Tissue-specific Stabilization of the Thyroid Hormone beta 1 Nuclear Receptor by Phosphorylation*

(Received for publication, July 24, 1996, and in revised form, December 2, 1996)

Yuan-Tsang Ting , Manoj Kumar Bhat , Rosemary Wong Dagger and Sheue-yann Cheng §

From the Laboratory of Molecular Biology, National Cancer Institute, and the Dagger  Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-4255

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The present study evaluated the expression and regulation of endogenous thyroid hormone receptors (TRs) in cultured cells. In COS-1 cells, the endogenous TR, subtype beta 1 (TRbeta 1), but not subtype beta 2 or alpha 1, was induced to express by okadaic acid (OA) in a concentration-dependent manner. The induced TRbeta 1 had immunoreactivity and partial V8 proteolytic maps similar to those of the transfected and in vitro translated human TRbeta 1 (h-TRbeta 1). The OA-induced expression of endogenous TRbeta 1 was, however, not observed in a variety of other cultured cell lines tested, indicating that the induction was cell type-dependent. TRbeta 1 induced by OA was a multisite phosphorylated protein, in which serine and threonine in a ratio of 10:1 were phosphorylated. The induced TRbeta 1 was functional as it could mediate the thyroid hormone-dependent transcriptional activity via several thyroid hormone response elements. The induction of endogenous TRbeta 1 expression by OA was not accompanied by an increase in mRNA levels but was the result of an increase in the stability of the TRbeta 1 protein. This is the first report to indicate that one of the mechanisms by which the TR isoforms are differentially expressed is via the tissue-specific stabilization of the TR isoform proteins. Furthermore, this selective stability of TRbeta 1 could be conferred by phosphorylation.


INTRODUCTION

Thyroid hormone nuclear receptors (TRs)1 are members of the steroid hormone/retinoic acid receptor superfamily. Two TR genes, TRalpha and TRbeta , have been identified which are located on chromosome 17 and chromosome 3, respectively. Four isoforms, beta 1, beta 2, alpha 1, and alpha 2, are generated from each of two TR genes, alpha  and beta , by alternative splicing (1, 2). They are transcription factors that regulate the expression of target genes by interacting with specific DNA sequences known as thyroid hormone response elements (TREs) in the promoter region of target genes (1, 2). The transcriptional activity of TRs is not only dependent on thyroid hormone 3,3',5-triiodo-L-thyronine (T3) but also on the type of TRE. Recent studies have indicated that the transcriptional activity of TRs is further modulated via interaction with four groups of cellular proteins: (a) members of the nuclear receptor superfamily, notably the retinoid X receptors (1, 2); (b) corepressors including p270/N-CoR (3), SMRT (4), TRUP (5), SHP (6), and TRACs (7); (c) coactivator SRC-1 (8); and (d) the tumor suppressor p53 (9). It is clear that regulation of the transcriptional activity of TRs is more complex than envisioned previously.

One important but less well studied mode of modulation of TR activity is the regulation of TR expression at the protein level. TRalpha 1 and TRbeta 1 are differentially expressed at different developmental stages and at different levels in various tissues (10-12). Moreover, using the specific T3 binding activity and immunoreactivity as measures of the expressed receptor proteins, it was found that there are marked variations in the TR protein:mRNA ratios in different tissues (11, 12), suggesting isoform- and tissue-dependent posttranslational modification of TRs. However, it is unknown how TRs could be modulated at the protein level. We studied this problem by first using the cultured rat growth hormone-producing cell line GH3, which has long been used as a model cell line for studying thyroid hormone action (13). Unfortunately, preliminary experiments indicate that the anti-TR antibodies currently available are not sensitive enough to detect the endogenous TRs in GH3 cells either by immunoprecipitation or Western blotting. However, we subsequently discovered that in COS-1 cells, endogenous TRbeta 1 could be induced to express by okadaic acid (OA), a potent and specific inhibitor of serine/threonine phosphoprotein phosphatases 1 and 2A (14). COS-1 cells are known to be functionally deficient in TRs and have been used widely as recipient cells for gene transfer to study TR action. We therefore characterized the process of induction of TRbeta 1 by OA as we hoped to use COS-1 cells as a model cell line to understand the regulation of expression of endogenous TRs. Thus, in the present paper, we studied the molecular basis of the induction of endogenous TRbeta 1 expression and found that the OA-induced TRbeta 1 was a phosphoprotein possessing the same characteristics and functions as transfected human (h)-TRbeta 1 in mediating thyroid hormone-dependent transactivation. Moreover, the induction of endogenous TRbeta 1 protein expression was cell type-dependent in that it did not occur in GH3 (13), neuro-2alpha (16), human choriocarcinoma JEG-3, human epidermal carcinoma A431, mouse fibroblast NIH3T3, and the human cervical carcinoma HeLa cells. The increase in TRbeta 1 protein expression by OA in COS-1 cells was not accompanied by increases in mRNA level or in the rate of synthesis of TRbeta 1 protein. Rather, our results showed that the increase in TRbeta 1 protein expression by OA was the result of increased stability of the TRbeta 1 protein most probably via phosphorylation and defined a novel role of phosphorylation in regulating the levels of TRbeta 1 in different cell types.


