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Endocrinology Vol. 143, No. 4 1538-1544
Copyright © 2002 by The Endocrine Society

RECEPTORS

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Cytosolic T3 Binding Protein as a Regulator for T3-Mediated Transactivation

Jun-ichirou Mori, Satoru Suzuki, Mutsuhiro Kobayashi, Takeshi Inagaki, Ai Komatsu, Teiji Takeda, Takahide Miyamoto, Kazuo Ichikawa and Kiyoshi Hashizume

Department of Aging Medicine and Geriatrics (J.-i.M., S.S., T.I., A.K., T.T., T.M., K.I., K.H.), Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390-8621, Japan; and Iida Municipal Hospital (M.K.), 438, Yawata, Iida, 395-0814, Japan

Address all correspondence and requests for reprints to: Satoru Suzuki, M.D., Ph.D., Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, Nagano, 390-8621, Japan. E-mail: . soutaro{at}hsp.md.shinshu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nicotinamide adenine dinucleotide phosphate (NADPH)- dependent cytosolic T3 binding protein (CTBP) plays a role in the regulation of nuclear transport of T3 in vitro. However, it is not known whether CTBP regulates the T3 action. In this study, we examined the effects of CTBP on cellular translocation of T3 and on transcriptional activation using established CTBP-expressing CHO or GH3 cells.

The expression of CTBP increased cellular and nuclear uptake of T3 in the CTBP-expressing cells. The efflux rate was decreased by induction of CTBP. Efflux from nuclei also inhibited by induction of CTBP.

Expression of CTBP suppressed the T3-regulated luciferase activity in GH3 cells. Suppression was observed to be related to the expression level of CTBP. T3 induction of rat GH mRNA was lower in the cells expressing CTBP than that in CTBP-null cells.

These results suggest that CTBP regulates the T3-induced gene expression, with which an increase in the nuclear content of the T3 is associated. Because we observed that a part of CTBP could be transported into nuclei and that acceptor protein for CTBP is present in nuclei as previously reported, interaction of CTBP with certain proteins, including transcription factors or nuclear T3 receptor, may contribute to the regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN OUR PREVIOUS studies, we demonstrated that nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cytosolic T3-binding protein (CTBP) plays an important role in the regulation of nuclear transport of T3 in vitro (1). Activation of CTBP with NADPH inhibits the nuclear entry of T3, whereas activation with NADP enhances the entry (2, 3). Further, we observed that acceptor protein for NADP-activated CTBP is present in nuclei (3, 4). These observations suggest that CTBP plays roles not only in regulation of nuclear T3 entry but also in regulation of T3-induced gene expression. However, it is not yet known whether CTBP regulates the T3 action.

In this study, we examined the effects of CTBP on cellular translocation of T3 and on transcriptional activation induced by T3 using CTBP-expressing CHO and GH3 cells. To determine whether CTBP affects the T3-induced transactivation, we transfected with thyroid hormone response element (TRE)-fused reporter gene and measured the reporter activity after adding T3 in CTBP-expressing GH3 cells. Further, we examined the effect of CTBP on expression of rat GH mRNA which is known as one of the T3 response genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of human CTBP and construction of plasmids
Full length of human CTBP cDNA was amplified by PCR using the following primers: sense, 5'-AGACTGAGGTTAGAAGGCACAGGTGGC-3'; antisense, 5'-CCTCAAGCATCCATCTCAACATCAAGT-3'. Human fetal brain Marathon Ready cDNA (CLONTECH Laboratories, Inc., Palo Alto, CA) was used as a template. Amplified fragment was sequenced and checked fidelity of the sequences (GenBank accession no. U85772).

The amplified fragment was cloned into TA cloning site of pT7-Blue (Novagen, Madison, WI). BamHI-SalI fragment of pT7-CTBP was ligated into pcDNA3.1 (Invitrogen, Carlsbad, CA), which is available for the expression in mammalian cells and in vitro transcription and translation (pcDNA-CTBP).

