced in brown fat and skeletal muscle of mice upon cold exposure (56). Kusuda et al. (57) found an increase in affinity of TR for T3 in the nuclear extract obtained from rats exposed to 4 C for 6 h. This suggested either a temperature-dependent modification of TRs or an interaction of TRs with another nuclear factor. Further investigation of nuclear proteins or factors modifying or complexing with TR, such as retinoid X receptor, that may transactivate TR or change its affinity for TH in a temperature-dependent manner is required.
The transcriptional activity of the delayed secondary TH response gene otc and of the early primary TH response genes tr and tr? were not altered in vivo in response to T3 at 4 C but were altered at 20 or 28 C. This finding was confirmed in the in vitro-cultured RBCs in the absence of serum. These results indicated that the low-temperature-sensitive process exists before or during the transcriptional activation of early primary TH response genes, and that this process was not mediated by extracellular signaling molecules, like in the case of the coactivator for peroxisome proliferator-activated receptor (PGC-1) in which noradrenaline is involved (58). The temperature sensitivity of tr? gene was greater than that of tr gene, especially at 20 and 28 C. Low temperature may differentially affect T3-signaling pathways mediated by TR and TR? at the metamorphic stages.
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Address all correspondence and requests for reprints to: Dr. Kiyoshi Yamauchi, Department of Biology, Faculty of Science, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. E-mail: sbkyama@ipc.shizuoka.ac.jp.
Abstract
We examined possible molecular mechanisms for the low-temperature arrest of T3-induced Rana catesbeiana metamorphosis. Scatchard plots revealed that the ratios of maximum binding capacity/dissociation constant for high-affinity sites of tadpole serum proteins for T3 at 20 and 28 C was 3.3–4.6 times less than that at 4 C, due to the decrease in maximum binding capacity values. Kinetic studies of T3 uptake into tadpole red blood cells demonstrated that the ratio of maximum uptake rate/Michaelis constant at 23 C was approximately 13 times greater than that at 4 C. The process of intracellular transport of T3 into the nucleus was not arrested at 4 C. The ratio of T3 incorporated into nuclei to that taken up into red blood cells was not significantly different at 4, 20, and 28 C, indicating the absence of temperature-sensitive sites in this process. T3 binding to the T3 receptors and ? were not temperature sensitive at least at 4 and 20 C. Transcription of the tr genes, early primary T3 response genes, was activated by 10 nM T3 at 20 and 28 C but was barely detected at 4 C. These results indicate that the major molecular event causing the low-temperature arrest of amphibian metamorphosis occurs after T3 entry into the nucleus but before or during the transcriptional activation of the tr genes. Plasma proteins binding T3 and the cellular thyroid hormone uptake system on the plasma membrane may contribute to the slowing of the incorporation of T3 into nucleus at 4 C by decreasing the uptake velocity of T3.
Introduction
TEMPERATURE IS AN important environmental factor that can affect the development, metabolism, and physical activity of cold-blooded animals. In homeotherms, thyroid hormones (THs) play an important role in calorigenesis: maintaining body temperature. Oxygen consumption, an indicator of the basal metabolic rate, increases with TH treatment. However, oxygen consumption does not increase during spontaneous amphibian metamorphosis that is obligatorily dependent on THs (1). This evidence suggests that THs do not influence the basal metabolic rate of amphibians (1). Rana catesbeiana tadpoles have an extended larval life that sometimes includes overwintering before metamorphosis. Overwintering occurs for 1 yr or more at high latitudes where tadpole development does not progress at air temperatures lower than 12.8 C (2). Huxley (3) demonstrated experimentally that the metamorphosis of R. temporaria tadpoles exposed to temperatures less than 5 C was suspended even if they were bred in water containing powder of grounded thyroid gland. It was later demonstrated using T4-treated hypophysectomized tadpoles that the metamorphic response at different temperatures was largely dependent on the sensitivity of peripheral tissues to TH (4). Tail regression, increased urea-N excretion and increased activities of the urea cycle enzymes, typical TH-induced responses during spontaneous and T3-induced amphibian metamorphosis, were absent in TH-treated tadpoles at 5 C (5, 6, 7, 8). When tadpoles that had been maintained for many weeks at 5 C after T3 injection were transferred to water at 25 C, a rapid metamorphic response occurred within a few days (6, 8). In contrast to the dramatic temperature sensitivity of the above responses, general metabolic pathways, such as whole protein and RNA synthesis, were not temperature dependent (9) with only some alteration in the amount and intracellular distribution of RNA in liver cells (10). These observations suggest that one or more processes responsible for low-temperature arrest of tadpole metamorphosis may exist in peripheral tissues.
At least eight possible aspects of TH homeostasis can be considered when determining the temperature-sensitive TH response described above: 1) transport of TH by specific binding protein(s) in blood plasma; 2) TH uptake into cells; 3) intracellular transport of TH into nucleus; 4) TH metabolism by cytoplasmic enzymes; 5) TH incorporation into nucleus; 6) TH binding to TH nuclear receptors (TRs); 7) transcription of TH-responsive target genes; and 8) translation of TH-responsive target gene transcripts. Three of the above processes, cellular uptake (11), incorporation into the nucleus (11, 12), and binding to TRs (13), were proposed to be temperature-sensitive. Griswold et al. (11) found that most of the T4 taken up into liver cells at 5 C was not distributed in nuclei. However, it was reported that a significant amount of T3 taken up by Xenopus (12) and Rana cells (14, 15) was distributed in the nuclei when the tadpoles were treated with T3 at 4–5 C. Although there is the possibility that the intracellular T4 transport and T3 transport systems are affected at different temperatures, the low-temperature arrest of metamorphosis occurs in both T4-treated and T3-treated tadpoles (5, 6).
