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Regulation of Thyrotropin Receptor Gene Expression in 3T3-L1 Adipose Cells is Distinct from Its Regulation in FRTL-5 Thyroid Cells

The Third Department of Internal Medicine, Yamanashi Medical University, Yamanashi 409–38, Japan

Address all correspondence and requests for reprints to: Toshimasa Onaya, M.D., Ph.D., Professor and Chairman, The Third Department of Internal Medicine, Yamanashi Medical University, 1110 Tamaho, Yamanashi 409–38, Japan. E-mail: onayat{at}res.yamanashi-med.ac.jp

	Abstract

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We previously have demonstrated that rat adipose tissue expresses TSH receptor (TSHR) messenger RNAs (mRNAs) at levels approaching those detected in the thyroid. Furthermore, we recently reported that TSHR mRNA is detected in fibroblast-like 3T3-L1 cells after their hormone-induced differentiation into adipocytes. TSH induces cAMP formation and lipolysis in differentiated 3T3-L1 cells. We now show that, in Northern blot analyses, TSH-induced down-regulation of TSHR mRNA levels, which can be duplicated by forskolin and dibutylyl cAMP, i.e. which is cAMP-mediated. We also have demonstrated that a ß-adrenergic stimulant, which stimulates cAMP formation in adipocytes, induces a down-regulation of TSHR mRNA levels in 3T3-L1 adipocytes. Nuclear run-on assays show that the ability of TSH/cAMP to decrease TSHR mRNA levels in 3T3-L1 cells reflects transcriptional regulation. This report also demonstrates that TSHR gene expression in 3T3-L1 adipocytes is regulated in a manner distinct from that observed in thyroid cells. Thus, in fully differentiated 3T3-L1 adipocytes, TSH-induced down-regulation of TSHR mRNA levels is evident within 1 h and is near maximum within 4 h after addition of TSH. A transient increase of TSHR gene expression, which has been demonstrated in FRTL-5 thyroid cells, was not observed in 3T3-L1 adipocytes. The down-regulation of TSHR gene expression induced by TSH/cAMP in 3T3-L1 cells is cycloheximide-insensitive, suggesting that continuous protein synthesis is not required for this process. In contrast, the down-regulation of TSHR gene expression observed in FRTL-5 cells is sensitive to cycloheximide. In both FRTL-5 thyroid cells and 3T3-L1 adipocytes, insulin or serum increased TSHR mRNA levels. Although insulin or serum was required for the TSH-induced down-regulation of TSHR mRNA levels in FRTL-5 thyroid cells, neither insulin nor serum was required for TSHR down-regulation in 3T3-L1 adipocytes. These findings demonstrate that TSH/cAMP regulates TSHR mRNA levels in adipocytes via a regulatory system distinct from that used in FRTL-5 cells. This report further demonstrates that adipose cells do not express thyroid transcription factor-1, which interacts with the TSHR promoter region in FRTL-5 cells, and that 3T3-L1 nuclear extracts exhibit a different binding activity to the cAMP-response element-like element in the TSHR promoter region compared with extracts from FRTL-5 cells.

	Introduction

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THE TSH receptor (TSHR) mediates the ability of TSH to affect the growth and function of thyroid cells (1, 2, 3, 4). Autoantibodies to the TSHR that increase thyroidal cAMP levels are present in patients with Graves’ disease and cause hyperfunctioning of the thyroid gland (5). Patients with Graves’ disease exhibiting exophthalmos and pretibial dermopathy often have high titer of thyroid-stimulating antibodies (6). Expression of TSHR in extrathyroidal cells may account for the extrathyroidal manifestations in Graves’ disease, especially because TSH binding activity in extrathyroidal tissues has been established (7, 8). The presence of TSHR messenger RNA (mRNA) in several extrathyroidal cells has been reported using the RT-PCR technique (9, 10, 11, 12).