Preparation and establishment of CTBP-expressing CHO cell line (CPC45) and GH3 cells
CHO-K1 cells, which do not possess NADPH-dependent T3 binding activity, were purchased from ATCC (Manassas, VA). The cells were transfected with pQBI-CTBP by calcium-phosphate method. After selection with 400 µg/ml G418, the cells were cloned (CPC45). Because parental CHO-K1 cell does not possess NADPH-dependent T3 binding activity, expression of CTBP was confirmed by the assay for NADPH-dependent T3 binding. pcDNA-CTBP or pQBI-25-fc2 plasmid (Quantum Biotechnology Inc., Québec, Canada), which induces green fluorescent protein (GFP), was transfected into GH3 cells by electroporation as previously described (5). The clones were selected by the incubation with 400 µg/ml G418. The expression of CTBP in CTBP-transfected cells was confirmed by NADPH-dependent T3 binding and Western blotting. A parental GH3 cell and a series of GH3-CTBP cells were cultured in DMEM without and with 100 µg/ml G418, respectively.

Preparation of polyclonal antibodies to CTBP
Synthetic peptides containing a part of human CTBP amino acid sequence were used in the immunization. Amino acid sequence (CNRTKENAEKFADTV) was chosen because of its high antigenicity index, determined by Epitope Adviser (Fujitsu, Shizuoka, Japan). The peptide was conjugated to form hapten with keyhole limpet hemocyanin. Antibodies were raised in rabbits obtained from Takara Inc. (Ohtsu, Japan). The antibody was purified by affinity column chromatography with the immunized peptide.

Western blotting
Cells were washed twice in ice-cold PBS and lysed by adding lysis buffer (0.05% SDS, 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.2, containing 1 mM phenylmethylsulfonyl fluoride). The lysate was boiled for 5 min, and stored at -80 C. Proteins were resuspended in lysis buffer containing 2% SDS, and samples were separated in 10% SDS-PAGE gels and transferred to immobilon-P membranes (Millipore Corp., Bedford, MA) by semidry electrophoretic transfer (Bio-Rad Laboratories, Inc., Richmond, CA). The membranes were blocked with TBS-T (100 mM NaCl, 10 mM Tris-HCl pH 7.5, and 0.1% Tween 20) containing 1% skim milk. Detection was done by measuring the enhanced chemiluminescence using a horseradish peroxidase-coupled mouse-antigoat IgG antibody (Amersham Pharmacia Biotech, Arlington Heights, IL).

Studies of uptake and efflux of T3
T3 uptake and diffusion were estimated by the method as previously described (6) with minor modification. The cells were grown in 24-well plates at 37 C in a humid atmosphere of 5% CO2 in air with exchanging the media every other day. After obtaining the late logarithmic phase of growth, the cells were cultured in the fresh media containing 10% resin-stripped FCS (7) for 24 h. After the incubation, the media were changed to the same media without FCS. Two times exchange of the media depleted the cells of the measurable amount of T3 (6), then 70 pM [125I] T3 (3,300 µCi/µg, Dupont NEN, Boston, MA) was added. After incubation for indicated times, the media were aspirated. After 1 min incubation with ice-cold Dulbeco’s PBS, it was replaced with fresh ice-cold PBS. After repeating this procedure three times, 2 ml of 0.25 M sucrose, 1 mM MgCl2, and 20 mM Tris-HCl (pH 7.4) containing 0.5% Triton X-100 was added. They were incubated for 10 min to lyse the cells. The suspension was divided into two 0.8-ml aliquots; one was for measurement of whole cell T3 uptake and the other was for measurement of nuclear uptake, respectively. The nuclear uptake was determined by measuring the radioactivity of the pellet obtained by centrifugation of the suspension at 1,500 x g for 10 min. Before measurement, the pellet was washed two times. Radioactive T3 uptake in the presence of 1 µM unlabeled T3 (nonspecific uptake) was less than 4% of total uptake.