Despite the above-detailed studies, evidence elucidating the molecular processes involved in the low-temperature arrest of TH-induced amphibian metamorphosis has not yet been obtained. In the present study, we investigated the possible temperature-sensitive processes in R. catesbeiana existing in the TH-signaling pathway after THs are secreted into the bloodstream including T3 binding to plasma proteins, T3 uptake into red blood cells (RBCs), T3 incorporation into nuclei, T3 binding to TRs and the induction of early primary TH response genes, the tr genes, and a delayed secondary TH-responsive gene, ornithine transcarbamylase (otc). A process in the T3-signaling pathway within the nucleus that occurs before or during the transcription of the tr gene, increasing the transcription of the tr gene, may be affected by temperature and contribute predominantly to the low-temperature arrest of T3-induced metamorphosis.
Materials and Methods
Preparation of serum and RBCs
Bullfrog R. catesbeiana tadpoles at stages X–XV (16) were collected from ponds in Saitama Prefecture and in Yaizu, Shizuoka Prefecture, Japan, from February 2000 to July 2004. The tadpoles were maintained in aerated, dechlorinated tap water at 20–25 C, unless otherwise noted, and fed boiled spinach three times a week. They were anesthetized by immersion in 0.2% ethyl 3-aminobenzoate methanesulfonic acid (Sigma-Aldrich, St. Louis, MO). The truncus arteries of the tadpoles were cut with scissors and blood samples were collected. After leaving the blood samples for several hours at room temperature, the serum was separated from the blood clot by centrifugation at 4000 x g for 15 min at 4 C. Before incubation at room temperature, a part of the blood sample was collected in heparinized tubes or tubes containing sodium citrate (final concentration 0.3%) to obtain RBCs. The RBCs were separated from the plasma by centrifugation at 1500 x g for 15 min at 4 C. After removal of the plasma and the buffy coat, the RBC fraction was washed six times with 70% Leibovitz’s L-15 medium (Sigma-Aldrich). The serum was used immediately or stored in aliquots at –20 C for later use, and the RBC suspension was kept on ice and used within a day.
Expression and purification of recombinant proteins
cDNAs encoding the ligand binding domain (LBD) of bullfrog TR (bTR), corresponding to amino acids 129–418 (17), and bTR?, corresponding to amino acids 87–373 (18), were amplified from a mixture of hepatic cDNAs, synthesized using RNA extracted from stage XIX tadpole livers, by PCR. The sense and antisense primers for amplifying the bTR LBD and bTR? LBD, designed with specific enzyme restriction sites, were 5'-AAGGATCCGCAATGGACCTTGTCCTGGATGATTC-3' and 5'-AAGTCGACTCACTGCCTTTTAGACTTCCTGGTCC-3' for bTR LBD, and 5'-AAGGATCCGCAACAGATTTGGTTTTGGACGACAG-3' and 5'-AACTCGAGCTGAAGCACAGTCTCTTCTAATCCTC-3' for bTR? LBD. The amplified cDNAs were ligated into the BamHI/XhoI or BamHI/SalI site of pGEX-6p-3 (Amersham Biosciences Corp., Piscataway, NY) to express glutathione-S-transferease (GST) bTR LBD or GST-bTR? LBD fusion proteins, respectively. cDNA encoding bTTR [bullfrog (b) transthyretin] (19) was ligated into the NdeI/BamHI site of pET3a (Novagen, Madison, WI). The resultant plasmids were introduced into Escherichia coli BL21. Bacteria were grown at 37 C until late-logarithmic phase, after which the recombinant proteins were expressed using 0.1 mM isopropyl-1-thio-?-galactopyranoside for 3 h at 28 C. The soluble fraction containing the recombinant proteins was obtained as described previously (20). bTTR was purified by affinity column chromatography using human retinal-binding protein coupled to Sepharose 4B as described by Larsson et al. (21). The GST-bTR LBD fusion proteins were purified by affinity column chromatography using glutathione coupled to Sepharose 4B (Amersham Biosciences) according to the manufacturer’s directions. Once purified, the proteins were stored in 10% glycerol at –85 C until required.
[125I]T3 binding assay
Serum proteins (8.2 μg/tube) or purified recombinant bTTR (10 ng/tube) were incubated in 250 μl of 20 mM Tris-HCl (pH 7.4), 93 mM NaCl, and 1 mM CaCl2 (TNC buffer), and GST-bTR LBD fusion proteins (50 ng/tube for GST-bTR LBD and 150 ng/tube for GST-bTR? LBD) were incubated in 250 μl of 10 mM Tris-HCl (pH 7.5), 1.5 mM EDTA, 1 mM dithiothreitol, and 10% (vol/vol) glycerol, with 0.1 nM [125I]T3, for 1 h and 1.5 h, respectively, unless otherwise noted, at 4, 20, or 28 C. Protein-bound [125I]T3 was separated from free [125I]T3 using the polyethyleneglycol method (22) for the TTR assay and the Dowex method (23, 24) for the TR assay. Radioactivity was measured in a counter (Auto Well System ARC-2000, Aloka, Japan). The amount of [125I]T3 bound nonspecifically was determined from the radioactivity of samples incubated with 5 μM unlabeled T3 for the TTR assay and with 1 μM unlabeled T3 for the TR assay. The amount of specifically bound [125I]T3 was calculated by subtracting the nonspecifically bound value from the total bound value. The dissociation constant (Kd) and maximum binding capacity (MBC) for T3 binding were determined from Scatchard plots for a single class (25) and for two classes of binding sites (26), as previously reported (20, 27).
