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Molecular Endocrinology 11 (4): 481-489
Copyright © 1997 by The Endocrine Society

The Thyrotrope-Restricted Isoform of the Retinoid-X Receptor-{gamma}1 Mediates 9-cis-Retinoic Acid Suppression of Thyrotropin-ß Promoter Activity

Bryan R. Haugen, Nicole S. Brown, William M. Wood, David F. Gordon and E. Chester Ridgway

Division of Endocrinology, Diabetes, and Metabolism Department of Medicine University of Colorado Health Sciences Center Denver, Colorado 80262


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TSHß is a subunit of TSH that is uniquely expressed and regulated in the thyrotrope cells of the anterior pituitary gland. Thyroid hormone receptors (TR) are known to mediate T3 suppression of TSHß gene expression at the level of promoter activity. The role of other nuclear receptors in regulation of this gene is less clearly defined. Retinoid X receptors (RXR) are a family of nuclear transcription factors that function both as 9-cis-retinoic acid (RA) ligand-dependent receptors and heterodimeric partners with TR and other nuclear receptors. Recently, the RXR isoform, RXR{gamma}, has been identified in the anterior pituitary gland and found to be restricted to thyrotrope cells within the pitutiary. In this report, we have further characterized the distribution of RXR{gamma}1, the thyrotrope-restricted isoform of RXR{gamma}, in murine tissues and different cell types. We have found that RXR{gamma}1 mRNA and protein are expressed in the TtT-97 thyrotropic tumor, but not the thyrotrope-variant {alpha}TSH cells or somatotrope-derived GH3 cells. Furthermore, we have studied the effects of RXR{gamma}1 on TSHß promoter activity and hormone regulation in these pituitary-derived cell types. Both T3 and 9-cis-RA independently suppressed promoter activity in the TtT-97 thyrotropes. Interestingly, the combination of ligands suppressed promoter activity more than either alone, indicating that these hormones may act cooperatively to regulate TSHß gene expression in thyrotropes. The RXR{gamma}1 isoform was necessary for the 9-cis-RA-mediated suppression of TSHß promoter activity in {alpha}TSH and GH3 cells, both of which lack this isoform. RXRß, a more widely distributed isoform, did not mediate these effects. Finally, we showed that the murine TSHß promoter region between -200 and -149 mediated a majority of the 9-cis-RA suppression of promoter activity in thyrotropes. This region is distinct from the T3-mediated response region near the transcription start site. These data suggest that retinoids can mediate TSHß gene regulation in thyrotropes and the thyrotrope-restricted isoform, RXR{gamma}1, is required for this effect.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TSH is a glycoprotein hormone that is produced only by the thyrotrope cells of the anterior pituitary gland (1). TSH contains two dissimilar, noncovalently associated subunits: the {alpha}-subunit, which is shared among other glycoprotein hormones, and the ß-subunit, which is functionally unique and has expression limited to thyrotropes. We have been investigating the cell-specific expression and regulation of the murine TSHß subunit gene. A number of factors including Pit-1, Pit-1T, thyroid hormone receptor (TR), and a yet undefined 50-kDa protein appear essential for TSHß promoter activity and its regulation in thyrotropes (2, 3, 4, 5, 6). Recently, our group (7) and Sugawara et al. (8) have studied a retinoid X receptor (RXR) isoform, RXR{gamma}, which is restricted to thyrotropes in the anterior pituitary gland as well as the thyrotrope-derived tumor, TtT-97. The RXRs belong to a large family of transcription factors and function as both 9-cis-retinoic acid (RA) ligand-dependent receptors and heterodimeric partners with thyroid hormone (TR), retinoic acid receptors, and vitamin D receptors (9). Mangelsdorf et al. (10) have demonstrated that RXR{alpha} and RXRß mRNA are widely expressed in developing embryo and adult murine tissues, while RXR{gamma} mRNA is more limited in distribution, including abundant expression in the anterior pituitary. RXR{gamma} has been further characterized as two isoforms, RXR{gamma}1 and RXR{gamma}2, which are generated by alternate splicing at the 5'-end of the gene (11). RXR{gamma}1 mRNA is restricted to brain and skeletal muscle. RXR{gamma}2 mRNA is found in heart, skeletal muscle, and liver. Pituitary mRNA has not been examined with an RXR{gamma}1 or {gamma}2-specific probe.

