The beta-2 isoform of the thyroid hormone receptor, TRB2, is only expressed in the anterior pituitary gland and brain. Within the pituitary only somatotropes and thyrotropes contain TRB2 mRNA. Several lines of evidence suggest that the TRB2 isoform is responsible for unique functional effects within somatotropes, such as activation of the rat GH gene. The first goal will be to determine the cis-acting regions of the TRB2 promoter that govern its activation in TtT-97 and GH3 cells. Preliminary data suggest that the Pit-l protein is crucially important, yet these two cells utilize different regions of the TRB2 promoter for activation. A variety of approaches will be used to dissect out critical cell-specific cis-acting elements. Secondly, the determinants involved in the T3 inhibition of TRB2 gene expression at the transcriptional level will be identified. Using DNase protection and gel shift studies, the role of TRs, thyroid receptor auxiliary proteins (TRAPS) and other factors on the unique negative regulatory element of the TRB2 gene will be evaluated. Thirdly, Pit-l function will be reconstituted in alphaTSH cells by stable transfection and analyze the phenotypic consequences of this change will be analyzed. The effects of replacing this critical transcription factor on TRB2 expression will be assessed, and the resultant impact on endogenous T3 responses on other processes, including cell growth and regulation of the TSH alpha-subunit promoter, will be tested.
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