Thyroid hormones (T3) and their receptors (TR) play a critical role in the function of the pituitary gland, particularly in thyrotropes, where they regulate expression of the alpha- and beta-subunits of TSH. Since the pituitary gland is composed of several cell types, we undertook a characterization of TR subtypes in a murine thyrotropic tumor (TtT-97), an excellent model in which to study thyroid hormone action in thyrotropes. We screened a thyrotrope cDNA library with rat TR alpha 1 and TR beta 1 cDNA probes and isolated cDNAs encoding the mouse TR alpha 1 and TR beta 1 isoforms as well as a partial clone corresponding to the non-T3 binding carboxy-terminal alpha 2 variant. The polymerase chain reaction was used to amplify additional cDNAs for the specific 5' domains of the mouse TR beta 1 and the pituitary-specific TR beta 2 amino-terminal variant. Using hybridization probes that discriminate between the alpha and beta isoforms and their variants, we demonstrated that thyrotropes contain TR alpha 1 and alpha 2 mRNAs as well as transcripts encoding Rev-erbA, which arise by transcription from the opposite strand of the TR alpha gene. In thyrotropes, the ratio of alpha 2 to TR alpha 1 mRNA levels more closely resembled the distribution in mouse brain than that in heart, where the mRNA levels of TR alpha 1 and alpha 2 are comparable. TR beta 1 and TR beta 2 mRNAs were detected in thyrotropes and were of similar size (approximately 6.4 kilobases). Despite the almost complete conservation between the rat and mouse TR beta 1 sequences at the protein level, the mouse and rat TR beta 2-specific N-terminal domains were less conserved, and the mouse protein was shorter by 39 amino acids at the N-terminus. Of the receptor species, only the mRNA encoding the TR beta 2 isoform, which was restricted to thyrotropes, was decreased by T3 treatment, although the mRNA for the alpha 2 variant was also reduced by T3 in thyrotropes and heart tissue. Levels of TR beta 1 mRNA were not changed in liver, but were increased in thyrotropic tumors and also somewhat in brain, an organ that is not responsive to T3 by classical criteria.

We constructed a murine genomic DNA library in lambda EMBL3 and have isolated and determined the nucleotide sequence of the murine thyrotropin beta-subunit (TSH beta) gene. The cloned gene was derived from a thyrotropic tumor and had no detectable rearrangements when compared to the murine TSH beta gene in total genomic DNA. The murine TSH beta gene is 5 kb in size and consists of five exons and four introns. The 5' untranslated region of the mRNA is encoded except for a single nucleotide by exons 1, 2, and 3. The protein-coding regions are encoded by exons 4 and 5 while the 3' untranslated region is entirely contained in exon 5. Primer extension analysis using an exon 1-specific primer was used to map the 5' end of the gene. Two transcriptional start sites are present in the murine TSH beta gene which appear to be positioned by two TATAAA sequences located 40 bp apart. In all, 99% of transcripts initiate at the downstream site. Transcription from both start sites is affected by thyroidal status in both murine pituitaries and in TtT97 thyrotropic tumors. Finally, sequences homologous with putative thyroid-responsive elements and cyclic AMP-responsive elements are present in the 5'-flanking region and may be important in regulating negative and positive effects on TSH beta gene expression.

We have isolated and determined the nucleotide sequence of the mouse alpha-subunit gene of the pituitary glycoprotein hormones. No detectable rearrangements were observed from the cloned gene when compared with total genomic DNA. The gene is approximately 13.5 kb in size and consists of 4 exons and 3 introns. The 5' untranslated region is encoded by exon 1 and 14 bp of exon 2. A large first intron of about 10 kb interrupts the 5' untranslated region. The coding region is present in exons 2, 3, and 4, while the 3' untranslated region is contained entirely within exon 4. A number of mouse B1 and B2 repeats are present in the 5'-flanking region, the first and third introns, as well as in the 3'-flanking region. In contrast to the mouse beta-subunit gene of thyrotropin, primer extension analysis revealed that the alpha-subunit gene has a single transcriptional start site and that the primary transcript does not exhibit alternative exon splicing. The transcriptional start site is identical in both mouse pituitaries and in TtT97 thyrotropic tumors and is responsive to thyroidal status. The 5'-flanking region contains a sequence that is homologous to a single copy of the duplicated 18-bp cyclic AMP-responsive element of the human alpha-subunit gene. Sequences homologous to putative thyroid-responsive and estrogen responsive elements are present in the 5'-flanking region and may be important in the multihormonal regulation of the alpha-subunit gene in throtrophs and gonadotrophs, respectively.