EXPERIMENTAL PROCEDURES

Immunoprecipitation

Monkey kidney COS-1 cells (8.2 × 105 cells/100-mm dish) were plated. Forty-eight h later, cells were preincubated in methionine or phosphate-free medium for 90 min and incubated with 100 µCi of [35S]methionine (ICN Biomedicals, Inc., Costa Mesa, CA) or 1 mCi (74 MBq) of carrier-free [32P]orthophosphate (Amersham Corp.) in the presence of OA (100-250 nM) for 3 h. For phosphorylation and metabolic labeling of the transfected h-TRbeta 1, cells were transfected with 8 µg of h-TRbeta 1 expression vector pCLC51 (16) by using LipofectAMINE according to the manufacturer's instructions (Life Technologies, Inc.). After labeling with either [35S]methionine or [32P]orthophosphate, labeled TRbeta 1 were immunoprecipitated with 5 µg of monoclonal antibody (mAb) C4 (17) or J52 (18) as described by Lin et al. (16). Identical conditions were used for detection of endogenous TRbeta 1 in GH3, neuro-2alpha , JEG-3, A431, NIH3T3, and HeLa cell lines.

Analysis of 35S-Labeled Peptides by V8 Protease Digestion

Gel proteolysis of 35S-labeled TRbeta 1 was performed according to Cleveland et al. (19). Briefly, the bands containing labeled TRbeta 1 were excised and loaded into the sample wells of a 15% acrylamide gel. Three µg of Staphylococcus aureus V8 protease (Boehringer Mannheim) in soaking solution was overlaid. Electrophoresis was performed, and the dried gel was autoradiographed.

Two-dimensional Analysis of Phosphoamino Acids and Tryptic Mapping

Two-dimensional analysis of phosphoamino acids and tryptic mapping was carried out according to Ref. 20.

Binding of [125I]T3 to Isolated Nuclei

COS-1 cells (2 × 106 cells/15-cm dish) were cultured in the presence or absence of 100 nM OA at 37 °C for 3 h. Nuclei were isolated according to Samuels et al. (21) and Zhao and Padmanabha (22). [125I]T3 binding was carried out as described by Samuels et al. (21) and Koerner et al. (23). Briefly, cells were scraped in buffer H (20 mM Hepes, pH 7.5, 5 mM KCl, 0.5 mM MgCl2, and 0.5 mM dithiothreitol) with or without 100 nM OA, and cells were pelleted at 800 × g for 10 min at 4 °C. Cells were lysed by passing back and forth (27 times) through a 1-ml syringe with a 25 × 1.56-cm needle. The lysed cell suspension was made isotonic by adding 1 ml of 0.5 M sucrose, and the nuclei were pelleted at 1,200 × g for 10 min. The nuclei was washed two times by resuspending in 2 ml of binding buffer B (0.32 M sucrose, 3 mM MgCl2 and 20 mM Tris, pH 7.8). The washed nuclei were incubated with 0.5 nM [125I]T3 in 200 µl of binding buffer B for 30 min at 37 °C. The binding was also carried out in the presence of 1 µM unlabeled T3 to determine the nonspecific binding. At the end of the incubation, an equal volume of 1% Triton X-100 was added, and the nuclei were pelleted. The nuclei were washed further with 1 ml of binding buffer B containing 0.5% Triton X-100, and the radioactivity bound to nuclei was determined in a gamma -counter. The nuclear protein concentrations were determined by Coomassie Plus Protein Assay Reagent (Pierce Chemical Co.) using aliquots of nuclei suspension.