TSHRs expressed in adipocytes have been shown to be functional (13, 14) and are suggested to be involved in the regulation of adipose function. Recently we reported that rat adipose tissue (15), as well as cultured rat preadipocytes (16), express similar amounts of TSHR mRNA as that detected in the thyroid gland. Marcus et al. (17) showed that human fat tissues also express TSHR and that TSH has significant effects on lipolysis in physiological concentrations (18). The maximum lipolytic effect of TSH was the same as that of isoproterenol in human adipocytes isolated from neonates (18). In addition, we recently showed that a full-length TSHR cDNA from rat fat cells was almost identical to that from the thyroid and that its function was indistinguishable from that in the thyroid (15). This clone differed by only one amino acid relative to the rat thyroid TSHR cDNA. When this fat TSHR cDNA was transfected into Chinese hamster ovary cells, TSH induced cAMP formation in a manner similar to that of Chinese hamster ovary cells transfected with thyroid TSHR cDNA (15). These observations suggest that TSHRs are involved in the regulation of adipose function and raise the question of whether TSH regulates TSHR gene expression in a manner similar to that observed in thyroid cells.

In rat FRTL-5 thyroid cells, TSH and the subsequent cAMP signal cause a time-dependent positive and then negative regulation of TSHR gene expression (19, 20). Insulin or insulin-like growth factor I is required for the autoregulation of TSHR gene expression by TSH/cAMP (20). Recently, the 5'-flanking region of the rat TSHR was cloned (21), and a minimal promoter region, -220 to -39 bp, that exhibits thyroid-specific expression and TSH/cAMP autoregulation of the TSHR was identified (21, 22, 23, 24, 25, 26). One regulatory element defined therein, -189 to -175 bp, binds thyroid transcription factor-1 (TTF-1) (24). The TTF-1 element contributes to thyroid-specific expression and to the cAMP autoregulation of the TSHR gene (24, 27). TSH/cAMP-increased TTF-1 phosphorylation results in up-regulation of TSHR gene expression (24, 27), whereas a TSH/cAMP-induced decrease in TTF-1 RNA levels is associated with down-regulation (24, 27). Additionally, single strand DNA-binding proteins (SSBPs) interact with an element contiguous with the 5'-end of the TTF-1 element (26). The SSBPs function conjointly with TTF-1 in the full expression and TSH/cAMP-induced negative regulation of TSHR in thyroid cells (26).

In contrast to thyroid cells, the control of TSHR gene expression in adipose cells is not understood. A suitable cell culture model for the study of adipocytes is 3T3-L1 cells, a fibroblast-like cell line derived from the Swiss mouse embryo that, upon reaching confluence, can be differentiated by hormonal induction into adipocytes (28, 29). We recently reported that a dramatic appearance of TSHR mRNA occurred after hormonal pulse-induced differentiation of 3T3-L1 cells into adipocytes by treatment with insulin, isobutylmethylxanthine (IBMX), and dexamethasone (30). This cultured cell system, therefore, is useful for studying the regulation of TSHR gene expression in adipocytes.

In the present study, we demonstrate that TSH and its cAMP signal down-regulate TSHR mRNA levels in differentiated 3T3-L1 cells in a manner distinct from that observed in thyroid cells. This report further explores the action of insulin on TSHR mRNA levels and TSH/cAMP-induced down-regulation in 3T3-L1 cells. These results suggest that TSHRs in adipose cells are autoregulated by TSH/cAMP, whose action is mediated by a different set of transcription factors from those used by thyroid cells. In this report, we also provide data showing differences between 3T3-L1 and FRTL-5 cells in nuclear proteins interacting with the TSHR promoter.

	Materials and Methods

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Cell culture
3T3-L1 cells [American Type Culture Collection (ATCC), Rockville, MD, No. CCL 92.1] were grown in high glucose DMEM and supplemented with 10% calf serum. Confluent 3T3-L1 preadipocytes were induced to differentiate into adipocytes, as previously described (30, 31). Briefly, 1 day after confluence, cells were treated with media containing 10% FBS, 10 µg/ml insulin, 0.2 µg/ml dexamethasone, and 0.5 mM IBMX for 3 days. After 3 days, this medium was replaced by media supplemented with 10 µg/ml insulin and 10% FBS, and two days later, the media was replaced with DMEM supplemented with 10% FBS. Cells were used for studies 9 or 10 days after induction of differentiation.

FRTL-5 rat thyroid cells (ATCC No. CRL 8305) were grown in Coon’s modified Ham’s F-12 medium supplemented with 5% calf serum and a mixture of six hormones containing bovine TSH (10 mU/ml), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml) (32).