RXRs are recognized as major TR-associated proteins in many positively regulated promoter systems (12, 13, 14). The role of RXRs in negative regulation of promoters such as TSHß, however, is less clear. Hallenbeck et al. (15) showed that RXRß interfered with TR{alpha}-mediated T3 suppression of a negative thyroid hormone response element (-24 to -1) of the murine (m)TSHß promoter in fibroblasts. 9-cis-RA did not have an impact on this effect. Carr and Wong (16) also showed that RXRß could interfere with both TR{alpha}- and TRß-mediated T3 suppression of a negative thyroid hormone response element (+11 to +27)of the rat (r)TSHß promoter in COS cells. This effect was also 9-cis-RA independent. In contrast, Cohen et al. (17) showed that RXR{alpha} interfered with TRß-mediated suppression of hTSHß promoter (-20 to +1) activity in JEG-3 cells, but this effect was 9-cis-RA dependent. Studies carried out by these groups used a small region of the TSHß promoter around the transcription start site, and none of these groups analyzed effects of the thyrotrope-restricted RXR{gamma} isoform.

Breen et al. (18) recently showed that RA-deficient rats had increased levels of TSHß mRNA in pituitary extracts. They also showed that treatment with retinyl palmitate lowered these message levels. Furthermore, they showed in gene transfer studies that all-trans-RA suppressed activity of a larger fragment of the rTSHß promoter (-800 to +150), suggesting that retinoids play a role in regulation of the TSHß gene.

In this report, we have further characterized the tissue and cell type distribution of the RXR{gamma} isoforms. We have also investigated the role of RXR{gamma}1 in T3- and 9-cis-RA hormone-mediated regulation of mTSHß promoter activity in thyrotropes and pituitary-derived cells using a large fragment (-390 to +40) of the mTSHß promoter that contains elements that mediate both thyrotrope-specific promoter activity and negative regulation by T3.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RXR Isoform mRNA in Various Cell Types
We first evaluated expression of the RXR isoform mRNAs in the two thyrotrope-derived cell types, TtT-97 and {alpha}TSH, by Northern blot analysis. Figure 1Go shows that while RXR{alpha} and RXRß mRNAs are present in both cell types, RXR{gamma} transcripts are abundantly expressed in TtT-97 thyrotropes, but are virtually undetectable in the {alpha}TSH cell line, which no longer expresses the TSHß gene and lacks T3 regulation (19). Liu and Linney (11) further characterized two subtypes of RXR{gamma}, {gamma}1 and {gamma}2, which are generated by alternate splicing at the 5'-end of the gene. We therefore generated oligonucleotide probes corresponding to the unique 5'-sequences of these two subtypes (Fig. 2Go) and repeated Northern blot analysis on RNA from our thyrotrope-derived cell types, as well as the pituitary-derived GH3 somatomammotropes and non-pituitary HeLa cells. Figure 3Go shows that only TtT-97 cells express RXR{gamma}1 mRNA, while all three pituitary-derived cell types contain RXR{gamma}2 mRNA. Non-pituitary HeLa cells lack both RXR{gamma} subtypes.



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  		Figure 1. Northern Blot Analysis for RXR Isoforms in TtT-97 and {alpha}TSH RNA

Ten micrograms of poly A+ RNA were size-separated and transferred to Nytran. The filter was hybridized separately with [32P]{alpha}dCTP-labeled probes specific for RXR{gamma}, RXR{alpha}, RXRß, and ß-actin. The probe was removed by treating the filter with boiling H20 between hybridization of each probe. The filter was exposed to radiographic film for 16 h at -70 C for each probe except RXRß, which was exposed for 40 h.

 


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  		Figure 2. Schematic of RXR{gamma}1 and RXR{gamma}2 Isoforms

Schematic comparison of RXR{gamma}1 and RXR{gamma}2 is outlined. Coding regions are in blocks of domains (A-E). The unique 5'-untranslated region of RXR{gamma}2 is highlighted. Specific oligonucleotides (————*), which were generated to unique regions of each isoform, are noted.

 


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  		Figure 3. Northern Blot Analysis for RXR{gamma}1 and RXR{gamma}2 Isoforms in RNA from Various Cell Types

Five micrograms of poly A+ RNA were size-separated and transferred to nytran. The filter was hybridized in succession with [32P]{gamma}dATP- labeled oligonucleotides specific for RXR{gamma}1 and RXR{gamma}2. The probe was removed by treating the filter with boiling H20 between hybridization of each probe. The filter was exposed to radiographic film for 16 h at -70 C for RXR{gamma}1 and 72 h at -70 C for RXR{gamma}2.