Uptake of [125I]T3 into bRBCs
RBCs used in this experiment were prepared from tadpoles collected during June and July, 2000–2004. All solutions were maintained at the temperature required before and during the experiment. Uptake was initiated by mixing the RBC suspension (2.5 x 106 cells) with [125I]T3 solution and adjusting the final concentration of [125I]T3 to 0.1 nM in the presence or absence of 5 μM T3. At the time point indicated, the cell-associated [125I]T3 and free [125I]T3 were immediately separated using oil centrifugation at 9100 x g for 2 min (28). The tip of the tube containing the cell pellet was cut off. The amount of [125I]T3 associated with cells and [125I]T3 remaining in the extracellular fraction was determined using a -counter.
Kinetic studies were carried out at either 4 or 23 C for 2 min. Kinetic parameters were determined by fitting the plot of initial velocity (Vi) vs. T3 concentration (S) to the Michaelis-Menten equation: Vi = Vmax/(1 + Km/S), where Vmax is the maximum uptake rate and Km is the Michaelis constant.
Incorporation of [125I]T3 into the nuclei of bRBCs
RBCs used in this experiment were prepared from tadpoles collected during June and July, 2000–2004. Initially, 2.5% tadpole serum (stage XV) was preincubated with 0.5 nM [125I]T3 in 70% Leibovitz’s L-15 medium for 1 h at 4, 20, or 28 C. RBCs (4.5 x 106 cells, stage XV), at 4, 20, or 28 C, were then added to 70% Leibovitz’s L-15 preincubation medium, at the equivalent temperature, adjusting the final volume to 450 μl/tube with the final concentration of tadpole serum and [125I]T3 1% and 0.2 nM, respectively. The cell suspensions were incubated for the various time periods at 4, 20, or 28 C. At the indicated time point, 200 μl of the cell suspension (2.0 x 106 cells) were removed to determine the amount of cell-associated [125I]T3 using the oil centrifugation described above. For determining the amount of nucleus-incorporated [125I]T3, after washing the remaining cells (2.5 x 106 cells) twice with TNC buffer, they were lysed with 2 ml of buffer containing 60 mM KCl, 15 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, 2 mM EDTA, 0.5 mM EGTA, 15 mM 2-mercaptoethanol, and 0.5% Triton X-100 (29). The isolated nuclei were collected by centrifugation at 1500 x g for 5 min and the amount of [125I]T3 incorporated into the nuclei was determined using a -counter.
RNA analyses
Tadpoles (stage X–XV), collected from June to July and from December to January, 2000–2004, were divided into two groups, one of which was maintained in aerated, dechlorinated tap water at 4 C and the other at either 20 or 28 C for 1 d before starting the experiments. For the in vivo study, tadpoles from each group were transferred into tap water with or without 10 nM T3 and were maintained at their respective temperatures for indicated time periods. After anesthetizing the animals, RBCs were prepared as described earlier. Livers were removed from the tadpoles, immediately deposited in liquid nitrogen and then stored at –85 C until used. For the in vitro studies, blood was collected from tadpoles maintained in aerated, dechlorinated tap water at 20–25 C within a horizontal laminar flow hood, and RBCs were prepared under sterile conditions. The cells (1.0 x 106 cells/35 mm dish) were cultured in 70% Leibovitz’s L-15 medium in the presence or absence of 10 nM T3 for either 2 or 5 d at 4, 20, or 28 C.
Total RNA was extracted from liver using the acid guanidinium thiocyanate-phenol-chloroform extraction procedure (30) and from RBCs using a QIAamp RNA Blood Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s directions. RNA (5–10 μg per lane) was size fractionated using a 1% agarose gel containing 2.6 M formaldehyde. After visualizing the 28S and 18S rRNAs by ethidium bromide staining and confirming the integrity and equal loading of the RNA samples, RNA was transferred onto a nylon membrane. Hybridization was performed with 0.1 μg/ml of digoxigenin-labeled RNA probe overnight at 68 C in a mixture of 0.75 M NaCl, 75 mM sodium citrate, 50% formamide, 0.02% sodium dodecyl sulfate (SDS), 0.1% N-lauroylsarcosine and 2% blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany). The antisense RNA probes were transcribed from cDNAs encoding bTTR (nucleotides 1–653) (19) and bOTC (nucleotides 1–1371) (31) using either T7 or T3 RNA polymerase (Roche Diagnostics) after linearizing the plasmids with the appropriate restriction enzymes. After hybridization, membranes were washed three times in a solution consisting of 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS at 68 C. The hybridized probe was detected colorimetrically using antidigoxigenin-alkaline phosphatase-conjugated antibody followed by the reaction with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, according to the manufacturer’s instructions (Roche Diagnostics). The band intensities were quantified using the NIH Image program, version 1.62 (National Institutes of Health, Bethesda, MD).