RNA isolation and Northern analysis
Total RNA was isolated from cells by the guanidine isothiocyanate extraction method (33). For Northern blot analyses, 20 µg of total RNA was electrophoresed on a 1% agarose gel containing 0.66 M formaldehyde and blotted onto a nitrocellulose filter, as previously described (20, 24). A mouse thyroid TSHR cDNA (base 18–1575) (34) was obtained by the RT-PCR technique (35) from a mouse thyroid cDNA library primed with oligo(dT). The PCR product was subcloned into pCRII vector (Invitrogen, San Diego, CA) and sequenced its identity. Rat ß-actin cDNA was kindly donated by Dr. L. D. Kohn (NIH, Bethesda, MD). The rat TTF-1 probe was a fragment from +1 to +331 bp excised from the TTF-1 expression vector, RcCMV-THA; it was the kind gift of Dr. R. Di Lauro (Stazione Zoologica A. Dohrn, Naples, Italy) (36). All probes were radiolabeled using a random primer labeling kit (Takara Shuzo Co., Kyoto, Japan). Blots were hybridized in 50% formamide, 2.5x Denhardt’s solution, 5x SSPE (0.6 M NaCl, 40 mM sodium phosphate, 4 mM EDTA, pH 7.4), 0.1% SDS, 0.1 mg/ml heat-denatured salmon sperm DNA, and 5% dextran sulfate. The filters were then washed three times at room temperature in 2x SSC (0.3 M NaCl, 30 mM sodium acetate, pH 7.0) containing 0.1% SDS, followed by three washes three times at 53 C in 0.1x SSC-0.1% SDS. The filters were exposed to an imaging plate and were analyzed using a BAS2000 image analyzer (Fuji Film Co., Tokyo, Japan).

In vitro nuclear run-on analysis
Nuclear run-on experiments were performed as described (37, 38). Briefly, nuclei isolated from 3T3-L1 cells, before or after induction of differentiation, were incubated at 26 C for 45 min in 100 µl reaction mixtures containing 16% glycerol, 20 mM HEPES (pH 8.0), 0.04 mM EDTA, 0.5 mM MnCl₂, 90 mM NH₄Cl, 5 mM MgCl₂, 2 mM dithiothreitol, and 0.4 mM each of ATP, cytidine 5'-triphosphate, and GTP, and 0.2 mCi [-³²P]uridine 5'-triphosphate. The reaction was stopped by the addition of 100 µl of a stop mixture containing 200 µg/ml ribonuclease (RNase)-free deoxyribonuclease I, 200 µg/ml proteinase K, 200 µg/ml yeast transfer RNA, 20 mM HEPES (pH 8.0), and 20 mM CaCl₂, followed by further incubation for 20 min at room temperature. Radiolabeled RNA was purified and hybridized to 4 µg DNA immobilized on nitrocellulose filters. Four micrograms each of mouse TSHR cDNA, rat ß-actin cDNA, and pGEM7Zf(+) (Promega, Madison, WI) plasmid DNA (to control for nonspecific binding) were immobilized on the nitrocellulose filters. Hybridization was performed at 45 C for 48 h in 50% formamide, 5x SSPE, 10x Denhardt’s solution, 0.1% SDS, 0.25 mg/ml heat-denatured salmon sperm DNA, 50 µg/ml poly(A), and 25 µg/ml yeast transfer RNA. Filters were then washed three times in 2x SSC-0.1% SDS for 30 min at room temperature, followed by washes in 0.1x SSC-0.1% SDS at 45 C. Filters were next washed twice in 0.2x SSC at 37 C for 5 min and incubated in 2x SSC containing 10 µg/ml RNase A and 350 U/ml RNase T1 at 37 C for 30 min. After incubation, the filters were washed twice in 0.2x SSC-0.1% SDS at room temperature and washed again in 0.2x SSC. Filters were exposed to an imaging plate and analyzed using a BAS2000 image analyzer (Fuji).

Nuclear extracts
Nuclear extracts were prepared exactly as previously described (21, 22, 23, 24, 25, 26, 27). 3T3-L1 cell extracts were prepared from cells 9 or 10 days after induction of differentiation. FRTL-5 nuclear extracts were from cells maintained in medium depleted of TSH for 7 days after they were grown to near confluency in medium containing TSH.

Electrophoretic mobility shift assays
Electrophoretic mobility shift assays were performed, as previously described (21, 22, 23, 24, 25, 26, 27), using a double-stranded oligonucleotide containing the rat TSHR promoter -146/-114 (relative to the translation start site) sequence (CCAGCGATGAGGTCACAGCCCCTTGGAGCCCTC; the underlining indicates the cAMP-response element (CRE)-like sequence).