Pituitary thyrotroph cells specialize in the synthesis of TSH, and thus represent a model to study cell-specific gene expression. We have used the murine TSH beta (mTSH beta) gene promoter and TSH-producing and nonproducing transplantable tumors derived from murine thyrotroph cells, referred to as TtT-97 and MGH 101A, respectively, to identify nuclear factors which selectively interact with the mTSH beta gene. DNase I protection analyses demonstrate that factors present in TtT-97 nuclear extracts bind with high affinity to five separate sites in the TSH beta promoter region, denoted as distal D1 (-253 to -227) and proximal, P1 (-76 to -68), P2 (-106 to -98), P3 (-126 to -112), and P4 (-142 to -131) footprints. By contrast, non-TSH beta expressing thyrotroph cell nuclear extracts and L-cell nonpituitary cell extracts did not appear to footprint the D1 site; whereas the nonpituitary nuclear extracts revealed minimal DNase I protection in the P1-P4 regions. These data show that the distal D1 site is thyrotroph specific and contains a 6 base pair direct repeat sequence (5'-AGATAT-3'). Factor occupancy of the D1 site is protein dependent, occurs rapidly (less than 15 sec), is destabilized by 170 mM KCl, and results in an associated DNase I hypersensitive region. A double-stranded oligonucleotide spanning the D1 footprint competes only the distal factor binding region. Transfection of plasmid constructs containing progressive 5'-deletions of the mTSH beta promoter linked to the reporter gene luciferase into primary TtT-97 cells demonstrate a marked decrease in activity between the regions -270 and -79, which contains the D1 region.(ABSTRACT TRUNCATED AT 250 WORDS)

Our studies are aimed at identifying the transcription factors that activate the glycoprotein hormone alpha-subunit promoter. Therefore, we performed a Southwestern screening of a thyrotropic (alphaTSH) cDNA expression library, using the region of the promoter from -490 to -310 that contains sequences critical for expression in thyrotrope cells. A clone was isolated corresponding to part of the coding sequence of Msx1, which is a homeodomain-containing transcription factor that has been found to play an important role in the development of limb buds and craniofacial structures. Northern blot analysis, using the cloned Msx1 cDNA fragment as a probe, demonstrated that alpha-subunit-expressing thyrotrope cells (alphaTSH cells and TtT97 tumors) contained Msx1 RNA transcripts of 2.2 kb, while somatomammotrope (GH3) cells that do not produce the alpha-subunit had barely detectable levels. The presence of Msx1 protein was demonstrated by Western blot analysis in alphaTSH cells. We also demonstrated that transcripts encoding the closely related Msx2 factor were not detectable by Northern blot analysis in either thyrotrope or somatomammotrope-derived cells. Subfragments of the region from -490 to -310 of the alpha-subunit promoter were used in a Southwestern blot assay using bacterially produced Msx1 and demonstrated that binding was localized specifically to the region from -449 to -421. Deoxyribonuclease I protection analysis, using purified Msx1 homeodomain, demonstrated structurally induced differences in DNA digestion patterns between -436 and -413, and sequence analysis of this region revealed a direct repeat of the sequence GXAATTG, which is similar to the Msx1 consensus-binding site. When nucleotides at both sites were mutated, Msx1 binding was dramatically reduced, and the activity of an alpha-subunit promoter construct decreased by approximately 50% in transfected thyrotrope (alphaTSH) cells. These studies suggest that Msx1 may play a role in the expression of the alpha-subunit gene in thyrotrope cells.