Determination of OA-induced Expression of TRbeta 1 on Transcription

COS-1 cells (3 × 105/60-mm dish) were plated 1 day before transfection. The cells were transfected with 1.5 µg of a reporter gene containing two copies of the palindromic TRE in tandem (pTK28m) or the chicken lysozyme TRE (pTKLys) upstream of the chloramphenicol acetyltransferase gene. pBluescript plasmid (Stratagene, La Jolla, CA) was used to adjust the total DNA to 3 µg/dish. Various concentrations of OA were added to the cells 3 h before the cells were lysed for analysis of chloramphenicol acetyltransferase activity, which was normalized to protein concentration.

Northern Analysis

Total RNA was isolated from COS-1 cells cultured in the presence or absence of 100 nM OA (RNeasy, Qiagen, Chatsworth, CA). Fifteen µg of RNA was electrophoresed on a 1% formaldehyde gel and blotted onto a nylon membrane, which was prehybridized at 68 °C with QuikHyb solution (Stratagene) for 3 h, followed by hybridization at 65 °C for 6 h with a radioactive rat TRbeta 1 cDNA probe prepared from a rat-TRbeta 1 expression plasmid (gift of Dr. R. Koenig, University of Michigan Medical Center). A human beta -actin cDNA probe was also prepared as an internal control. The membrane was washed under stringent conditions with 0.2 × SSPE at 60 °C for 10 min. The labeled filters were analyzed by exposure to x-ray film, stripped, and reprobed with the beta -actin cDNA.

Reverse Transcriptase-Polymerase Chain Reaction (PCR)

2 µg of total RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Clontech, Palo Alto, CA) after treatment with RNase-free DNase I (Boehringer Mannheim). 5-20 µl of the resultant 100-µl cDNA reaction was used for PCR to ensure linearity of response. Primers identical to sequences common to human and mouse TRbeta 1 were used to amplify a 378-base pair cDNA fragment. h-TRbeta 1 was also used as a template for PCR to serve as a control. PCR products were assessed by Southern analysis using the above rat TRbeta 1 cDNA probe.

Determination of the Stability of TRbeta 1 Protein

COS-1 cells were transfected with h-TRbeta 1 expression vector (pCLC51) and incubated in methionine-free medium in the presence or absence of OA. Cells were pulse labeled with 50 µCi of [35S]methionine for 30 min and chased for various time periods. Cell lysates were prepared, and immunoprecipitation was performed as described above.


RESULTS

Cell Type- and TR Isoform-dependent Induction of Expression of Endogenous TRbeta 1 Protein by OA

When COS-1 cells were treated with 250 nM OA, we found that a protein band with a molecular weight identical to that of transfected h-TRbeta 1 was immunoprecipitated with monoclonal anti-h-TRbeta 1 J52 (mAb J52; 19) (lane 8 versus lane 9 of Fig. 1A). This band was not present when the cells were not treated with OA as shown in lane 1 (Fig. 1A). As a control for specific expression of transfected h-TRbeta 1, lane 2 shows that when an identical expression plasmid lacking the coding sequence of h-TRbeta 1 was used, no expression of h-TRbeta 1 was detected. Transfection of this TRbeta 1-less vector had no effect on the induction of TRbeta 1 in COS-1 cells by OA, as the intensity of the TRbeta 1 band seen was identical to that in its absence (lane 8 versus lane 7). The intensity of the TRbeta 1 band in the presence of OA (lane 9) was slightly higher than that in the absence of OA (lane 3), reflecting the combined intensities of the endogenous and the transfected TRbeta 1. The minor lower bands in lanes 3 and 9 probably represented the degradation products. The epitope for mAb J52 is in the middle of the A/B domain (18). To be certain that the band detected in lane 7 was TRbeta 1, we also used another monoclonal antibody, C4, whose epitope is located at the COOH-terminal Glu457-Val-Phe-Glu-Asp461 (17). As shown in lane 10, a protein band with the same electrophoretic mobility as for transfected h-TRbeta 1 (lanes 12 and 6) was detected which was not seen in the absence of OA (lane 4). Lanes 13-15 are controls using an irrelevant mAb that did not immunoprecipitate TRbeta 1 to confirm the specificity of TRbeta 1 bands seen in lanes 7 and 10.


