The alpha-subunit of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Over- production of free alpha-subunit occurs in human pituitary tumors which secrete TSH, LH and FSH, as well as those secreting only alpha-subunit. This group of tumors constitute a significant proportion of human pituitary tumors. In addition, production of alpha-subunit by pituitary tumor cells of other lineages has been described, particularly those which produce growth hormone. Ectopic expression of alpha-subunit has also been demonstrated in cancerous tissues, where the mechanism of alpha-subunit production and its relationship with abnormal cell growth has yet to be elucidated. In each of the cell types where alpha-subunit is normally expressed, it is associated with a different beta-subunit. In thyrotropes, alpha-subunit gene expression and regulation is coordinated with expression of the TSHbeta-subunit gene. Secretion of active TSH is important in maintaining growth and function of the thyroid gland. The mechanism of expression of the TSH-subunit genes is not yet fully understood. Strong evidence now supports the notion that unique transcription factors determine the expression of the TSH-subunit genes. The goal of the research project proposed in this grant is to identify the factors responsible for alpha-subunit gene expression in thyrotropes. Knowledge of the cis-acting elements of the murine alpha-subunit promoter which enhance expression in thyrotropic cells and availability of a pure murine alpha-secreting thyrotropic cell line are crucial to attaining this goal. The investigator proposes to develop three specific aims. First, the trans-acting factors which interact with alpha-subunit cis- acting elements will be identified. This will be approached by """"""""ligand affinity screening"""""""" using strongly footprinted sequences of the alpha- subunit gene between -480 and -400, which have been found to be functionally important for expression only in thyrotropes. Second, alpha-subunit expression will be reconstituted in non-alpha-subunit expressing cell systems by using transfection and in vitro transcription assays to establish the functional competence of newly identified factors. Third, the contribution of GHF1/Pit1 and TEF, two pituitary transcription factors, on alpha-subunit expression will be assessed, by evaluating their interactions with the alpha-subunit promoter and testing their function by transfection and in vitro transcription. Acquisition of new knowledge from this proposal will be important in the understanding not only of TSH physiology, but also of pituitary tumors which secrete alpha-subunit, and may elucidate mechanisms that contribute to alpha-subunit expression in non-pituitary neoplasms.