 
To further analyze for the presence or absence of the RXR{gamma}1 mRNA in various mouse tissues and cell types, we performed sensitive RT-PCR on total RNA (Fig. 4Go). RT-PCR of total RNA from different murine tissues shows that RXR{gamma}1 mRNA is present in pituitary tissue, and a much smaller amount is present in brain and cardiac tissue. Lung RNA yeilded no detectable message for RXR{gamma}1. TtT-97 RNA gave a product for RXR{gamma}1, while {alpha}TSH cell RNA showed a barely detectable band, suggesting that while RXR{gamma}1 message is detectable in {alpha}TSH cells with this sensitive technique, it is at a much lower level than found in TtT-97 thyrotropes. GH3 mRNA had no detectable RXR{gamma}1 mRNA. A control RT-PCR with glyceraldehyde 3-phosphate dehydrogenase (G3PDH) showed that relatively similar amounts of RNA were used.



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  		Figure 4. RT-PCR of RNA for RXR{gamma}1 in Various Tissues and Cell Types

Total RNA (2.5 µg) from different murine tissues and cell types underwent reverse transcription with random hexamers. The sample was divided into two reactions for PCR using oligonucleotides specific for RXR{gamma}1 or G3PDH over 30 cycles under conditions described in Materials and Methods. One-tenth of the reaction volume was size-separated on a 1% agarose gel containing ethidium bromide and photographed on an UV light source.

 
Western Blot Analysis of Nuclear Extracts from TtT-97 and {alpha}TSH Cells
To evaluate for the presence of RXR{gamma}1 protein in the thyrotrope-derived cells, we performed Western blot analysis of nuclear protein extracts from two separate TtT-97 tumors as well as {alpha}TSH cells. The RXR{gamma}1-specific antiserum was kindly provided by Dr. W. W. Chin. Figure 5Go shows that no RXR{gamma}1 protein is present in {alpha}TSH cells while two imunoreactive bands are seen in both TtT-97 thyrotrope extracts. The lower band (~50 kDa) corresponds to the form previously reported by Sugawara et al. (8), while the upper band may represent a different phosphorylation state, an alternate form of RXR{gamma}1, or a protein that cross-reacts with the RXR{gamma}1 antiserum.



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  		Figure 5. Western Blot Analysis of TtT-97 and {alpha}TSH Nuclear Extracts

Twenty milligrams of nuclear extracts from two separate TtT-97 tumor preparations and {alpha}TSH cells were size-separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. After blocking with 7.5% nonfat milk in PBS, the filter was hybridized with antiserum generated against a peptide specific for RXR{gamma}1, then hybridized with a secondary antibody linked to horseradish peroxidase. Size standards are shown at the left.

 
Effects of RXR Isoforms on the TSHß Promoter in Pituitary-Derived Cells
To prove that functional receptors were expressed in transiently transfected cells, we cotransfected each receptor with a TREpal-TK-luciferase plasmid (kindly provided by Dr. J. L. Jameson) into GH3 cells (Fig. 6Go). Both RXR{gamma}1 and RXRß stimulated the TREpal element in the presence of the RXR ligand 9-cis-retinoic acid (RA), indicating that functional receptors are indeed expressed. Interestingly, equal amounts of transfected plasmids for the RXR isoforms mediated a similar promoter stimulation, suggesting that functionally equivalent amounts of these receptors are being expressed.



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  		Figure 6. Stimulation of TREpal TK-Luciferase with 9-cis-RA in the Presence or Absence of RXR Isoforms in GH3 Cells

Transient transfections were carried out as described in Materials and Methods. Twenty micrograms of a TREpal (2x) TK promoter-luciferase promoter DNA with 1 µg of pCMV or pCMV-RXR isoforms was transfected by electroporation into GH3 cells. Results are expressed as fold stimulation of promoter activity in the presence of 2 µM 9-cis-RA vs. no ligand.

 
We then cotransfected RXR{gamma}1 or RXRß with the mTSHß (-390 to +40) promoter-luciferase reporter in GH3 and {alpha}TSH cells, both of which lack the RXR{gamma}1 isoform. Neither isoform stimulated the TSHß promoter in these pituitary-derived cells (data not shown). Addition of more RXR isoform plasmids (10 µg) also had no stimulatory effect on the TSHß promoter.