Fig. 1. Panel A, effect of OA on the expression of endogenous TRbeta 1 in COS-1 cells. Untransfected COS-1 cells (2 × 106 cells/100-mm dish) (lanes 1, 4, 7, 10, and 13) and cells containing transfected h-TRbeta 1 expression vector (lanes 3, 6, 9, 12, 15) or empty vector (lanes 2, 5, 8, 11, and 14) were labeled with 100 µCi [35S]methionine in the absence of OA (lanes 1-6) or in the presence of 250 nM OA (lanes 7-15). After lysis of cells, immunoprecipitation was carried out with mAb 52 (lanes 1-3, 7-9), mAb C4 (lanes 4-6, 10-12), a control antibody MOPC (lanes 13-15). Panel B, partial peptide maps of TRbeta 1. The gel slices containing immunoprecipitated endogenous TRbeta 1 (lane 3), transfected h-TRbeta 1 (lane 2), in vitro translated h-TRbeta 1 (lane 4), and the cytosolic thyroid hormone-binding protein, p58 protein (lane 1) were digested with S. aureus V8 protease according to "Experimental Procedures."
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To support further the conclusion that the protein induced by OA was indeed TRbeta 1, we carried out one-dimensional partial V8 protease digestion. The peptide map of the immunoprecipitated [35S]methionine-labeled TRbeta 1 derived from OA-treated cells (Fig. 1B, lane 3) was compared with the maps obtained from either transfected h-TRbeta 1 (lane 2) or in vitro translated h-TRbeta 1 (lane 4). An identical pattern of four peptides, a, b, c and d, with molecular masses of 14, 12.5, 11, and 9 kDa, respectively, was seen in lanes 2-4. The weaker band (peptide a) and the stronger band (peptide d) seen in lane 3 compared with those in lanes 2 and 4 probably were due to more extensive proteolysis of peptide a to peptide d in TRbeta 1 derived from COS-1 cells. The differences in the protease susceptibility of peptide a could reflect the differences in local aggregated charge or structure surrounding the V8 cleavage sites due to minor differences in the primary sequences of human and monkey TRbeta 1. However, a completely different protein (p58, a cytosolic thyroid hormone-binding protein; 24) yielded a completely different peptide map, as shown in lane 1. Therefore, based on the immunoreactivity and peptide mapping, we have clearly established that in COS-1 cells, OA induced the expression of TRbeta 1.

mAb C4 recognizes both TRbeta 2 and TRalpha 1 because of the common conserved epitope at the COOH terminus. However, examination of lanes 10 and 11 of Fig. 1A further shows that no protein band larger or smaller than TRbeta 1 (molecular mass ~55,000) was detected, indicating that neither TRbeta 2 (molecular mass ~58,000) nor TRalpha 1 (molecular mass ~47,000) was induced to express by OA. To assess whether OA could also affect the expression of TRbeta 1 in other cell types, we similarly treated the cells derived from different tissues with OA. Fig. 2A shows that no OA-induced expression of TRbeta 1 was detected in GH3, neuro-2alpha , JEG-3, A431, NIH3T3, and HeLa cells. These results indicate that the induction of the expression of TRbeta 1 was TR isoform- and cell type-dependent.


