Proc. Nati. Acad. Sci. USA Vol. 89, pp. 9277-9281, October 1992 Cell Biology
Regulation of thyroid hormone receptor-mediated transcription by a cytosol protein KIYOTo ASHIZAWA AND SHEUE-YANN CHENG* Gene Regulation Section, Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 4BO9, Bethesda, MD 20892
Communicated by Joseph E. Rall, June 25, 1992 (received for review February 25, 1992)
ABSTRACT Thyroid hormone receptors (TRs) are members of the steroid hormone/retinoic acid receptor superfamily, which regulate homeostasis, development, and differentiation. Their transcriptional activity is modulated by the thyroid hormone 3,3',5-triiodo-L-thyronine (T3). The present study evaluated the effect of the availability of cytoplasmic T3 on the modulation of transcriptional responses of the TRs. In human choriocarcinoma JEG-3 and monkey COS-1 cells, the cytosolic thyroid hormone binding protein is a monomer of the tetrameric pyruvate kinase, subtype M2, which does not bind T3. The in vivo monomer-tetramer interconversion is regulated by glucose via fructose 1,6-bisphosphate. At the physiological T3 concentration, lowering the glucose concentration led to an increase in the cellular concentration of the cytosolic thyroid hormone binding protein. By using a transient transfection system, a concomitant reduction in the transcriptional activity of the human 131 thyroid hormone receptor was detected in both cell lines. In the absence of glucose, the transcriptional activity of the human 131 thyroid hormone receptor in JEG-3 and COS-1 cells was reduced by 65-75% and 90-95%, respectively. However, glucose had no effect on the basal transcriptional activity. These rmdings demonstrate an important prenuclear step in the modulation of the gene regulating activity of the TRs.
the monomer-tetramer interconversion can be manipulated by the glucose concentration in the medium provided us with a tool to assess the critical role of the cytoplasmic level of T3 on the gene regulatory activity of the TRs. In the present studies, using a transient transfection system, we found that the increase in the cellular p58-M2 leads to a reduction of the transcriptional activity of the TRs. Thus, p58-M2 plays a critical role in the regulation of the transcriptional responses of the TRs.
MATERIALS AND METHODS
D-threo-[dichloroacetyl-1-14C]Chloramphenicol (2.04 GBq/
mmol; 55 mCi/mmol) was purchased from Amersham. Affinity-purified goat anti-mouse IgG conjugated to rhodamine or peroxidase was from Jackson ImmunoResearch. T3, 3,3',5-triiodothyroacetic acid (Triac), 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid, and monoclonal antibody (mAb) MOPC were from Sigma. Acetyl coenzyme A was obtained from Pharmacia. Thyroid hormone-depleted serum was prepared as described by Kitagawa et al. (6). Cell Lines. Human choriocarcinoma JEG-3 cells were kindly provided by R. Koenig (University of Michigan) and cultured in minimal essential medium. Monkey COS-1 cells were a generous gift of B. Howard (National Institutes of Health). COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10%o (vol/vol) fetal calf serum. Immunocytochemistry. Cultured cells were processed for immunofluorescence as described (5). Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min at 230C and incubated with mAb J13 or J11 at 1 ,ug/ml in PBS containing 0.1% saponin and normal goat globulin at 4 mg/ml for 30 min at room temperature. After being washed with PBS, the cells were incubated with affinity-purified goat anti-mouse IgG conjugated to rhodamine (25 pug/ml) for 30 min. Cells treated with mAb MOPC were used as a control. The cells were viewed by using a Zeiss RA microscope (Carl Zeiss Instruments, Hanover, MD) equipped with rhodamine epifluorescence optics. mAb J11 and J13 were affinity purified as described (5). In Situ Quantitative Immunoassay of Cellular p58-M2. For the determination of p58-M2 in intact cells, a sensitive in situ quantitative immunoassay was used (7). Overnight cultures of JEG-3 or COS-1 cells (6 x 105 cells per 35-mm dish) were fixed with 3.7% formaldehyde. After being washed with PBS, cells were incubated with 1 ml of affinity-purified antibody (1 ,ug/ml). After being washed, cells were incubated with 1 ml of peroxidase conjugated to affinity-purified goat anti-mouse IgG (5 lug/ml). After washing the cells with PBS, 1 ml of