Hormonally Induced Effects of RXR Isoforms on the TSHß Promoter in Pituitary-Derived Cells
To examine the influence of RXR{gamma}1 on T3- and 9-cis-RA-mediated TSHß promoter activity, we first analyzed the effects of T3, 9-cis-RA, and a combination of the two on mTSHß promoter activity in TtT-97 thyrotropes that contain RXR{gamma}1. As shown in Fig. 7Go, T3 (10 nM) suppressed promoter activity by 60%. 9-cis-RA (2 µM) also inhibited promoter activity by 60%. Interestingly, a combination of T3 and 9-cis-RA suppressed promoter activity more (by 80%), which was significantly greater (P < 0.05) than either ligand alone.



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  		Figure 7. Murine TSHß Promoter Activity in TtT-97 Cells in the Presence of T3 or 9-cis-RA

Transient transfections were carried out as described in Materials and Methods. Twenty micrograms of mTSHß (-390 to +40) promoter-luciferase reporter DNA were transfected into 5–10 million TtT-97 cells. Cells were incubated in the absence or presence of T3 (10 nM), 9-cis-RA (2 µM) or both. Results are expressed as percent promoter activity in the presence of ligand vs. no ligand. Asterisk denotes a significant difference (P < 0.05) between the T3/9-cis-RA group and the other two groups.

 
To determine whether RXR{gamma}1 is required for 9-cis-RA or T3 suppression of TSHß promoter activity, we next examined the effects of T3 and 9-cis-RA on promoter activity in GH3 somatomammotropes that respond to T3, but lack RXR{gamma}1. These cells do, however, contain RXR{alpha} and RXRß (8). T3 alone suppressed TSHß promoter activity by approximately 50% (Fig. 8AGo). Cotransfection of RXR{gamma}1 caused only a 60% suppression of promoter activity, indicating that this isoform does not influence the T3 response. Interestingly, cotransfection of RXRß appeared to block T3-mediated inhibition of TSHß promoter activity, which was also shown by Hallenbeck and co-workers (15) in mouse fibroblasts as well as by Carr and Wong (22) in COS cells. Figure 8BGo shows that 9-cis-RA alone mediated only a modest suppression of TSHß promoter activity (20%). Cotransfection of RXR{gamma}1 mediated a 55% suppression of promoter activity with 9-cis-RA, while RXRß had no effect. These data suggest that RXR{gamma}1 is necessary for the 9-cis-RA-mediated suppression of TSHß promoter activity seen in thyrotropes, and that RXR{gamma}1 and RXRß function quite differently in pituitary-derived cells. The combination of T3 and 9-cis-RA suppressed TSHß promoter activity only to a level seen with T3 alone in these GH3 cells (Fig. 8CGo). However, cotransfection of RXR{gamma}1 augmented this suppression in the presence of both ligands, while RXRß had no additional effect. This model suggests that the thyrotrope-restricted RXR{gamma}1 isoform is acting in an isoform-specific manner to mediate ligand-dependent suppression of TSHß promoter activity in pituitary-derived cells.



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  		Figure 8. Murine TSHß Promoter Activity in GH3 Cells Influenced by RXR Isoforms, T3, and 9-cis-RA

Transient transfections were carried out as described in Materials and Methods. Twenty micrograms of mTSHß (-390 to +40) promoter-luciferase reporter DNA were transfected into 5 million GH3 cells with 1 µg of pCMV or pCMV-RXR isoform DNA. Results are expressed as percent promoter activity in the presence of ligand vs. no ligand. A, T3 10 nM. Asterisk denotes a significant difference (P < 0.05) between RXR{gamma}1 and RXRß. B, 9-cis-RA 2 µM. Asterisk denotes a significant difference (P < 0.05) between RXR{gamma}1 and both pCMV and RXRb. C, T3 (10 nM) + 9-cis-RA (2 µM). Asterisk denotes a significant difference (P < 0.05) between RXR{gamma}1 and both pCMV and RXRß.

 
Finally, we examined the effects of T3 and 9-cis-RA on TSHß promoter activity in the thyrotrope-derived {alpha}TSH cells that do not respond to T3 (V. D. Sarapura, unpublished) and lack RXR{gamma}1. Like GH3 cells, {alpha}TSH cells contain RXR{alpha} and RXRß (Fig. 1Go). T3 only modestly suppressed activity of the TSHß promoter in these cells (Fig. 9AGo). Cotransfection of either RXR{gamma}1 or RXRß did not influence this lack of suppression. 9-cis-RA alone modestly suppressed TSHß promoter activity in these cells (Fig. 9BGo), but cotransfection of RXR{gamma}1 mediated a 60% suppression of promoter activity, which is similar to that observed in GH3 cells. Cotransfection of RXRß was not different than addition of 9-cis-RA alone. Furthermore, cotransfection of the RXR{gamma}2 isoform in these {alpha}TSH cells had no effect on promoter activity with 9-cis-RA (data not shown). The combination of T3 and 9-cis-RA behaved in a similar fashion to 9-cis-RA alone (Fig. 9CGo). These data suggest that RXR{gamma}1 may be the primary RXR isoform required for the 9-cis-RA suppression of TSHß promoter activity in thyrotropes.