	Results

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TSH down-regulates TSHR mRNA levels in differentiated 3T3-L1 adipocytes
Fibroblast-like 3T3-L1 cells were fully differentiated into adipocytes in the presence of insulin, dexamethasone, and IBMX. We previously demonstrated that the steady-state TSHR mRNA level increases during differentiation of 3T3-L1 cells. Northern blot analysis demonstrated that TSHR mRNA is not detectable in undifferentiated 3T3-L1 cells and does not appear until the 5th day after the initiation of differentiation (Fig. 1A, lane 2, and Ref.30). TSHR mRNA becomes evident by day 7 and reaches a maximum sustained level at day 11 (30). It is important to note that the mRNA remains at maximum levels for 5 days after the inducing hormones are removed from the cells (Fig. 1A, lanes 1 and 3). Despite this stable expression, treatment of differentiated cells with TSH (10 mU/ml) for 24 h caused a decrease in TSHR mRNA levels (Fig. 1). Down-regulation of TSHR mRNA levels could be duplicated by forskolin (10 µM) or dibutyryl cAMP (1 mM) treatment, both of which increased cAMP levels (Fig. 1).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effects of TSH, forskolin (FSK), dibutylyl cAMP (DBC), and isoproterenol on expression of TSHR and ß-actin mRNA expression in 3T3-L1 cells. Equal amounts of total RNA (20 µg per lane) are subjected to sequential Northern analyses using mouse TSHR and ß-actin cDNA probes (A). Confluent 3T3-L1 cells (preadipo, lane 2), grown in DMEM with 10% calf serum, are induced to differentiate into adipocytes using two different protocols. Cells are either treated with insulin (I) and 10% FBS (F) (IF) for 7 days, preceded by 3 days of treatment with insulin, dexamethasone (D), IBMX (X), and FBS (IDXF) (lane 1). Alternatively, cells (adipo) are treated with FBS for 5 days followed by treatments with IDXF (3 days) and IF (2 days), as described in the Materials and Methods (lane 3), and then exposed to TSH (10 mU/ml) (lane 4), forskolin (10 µM) (lane 5), or DBC (1 mM) (lane 6) for 24 h. The periods of incubation with hormones are indicated in parentheses (days). B, To investigate the effect of isoproterenol on TSHR mRNA levels in differentiated 3T3-L1 cells, RNA is prepared from cells exposed to the indicated concentrations of isoproterenol for 4 h, and equal amounts (20 µg/lane) are subjected to sequential Northern blot analyses using the TSHR and ß-actin probes. Quantitation is performed using a BAS2000 image analyzer (Fuji). The TSHR/ß-actin ratio is calculated and compared with the control (lane 3, adipo). Values are expressed as a percent of the control and are means ± SEM of three separate experiments with different batches of cells.

Transcriptional rate (nuclear run-on) assays revealed that both the differentiation-induced increase and the TSH/cAMP-induced down-regulation of TSHR mRNA levels occurs at the level of gene transcription. Nuclei were isolated from undifferentiated 3T3-L1 cells or 10 days after the initiation of differentiation in the presence or absence of TSH or forskolin for 4 h. TSHR transcripts were barely detectable in nuclei isolated from undifferentiated cells, whereas transcripts were abundant in nuclei isolated from fully differentiated cells (Fig. 2A). Treatment of differentiated cells with TSH or forskolin for 4 h caused a decrease in TSHR transcripts. No change was detected in levels of ß-actin transcripts (Fig. 2A). Although [³²P] uridine 5'-triphosphate (UTP)-labeled nuclear RNA isolated from differentiated cells hybridized slightly to the control plasmid, the differentiation-induced increase and TSH, as well as forskolin-induced down-regulation of TSHR mRNA transcription, were still statistically significant when the intensities generated by nonspecific hybridization were subtracted (Fig. 2B). These results demonstrate that TSH down-regulates the transcription rate of the TSHR gene via its cAMP signal in differentiated 3T3-L1 adipocytes.