Fig. 2. Panel A, effect of OA on the expression of endogenous TRbeta 1 in different cell types. Cells were labeled in the absence (odd numbered lanes) or presence of 100 nM OA (even numbered lanes), and the lysates were immunoprecipitated using mAb C4. Panel B, dose dependence of OA-induced TRbeta 1 expression. COS-1 cells were labeled as described in Fig. 1A either in the absence of OA (lane 1) or in the presence of 25, 50, 100, and 250 nM OA for lanes 2, 3, 4, and 5, respectively. Immunoprecipitation using mAb C4 was carried out as described in Fig. 1A.
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We further examined the effect of the concentration of OA, ranging from 25 to 250 nM, on the induction of TRbeta 1 in COS-1 cells. Lane 2 of Fig. 2B shows that at 25 nM, no TRbeta 1 was induced. At 50 nM (lane 3), a low level of TRbeta 1 was expressed, and a maximal level of TRbeta 1 was expressed at 100 nM of OA (lane 4). A further increase in the OA concentration (250 nM) led to a fall in the expression of TRbeta 1.

TRbeta 1 Induced by OA Was a Phosphoprotein

Previously we have shown that transfected h-TRbeta 1 is a phosphoprotein and that phosphorylation is enhanced by OA (16, 25). Therefore we asked the question of whether the induced TRbeta 1 was also a phosphoprotein. Fig. 3 shows that endogenous TRbeta 1 induced by OA was a phosphoprotein. The extent of phosphorylation was similar at 100 nM (lane 2 of Fig. 3) and 250 nM of OA (lane 3 of Fig. 3). Lane 4 is a negative control to indicate that when an irrelevant antibody was used, no phosphorylated TRbeta 1 was immunoprecipitated. However, it is important to point out that the TRbeta 1 induced at 250 nM OA was about 1/25 of that at 100 nM (see Fig. 2 B). Therefore, the extent of phosphorylation of TRbeta 1 at 250 nM would be expected to be higher than that seen for TRbeta 1 induced at 100 nM OA (lane 3 versus lane 2 of Fig. 3). These results are consistent with earlier observations that phosphorylation of transfected h-TRbeta 1 was enhanced when the OA concentration was increased from 100 to 250 nM (16). Similar to the transfected h-TRbeta 1 (16), phosphorylated serine and threonine in a ratio of 10:1 were detected for the induced endogenous TRbeta 1 (data not shown). Two-dimensional tryptic mapping indicates one major phosphopeptide, corresponding to 60% of total counts, and five other minor phosphopeptides (data not shown), suggesting that multiple phosphorylation sites were present in phosphorylated TRbeta 1 induced by OA.


Fig. 3. Effect of OA on the phosphorylation of endogenous TRbeta 1 in COS-1 cells. COS-1 cells were labeled with 1 mCi of [32P]orthophosphoric acid in the absence of OA (lane 1) or in the presence of 100 nM (lane 2) and 250 nM (lanes 3 and 4) OA. After lysis of cells, immunoprecipitation was carried out using mAb C4 (lanes 1-3) or control mAb MOPC (lane 4).
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OA Increased T3 Binding and Enhanced Endogenous TRbeta 1-mediated Transcription

Whether the TRbeta 1 induced by OA was functional was first assessed by T3 binding. Nuclei were isolated from COS-1 cells cultured in the presence or absence of OA and incubated with [125I]T3. As shown in Table I, the nuclei isolated from cells treated with OA had nearly 2-fold more T3 binding than those not treated with OA, indicating that OA-induced TRbeta 1 was functional as a T3 binder.

Table I.

Comparison of [125I]T3 binding to isolated nuclei of COS-1 cells cultured in medium with or without OA

COS-1 cells (2 × 106 cells/15-cm dish) were cultured in the presence or absence of 100 nM OA for 3 h at 37 °C. Nuclei isolated as described under "Experimental Procedures" were incubated with 0.5 nM [125I]T3 for 30 min at 37 °C. An equal volume of 1% Triton X-100 in the binding buffer was added, and the nuclei were subsequently washed with binding buffer containing 0.5% Triton X-100. [125I]T3 bound to the nuclei were counted in a gamma -counter, and the proteins were determined as described under "Experimental Procedures." The nonspecific binding was obtained by incubating nuclei with 0.5 nM [125I]T3 in the presence of 1 µM unlabeled T3. The nonspecific binding was ~15% of total binding. Data are averages of three independent experiments each with duplicates (mean ± S.D., n = 3).
OA Specific [125I]T3 bound