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  		Figure 9. Murine TSHß Promoter Activity in {alpha}TSH Cells Influenced by RXR Isoforms, T3, and 9-cis-RA

Transient transfections were carried out as described in Materials and Methods. Twenty micrograms of mTSHß (-390 to +40) promoter-luciferase reporter DNA were transfected into 3 million {alpha}TSH cells with 1 µg of pCMV or pCMV-RXR isoform DNA. Results are expressed as percent promoter activity in the presence of ligand vs. no ligand. A, T3 10 nM B) 9-cis-RA 2 µM. Asterisk denotes a significant difference (P < 0.05) between RXR{gamma}1 and pCMV. C, T3 (10 nM) + 9-cis-RA (2 µM). Asterisk denotes a significant difference (P < 0.05) between RXR{gamma}1 and both pCMV and RXRß.

 
5'-Deletion Mapping of the 9-cis-RA Effect on TSHß Promoter Activity in Thyrotropes
To identify regions of the TSHß promoter responsible for the 9-cis-RA- mediated suppression of promoter activity in thyrotropes, we cotransfected 5'-deletion mutants of the TSHß promoter in the pA3 luciferase plasmid into TtT-97 thyrotropes in the presence and absence of 2 mM 9-cis-RA. Figure 10AGo shows that deletions to -200 had no effect on the 9-cis-RA-mediated suppression (60–65%) of promoter activity. A 5'-deletion of the TSHß promoter between -200 and -149 greatly reduced the 9-cis-RA response while a deletion to -117 had no further effect, suggesting that the -200 to -149 region of the mTSHß promoter contains elements responsible for a majority of the 9-cis-RA-mediated suppression of promoter activity in thyrotropes. Interestingly, our lab and others have mapped the thyroid hormone-response region of the TSHß promoter to a different area near the transcription start site (3, 21, 22, 23). Sequence analysis of the -200 to -149 promoter region reveals a number of putative hormone response element half-sites (Fig. 10BGo).



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  		Figure 10. 5'-Deletion Mapping of the 9-cis-RA Effect on TSHß Promoter Activity in Thyrotropes

A, Transient transfections were carried out as described in Materials and Methods. Twenty micrograms of mTSHß promoter-luciferase reporter DNA were transfected into 5–10 million TtT-97 cells with 1 µg pCMV ßgal. Each 5'-promoter deletion is listed at the bottom; the 3'-promoter end was kept constant at +40. Results are expressed as percent promoter activity in the presence of 2 µM 9-cis-RA vs. no ligand. B, Murine TSHß promoter sequence between -202 and -139. Arrows represent potential hormone response element half-sites.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this report we have analyzed the distribution of RXR{gamma}1 in thyrotrope cells compared with other cell lines and tissues. We have also characterized a ligand-dependent role for the RXR{gamma}1 isoform in ligand-mediated suppression of the TSHß promoter in pituitary-derived cells. Our studies show that both thyroid hormone and 9-cis-RA suppress TSHß promoter activity in thyrotropes that contain both TRs and all three RXR isoforms. Furthermore, these studies suggest that the suppressive effect of 9-cis-RA on TSHß promoter activity is dependent on the RXR{gamma}1 isoform that is restricted to thyrotrope cells in the anterior pituitary.