In some cases, RNA, after being treated with reverse transcriptase (TaqMan Reverse Transcription Reagents, Applied Biosystems), was analyzed by real-time PCR using SYBR Green Master Mix and ABI Prism 7000 (Applied Biosystems, Foster City, CA). Each PCR was run in triplicate to control for PCR variation. The endpoint used in real-time PCR quantification, Ct, is defined as the PCR cycle number that crosses an arbitrarily placed signal threshold and is a function of the amount of target cDNA present in the starting material. Quantification was determined by applying the 2-Ct formula and calculating the average of the three values obtained for each sample. To standardize each experiment, the amount of TR transcript was divided by the amount of 18S rRNA present in the same sample. Primer sequences used were as follows: sense 5'-GCGTCGGAAGGAGGAGATG-3' (nucleotide numbers 476–494) and antisense 5'-TCCCACTCCTCGCTGCTT-3' (524–541) for bTR (17); sense 5'-AAGTCACTGGAAACAGAAACGAAAA-3' (444–468) and antisense 5'-CCCTCCGGTGCATTAACTATAGG-3' (496–518) for bTR? (18); and sense 5'-TGTGCCGCTAGAGGTGAAATT-3' (908–928) and antisense 5'-TGGCAAATGCTTTCGCTTT-3' (952–970) for bullfrog 18S rRNA, which were designed using the corresponding Xenopus laevis and human 18S rRNA sequences (32).
Western blotting and protein determination
Serum for Western blotting and protein determination was collected from the tadpoles used for RNA analysis. After boiling for 5 min under reducing conditions, 2 μl of serum for each tadpole was electrophoresed in a SDS 15% polyacrylamide gel (33). Proteins in the gel were then transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) at 1.75 mA/cm2 for 1 h. After blocking with 10% (wt/vol) skimmed milk in 50 mM Tris-buffered saline overnight at 4 C, the nitrocellulose membrane was immunoblotted for 1 h at room temperature with a rabbit polyclonal antibody against the purified bTTR [1:500 diluted with 50 mM Tris-buffered saline (pH 7.6), containing 1% skimmed milk] as described previously (28). Binding was visualized using a detection kit for alkaline phosphatase according to the manufacturer’s directions (Promega, Madison, WI). The band intensities were quantified using NIH Image, version 1.62. Protein concentration was measured by the Bradford dye-binding method using bovine -globulin as the standard (34).
Statistical analysis
The data are presented as mean ± SEM. Differences between groups were analyzed using either the Student’s t test or the Cochran-Cox test to evaluate the significance of the differences. P < 0.05 was considered statistically significant.
Results
Effects of temperature on T3 binding to plasma TH-binding proteins and the expression of TTR mRNA in liver
The TH-binding protein responsible for binding the majority of T3 in tadpole plasma is TTR (22). To examine whether the incubation temperature influenced T3 binding to tadpole plasma proteins, Scatchard analysis of T3 was performed using tadpole serum at 4, 20, or 28 C (Fig. 1A). Two classes of T3 binding sites were detected at these temperatures. The Kd values for high-affinity sites at 20 C, 1.13 nM, and at 28 C, 0.97 nM, were similar to the value determined at 4 C, 0.98 nM. However, the MBC values for high-affinity sites at 20 C, 1.8 pmol/mg protein, and at 28 C, 2.1 pmol/mg protein, were 4.0 times less (P < 0.05) and 3.4 times less (P < 0.01), respectively, than that determined at 4 C, 7.2 pmol/mg protein (Table 1). There were no significant temperature effects on both Kd and MBC values for low-affinity sites. To exclude the possibility that the plasma TH-binding proteins partially and/or irreversibly lost their activity during incubation at 20 or 28 C, all serum samples for the 4 C experiments were used after preincubation at 20 or 28 C for 1 h. This pretreatment did not change the T3 binding activity.
FIG. 1. Effect of temperature on T3 binding to serum proteins or purified TTR. Proteins were incubated with 0.1 nM [125I]T3 in the presence of various concentrations of unlabeled T3 for 1 h at 4 (closed circles), 20 (closed triangles), and 28 C (closed squares). Data are presented as Scatchard plots. Broken lines represent the Scatchard plots resolved into two lines showing the high-affinity and low-affinity sites. A, Serum proteins obtained from the stage X–XV tadpoles; B, purified recombinant TTR. Each point represents the mean of triplicate determinations. These experiments were repeated at least three times.
TABLE 1. Effects of temperature on T3 binding to tadpole plasma proteins and on T3 uptake into tadpole RBCs
Similar results were obtained from the Scatchard analysis of T3 binding to the purified recombinant bTTR (Fig. 1B). The Kd values of bTTR at 20 and 28 C, which were four times greater than those for the high-affinity sites of serum proteins, were not significantly different from that determined at 4 C, whereas the MBC values at 20 and 28 C were 1.7 times less (P < 0.05) and 2.0 times less (P < 0.01) than that determined at 4 C, respectively (Table 1).
