An Unliganded Thyroid Hormone Nuclear β Receptor Induces Pituitary Tumors

Furumoto H, Ying H, Chandramouli GV, Zhao L, Walker RL, Meltzer PS, Willingham MC, and Cheng SY. An unliganded thyroid hormone beta receptor activates the cyclin D1/cyclin-dependent kinase/retinoblastoma/E2F pathway and induces pituitary tumorigenesis. Mol Cell Biol 25: 124–5, 2005.

hyroid stimulating hormone–secreting pituitary tumors (TSH-omas) represent about 2% of all pituitary adenomas in humans. Because diagnosis occurs late in the natural course, curative surgical resection of TSH-omas remains under 50%. Our study focused on understanding the molecular genetic events underlying the pathogenesis of TSH-omas and on identifying potential molecular targets for their early diagnosis.

Clues concerning genetic alterations leading to TSH-omas emerged when dominantly negative mutated thyroid hormone β receptors (TRβ) were identified in several patients with TSH-omas (Ando S et al. J Clin Endocrinol Metab 86: 5572–6, 2001; Ando S et al. Mol Endocrinol 15: 1529–38, 2001). TRβ and TRα are ligand-dependent transcription factors that mediate the biological activities of thyroid hormone (T3) in growth, differentiation, and development. Our creation of a TRβ knockin mutant mouse harboring a targeted dominantly negative PV mutation in the TRβ gene locus has provided an opportunity to address the role of TRβ mutants in the pathogenesis of TSH-omas. The PV mutation was identified in a patient with resistance to thyroid hormone. It has a frame-shift mutation in the last carboxyl-terminal 14 amino acids that results in the loss of T3 binding and transcription activities. As TRβPV/PV mice age, they spontaneously develop TSH-omas with enlarged and highly vascular pituitaries. Pathohistologic analyses indicate that TSH-containing thyroprival cells (enlarged, growth-stimulated, TSH-producing cells with large nuclei) are in the pituitaries of TRβPV/PV mice, beginning at the age of approximately 3 months. As these mice age further (beyond an additional 3 months), adenomas staining positively for TSH and varying in size from microscopic foci (microadenomas) to almost the entire size of the adenohypophysis (with only a thin rim of remaining normal tissue) also become detectable in the pituitaries.

We have found that TRβPV/PV mice exhibit severe dysregulation of the hypothalamic-pituitary-thyroid axis, having a 9- to 15-fold increased thyroid hormone level that is associated with a 400- to 500-fold elevated circulating serum TSH level. To address whether persistent elevation of TSH due to the loss of the negative regulation by thyroid hormone underlies the development of TSH-omas, we took advantage of mice devoid of all TRs (TRα1–/–TRβ–/– mice) that also exhibited severe dysfunction of the pituitary-thyroid axis. In contrast to TRβPV/PV mice, the size of the pituitaries in the TRα1–/–TRβ–/– mice was significantly reduced (20% to 30%), despite a virtually identical loss of the negative regulation of TSH by thyroid hormone in both mutant mice, as compared with the age-matched, wild-type mice. Moreover, histological examination of the pituitary indicated no apparent focal adenomas. These results suggest that the loss of the negative regulation of TSH by thyroid hormone alone is not sufficient to induce TSH-omas.

The distinction between the pituitary growth phenotypes of TRβPV/PV and TRα1–/–TRβ–/– mice provides us with a tool to identify genes that are differentially expressed to promote tumor growth in TRβPV/PV mice. Indeed, using cDNA microarrays, we identified cyclin D1 as one of 12 genes involved in growth and cell proliferation pathways whose expression is mostly activated in TRβPV/PV mice but is repressed or not significantly changed in TRα1–/–TRβ–/– mice. Additional biochemical and cell-based studies indicated that the increased expression of cyclin D1 at both the mRNA and protein levels leads to the activation of the cyclin-dependent kinase (CDK)/retinoblastoma (Rb)/E2F pathway that mediates, at least in part, the aberrant proliferation of thyrotrophs in TRβPV/PV mice.

How does a TRβ mutant (PV) protein activate the expression of cyclin D1, a critical regulator of the cell cycle and tumorigenesis? A cell-based transcriptional study showed that the liganded wild-type TRβ repressed the cyclin D1 promoter activity, whereas PV, which does not bind T3, failed to do so, resulting in constitutive activation of the promoter activity. Analysis of the cyclin D1 promoter revealed the absence of apparent thyroid hormone response elements, but the presence of DNA binding sites for transcription factors AP1, E2F1, SP1, TCF/LEF, and cyclic AMP response element binding protein (CREB). These results suggest that wild-type TRβ and mutant PV could regulate the cyclin D1 promoter activity via protein-protein interaction with these transcription factors. Indeed, in vitro GST pull-down and cell-based co-immunoprecipitation assays demonstrated the physical interaction of TRβ and mutant PV with CREB. Promoter deletion analysis indicates that when the CREB binding site is deleted, the repression by the liganded TRβ or the activation by PV is lost. This suggests that PV, like TRβ, is tethered to the CREB-containing promoter through the physical interaction with CREB on the cyclin D1 promoter, resulting in constitutive activation of cyclin D1 expression (Figure 1).

Figure 1. An unliganded TRβ mutant constitutively activates the expression of cyclin D1 promoter, leading to aberrant growth of thyrotrophs in TRβPV/PV mice. A) A T3-bound wild-type TRβ acts to repress the expression of the cyclin D1 promoter via tethering to DNA-bound cyclic AMP response element binding protein (CREB) on the promoter of cyclin D1. B) PV protein, a TRβ mutant that has lost T3 binding activity, tethering to DNA-bound CREB, cannot function to repress the cyclin D1 promoter, leading to constitutive activation of the cyclin D1 promoter. The increased expression of cyclin D1 activates the cyclin-dependent kinase (CDK)/retinoblastoma (Rb)/E2F pathway, resulting in aberrant proliferation of thyrotrophs and tumors in the pituitaries of TRβPV/PV mice.

Thus, the in vivo evidence suggests that mutation of the TRβ gene is one of the genetic events mediating the pathogenesis of this disease. Importantly, the present study reveals a novel molecular mechanism by which an unliganded TRβ mutant induces pituitary tumorigenesis in vivo and provides mechanistic insights into the pathogenesis of TSH-omas in patients. This study also raises the possibility that the mutated TRβ could serve as a molecular marker for diagnosis.

Sheue-Yann Cheng, PhD
Principal Investigator
Laboratory of Molecular Biology
NCI-Bethesda, Bldg. 37/Rm.5128
Tel:301-496-4280
Fax:301-480-9676
sycheng@helix.nih.gov