Diffusion of T3 was studied by using the cells in which 70 pM [125I] T3 was incubated for 24 h as described for the uptake study. After washing two times with warmed resin-stripped media, the cells were incubated with fresh resin-stripped media. The contents of radioactivity in whole cells and in nuclear pellet were measured by the same procedures for the uptake study.

T3 binding assay
T3 binding activity of CTBP was measured as previously described (8). CTBP containing fractions were incubated with TED buffer (10 mM Tris-HCl, pH 7.4, containing 0.5 mM EGTA, and 1 mM DTT) to make a final volume of 200 µl. Incubation was performed in the absence or presence of 100 µM NADPH. After appropriate incubation times, bound and free hormones were separated by dextran-coated charcoal. The dissociation constant and the maximal binding capacity were estimated by the method of Scatchard (9). The concentration of the protein and DNA were measured by the methods of Bradford (10) and of Burton (11), respectively.

Nuclear T3 binding was measured as previously described (12). The characteristics of T3 binding was determined by Scatchard analysis. Radioactive T3 binding to nuclei prepared from GH3 cells was displaced by unlabeled T3 or its analogues. Triiodo-L-thyroacetic acid was the most potent to displace, which was followed by T3, triiodo-D-thyronine, and L-T4.

Luciferase assay
GH3 cells and cloned cells were treated with trypsin for 48 h before transfection and were plated into 10-cm dishes. Twenty-four hours before transfection, the medium was changed to DMEM with 10% serum pretreated with resin. The cells were electroporated with 10 µg 2xPAL-TK-Luc (13), and 1.0 µg pSV-ß-galactosidase vector (Promega Corp., Madison, WI) as previously described. The cells were distributed into 24 wells with DMEM containing serum pretreated with 10% resin. Twelve to 16 h after incubation, the medium was changed to fresh DMEM containing 10% serum pretreated with resin and various concentrations of T3. After additional 24 h incubation, the cells were harvested. Luciferase activity was determined by the Promega Corp. Luciferase Assay System according to the protocol using Berthold Lumat (E.G. & G., Berthold, Evry, France). ß-Galactosidase activity was measured by the method previously described (5), and all luciferase data were corrected for ß-galactosidase activity to account for variations in transfection efficacy.

RNA analysis
Total RNA corresponding to 106 cells was extracted and eluted by using Midi kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. The cDNA fragments of rat GH and elongation factor 1{alpha} (EF1) (14) were amplified by reverse transcriptase PCR using GH3 cell-derived mRNA as template. The primers used were 5'-ATGGCTGCAGACTCTCAG-3' and 5'-GAAAGCACAGCTGCTTTC-3' for rat GH, and 5'-TCCCAGTGGTCATCACCATG-3' and 5'-ATGGACAATTTGGCACCT-3' for EF1. After cloning to pGEM-T easy vector (Promega Corp.), fidelity of each fragment was confirmed by sequencing. The probes were obtained after digestion with EcoRI. Autoradiographic signals were quantitated by a bio-imaging analyzer BAS-1500 (Fuji Photo Film Co., Ltd., Kanagawa, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of expression of CTBP on cellular and nuclear T3 uptakes
Three different clones (clones 3, 7, and 8) that can express CTBP were obtained. As shown in Fig. 1Go, high, intermediate, and low expressions were observed in clones 8, 7, and 3, respectively. The maximal T3 binding capacity (MBC) and affinity constant (Ka) for T3 were estimated in each cell line. As shown in Table 1Go, MBC, which was determined in the presence of NADPH, was related to the levels of CTBP expressed. In contrast to the MBC, Ka was not significantly different among each cell line.



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  		Figure 1. Expression of CTBP in established cell lines derived from GH3 cells. GH3 cells were transfected with cDNAs coding for CTBP. Twenty-four hours after incubation, media were changed which contained 400 µg/ml of G418 to select the CTBP-expressing clones. Cell extracts from the obtained clones were submitted to Western blot analysis with an antibody (CTBP168). The intensity of the band was measured by the densitometer. The value obtained from the lane of GH3 was subtracted from that of each cell line. The relative expression levels were calculated from the data in which the expression level in clone C3 was defined as 1.