fmol/50 µg nuclear proteins
 - 0.34  ± 0.06
+ 0.58  ± 0.07

We further examined the transcriptional activity of OA-induced endogenous TRbeta 1. Two reporter genes, one containing palindromic TRE and the other containing chicken lysozyme TRE, were transfected into cells. As shown in Fig. 4, the T3-dependent transcriptional activity of TRbeta 1 was OA-dependent. An approximately 1.8- and 2.5-fold increase in the transcriptional activity on chicken lysozyme TRE was observed at 100 and 250 nM OA, respectively. On palindromic TRE, the increase was slightly lower (~1.5- and 1.8-fold increase at 100 and 250 nM, respectively). These findings are similar to that observed previously of a 2-fold increase in the transcriptional activity of the transfected TRbeta 1 by OA (17). Thus, the induced TRbeta 1 was functional, and its transcriptional activity was enhanced by phosphorylation.


Fig. 4. Regulation of endogenous TRbeta 1-mediated transcription by OA. COS-1 cells (3 × 105 cells/60-mm dish) were transfected with pTK28m (open bars; palindromic TRE or Pal) or pTKLys (shaded bars, chicken lysozyme TRE, or Lys). 24 h later, cells were treated with medium containing thyroid hormone-depleted serum with or without 100 nM T3 for an additional 24 h. In the last 3 h, various concentrations of OA as indicated were added to the medium. Cells were lysed, and chloramphenicol acetyltransferase (CAT) activity was measured. Data are the average of three experiments each with duplicates (mean ± S.D. n = 3). The means between the chloramphenicol acetyltransferase activities in the presence or absence of OA were analyzed by Student's t test, which indicates that the differences are very significant with p < 0.01.
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mRNA Level of TRbeta 1 Was Decreased by OA

The induction of expression of TRbeta 1 protein by OA may occur at either the level of transcription or translation. To understand the molecular basis of the induction, we first determined the TRbeta 1 mRNA levels in cells treated with or without OA by Northern analysis. Although it was possible to detect TRbeta 1 mRNA from total RNA (Fig. 5A, lane 1) and mRNA (lane 2) prepared from control cells (GH3), no TRbeta 1 mRNA was detectable from total RNA (lanes 3 and 4) and mRNA (lanes 5 and 6) prepared from COS-1 cells in the presence (lanes 4 and 6) or absence (lanes 3 and 5) of OA. beta -Actin mRNA was detectable in GH3 cells (lane 2 of Fig. 5B) and also in COS-1 cells either from total RNA (lanes 3 and 4 of Fig. 5B) or at a much greater abundance using the mRNA (lanes 5 and 6 of Fig. 5B), confirming the presence of intact RNA. Comparing the intensities of the beta -actin bands in lanes 3 and 4 or 5 and 6, it can be seen that OA had no effect on the expression of beta -actin mRNA.


Fig. 5. Panel A, detection of TRbeta 1 RNA in GH3 and COS-1 cells by Northern analysis. Total and poly(A)+ RNA from the following samples were loaded onto a 1.0% formaldehyde gel and probed with a rat TRbeta 1 cDNA probe: lane 1, GH3 total RNA (20 µg); lane 2, GH3 poly(A)+ RNA (10 µg); lane 3, COS-1 total RNA without OA (20 µg); lane 4, COS-1 total RNA with OA (20 µg); lane 5, COS-1 poly(A)+ without OA (20 µg); lane 6, COS-1 poly(A)+ with OA (20 µg). Hybridization was carried out to a rat TRbeta 1 cDNA probe, and autoradiography was performed for 9 h. Panel B, the blot from above was then stripped and reprobed with a beta -actin cDNA probe. Panel C, effect of OA treatment on TRbeta 1 mRNA from COS-1 cells. 2 µg of total RNA prepared from COS-1 cells not treated with OA or treated with increasing concentrations of OA (25-250 nM) was reverse transcribed (RT). 15 µl of the resultant 100 µl of synthesized cDNA was taken for PCR after establishing that this volume was within the linear range for PCR. Lanes 1-5 show RNA samples treated with reverse transcriptase; lanes 6-10, without reverse transcriptase. Lane 11 shows the position of the amplified fragment from h-TRbeta 1 plasmid which was used for PCR. Southern analysis was then performed to detect the amplified cDNA.
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In light of the inability to detect TRbeta 1 mRNA in COS-1 cells by Northern analysis, we performed reverse transcriptase-PCR, and the resultant cDNA was amplified by PCR and identified by Southern analysis. In preliminary experiments, we had first titrated the amounts of RNA so that RNA used in reverse transcriptase-PCR was in the linear range, and the resultant cDNA can be compared quantitatively. Lane 1 of Fig. 5C shows that at 25 nM OA, TRbeta 1 mRNA was similar to that without OA treatment. Further increases in OA concentration led to a dose-dependent reduction of mRNA up to 100 nM, which then leveled off at 250 nM. Lanes 6-10 are controls to indicate that when no reverse transcription was carried out, no band was detected. These results indicate that OA affected the expression of endogenous TRbeta 1 at the mRNA level.