The thyroid hormone 3,3',5-triiodo-L-thyronine (T3) plays an important role in the growth, development, and metabolism of vertebrates. Abundant evidence demonstrates that most if not all of the T3 biological effects are mediated through the thyroid hormone nuclear receptors (TRs). Recently, cDNAs encoding several isoforms of TRs have been isolated and characterized. In the transient gene expression studies, the transcriptional activity of TRs have been shown to be modulated by T3 (1). Thus, regulation in the availability of cytoplasmic T3 to the receptors in the nucleus could be a key step in the modulation of transcriptional responses of the TRs. Previously, we have purified a cytosolic thyroid hormone binding protein (p58-M2) from human epidermoid carcinoma A431 cells (2). It has a molecular weight of 58,000. Analysis of its cDNA sequence indicates that it is a subunit of pyruvate kinase, subtype M2 (PKM2). p58-M2 binds T3 and exhibits T3 analog specificity. The tetrameric PKM2 does not bind T3 (3). In vitro, fructose 1,6-bisphosphate, a metabolite of the glycolytic pathway, stimulates the association of monomeric p58-M2 to form tetrameric PKM2 (3, 4). In vivo, at the physiological concentration of glucose, 30-35% of PKM2 exist as a monomer. However, PKM2 dissociates into monomer upon deprivation of glucose due to the reduction of intracellular fructose 1,6-bisphosphate (5). The increase in the p58-M2 is expected to bind more cytoplasmic T3, rendering less free T3 to be available to the TRs. The findings that
Abbreviations: T3, 3,3',5-triiodo-L-thyronine; Triac, 3,3',5-triiodothyroacetic acid; TR, thyroid hormone nuclear receptor; CAT,
chloramphenicol acetyltransferase; p58-M2, cytosolic thyroid hormone binding protein; PKM2, pyruvate kinase, subtype M2; mAb, monoclonal antibody; h-TR,81, human ,31 thyroid hormone receptor. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9277
9278
Cell Biology: Ashizawa and Cheng
2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (1 mM) in citrate acid buffer (pH 4.4) and 30% H202 (1 pul/ml) were added to the cells. The reaction was stopped by the addition of 1 ml of 10%o acetic acid. The intensity of the color was determined in a Vmax microplate reader (Molecular Devices, Palo Alto, CA). Determination of the T3-Dependent Transcriptional Activity of Human (31 Thyroid Hormone Receptor (h-TR.31) as Assessed by Chloramphenicol Acetyltransferase (CAT) Activity. JEG-3 cells or COS-1 cells were transfected with an h-TR,81 expression plasmid (pCLC51; 2 ug) and a reporter plasmid (pTK28m; 2.5 ,ug) that contained two copies of the perfect inverted T3-response element in tandem fused to the herpes simplex virus thymidine kinase promoter (8). Cells were also cotransfected with a human growth hormone expression plasmid (pXGH5; 1 jug) as an internal control for transfection efficiency (9). Transfection was carried out 24 hr after plating the cells (4.7 x 105 cells per 60-mm dish) using the calcium phosphate precipitation method in medium containing 10%1 thyroid hormone-depleted serum. Twenty-four hours after transfection, increasing concentrations of T3 or Triac (0.05 nM-i ,uM) were added to the thyroid hormone-depleted medium with or without 5 mM glucose. CAT activity was
determined 18 hr later. Binding of T3 to p58-M2. JEG-3 cells (8 x 106 cells per 150-mm dish) were plated and cultured for 24 hr. After being washed with PBS (Ca2+ and Mg2e free), cells were scraped by a rubber policeman. After centrifugation (500 x g, 5 min), the cell pellet was suspended in PBS containing 1 mM EDTA, leupeptin at 1 jg/ml, and 0.1 mM phenylmethylsulfonyl fluoride and sonicated twice for 10 sec (100 W). After centrifugation (80,000 x g, 20 min), the supernatant, which contained p58-M2, was used for binding to T3. Binding of p58-M2 to T3 was carried out as described in refs. 2 and 3. The binding data were analyzed by the LIGAND program (10).