View larger version (12K): [in this window] [in a new window]   Figure 2. Transcriptional nuclear run-on analysis of the TSHR gene in undifferentiated and differentiated 3T3-L1 cells. Nuclei are isolated from 3T3-L1 cells before (preadipo) and after induction of differentiation (adipo) with the same hormonal treatment as in Fig. 1A, lane 3. Nuclei are then isolated from differentiated 3T3-L1 cells exposed to TSH (10 mU/ml) or forskolin (FSK) (10 µM) for 4 h. [32P]UTP-labeled RNA transcripts isolated from nuclei are used in hybridization reactions with either mouse TSHR cDNA, control ß-actin cDNA, or control empty vector pGEM7Z plasmid DNA as indicated. All filters are hybridized and washed, as described in Materials and Methods. Quantitation is performed using a BAS2000 image analyzer (Fuji). The TSHR/ß-actin ratio is calculated after subtraction of values generated by nonspecific hybridization to control empty plasmid DNA. The ratio from differentiated 3T3-L1 cells (adipo) not exposed to TSH or forskolin is set at 100% (B). Data are mean ± SEM of three separate experiments with different batches of cells.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of different concentrations of TSH on expression of TSHR mRNA in 3T3-L1 adipocytes and FRTL-5 thyroid cells. 3T3-L1 cells are induced to differentiate into adipocytes, as described in the Materials and Methods. FRTL-5 cells are maintained for 7 days without any TSH in the medium. RNA is prepared from cells exposed to the indicated concentrations of TSH for 24 h, and equal amounts (20 µg/lane) are subjected to sequential Northern blot analysis using the TSHR and ß-actin probes. The TSHR/ß-actin ratios are expressed as a percent of control and are means ± SEM of three separate experiments with different batches of cells.

View larger version (30K): [in this window] [in a new window]   Figure 4. Kinetics of TSH effects on TSHR mRNA levels in 3T3-L1 adipocytes and FRTL-5 thyroid cells. 3T3-L1 cells are induced to differentiate into adipocytes, as described in the Materials and Methods. FRTL-5 cells are maintained for 7 days with no TSH in the medium. RNA is prepared from the cells exposed to TSH for the indicated times, and equal amounts (20 µg/lane) are subjected to sequential Northern analysis using the TSHR and ß-actin probes. The TSHR/ß-actin ratios are expressed as a percent of control and are means ± SEM of three separate experiments with different batches of cells. The inset shows a typical experiment using differentiated 3T3-L1 cells.

View larger version (33K): [in this window] [in a new window]   Figure 5. Effect of cycloheximide (CHx) on the TSH- and forskolin (FSK)-induced down-regulation of TSHR mRNA levels. 3T3-L1 cells are induced to differentiate into adipocytes, as described in Materials and Methods. FRTL-5 cells are maintained 7 days with no TSH in the medium. Cells are exposed to TSH (10 mU/ml) or forskolin (10 µM) is exposed in the presence or absence of cycloheximide (10 µM) for 4 h (3T3-L1) or 24 h (FRTL-5). Northern analyses are performed and quantitated as described. The data are presented as TSHR mRNA levels per µg of total RNA applied to the gels; ethidium bromide staining confirmed that equal amounts of RNA were applied to each lane, because TSH induces a marked decrease in ß-actin mRNA levels when FRTL-5 cells were incubated with cycloheximide (21). Data are means ± SEM of three separate experiments with different batches of cells.

View larger version (19K): [in this window] [in a new window]   Figure 6. Effects of insulin and serum on the TSH- or forskolin (FSK)-induced down-regulation of TSHR mRNA levels. A) Eight days after induction of differentiation, media is replaced with either medium without insulin or FBS, medium with insulin only, or medium with FBS only. Cells are maintained in this media for 2 days and are exposed to TSH (10 mU/ml) for 24 h. B) FRTL-5 cells are maintained for 6 days in medium containing 0.2% calf serum without TSH and insulin (basal) or in media containing insulin or 5% calf serum and then exposed to TSH for 24 h. RNA is isolated, and Northern blot analyses are performed as described. The ratio of TSHR to ß-actin mRNA levels is calculated with the ratio of TSHR mRNA to ß-actin mRNA in cells maintained in medium alone set at 100%. Data are means ± SEM of three separate experiments with different batches of cells.