In previous studies (19, 28), it was demonstrated that the amount of TTR in plasma and its mRNA in liver decreased during the climax stages of spontaneous metamorphosis. However, it is not known whether the expression of the ttr gene is controlled by a developmentally programmed process or is down-regulated by TH. Premetamorphic tadpoles at stages X–XV, collected during summer (panel A) and winter (panel B), were bred in dechlorinated tap water with or without 10 nM T3 for 0–6 d at 4 or 28 C. The amount of TTR mRNA in liver did not change significantly with T3 treatment at either 4 or 28 C in both summer and winter tadpoles, suggesting that the expression of the ttr gene is not down-regulated by T3-treatment for at least 6 d (Fig. 2, A and B). The amount of TTR in sera, prepared from these same tadpoles, did not change with T3-treatment at both temperatures, although slightly higher amount of TTR was found in sera from winter tadpoles bred at 28 C (Fig. 2, C and D).
FIG. 2. Effects of temperature on the amounts of TTR mRNA in liver and of TTR in serum. Stage X–XV tadpoles were collected in summer (A and C) and winter (B and D) and then bred in dechlorinated tap water with or without 10 nM T3 for specified periods at 4 and 28 C. A and B, Total RNA was extracted from the stocked frozen livers of the two groups at the same time, and was analyzed by Northern blot hybridization (10 μg/lane) with digoxygenin-labeled cRNAs for TTR. The quantity of RNA loaded in each lane was standardized using ethidium bromide staining of rRNAs. Positions of TTR mRNA and 28S and 18S rRNAs are shown on the left. C and D, Blood was collected from the same tadpoles that were used for Northern blot analysis. Serum was prepared as described. Two μl of the serum was mixed with the same volume of 2x SDS sample buffer, and the mixture was immediately boiled for 5 min under reducing conditions. After SDS-PAGE, the immobilized proteins were immunoblotted with a polyclonal antibody against the purified bTTR as described. These experiments were repeated two times with similar results.
Temperature-sensitive T3 uptake into the tadpole RBCs
To examine whether the T3 uptake system is a temperature-dependent process, a time course of [125I]T3 uptake into tadpole RBCs was investigated. [125I]T3 uptake was linear up to 6 min at 4 C and 3 min at 23 C (Fig. 3A). Uptake was saturable. Nonspecific uptake at 4 and 23 C, measured in the presence of 5 μM unlabeled T3, was 36–43% and 17–21% of total [125I]T3 uptake, respectively. Approximately 11% of the [125I]T3 added to the incubation medium was taken up by the RBCs after 9 min at 23 C by a saturable process. However, only 2% of the [125I]T3 added was taken up by the RBCs after 9 min at 4 C.
FIG. 3. Effect of temperature on [125I]T3 uptake into tadpole RBCs. A, Time course of T3 uptake by tadpole RBCs. RBCs were incubated with 0.1 nM [125I]T3 at 4 (open circles) and 23 C (open squares) for indicated time periods. Nonsaturable cell-associated [125I]T3 was measured in the presence of 5 μM unlabeled T3 at 4 (closed circles) and 23 C (closed squares). Each point represents the mean ± SEM of triplicate determinations. Deviations less than the size of the symbols are not shown. B, Hanes plots of T3 uptake. The initial velocity (Vi) of [125I]T3 uptake was measured after 2 min incubation with increasing unlabeled T3 concentrations (S) (0–500 nM) at 4 (closed circles) and 23 C (closed squares). The graphs are drawn after subtraction of the amounts of nonsaturable cell-associated T3. Each point represents the mean of triplicate determinations. Kinetic constants were obtained by linear regression. The abscissa intercepts of the lines correspond to –Km, the ordinate intercepts to Km/Vmax and the slopes to 1/Vmax. These experiments were repeated four to six times.
In the following experiments, the Vi values of T3 uptake were measured routinely after a 2-min incubation at 4 or 23 C. The curves obtained in the presence of 5 μM unlabeled T3 did not extrapolate to zero, indicating an instantaneous and unsaturable adsorption of T3 to the RBC membrane. The Km and Vmax for T3 were determined by plotting S/Vi as a function of T3 concentration (S). Hanes plots of the data were linear, yielding a Km of 199 nM and a Vmax for transport of 207 fmol/min/106 cells at 4 C, and a Km of 169 nM and a Vmax for transport of 2310 fmol/min/106 cells at 23 C (Fig. 3B and Table 1). Low temperature decreased the Vmax value by 11-fold but did not significantly affect the Km value.
Effect of temperature on intracellular transport of T3 in tadpole RBCs
To discover whether an intracellular T3 transport system in tadpole RBCs showed temperature dependency, time courses of T3 uptake into cells and of T3 incorporation into nuclei were investigated at 4, 20, or 28 C. The amount of cell-associated T3 reached a plateau after 6 h at 20 C (Fig. 4A) and after 3 h at 28 C (Fig. 4C), and it continued to increase even after 5 d at 4 C (Fig. 4C). Similar results were obtained for T3 incorporation into nuclei at these temperatures. Figure 4, B and D, shows the ratios of T3 uptake into cells to T3 incorporation into nuclei, at 4, 20, or 28 C, as a function of the incubation time. The ratio reached a plateau by 2 d at 28 C, by 1 d at 20 C and by 2–5 d at 4 C. The value of the ratio when it reached a plateau was similar for each temperature, 13–16%. It suggests the absence of a temperature-sensitive process in the intracellular T3 transport system in tadpole RBCs, and that a significant amount of T3 is transported into the nucleus even at 4 C.