RESULTS Interconversion of the Monomeric p58-M2 and the Tetrameric PKM2 Is Regulated by Glucose in JEG-3 Cells. To establish that p58-M2 plays a regulatory role in the transcriptional activity of h-TRj31, we chose human choriocarcinoma JEG-3 cells for our studies. JEG-3 cells are functionally deficient in the TRs, and the transfected TRs in this cell line have been shown to be modulated by T3 (11). Thus, it is a suitable cell line to test our hypothesis. We first demonstrated that p58-M2 is present in JEG-3 cells and that its association to PKM2 is regulated by glucose. Fig. 1A shows that PKM2 is present in the cytosol ofJEG-3 because bright fluorescence was seen by using a mAb that recognizes both the monomeric p58-M2 and the tetrameric PKM2 (5). In the presence of the glucose-containing medium, only a low level of cytoplasmic fluorescence was seen with the monomer-specific monoclonal antibody J13 (Fig. 1B) (5), indicating the presence of a small amount of p58-M2 in equilibrium with PKM2 at the physiological concentration of glucose. Upon deprivation of glucose for 2 hr, PKM2 dissociated to form the monomeric p58-M2 as demonstrated with antibody J13 (Fig. 1D). Fig. 1E shows that after 18 hr no significant change in the level of p58-M2 was seen. When glucose was added after deprivation of glucose for either 2 hr or 18 hr, within minutes the fluorescence pattern shown in Fig. 1B was again observed (data not shown). Therefore, as previously described for human A431, HepG2, fibroblasts, and monkey COS-1 cells (5), the interconversion of p58-M2 and PKM2 in JEG-3 is
regulated by glucose. p58-M2 Regulates the T3-Dependent Transcriptional Activity of h-TR31. To assess the effect of changing the cellular p58-M2 level on the transcriptional activity of TR, we co-
transfected the h-TRp1 expression plasmid and a reporter
Proc. Natl. Acad Sci. USA 89 (1992)
Glucose-Containing Medium
A
mAb
13
Ji 1
:
13
MOPC
Glucose-Free Medium
F
mAb:
J13
113
MOPC
FIG. 1. Visualization of monomeric p58-M2 and tetrameric PKM2 by fluorescence. JEG-3 cells were fixed, permeabilized using saponin, and processed for immunofluorescence similar to that described by Ashizawa et al. (5). JEG-3 cells were cultured in glucosecontaining medium (A-C and A'-C') or glucose-free medium for 2 (D and D') or 18 (E, E', F, and F') hr and incubated with mAbs Jil, J13, or MOPC (control antibody) at 1 itg/ml as indicated. (A-F) Fluorescence micrographs. (A'-F') Corresponding phase contrast micrographs. (x400.)
gene that contained two copies of palindromic T3 response elements in tandem upstream ofthe CAT gene. Fig. 2A shows that the transcriptional activity of h-TR(1 is T3 dependent and is maximal at 5-10 nM T3. In the absence of glucose, transcriptional activity was reduced by 65-75% at the physiological concentration of T3 (
Regulation of thyroid hormone receptor-mediated transcription by a cytosol protein KIYOTo ASHIZAWA AND SHEUE-YANN CHENG* Gene Regulation Section, Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 4BO9, Bethesda, MD 20892
Communicated by Joseph E. Rall, June 25, 1992 (received for review February 25, 1992)
ABSTRACT Thyroid hormone receptors (TRs) are members of the steroid hormone/retinoic acid receptor superfamily, which regulate homeostasis, development, and differentiation. Their transcriptional activity is modulated by the thyroid hormone 3,3',5-triiodo-L-thyronine (T3). The present study evaluated the effect of the availability of cytoplasmic T3 on the modulation of transcriptional responses of the TRs. In human choriocarcinoma JEG-3 and monkey COS-1 cells, the cytosolic thyroid hormone binding protein is a monomer of the tetrameric pyruvate kinase, subtype M2, which does not bind T3. The in vivo monomer-tetramer interconversion is regulated by glucose via fructose 1,6-bisphosphate. At the physiological T3 concentration, lowering the glucose concentration led to an increase in the cellular concentration of the cytosolic thyroid hormone binding protein. By using a transient transfection system, a concomitant reduction in the transcriptional activity of the human 131 thyroid hormone receptor was detected in both cell lines. In the absence of glucose, the transcriptional activity of the human 131 thyroid hormone receptor in JEG-3 and COS-1 cells was reduced by 65-75% and 90-95%, respectively. However, glucose had no effect on the basal transcriptional activity. These rmdings demonstrate an important prenuclear step in the modulation of the gene regulating activity of the TRs.
the monomer-tetramer interconversion can be manipulated by the glucose concentration in the medium provided us with a tool to assess the critical role of the cytoplasmic level of T3 on the gene regulatory activity of the TRs. In the present studies, using a transient transfection system, we found that the increase in the cellular p58-M2 leads to a reduction of the transcriptional activity of the TRs. Thus, p58-M2 plays a critical role in the regulation of the transcriptional responses of the TRs.