 

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  		Table 1. MBC and Ka of NADPH-dependent T3 binding in the CTBP-expressing cell lines and in various rat tissues

 
Effect of CTBP expression on T3 uptake was examined in GH3 cells, which are abundant in nuclear T3 receptors (15) but do not express CTBP. The uptake was estimated by measuring the radioactivity after exposure of 70 pM 125I T3 in parental and series of CTBP-expressing cells. The maximal uptake in whole cell was larger in CTBP-expressing cells than that in CTBP-null cells. Subtraction of CTBP-independent T3 uptake revealed that the level of expression of CTBP well correlated to the level of T3 uptake (Fig. 2Go, left panel). Not only whole cell uptake but also nuclear uptake was enhanced by expressing CTBP. The increase in nuclear uptake was also dependent on the levels of CTBP expression (Fig. 2Go, right panel). The MBCs of CTBP positively related to the uptakes in whole cells and nuclei of established cell lines (Fig. 3Go).



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  		Figure 2. Effects of CTBP expression on cellular and nuclear T3 uptakes. A, Whole cell uptake of T3. CTBP-expressing clones (clones 3, 7, and 8) were obtained as described in Fig. 1Go. The cells were preincubated in serum free media for 24 h to deplete T3 endogenously presents in the cells, then 70 pM [125I] T3 were added to each incubation. After appropriate incubation times, radioactivities incorporated into the cells were measured. The CTBP-dependent uptakes which were obtained by subtraction of the radioactivity incorporated into GH3 cells (CTBP-independent uptake) are shown. Inset shows the total incorporation of radioactive T3 in each cells. Abscissa shows the time after adding radioactive T3. Each datum indicates the mean of triplicate determinations. B, Nuclear uptake of T3. After incubation of the cells with 70 pM [125I] T3 as described in A, the nuclear fractions were prepared as described in Materials and Methods. The CTBP-dependent nuclear uptake was obtained by subtraction of CTBP-independent nuclear uptake, which was obtained in CTBP nonexpressing GH cells, from the uptake obtained in each CTBP-expressing cells as mentioned in (A). Inset shows the total nuclear uptake into each cells. Each datum indicates the mean of triplicate determinations, which did not vary by more than 5%.

 


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  		Figure 3. Relation between MBC and T3 uptake in CTBP-expressing cell lines. MBC and T3 uptake demonstrated in Tables 1Go and 2Go, respectively, were plotted. Closed squares and circles indicated the mean values of whole cellular and nuclear uptake, respectively. The bars represented SD. Dotted lines were constructed by linear regression analysis.

 
To clarify the effect of CTBP expression on T3 uptake in other cell line, we established CTBP-expressing CHO cells line. In contrast to the GH3 cells, which are derived from rat pituitary gland and are abundant in nuclear T3 receptors (15) but are quite low in NADPH-dependent T3 binding activity, CHO cells possess neither nuclear T3 binding nor NADPH-dependent T3 binding. As shown in Table 2Go, the specific whole cell uptake was larger in CTBP-expressing CHO cells than that in CTBP-null CHO cells. Thus, it is possible that CTBP plays a role in the retention of whole cell T3 uptake not only in GH3 but also CHO cells.


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  		Table 2. Total uptakes of T3 in CTBP-CHO cells and a series of CTBP-expressing GH3 cells

 
Effects of CTBP expression on T3 effluxes
Export of T3 from whole cell to media and from nuclei to cytoplasm was examined in GH3 and CTBP-expressing cloned GH3 cells. The cells were incubated in the presence of radiolabeled T3 for 48 h, and the export was estimated by measuring contents of radiolabeled T3 in whole cells (Fig. 4Go inset, left panel) and in nuclei (Fig. 4Go inset, right panel) after deprivation of T3 from media. The first-order dissociation kinetics was calculated from data obtained within 7.5 h from the beginning of incubation. The rate of whole cellular efflux was significantly decreased by expressing CTBP. The rate of nuclear efflux was also decreased by expressing CTBP. Calculated half life of nuclear T3 content was longer in the cells expressing CTBP than that observed in CTBP-null cells (Table 3Go). The prolongation of the half-life was correlated to the expression level of CTBP (Fig. 4Go).