FIG. 4. Time courses of [125I]T3 uptake into RBCs and of [125I]T3 incorporation into nuclei at 4, 20, and 28 C. RBCs (4.5 x 106 cells, stage XV) were incubated with 0.2 nM [125I]T3 in the presence of 1% tadpole plasma (stage XV) for the indicated periods (A and C). The cell-associated [125I]T3 at 4 (open circles), 20 (open triangles), and 28 C (open squares), and the nuclei-incorporated [125I]T3 at 4 (closed circles), 20 (closed triangles), and 28 C (closed squares), were determined as described. Each point represents the mean ± SEM of triplicate determinations. B and D, The ratio of the amount of [125I]T3 incorporated into the nuclei to the amount of [125I]T3 associated with RBCs is plotted against the incubation time: 4 (closed circles), 20 (closed triangles), and 28 C (closed squares).
Effect of temperature on T3 binding to LBDs of bTRs
Recombinant GST-bTR (Fig. 5A) and GST-bTR? (Fig. 5B) LBD fusion proteins were incubated with 0.1 nM [125I]T3 for the indicated time periods at 4, 20, or 28 C. T3 binding to the recombinant proteins was saturable. Nonspecific binding was less than 26% of total binding at these temperatures. The T3 binding activity of GST was negligible when purified GST, which was expressed from pGES-6p-3 in E. coli, was substituted for the GST-bTR LBD fusion proteins in the assay. T3 binding to bTR and ? LBDs increased gradually and reached a plateau within 1.5 h at 4 and 20 C. No significant difference was found in the amount of bound [125I]T3 between samples incubated at 4 and 20 C. [125I]T3 binding activity of both bTR LBDs at 28 C was lost due to severe protein denaturation especially after 15 min incubation. This suggests that T3 binding to bTRs is a temperature-independent process at least in the range of 4–20 C. Scatchard analysis revealed a single class of T3 binding sites with Kd values, at 4 C, of 0.25 ± 0.02 nM (n = 3) for bTR LBD (Fig. 5A, inset) and 0.76 ± 0.20 nM (n = 3) for bTR? LBD (Fig. 5B, inset), which were one order of magnitude greater than those for in vitro-translated bTRs (17, 18).
FIG. 5. [125I]T3 binding to GST-fused LBDs of bTRs. A and B, GST-bTR LBD (A, 50 ng/250 μl) or GST-bTR? LBD (B, 150 ng/250 μl) fusion proteins were incubated with 0.1 nM [125I]T3 in the presence or absence of unlabeled 1 μM unlabeled T3 for the indicated periods at 4 (closed circles), 20 (closed triangles), and 28 C (closed squares). Nonspecific binding was subtracted from total binding. Each point represents the mean ± SEM of triplicate determinations. Deviations less than the size of the symbols are not shown. Inset, Purified recombinant proteins were incubated with 0.03–1.0 nM [125I]T3 in the presence or absence of 1 μM unlabeled T3 for 1.5 h at 4 C. Data were represented as Scatchard plots. Each value is the mean of triplicate determinations. These experiments were repeated at least three times.
Effect of temperature on T3-activated genes
To examine whether low temperature affects the activation of gene transcription for the urea cycle enzyme OTC by T3, Northern blot analysis was performed (Fig. 6). The premetamorphic tadpoles, which were collected in summer (panel A) and winter (panel B), were bred in dechlorinated tap water with or without 10 nM T3 at 4 or 28 C for 0–6 d. The OTC mRNA was up-regulated in T3-treated tadpole livers by 2–4 d at 28 C. Its amount increased 2- to 3-fold by d 6 at 28 C; however, no change in OTC mRNA levels was detected at 4 C even in the 6-d T3-treated tadpoles. There was no significant difference in the amount of T3-induced OTC mRNA at 28 C between the summer and winter tadpoles, suggesting that the winter tadpole livers are as TH sensitive as the summer tadpoles livers. The genes encoding the urea cycle enzymes in tadpole liver are delayed secondary TH response genes (35, 36). Therefore, the major temperature-sensitive site in the tadpole TH-signaling pathway might exist before the delayed secondary TH response genes are transcribed.
FIG. 6. Effects of temperature on the delayed secondary TH-responsive gene transcript for OTC in tadpole liver. Stage X–XV tadpoles were collected in summer (A) and winter (B) and then bred in dechlorinated tap water with or without 10 nM T3 for indicated periods at 4 and 28 C. Total RNA was extracted at the same time from the stocked frozen livers of the two groups and was analyzed by Northern blot hybridization (10 μg/lane) with digoxygenin-labeled cRNAs for OTC. The quantity of RNA loaded in each lane was standardized using ethidium bromide staining of rRNAs. Positions of rRNAs (28S and 18S) and mRNA for OTC are shown on the left.
Next, the effect of temperature on the activation of early primary TH response genes was examined. TR? is an early primary TH response gene in tadpoles (35) and its expression is up-regulated in tadpole liver during spontaneous and TH-induced metamorphosis (37, 38). The TR gene is also an early primary TH response gene in bullfrog tadpole RBCs (39, 40) and is up-regulated by TH in vivo and in vitro (39, 41). TR and ? genes were confirmed as early primary TH response genes by showing cycloheximide-insensitive T3-induction of their transcripts in tadpole RBCs (left part of Fig. 7A) and liver (left parts of Fig. 7, B and C). The TR? transcript was not detected in tadpole RBCs by real-time PCR, in agreement with a previous report (17).
