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The glycoprotein hormone alpha-gene is regulated by multiple hormones in different pituitary and placental cell types. In thyrotropes, the alpha-gene is stimulated by TRH and repressed by thyroid hormone (T3). We used transient expression assays in primary cultures of rat pituitary cells to examine regulation of the alpha-promoter (alpha Luc) by TRH and T3. The -846 alpha Luc activity was stimulated 3.4-fold by TRH and repressed 44% by T3. GnRH and cAMP stimulated -846 alpha Luc by 8.3- and 8.6-fold, respectively. T3 blocked TRH stimulation, but it had no effect on stimulation by GnRH or cAMP, suggesting that the T3-mediated effects are thyrotrope specific. TRH and T3 responsiveness was preserved with deletions to -346 basepairs (bp). TRH responsiveness was lost after deletion to -280 bp, whereas T3-mediated repression was eliminated by further deletion to -180 bp. A series of DNA fragments between -420 and -180 was linked to -132 alpha Luc to study TRH and T3 responses in greater detail. Sequences between -346 to -180 bp conferred TRH responsiveness and T3 inhibition. TRH responsiveness was not seen after 3'-deletions of this fragment to -244 or -280 bp. These results together with the 5'-deletions provide evidence for two interdependent TRH regulatory regions: one between -346 to -280 bp and another between -244 to -180 bp. T3-dependent repression only requires sequences between -244 and -180 bp. Site-directed cluster mutations were created in each of these two regulatory domains. A mutation in region 1 (-346 to -328 bp) eliminated TRH stimulation, but retained basal suppression by T3.(ABSTRACT TRUNCATED AT 250 WORDS)

molecular endocrinology

Localization of promoter sequences required for thyrotropin-releasing hormone and thyroid hormone responsiveness of the glycoprotein hormone alpha-gene in primary cultures of rat pituitary cells.