OA Increased the Stability of TRbeta 1 Protein

Because the expression level of endogenous TRbeta 1 was virtually nondetectable under the present experimental conditions (Fig. 1A and lane 1 of Fig. 2A), it was not possible to compare the effect of OA on the expression of endogenous TRbeta 1 at the translation level. However, since we have shown that the characteristics and functions of endogenous TRbeta 1 were indistinguishable from those of transfected TRbeta 1 (see above), we used transfected TRbeta 1 to study the effect of OA on the stability of TRbeta 1. The stability of TRbeta 1 was examined by a brief pulse with [35S]methionine followed by a chase for different lengths of time in the presence (Fig. 6A, lanes 8-14) or absence of 100 nM OA (lanes 1-7). Comparison of the intensities of labeled TRbeta 1 at each time point indicates that TRbeta 1 from cultures in the presence of OA was more stable than without OA treatment. Quantitation analyses indicate that t1/2 of TRbeta 1 was ~1.88 ± 0.07 and 0.96 ± 0.02 h (Fig. 6B) in the presence of OA or absence of OA, respectively, indicating that treatment of cells with OA led to a 2-fold increase in the stability of TRbeta 1.


Fig. 6. Panel A, determination of the effect of OA on the half-life of TRbeta 1. COS-1 cells were transfected with pCLC51 (8 µg). Two days later, 100 nM OA was added to the cultures for 3 h, and cells were labeled with 50 µCi of [35S]methionine for 30 min and chased for 1.5, 3.5, 5.5, 7.5, 9.5, and 16.5 h. Cell lysates were prepared, and immunoprecipitation was performed using mAb C4 as described in Fig. 1A. Panel B, half-life of TRbeta 1 protein. The intensities of the TRbeta 1 bands from the gel above were quantified by PhosphorImager analysis and plotted. Panel C, effect of OA on the rate of synthesis of TRbeta 1. Two days after transfection with 8 µg TRbeta 1 expression plasmid, COS-1 cells were incubated with 100 nM OA for 3 h followed by labeling with 50 µCi of [35S]methionine for 5, 10, 15, 20, 25, and 30 min. The lysates were prepared, and immunoprecipitation was performed as described in Fig. 1A.
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We further examined the effect of OA on the rate of synthesis of transfected TRbeta 1 by labeling cells with [35S]methionine for various lengths of time in the presence (Fig. 6C, lanes 7-12) or absence (lanes 1-6) of OA. The intensities of bands were quantified, and the rates of synthesis were calculated to be 3,546 ± 607 and 3,402 ± 250 counts/min for TRbeta 1 synthesized in the absence or presence of OA, respectively. These data indicate that OA had no effect on the rate of synthesis of TRbeta 1, suggesting that the increased stability of the TRbeta 1 protein was the major factor responsible for the OA induction of the TRbeta 1 protein.


DISCUSSION

The mRNAs of the TR isoforms and their encoded receptor proteins are known to be expressed differentially in a tissue-specific manner. The ratios of the mRNA versus the protein levels vary widely from tissue to tissue (11, 12). Moreover, the timing of the appearance of the various TR isoforms and the ratios of these isoforms are regulated tightly during development (10, 11). However, the mechanisms underlying this tissue-specific and developmental regulation of TRs are largely unknown. In the present study we discovered that the expression of endogenous TRbeta 1 protein, but not TRbeta 2 or TRalpha 1, could be induced by OA in COS-1 cells, which are believed to be functionally deficient in response to T3. However, this induction is unique among the cell lines examined including T3 functionally competent GH3 cells (13) and neuro-2alpha cells (15) and T3 functionally deficient cells (JEG-3, NIH3T3, HeLa, and A431 cells). Therefore, our findings highlight a potentially novel mechanism explaining the tissue-specific expression of TR isoforms.