FIG. 7. Effects of temperature on the early primary TH-responsive gene transcripts for TRs in tadpole RBCs and liver. In in vivo studies, tadpoles were treated with or without 10 nM T3 for 4–5 d at 4, 20, and 28 C, whereas, in the in vitro studies, the isolated RBCs were treated with and without 10 nM T3 for 2 d at 4 and 28 C. RNAs were prepared from RBCs (A) and liver (B and C), and TR (A and B) and ? (C) transcripts were analyzed by real-time PCR. Some tadpoles were treated with 1.0 μM (closed bars) cycloheximide (Chx) in the presence or absence of 10 nM T3 for 4 d at 28 C, and some RBCs were cultured with 0.5 μM (hatched bar) and 3.0 μM Chx (cross-hatched bar) in the presence or absence (open bar) of 10 nM T3 for 2 d at 28 C. Vertical axis represents the ratio of TR transcripts of T3-treated samples to those of T3-untreated samples. Each number of repeated experiments was shown in parentheses. Each value is the mean ± SEM. Statistically significant differences were: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
The amount of the TR mRNA was 7–19 times higher in T3-treated RBCs than in T3-untreated RBCs when stage X–XV tadpoles were immersed for 4–5 d in chlorinated tap water with or without 10 nM T3 at 20 or 28 C (Fig. 7A). The same result was obtained when stage X–XV tadpole RBCs were cultured for 2 d in 70% Leibovitz’s L-15 culture medium with or without 10 nM T3. Low temperature (4 C) almost completely abolished the T3 responses found at 20 and 28 C in both the in vivo and in vitro studies. When tadpole RBCs were cultured with T3 in vitro at 4 C even for 5 d, the amount of TR transcript did not increase (data not shown). Similar experiments were conducted examining the low-temperature effect on TR and ? mRNAs in tadpole livers (right parts of Fig. 7, B and C). The amount of the TR mRNA was seven to nine times higher in T3-treated tadpole livers than in T3-untreated ones, at 20 and 28 C in the in vivo study, which was similar to the response found in RBCs. The amount of the TR? mRNA increased approximately 30 and 200 times in T3-treated tadpole liver at 20 and 28 C, respectively. The induction of TR gene transcription in liver by T3 was almost completely suppressed by exposing tadpoles to 4 C for 4–5 d. The in vivo T3 response at 20 C was comparable with that found at 28 C for the amount of TR transcript, and the amount of TR? transcript at 20 C was one order of magnitude greater than that found at 4 C and one order of magnitude less than that found at 28 C. Therefore, it is likely that the most critical site for the low-temperature arrest of T3-induced metamorphosis exists in T3-signaling pathway in the nucleus, probably at the induction of the early primary TH response genes.
Discussion
In this study, we investigated the effect of temperature on the TH-signaling pathway during T3-induced tadpole metamorphosis and found that the low-temperature arrest of amphibian metamorphosis was most likely due to a TH-dependent process within the nucleus that occurred before or during the transcriptional activation of the tr genes. T3 binding to serum proteins and cellular T3 uptake were also temperature dependent and may slow the rate of T3 incorporation into the nucleus at low temperatures resulting in the delayed progress of metamorphosis. TH binding activity in R. catesebeiana plasma dramatically changes during metamorphosis. Miyauchi et al. (42) demonstrated that high-affinity sites for T3 rather than for T4 were present in plasma at the premetamorphic stage, and that their amount decreased after the beginning of metamorphic climax stage (stage XX) and disappeared at the end of metamorphic climax stage (stage XXV). These changes in the TH-binding activity of plasma were shown to be due to changes in the concentration of a major TH-binding protein, later identified as TTR (22, 28). bTTR was found to have 102 times higher affinity for T3 than for T4 (22, 43). Conversely, human TTR has 10 times higher affinity for T4 than for T3 (44). This suggested that TTR has a regulatory role in the free concentration of tadpole plasma T3.
Low temperature increased the MBC of high-affinity sites of plasma proteins for T3 with no significant differences in the Kd values (Fig. 1A and Table 1). Similar results were obtained when we used the purified recombinant bTTR instead of tadpole serum (Fig. 1B and Table 1), which was in agreement with a previous report (22). It is likely that low temperature affects the tertiary structure of TTR subunits, subunit-subunit interactions or a dimer-dimer interaction making a higher affinity T3-binding pocket, although we cannot fully explain how low temperature changes the apparent MBC. Quantitative analysis of temperature sensitivity using the ratio of MBC/Kd at different temperatures (see Table 1) revealed that the ratio (4 C/20 C) was 4.6 for tadpole serum and 2.2 for the purified bTTR, whereas the ratio (20 C/28 C) was 0.7 for tadpole serum and 1.2 for the purified bTTR. This result suggests that tadpole plasma creates a reserve of T3 at 4 C three to five times greater than that at 20 and 28 C, and that this reserve is predominantly created by the major T3-binding protein in tadpole plasma, TTR. Therefore, TTR might affect plasma TH homeostasis in a temperature-dependent manner, altering the equilibrium of free T3/ total T3 in plasma that determines the amount of T3 available for target cells (45, 46). Frieden et al. (6) indicated that a decrease in breeding temperature from 30 to 20 C slightly slowed the progress of tadpole metamorphosis, determined by tail length and urea-N/NH3-N excretion, and that breeding tadpoles at 15 and 10 C dramatically decreased the progress of tadpole metamorphosis. This temperature dependency may be explained in part by the thermo-sensitive characteristic of high-affinity sites for T3 present in tadpole plasma. Greater T3 binding to plasma proteins in tadpoles may decrease the free T3 concentration in plasma when they are exposed to low temperature. It is doubtful whether this decrease is compensated by a negative feedback loop of the hypothalamus/pituitary/thyroid system as found in homeotherms, because low temperature also decreases the activity of the thyroid in tadpoles (47, 48).