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  		Figure 4. Effects of CTBP expression on cellular and nuclear T3 efflux. MBC and half-life demonstrated in Tables 1Go and 3Go, respectively were plotted. Open squares and circles indicated the mean values of half-life obtained from the whole cells and the nuclei, respectively. The abscissa represents semilogarithmic plot of MBC. The ordinate indicates half-life. Inset, Cells were incubated with 70 pM [125I]T3 for 48 h in resin-stripped media. After further incubation for appropriate times in the absence of radiolabeled T3 in resin-stripped media, cells were harvested, and radioactivities in cell homogenate (left panel) and in nuclear pellet (right panel) were measured. Closed square, circle, triangle, and cross represent the radioactivities in GH3, C3, C7, and C8, respectively. The abscissa indicates the time after depletion of radiolabeled T3, and the ordinate represents semilogarithmic plot of radioactivity. Each datum indicates the mean of triplicate determinations, which did not vary by more than 5%.

 

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  		Table 3. Half-life of [125I] T3 contents in whole cells and nuclei, after deprivation of T3 from the media

 
Effect of CTBP on T3-mediated gene expression
Above results suggested that CTBP expression influences the gene expression regulated by T3 because the T3 content in nuclei could be augmented by CTBP expression. We estimated the effect of CTBP on T3-mediated transcription in CTBP-expressing and nonexpressing GH3 cell lines. We transfected TRE containing luciferase reporter gene (TREx2-TK-Luc) into GH3 cells and cloned cells (C3, C7, and C8). The levels of nuclear T3 receptor were not different among these cell lines (Table 4Go). T3 increased luciferase activity in CTBP nonexpressing GH3 cells. The luciferase activity induced by T3 was significantly lower in CTBP-expressing cells than that in nonexpressing cells. The diminishment of luciferase activity correlated to the expression level of CTBP (Fig. 5Go).


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  		Table 4. MBC and Ka of nuclear T3 receptors in the parental GH3 and CTBP-expressing cell lines

 


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  		Figure 5. Effect of CTBP expression on trans-activation induced by T3 in GH3 cells. 2xPAL-TK-Luc reporter gene and 1.0 µg pSV-ß-galactosidase vector were cotransfected into GH3 cells and cloned cells (C3, C7, and C8), and incubated with various concentrations of T3 for 24 h. After cell harvest, luciferase and ß-galactosidase activities were measured. All luciferase activities were corrected for ß-galactosidase activities. Data were expressed as fold induction by T3. Each value represents the mean ± SD of five separate determinations. Asterisk indicates statistical significance (P < 0.05). N.S., No significance.

 
Effect of CTBP on T3 response gene expression
Above results suggested that CTBP possesses suppressive effect on T3-induced mRNA expression. To evaluate the effect of CTBP on the TR activation-induced transcription, we measured the amount of rat GH mRNA induced by T3 in GH3 and the cloned cells. Twenty-four hours after culture of these cells with 10 nM T3, rat GH mRNA levels were estimated by Northern blot analysis. As shown in Fig. 6AGo, T3 increased the level of rat GH mRNA in a dose-dependent manner. The T3-induced increase in GH mRNA expression was inhibited by expressing CTBP (Fig. 6Go, A and B). To exclude the possibility that overexpressed protein may influence the T3-induced mRNA expression (nonspecific expression), we developed GFP-expressing GH3 cells, and the changes in rat GH mRNA expression levels were examined. As shown in Fig. 6Go, C and D, overexpression of GFP did not affect the expression of rat GH mRNA.