A CRE-like element, TGAGGTCA, exists in the minimal promoter region of the TSHR gene and contributes to its constitutive expression in thyroid cells (22). It also has been revealed that the enhancer activity of TTF-1 on the TSHR promoter totally depended on the CRE-like site (24). Furthermore, a recent report (41) showed that the CRE-like site also was involved in TSH-induced regulation of TSHR gene via the induction of the inducible cAMP early repressor (ICER) in FRTL-5 cells. To investigate nuclear proteins interacting with the CRE-like sequence in adipose cells, we performed electrophoretic mobility shift assays. Nuclear extract from differentiated 3T3-L1 cells exhibited protein/DNA complexes (A and C complexes) similar to those formed with FRTL-5 nuclear extract. However, a protein/DNA complex (B complex), which was not detectable in the FRTL-5 lane, was formed in the 3T3-L1 lane. Although the functional role of this nuclear protein is unclear at this time, we hypothesize that the CRE-like element in adipocytes may exhibit activities different from those in FRTL-5 cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present report, the down-regulation of TSHR gene expression induced by TSH in 3T3-L1 adipocytes was investigated. We have shown previously that treatment of fibroblast-like 3T3-L1 cells with insulin, after exposure to insulin, dexamethasone, and IBMX, induced expression of TSHR mRNA. TSHR mRNA expression was observed concurrently with morphologic differentiation, as assessed by the formation of lipid droplets in the cells (30). We now show that expression of the TSHR gene is irreversibly induced during hormonal differentiation in 3T3-L1 cells. The nature of this irreversible switch is underscored by the fact that expression of the TSHR gene, as well as the adipocytic phenotype, does not depend on continued hormonal stimulation. TSH causes an increase in cAMP levels in differentiated 3T3-L1 adipocytes within 30 min (30). Despite the stable expression of the TSHR gene after a hormonal pulse that included the cAMP-generating agent, IBMX, exposure of the differentiated 3T3-L1 cells to TSH results in a decrease in TSHR mRNA levels. Because the TSH-induced down-regulation also can be mimicked by a cAMP analog and forskolin, this TSH action is presumed to be cAMP mediated. We demonstrate that the increase in TSHR mRNA during 3T3-L1 cell differentiation and the TSH/cAMP-mediated decrease in TSHR mRNA levels occur at the level of transcription.

In FRTL-5 thyroid cells, the hormonal regulation of TSHR gene expression has been well characterized (20). As previously reported (20), short exposures to TSH (2 h) increases TSHR mRNA levels; however, TSHR mRNA levels decline thereafter. Saji et al. (20) also have demonstrated that the TSH-induced up-regulation, as well as the down-regulation, is transcriptional. In contrast, in 3T3-L1 adipocytes, TSH treatment resulted in a decrease in TSHR mRNA levels without any transient increase at early time points (Fig. 4). This difference may be caused by the lack of TTF-1 expression in 3T3-L1 adipocytes (Fig. 7A). The minimal promoter region of the TSHR gene has a TTF-1-binding element that plays an important role in TSHR gene expression in thyroid cells (24) and is, at least in part, involved in the TSH/cAMP-induced autoregulation of TSHR gene expression (24, 26, 27). TSH causes a time-dependent increase, and then decrease, in the amount of TTF-1/DNA complex formed in gel mobility shift assays using nuclear extracts from FRTL-5 thyroid cells (24, 27). It has been hypothesized that the early increases in TSHR gene expression are associated with increased TTF-1 binding to the TSHR promoter mediated by the activation of protein kinase A (PKA) (24, 27). However, the hypothesis that TTF-1 is involved in TSH/cAMP regulation is still a matter of controversy. Christophe-Hobertus et al. (42) reported that TTF-1 is not involved in the regulation of thyroglobulin gene expression by TSH/cAMP. Kambe et al. (43) recently showed that TSH increased DNA-binding activity of TTF-1 in whole cell extracts prepared without dithiothreitol.

View larger version (35K): [in this window] [in a new window]   Figure 7. Differences in transcription factors interacting with the TSHR promoter region in 3T3-L1 adipocytes and FRTL-5 thyroid cells. (A) Total RNAs are prepared from rat liver, rat epididymal fat pad, 3T3-L1 cells before (preadipo) and after (adipo) the induction of differentiation, and FRTL-5 cells maintained in the medium without TSH for 7 days. Equal amounts of total RNA (20 µg per lane) are subjected to sequential Northern analyses using rat TTF-1 and ß-actin cDNA probes. (B) One µg of nuclear extracts from FRTL-5 cells maintained in the absence of TSH and differentiated 3T3-L1 cells are used in electrophoretic mobility shift assays. Synthetic oligonucleotide, identical to the promoter region of rat TSHR gene spanning -146 to -114 bp, is used as a probe.