MATERIALS AND METHODS
D-threo-[dichloroacetyl-1-14C]Chloramphenicol (2.04 GBq/
mmol; 55 mCi/mmol) was purchased from Amersham. Affinity-purified goat anti-mouse IgG conjugated to rhodamine or peroxidase was from Jackson ImmunoResearch. T3, 3,3',5-triiodothyroacetic acid (Triac), 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid, and monoclonal antibody (mAb) MOPC were from Sigma. Acetyl coenzyme A was obtained from Pharmacia. Thyroid hormone-depleted serum was prepared as described by Kitagawa et al. (6). Cell Lines. Human choriocarcinoma JEG-3 cells were kindly provided by R. Koenig (University of Michigan) and cultured in minimal essential medium. Monkey COS-1 cells were a generous gift of B. Howard (National Institutes of Health). COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10%o (vol/vol) fetal calf serum. Immunocytochemistry. Cultured cells were processed for immunofluorescence as described (5). Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min at 230C and incubated with mAb J13 or J11 at 1 ,ug/ml in PBS containing 0.1% saponin and normal goat globulin at 4 mg/ml for 30 min at room temperature. After being washed with PBS, the cells were incubated with affinity-purified goat anti-mouse IgG conjugated to rhodamine (25 pug/ml) for 30 min. Cells treated with mAb MOPC were used as a control. The cells were viewed by using a Zeiss RA microscope (Carl Zeiss Instruments, Hanover, MD) equipped with rhodamine epifluorescence optics. mAb J11 and J13 were affinity purified as described (5). In Situ Quantitative Immunoassay of Cellular p58-M2. For the determination of p58-M2 in intact cells, a sensitive in situ quantitative immunoassay was used (7). Overnight cultures of JEG-3 or COS-1 cells (6 x 105 cells per 35-mm dish) were fixed with 3.7% formaldehyde. After being washed with PBS, cells were incubated with 1 ml of affinity-purified antibody (1 ,ug/ml). After being washed, cells were incubated with 1 ml of peroxidase conjugated to affinity-purified goat anti-mouse IgG (5 lug/ml). After washing the cells with PBS, 1 ml of
The thyroid hormone 3,3',5-triiodo-L-thyronine (T3) plays an important role in the growth, development, and metabolism of vertebrates. Abundant evidence demonstrates that most if not all of the T3 biological effects are mediated through the thyroid hormone nuclear receptors (TRs). Recently, cDNAs encoding several isoforms of TRs have been isolated and characterized. In the transient gene expression studies, the transcriptional activity of TRs have been shown to be modulated by T3 (1). Thus, regulation in the availability of cytoplasmic T3 to the receptors in the nucleus could be a key step in the modulation of transcriptional responses of the TRs. Previously, we have purified a cytosolic thyroid hormone binding protein (p58-M2) from human epidermoid carcinoma A431 cells (2). It has a molecular weight of 58,000. Analysis of its cDNA sequence indicates that it is a subunit of pyruvate kinase, subtype M2 (PKM2). p58-M2 binds T3 and exhibits T3 analog specificity. The tetrameric PKM2 does not bind T3 (3). In vitro, fructose 1,6-bisphosphate, a metabolite of the glycolytic pathway, stimulates the association of monomeric p58-M2 to form tetrameric PKM2 (3, 4). In vivo, at the physiological concentration of glucose, 30-35% of PKM2 exist as a monomer. However, PKM2 dissociates into monomer upon deprivation of glucose due to the reduction of intracellular fructose 1,6-bisphosphate (5). The increase in the p58-M2 is expected to bind more cytoplasmic T3, rendering less free T3 to be available to the TRs. The findings that
Abbreviations: T3, 3,3',5-triiodo-L-thyronine; Triac, 3,3',5-triiodothyroacetic acid; TR, thyroid hormone nuclear receptor; CAT,
chloramphenicol acetyltransferase; p58-M2, cytosolic thyroid hormone binding protein; PKM2, pyruvate kinase, subtype M2; mAb, monoclonal antibody; h-TR,81, human ,31 thyroid hormone receptor. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9277
9278
Cell Biology: Ashizawa and Cheng