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  		Figure 6. Effect of CTBP expression on GH mRNA levels in GH3 cells. GH3 cells and cloned cells (C3, C7, and C8) were prepared as described. Twenty-four hours after incubation with DMEM with 10% resin-stripped serum, various concentrations of T3 (A) or 10 nM T3 (B) were added. After incubation for 24 h (A) or appropriate time (B), total RNAs were prepared. Eight micrograms of total RNA were analyzed by Northern blotting. Autoradiography of each blot hybridized with 32P-labeled cDNA probes of rat GH or EF1 was carried out by BAS-1500. Similar results were obtained in more than three experiments. C, pQBI-25-fc2 plasmid was transfected into GH3 cells by electroporation. The clones were selected by the incubation with 400 µg/ml G418. Cell extracts from the parental GH3 cells and the obtained clone were applied to Western blot analysis with anti-GFP antibody. D, Eight micrograms of total RNA, extracted from GH3 and GFP-GH3 cells after 24 h incubation with or without 10 nM T3, were analyzed by Northern blotting with rat GH and EF1 as probes. Similar results were obtained in more than three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously suggested that CTBP plays roles not only in the cytoplasmic T3 reservoir but also in the regulation of T3 nuclear transport or in shuttling T3 between cytoplasm and nucleus (3). The levels of expression of CTBP correlated to the MBC of T3 in CTBP-expressing GH3 cells prepared in this study. The Kas for T3 in CTBP expressed in these cells were similar to those obtained in CTBP prepared from rat tissues, indicating that the characteristics of T3-binding in CTBP expressed in GH3 cell are similar to those of native CTBP.

Although we have demonstrated the T3 binding of purified CTBP in the presence of NADPH or NADP+ in vitro (3), it was not determined whether the expression of CTBP molecule alters the T3 content in living cells. The positive co-relation between the expression of CTBP and T3 contents in established cell lines indicates that CTBP holds T3 in living cells. These data imply that physiologically, CTBP affects the tissue content of T3 in vivo.

We observed that maximal level of T3 uptake or the efflux from nucleus to cytoplasm was correlated to the expression level of CTBP in GH3 cells. These results suggest that CTBP plays a role not only in cytoplasmic reservoir but also in nuclear retention of T3. In our previous study, we observed that CTBP acceptor sites present in nuclei (4), and NADP-activated form of CTBP can accelerate the nuclear import of T3 (2). Thus, the observation that increase in nuclear uptake induced by expression of CTBP may reflect the acceleration of nuclear import induced by NADP-dependent activation of CTBP. A delayed efflux from nucleus, induced by expression of CTBP, may reflect the presence of CTBP-acceptor interaction in nucleus. We found that T3 was passively or actively transferred into nuclei of CTBP-null GH3 cells, indicating that free T3 or T3-bounded other proteins may also be transferred into nuclei. Thus, not only the expression of CTBP, but also other mechanisms may affect the nuclear content of T3 in the CTBP-expressing cells.

These observations suggest that nuclear events induced by thyroid hormone may be also influenced by expression of CTBP. As is shown, the T3-induced gene expression, evaluated by estimation of reporter gene expression, was suppressed by expression of CTBP in vitro. Further, level of rat GH mRNA, which is one of the T3-responsive genes, was decreased in GH3 cells expressing CTBP. Although we did not examine other thyroid hormone response genes, these results suggest that CTBP strongly influences the T3 action mediated through T3 receptor-TRE interaction.

Because we artificially expressed CTBP in CTBP-null GH3 cells, it was possible that the transcriptional activity was affected by the artificial expression of CTBP, namely squelching effect. T3 response, however, was not suppressed in the GFP-expressed GH3 cell line as a negative control, and physiological amount of CTBP was present in a series of CTBP-expressed cell lines, suggesting the possibilities of artificial modification may be low.