The T3-treated summer tadpoles maintained at low temperature had the same amount of serum TTR as those maintained at 28 C (Fig. 2C). The T3-treated winter tadpoles maintained at 28 C had a slightly higher concentration of serum TTR than those maintained at 4 C (Fig. 2D). The amounts of TTR mRNA in the liver did not change significantly during the 6 d of T3 treatment (Fig. 2, A and B). It was previously found that acclimatization (winter vs. summer) and thermal acclimation for short periods in the laboratory resulted in somewhat different hormonal responses in bullfrog tadpoles (48). However, in our study, the amount of TTR in serum and of TTR mRNA in liver did not vary between samples from acclimatization and thermal acclimation. It is therefore unlikely that an increase in the concentration of plasma TTR causes the low-temperature arrest of amphibian metamorphosis.
[125I]T3 uptake experiments in the absence of serum demonstrated that T3 uptake into tadpole RBCs was a temperature-dependent process (Fig. 3). The Vmax value at 4 C was 11 times less than that at 23 C, whereas there was no significant difference in Km values at 4 C and at 23 C. Because the time required for the cellular T3 level to reach equilibrium was longer at 4 C than at 23 C, the T3 uptake system may contribute in part to the delayed progress of T3-induced metamorphosis at low temperature. The Km values for T3 uptake, 169 nM at 23 C and 199 nM at 4 C, were in the range of those for RBCs from the same species (49) or other species (50, 51, 52, 53). The ratio of Vmax/Km at 23 C to Vmax/Km at 4 C was approximately 13 (the ratio per 10 C difference, [Vmax/Km]10 = 3.9). The [Vmax/Km]10 value for bullfrog RBCs was similar to those reported for rat RBCs, rat hepatocytes and human RBCs: 40 at 37 C/at 0 C ([Vmax/Km]10 = 2.7) (50), 50 at 25 C/at 0 C([Vmax/Km]10 = 4.7) (54) and 26 at 37 C/at 0 C ([Vmax/Km]10 = 2.4) (51), respectively. Therefore, temperature-sensitive [125I]T3 uptake into RBCs is a characteristic common to the above animal cells rather than a characteristic specific to amphibian cells.
We found that a significant proportion of [125I]T3 taken up into RBCs in vitro was transported into the nucleus at 4, 20, and 28 C and that the amount of [125I]T3 incorporated into the nucleus did not differ at these temperatures after incubation for 2–5 d. Translocation, however, was slower at 4 C than at 20 and 28 C (Fig. 4), which was in agreement with previous in vivo (12, 14, 15) and in vitro studies (55). It is unlikely that the intracellular transport of TH plays an important role in the low-temperature arrest of amphibian metamorphosis. The amounts of T3 incorporated into nuclei at 4 C did not reach a plateau even after incubation for 5 d; however, the ratio of the amount of T3 incorporated into the nucleus to the amount of T3 taken up into RBCs reached a plateau at 1–5 d. This suggests that a limiting process responsible for the slow T3 incorporation into nucleus at 4 C would be a cellular T3 uptake system but not an intracellular T3 transport system into nucleus.
We found no temperature dependency during a time course study of in vitro [125I]T3 binding to purified bTRs at 4 or 20 C (Fig. 5), although their T3 binding activity at 28 C was inhibited during incubations longer than 15 min. It may be concluded that T3 binding to bTRs is not critical in the low-temperature arrest of amphibian metamorphosis. However, our result does not exclude the possibility of other temperature-dependent factors that may interact with the TH-signaling pathway in the nucleus, such as the cold-inducible coactivator for peroxisome proliferator-activated receptor (PGC-1), which is dramatically induced in brown fat and skeletal muscle of mice upon cold exposure (56). Kusuda et al. (57) found an increase in affinity of TR for T3 in the nuclear extract obtained from rats exposed to 4 C for 6 h. This suggested either a temperature-dependent modification of TRs or an interaction of TRs with another nuclear factor. Further investigation of nuclear proteins or factors modifying or complexing with TR, such as retinoid X receptor, that may transactivate TR or change its affinity for TH in a temperature-dependent manner is required.
The transcriptional activity of the delayed secondary TH response gene otc and of the early primary TH response genes tr and tr? were not altered in vivo in response to T3 at 4 C but were altered at 20 or 28 C. This finding was confirmed in the in vitro-cultured RBCs in the absence of serum. These results indicated that the low-temperature-sensitive process exists before or during the transcriptional activation of early primary TH response genes, and that this process was not mediated by extracellular signaling molecules, like in the case of the coactivator for peroxisome proliferator-activated receptor (PGC-1) in which noradrenaline is involved (58). The temperature sensitivity of tr? gene was greater than that of tr gene, especially at 20 and 28 C. Low temperature may differentially affect T3-signaling pathways mediated by TR and TR? at the metamorphic stages.
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