2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (1 mM) in citrate acid buffer (pH 4.4) and 30% H202 (1 pul/ml) were added to the cells. The reaction was stopped by the addition of 1 ml of 10%o acetic acid. The intensity of the color was determined in a Vmax microplate reader (Molecular Devices, Palo Alto, CA). Determination of the T3-Dependent Transcriptional Activity of Human (31 Thyroid Hormone Receptor (h-TR.31) as Assessed by Chloramphenicol Acetyltransferase (CAT) Activity. JEG-3 cells or COS-1 cells were transfected with an h-TR,81 expression plasmid (pCLC51; 2 ug) and a reporter plasmid (pTK28m; 2.5 ,ug) that contained two copies of the perfect inverted T3-response element in tandem fused to the herpes simplex virus thymidine kinase promoter (8). Cells were also cotransfected with a human growth hormone expression plasmid (pXGH5; 1 jug) as an internal control for transfection efficiency (9). Transfection was carried out 24 hr after plating the cells (4.7 x 105 cells per 60-mm dish) using the calcium phosphate precipitation method in medium containing 10%1 thyroid hormone-depleted serum. Twenty-four hours after transfection, increasing concentrations of T3 or Triac (0.05 nM-i ,uM) were added to the thyroid hormone-depleted medium with or without 5 mM glucose. CAT activity was
determined 18 hr later. Binding of T3 to p58-M2. JEG-3 cells (8 x 106 cells per 150-mm dish) were plated and cultured for 24 hr. After being washed with PBS (Ca2+ and Mg2e free), cells were scraped by a rubber policeman. After centrifugation (500 x g, 5 min), the cell pellet was suspended in PBS containing 1 mM EDTA, leupeptin at 1 jg/ml, and 0.1 mM phenylmethylsulfonyl fluoride and sonicated twice for 10 sec (100 W). After centrifugation (80,000 x g, 20 min), the supernatant, which contained p58-M2, was used for binding to T3. Binding of p58-M2 to T3 was carried out as described in refs. 2 and 3. The binding data were analyzed by the LIGAND program (10).
RESULTS Interconversion of the Monomeric p58-M2 and the Tetrameric PKM2 Is Regulated by Glucose in JEG-3 Cells. To establish that p58-M2 plays a regulatory role in the transcriptional activity of h-TRj31, we chose human choriocarcinoma JEG-3 cells for our studies. JEG-3 cells are functionally deficient in the TRs, and the transfected TRs in this cell line have been shown to be modulated by T3 (11). Thus, it is a suitable cell line to test our hypothesis. We first demonstrated that p58-M2 is present in JEG-3 cells and that its association to PKM2 is regulated by glucose. Fig. 1A shows that PKM2 is present in the cytosol ofJEG-3 because bright fluorescence was seen by using a mAb that recognizes both the monomeric p58-M2 and the tetrameric PKM2 (5). In the presence of the glucose-containing medium, only a low level of cytoplasmic fluorescence was seen with the monomer-specific monoclonal antibody J13 (Fig. 1B) (5), indicating the presence of a small amount of p58-M2 in equilibrium with PKM2 at the physiological concentration of glucose. Upon deprivation of glucose for 2 hr, PKM2 dissociated to form the monomeric p58-M2 as demonstrated with antibody J13 (Fig. 1D). Fig. 1E shows that after 18 hr no significant change in the level of p58-M2 was seen. When glucose was added after deprivation of glucose for either 2 hr or 18 hr, within minutes the fluorescence pattern shown in Fig. 1B was again observed (data not shown). Therefore, as previously described for human A431, HepG2, fibroblasts, and monkey COS-1 cells (5), the interconversion of p58-M2 and PKM2 in JEG-3 is
regulated by glucose. p58-M2 Regulates the T3-Dependent Transcriptional Activity of h-TR31. To assess the effect of changing the cellular p58-M2 level on the transcriptional activity of TR, we co-
transfected the h-TRp1 expression plasmid and a reporter
Proc. Natl. Acad Sci. USA 89 (1992)
Glucose-Containing Medium
A
mAb
13
Ji 1
:
13
MOPC
Glucose-Free Medium
F
mAb:
J13
113
MOPC
FIG. 1. Visualization of monomeric p58-M2 and tetrameric PKM2 by fluorescence. JEG-3 cells were fixed, permeabilized using saponin, and processed for immunofluorescence similar to that described by Ashizawa et al. (5). JEG-3 cells were cultured in glucosecontaining medium (A-C and A'-C') or glucose-free medium for 2 (D and D') or 18 (E, E', F, and F') hr and incubated with mAbs Jil, J13, or MOPC (control antibody) at 1 itg/ml as indicated. (A-F) Fluorescence micrographs. (A'-F') Corresponding phase contrast micrographs. (x400.)
gene that contained two copies of palindromic T3 response elements in tandem upstream ofthe CAT gene. Fig. 2A shows that the transcriptional activity of h-TR(1 is T3 dependent and is maximal at 5-10 nM T3. In the absence of glucose, transcriptional activity was reduced by 65-75% at the physiological concentration of T3 (