RXRs are recognized as the major TR-associated proteins on many promoters and belong to a large family of nuclear transcription factors (24). They function as both 9-cis-RA ligand-dependent receptors and heterodimeric partners with TRs, retinoic acid receptors, and vitamin D receptors (9). The essential role for these receptors is illustrated in studies of targeted disruption of the RXR{alpha} (25, 26) and RXRß (27) genes, resulting in significant developmental abnormalities and death, particularly in the RXR{alpha}-deficient animals. Mangelsdorf et al. (10) demonstrated that RXR{alpha} and RXRß mRNA were widely expressed in developing embryo and adult murine tissues, while RXR{gamma} mRNA was more restricted in its distribution, including abundant expression in the anterior pituitary. We investigated the expression of the RXR isoforms in our thyrotrope-derived cell types and found that while RXR{gamma} mRNA was highly expressed in TtT-97 thyrotropes, this message was virtually undetectable by Northern blot analysis and barely detectable by RT-PCR in {alpha}TSH cells, which do not express the TSHß subunit gene and lack T3 regulation. In a recent report, Sugawara and colleagues (8) further localized RXR{gamma} almost exclusively to the thyrotropes of the rat anterior pituitary gland. These data suggest that the thyrotrope-restricted RXR{gamma} isoform may play a unique role in thyrotrope development, mature thyrotrope phenotype, or TSHß gene regulation by factors such as T3 or retinoic acid.

RXR{gamma} exists as two isoforms, RXR{gamma}1 and RXR{gamma}2 (11), which differ at the N terminus. RXR{gamma}1 mRNA is restricted to brain and skeletal muscle, while RXR{gamma}2 is found in heart, skeletal muscle, and liver. In this study, we show that RXR{gamma}1 is the thyrotrope-restricted isoform. Testing pituitary-derived cells, we also showed that RXR{gamma}1 message is most prominent in TtT-97 thyrotropes and is barely detectable or undetectable by the sensitive RT-PCR technique in the thyrotrope-derived {alpha}TSH cells and somatotrope-derived GH3 cells. Since RXR{gamma}1 is restricted to the thyrotropes in the anterior pituitary gland and is lacking in {alpha}TSH and GH3 cells, we used these pituitary-derived cells to investigate the role of RXR{gamma}1 on TSHß promoter function.

While information regarding TR/RXR interaction on positively regulated elements is well known (12, 13, 14), data for the role of RXRs in negative regulation of the TSHß promoter is less clear. Using an oligonucleotide corresponding to the -24 to -1 region of the mouse TSHß promoter, Hallenbeck et al. (15) showed that RXRß interfered with TR{alpha}1-mediated T3 suppression in mouse fibroblasts in a 9-cis-RA ligand-independent manner. Similarly, Carr and Wong (16) in studies with COS cells using the +11 to +27 and +18 to +27 rTSHß fragments showed that both fragments could specifically confer T3-mediated suppression of promoter activity with both TR{alpha} and TRß. Addition of RXRß interfered with this response in a 9-cis-RA ligand-independent manner. Our data, using a larger TSHß promoter fragment (-390 to +40), agree with these studies using RXRß. In contrast, Cohen et al. (17) showed that RXR{alpha} interfered with TRß-mediated T3 suppression of the mTSHß promoter (-20 to +1) in a 9-cis-RA ligand-dependent manner in JEG-3 cells. They further showed that TRß interacted with the TSHß promoter (-18 to +37) as a monomer, and that this protein-DNA interaction was disrupted by addition of RXR{alpha}, suggesting that RXR interferes with TR-mediated T3 suppression of the TSHß promoter by forming heterodimers in solution, and these protein-protein interactions are 9-cis-RA dependent. Our results showing that RXRß interferes with the T3 response in a ligand-independent manner may differ from these results due to our use of a larger TSHß promoter fragment or isoform differences between RXR{alpha} and RXRß. TR/RXR synergy on the TRH promoter, which is negatively regulated by T3, has been recently reported (28). In both CV-1 and JEG-3 cells, addition of RXR{alpha} or RXRß augmented T3-mediated suppression of the hTRH promoter by TRß. The authors further demonstrated that TR/RXR heterodimers could form on a putative thyroid hormone response element half-site, supporting the concept that TR and RXR can functionally interact on promoter elements outside the classical DR4.