Based on the findings in this study, it is possible that nuclear content of T3 is high in nucleus of the cells expressing CTBP. Nevertheless, the T3 action was inhibited in these cells. Precise mechanism of this phenomenon could not be solved in this study. There are several possibilities to explain molecular mechanisms of the data. 1) CTBP may increase T3 catabolism in CTBP-expressing cells. 2) CTBP may increase turnover of rat GH mRNA in CTBP-expressing cells. 3) CTBP may compete for T3 binding with classic nuclear receptors. However, a noncompetitive inhibition is seen in Fig. 6Go, suggesting that the third possibility is less likely.

The fourth possibility is that CTBP may directly enter into nucleus because we could isolate the CTBP acceptor in nuclei and we observed the entry of CTBP when the protein is activated by NADP (2). We observed that CTBP molecule can make homodimer in vitro, which indicated that the protein may interact with other proteins even in nucleus (16). These considerations lead us to a hypothesis that CTBP entered into nucleus may regulate the redundant functions, including the transcriptional functions of nuclear receptor or cofactor(s) for the transactivation. However, the precise mechanism of the regulation induced by CTBP is remained to be elucidated.


   Footnotes
 
Abbreviations: CTBP, Cytosolic T3 binding protein; EF1, elongation factor 1{alpha}; GFP, green fluorescent protein; Ka, affinity constant; MBC, maximal T3 binding capacity; NADPH, nicotinamide adenine dinucleotide phosphate; TRE, thyroid hormone response element.

Received September 11, 2001.

Accepted for publication December 14, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. C. H. Friesema, J. Jansen, J.-w. Jachtenberg, W. E. Visser, M. H. A. Kester, and T. J. Visser
Effective Cellular Uptake and Efflux of Thyroid Hormone by Human Monocarboxylate Transporter 10
Mol. Endocrinol., June 1, 2008; 22(6): 1357 - 1369.
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 Mol. Endocrinol.Home page
S. Suzuki, N. Suzuki, J.-i. Mori, A. Oshima, S. Usami, and K. Hashizume
{micro}-Crystallin as an Intracellular 3,5,3'-Triiodothyronine Holder in Vivo
Mol. Endocrinol., April 1, 2007; 21(4): 885 - 894.
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 Protein Sci.Home page
Z. Cheng, L. Sun, J. He, and W. Gong
Crystal structure of human {micro}-crystallin complexed with NADPH
Protein Sci., February 1, 2007; 16(2): 329 - 335.
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 J. Med. Genet.Home page
A Oshima, S Suzuki, Y Takumi, K Hashizume, S Abe, and S Usami
CRYM mutations cause deafness through thyroid hormone binding properties in the fibrocytes of the cochlea.
J. Med. Genet., June 1, 2006; 43(6): e25 - e25.
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 Hum Reprod UpdateHome page
J. D. Hennebold
Characterization of the ovarian transcriptome through the use of differential analysis of gene expression methodologies
Hum. Reprod. Update, May 1, 2004; 10(3): 227 - 239.
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T. Inagaki, S. Suzuki, T. Miyamoto, T. Takeda, K. Yamashita, A. Komatsu, K. Yamauchi, and K. Hashizume
The Retinoic Acid-responsive Proline-rich Protein Is Identified in Promyeloleukemic HL-60 Cells
J. Biol. Chem., December 19, 2003; 278(51): 51685 - 51692.
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 EndocrinologyHome page
M. Moreno, E. Silvestri, A. Lombardi, T. J. Visser, F. Goglia, and A. Lanni
Identification of 3,5-Diiodo-L-Thyronine-Binding Proteins in Rat Liver Cytosol by Photoaffinity Labeling
Endocrinology, June 1, 2003; 144(6): 2297 - 2303.
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 EndocrinologyHome page
C. Ko, N. A. Grieshaber, I. Ji, and T. H. Ji
Follicle-Stimulating Hormone Suppresses Cytosolic 3,5,3'-Triiodothyronine-Binding Protein Messenger Ribonucleic Acid Expression in Rat Granulosa Cells
Endocrinology, June 1, 2003; 144(6): 2360 - 2367.
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