Cycloheximide inhibited the ability of TSH and forskolin to down-regulate TSHR gene expression in FRTL-5 cells (Fig. 5 and Ref.20). The TSH-induced decrease in TTF-1/TSHR complex formation, which coincides with TSH-induced down-regulation of TTF-1 and TSHR mRNA levels in FRTL-5 cells, is a cycloheximide-sensitive process (24). Although 3T3-L1 adipocytes do not express TTF-1, TSH and a cAMP-mediated signal down-regulate TSHR gene expression in 3T3-L1 adipocytes. Thus, it was of interest that the TSH/cAMP-induced down-regulation in 3T3-L1 adipocytes is not affected by cycloheximide (Fig. 5). This result suggests that, in 3T3-L1 cells, the TSH/cAMP-induced down-regulation of TSHR mRNA levels does not require continuous protein synthesis, or that the TSH-activated inhibitory factor in 3T3-L1 cells may have a longer half-life than the same factor in FRTL-5 cells. TSHR suppressor element-binding protein-1, which is ubiquitously expressed, has been cloned (44). This protein suppresses TSHR promoter activity, and its DNA-binding activity is enhanced by PKA-induced phosphorylation (44). Although it is not yet clarified, the TSH/cAMP-induced down-regulation of TSHR gene expression in 3T3-L1 adipocytes may be the result of interactions with TSHR suppressor element-binding protein-1, as well as nuclear factors expressed only in fat cells.

The present report shows further that insulin and serum are positive regulators of TSHR mRNA levels in 3T3-L1 adipocytes. In FRTL-5 cells, it has been revealed that insulin, insulin-like growth factor I, and serum up-regulated the transcription of TSHR gene (20). The difference, however, is that insulin/serum is required for TSH/cAMP to down-regulate TSHR gene expression in FRTL-5 cells (Fig. 6B and Ref.20) but not in 3T3-L1 adipocytes (Fig. 6A). The region immediately upstream of the TTF-1 site in TSHR gene was identified as a insulin-response element (IRE) (25). This IRE seems to function in a thyroid-specific manner, because mutation of the TTF-1 site results in the loss of IRE function in FRTL-5 cells (25). These findings lead us to a hypothesis that insulin-regulating factors distinct from those in FRTL-5 may be involved in the transcriptional regulation of TSHR gene by insulin/serum in 3T3-L1 adipocytes.

Catecholamines play a major role in lipid metabolism via ß-adrenergic receptors. It has been reported that increased levels of cAMP in adipose cells induce an increase in ß₃-adrenergic receptors (40) and a loss of ß₁- and ß₂-adrenergic receptors (39). The present study confirms that the cAMP signal generated by a ß-adrenergic agonist induces a down-regulation of TSHR mRNA levels. This result suggests that TSHR and ß-adrenergic receptors are functionally interrelated in adipose cells. In contrast, this was not found in thyroid cells, because we have shown previously that cultured thyroid cells (FRTL) do not express ß-adrenergic receptors (45). In FRTL-5 cells, a ß-adrenergic agonist also failed to induce cAMP formation (46)

Transcriptional regulations mediated by cAMP signal in adipose cells have been reported in several genes. cAMP mediates the up-regulation of the ß₃-adrenergic receptor (40), GLUT1 (47), adipose fatty acid-binding protein (48), and uncoupling protein genes (49), as well as the down-regulation of ß₁- and ß₂-adrenergic receptor (39), GLUT4 (47), and S14 genes (50). However, most of the regulatory mechanisms in these genes remain unclear. This report shows that TSH and the cAMP-induced pathway down-regulate TSHR gene expression in differentiated 3T3-L1 cells in a manner distinct from that observed in thyroid cells. Although some of the transcription factors interacting with the TSHR promoter may be common in adipose and thyroid cells, it is reasonable to predict that a set of transcription factors distinct from those used in thyroid cells will mediate the hormonal regulation of TSHR gene expression, as well as its differentiation-dependent expression, in adipocytes. In this report, we confirmed no expression of TTF-1 in adipose cells. The present report also identified that the CRE-like sequence in the minimal TSHR promoter region and nuclear extract from 3T3-L1 adipocytes formed a protein/DNA complex that was not detectable with the FRTL-5 nuclear extract. These results reinforce our hypothesis that adipose cells have different regulatory mechanisms for the TSHR gene expression from those in FRTL-5 cells. Characterization of the TSHR promoter in adipose cells is under current investigation.

Received August 23, 1996.

	References

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