Our data indicate that the molecular mechanisms of the induction of TRbeta 1 expression by OA were complex. The induction was mediated at both the transcriptional and translational levels. The amounts of mRNA of TRbeta 1 were reduced as the concentration of OA was increased. However, paradoxically, although OA treatment led to a reduction of mRNA, the TRbeta 1 proteins were increased by OA except at a high concentration of 250 nM. The sharp increase in the level of TRbeta 1 protein from 50 to 100 nM (Fig. 2B) was not proportional to the corresponding decrease in mRNA (Fig. 5C), reminiscent of what has been observed in the in vivo studies reported by Schwartz et al. (12) in that there are large variations in the mRNA:TR protein ratios, suggesting additional regulation at the posttranslational level. Indeed, we found that although OA had no effect on the synthesis of TRbeta 1, OA increased its stability by nearly 2-fold. Therefore, the increase in the stability of TRbeta 1 plays a major role in the function of TRs. This is the first clear report demonstrating that one of the mechanisms of the regulation of TR expression is at the posttranslational level.

The reasons for the increased stability of TRbeta 1 protein by OA are not entirely clear. OA is an inhibitor of phosphoprotein phosphatase 1 and 2A and has been used widely as a tool to study the functions of phosphorylation in cellular processes (14). We have demonstrated that OA not only induced TRbeta 1 expression but also increased dramatically the extent of phosphorylation. The induced TRbeta 1 was a phosphoprotein that was phosphorylated at multiple Ser and Thr sites. Although the phosphorylation sites are unknown, it is reasonable to assume that phosphorylation of Ser or/and Thr residues by the treatment of cells with OA could change the conformation of TRbeta 1, thereby decreasing its susceptibility to proteolytic degradation. Phosphorylation-induced structural modification is not without precedent. Structural changes in glycogen phosphorylase have been studied by x-ray crystallography (26). As a result of phosphorylation of Ser-14, the interaction between subunits is strengthened, and the binding sites for allosteric effectors and substrates are altered (26). However, conformational changes are not obligatory for enhanced susceptibility of TRbeta 1 to proteolytic degradation. The increased phosphorylation of Ser or/and Thr sites could alter the interaction of TRbeta 1 with cellular proteases, thus altering the kinetics of degradation. Alteration of the stability of proteins by phosphorylation has been demonstrated directly for human Ikappa B-alpha (27), nicotinic acetylcholine receptors (28), and the Drosophila yan protein (29). Similar to the present studies of TRbeta 1, the changes in the stability of these proteins led to functional consequences (27-29).

Phosphorylation of TRs is not only limited to the beta 1 subtype. Chicken TRalpha 1 (30, 31) and rat TRalpha 2 (32) were also found to be phosphorylated. Therefore the different extents of phosphorylation at different sites in TR isoforms could affect their stability differently. The present study thus raises the interesting possibility that variations in the protein stability of TRbeta 1 may be one mechanism by which the tissue-specific and development-specific expression of the TR isoforms are regulated. Although it remains to be established that the stabilities of other TR isoforms are regulated by phosphorylation, what is clear from our study is that differential phosphorylation of TR isoforms in different cell types adds yet another level of fine tuning to the already multifaceted and complex mechanisms of regulation of TR-dependent gene transcription.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Laboratory of Molecular Biology, NCI, National Institutes of Health, Bldg. 37, Rm. 2D24, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-496-4280; Fax: 301-480-9676.
1    The abbreviations used are: TR(s), thyroid hormone receptor(s); T3, 3,3',5-triiodo-L-thyronine; h-TRbeta 1, human TR subtype beta 1; TRalpha 1, human TR subtype alpha 1; TRE, thyroid hormone response element; OA, okadaic acid; mAb, monoclonal antibody; PCR, polymerase chain reaction.

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