Because RXR{gamma}1 was identified as the thyrotrope-restricted isoform, we examined the ligand-independent and dependent effects of RXR{gamma}1 on a segment of the mTSHß promoter (-390 to +40) containing the thyrotrope-specific elements in addition to the region(s) that mediate response to T3 in pituitary-derived cells. RXR{gamma}1 did not appear to alter promoter activity in a ligand-independent manner in GH3 or {alpha}TSH cells, which lack RXR{gamma}1 but contain endogenous RXR{gamma}2, suggesting that RXR{gamma}1 is not sufficient to stimulate promoter activity in these cells. While investigating ligand-dependent effects of RXR isoforms, we found that RXRß interfered with T3-mediated suppression of TSHß promoter activity in GH3 cells, which is consistent with results by Hallenbeck et al. (15) and Carr and Wong (16). RXR{gamma}, however, did not interfere with this response in these cells, suggesting functional differences between the two isoforms. Furthermore, 9-cis-RA had the most profound suppression of promoter activity in both GH3 and {alpha}TSH cells only in the presence of RXR{gamma}, and not with RXRß. These data strongly suggest that RXR isoform differences play a major role in the modulation of hormone responses in this system. Finally, we showed that both T3 and 9-cis-RA can independently mediate suppression of TSHß promoter activity in TtT-97 thyrotropes. This combined effect is not as pronounced in GH3 cells, which may be explained by the overexpression of Pit-1T to stimulate promoter activity. We have further shown that the promoter region responsible for the 9-cis-RA effect (-200 to -149) is distinct from the T3- responsive region identified by others (3, 21, 22, 23).

In summary, our data suggest that both thyroid hormone and retinoids suppress TSHß promoter activity in thyrotropes. Both ligands appear necessary for maximal inhibition. The thyrotrope-restricted isoform, RXR{gamma}1, appears to mediate 9-cis-RA suppression of promoter activity in thyrotropes. Identification of RXR{gamma}1 partners and cis-acting elements responsible for this effect will increase our understanding of complex interactions of hormones on regulation of gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals and Cell Culture
All LAF-1 mice used in these studies for the TtT-97 tumors were treated in accordance with the NIH guidelines on animal use and care. All protocols were reviewed and approved by the University of Colorado Health Sciences Center Committee on Use and Care of Animals.

Monolayer cultures of GH3 (ATCC CCL 82.1) and {alpha}TSH cells were maintained in DMEM supplemented with 10% FCS. Replacement with the same medium containing charcoal-stripped FCS, which was lacking detectable T4 and T3 levels, was done 48 h before transfection.

Plasmids
mRXR{alpha}, ß, and {gamma} cDNAs were generously provided by Dr. R. M. Evans. A NotI to BstX1 RXRb fragment was gel purified and subcloned into the NotI site of a pCMV ß-galactosidase vector (Clontech, Palo Alto, CA) from which the ß-galactosidase coding region was removed. The RXRß fragment and vector were blunted with T4 DNA polymerase (Promega, Madison, WI) and ligated (29). A fragment corresponding to the RXR{gamma}1 coding region was generated by PCR from the plasmid supplied by R. M. Evans using oligonucleotides corresponding to the start (5'-GCGGATCCATGTATGGAAATTATTCC-3') and termination sites (5'-GCGAATTCTCAGGTGATCTGCAGTGGGGT-3') of RXR{gamma}1. PCR conditions were 30 cycles at 94 C for 1 min, 45 C for 1 min, and 72 C for 1 min. Final PCR extension was at 72 C for 7 min. The gel-purified fragment was blunted and ligated into the pCMV vector as described for RXRß. Complementary DNA orientation in the plasmids was verified by sequencing. TSHß promoter-luciferase reporter constructs and 5'-deletion mutants were prepared as previously described (5).

Northern Blot and RT-PCR Analysis
RNA was prepared from cells and tissues, and polyadenylated RNA was enriched as previously described (30). Ten micrograms of poly A+ RNA from TtT-97 and {alpha}TSH cells were size-separated on a 1% agarose, 6% formaldehyde gel, transferred to a nytran filter, and covalently bound by UV irradiation (Fischer Biotech, Pittsburgh, PA, 120 mJ). The filter was hybridized overnight with a [{alpha}32P]dCTP-radiolabeled RXR{gamma} HindII (450 bp) fragment, washed, and exposed to radiographic film for 16 h at -70 C (31). The filter was then washed twice with sterile water at 90 C, rehybridized with a radiolabeled RXR{alpha} EcoRV (510 bp) fragment, washed, and exposed to radiographic film. This procedure was then repeated with an RXRß NcoI (1400 bp) and a mouse ß-actin fragment.

Five micrograms of poly A+ RNA from TtT-97, {alpha}TSH, GH3, and HeLa cells (Fig. 2Go) were size-separated as above. The subsequent filter was hybridized with a [{gamma}32P]ATP kinase-labeled antisense oligonucleotide corresponding to the unique RXR{gamma}1 5'-sequence (5'-CCATACATGTTGGCTGCTCAGTT-3'). After washing, the filter was exposed to radiographic film overnight at -70 C. The probe was removed by treating the filter with sterile water at 90 C two times and rehybridized with a labeled antisense oligonucleotide corresponding to the unique 5'-untranslated RXR{gamma}2 sequence (5'-CAGTGGCCAGTTCCCACAGACCCAGCGC-3'). After washing, the filter was exposed to film for 3 days at -70 C.

RT-PCR was performed as previously described (4). Briefly, RT-PCR was carried out by reverse transcription of 2.5 µg of total RNA with random hexamers (600 ng) and avian myeloblastosis virus reverse transcriptase (Promega). The product was then divided into three PCR reactions for RXR{gamma}1, and G3PDH. Reactions were carried out with 250 ng of a sense oligonucleotide coresponding to unique sequences for RXR{gamma}1 (5'-GCGGATCCATGTATGGAAATTATTCC-3') and an antisense oligonucleotide corresponding to the termination sequence of RXR{gamma} (5'-GCGAATTCTCAGGTGATCTGCAGTGGGGT-3'). G3PDH PCR was performed using commercially supplied oligonucleotides (Clontech). PCR reactions were performed with 2.5U Taq polymerase (Boehringer, Indianapolis, IN) at 94 C for 1 min, 52.5 C for 1 min, and 72 C for 1 min over 30 cycles. One-tenth of the reaction was size-separated on a 1% agarose gel containing ethidium bromide and exposed to UV light for photography.

Western Blot Analysis
Western blot analysis was carried out as previously described (4). Briefly, 20 µg of nuclear protein extracts were size-separated on a 10% polyacrylamide-SDS gel and transferred to nitrocellulose. The filter was blocked with 7.5% nonfat milk and hybridized for 1 h with a rabbit antiserum raised against an oligonucleotide specific for RXR{gamma}1 (kindly provided by W. W. Chin) diluted 1:1000 in PBS (PBS 20 mM Na2HPO4/NaH2PO4, pH 7.4, 100 mM NaCl) with 0.2% Tween-20. After washing, the filter was hybridized with a goat anti-rabbit IgG antiserum labeled with horseradish peroxidase (BRL Life Technologies, Inc., Gaithersburg, MD) at a 1:4000 dilution in 1% nonfat milk 0.2% Tween-20 PBS. The filter was then washed, a chemiluminescent assay performed (Amersham Technologies., Arlington Heights, IL) and exposed to radiographic film for 2 min.

Transient Transfection Studies
Transient transfection assays have been previously outlined (3, 5). Briefly, 20 µg of the mTSHß (-390 to +40) promoter-luciferase reporter DNA, 1 µg pCMV-RXR isoform or pCMV empty vector DNA, and 1 µg pCMV ß-galactosidase DNA as an internal control for transfection efficiency were transfected by electroporation into 5–10 million TtT-97, 5 million GH3, or 3 million {alpha}TSH cells. Five micrograms of pCMV-Pit-1T were cotransfected in all GH3 experiments for stimulation of TSHß promoter activity (5). Cells were incubated at 37 C for 40 h in DMEM with 10% charcoal-stripped FCS and presence or absence of 10 nM T3 (Sigma Chemical Co., St. Louis, MO) and/or 2 µM 9-cis-RA (provided by A. Levin, Hoffman-LaRoche, Nutley, NJ). After harvest, cells were subjected to freeze-thaw extraction and assayed for both luciferase and ß-galactosidase activity as previously described (4).

Statistical Analysis
Statistical comparisons of transfection studies were carried out using Kruskal-Wallis nonparametric testing.


   ACKNOWLEDGMENTS
 
We thank the Tissue Culture Core Laboratory for maintainence of the GH3 and {alpha}TSH cells. We would also like to thank Dr. Ronald M. Evans (Salk Institute, San Diego, CA) for the RXR isoform cDNAs, as well as Dr. William W. Chin (Brigham and Women’s Hospital, Boston, MA) for the RXR{gamma} antiserum. We thank Dr. A. Levin (Hoffman-LaRoche, Nutley, NJ) for the 9-cis-RA.


   FOOTNOTES
 
Address requests for reprints to: Bryan R. Haugen, M.D., B151 4200 East Ninth Avenue, Denver, Colorado 80262.

This work was supported by NIH Grants DK-02331, CA-46934, and DK-36842 as well as a grant from the Thyroid Research Advisory Council (Knoll Pharmaceutical). This work was also supported by a generous gift from the Lucille P. Markey Charitable Trust.

Received for publication June 24, 1996. Revision received December 19, 1996. Accepted for publication January 7, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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