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

Abstract

The glycoprotein hormone α-gene is regulated by multiple hormones in different pituitary and placental cell types. In thyrotropes, the α-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 α-promoter (αLuc) by TRH and T3. The -846α Luc activity was stimulated 3.4-fold by TRH and repressed 44% by T3. GnRH and cAMP stimulated -846 αLuc 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 αLuc 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. A mutation in region 2 (-219 to -197 bp) eliminated TRH and T3 responsiveness. Both mutants still responded to cAMP. We conclude that TRH response elements are distinct from previously defined cAMP regulatory elements and involve two domains in the up-stream region of the α-promoter. The region between -219 and -197 bp forms a complex regulatory element that mediates positive regulation by TRH and negative regulation by T3. The sequences required for T3 repression in pituitary cells are distinct from the T3 receptor-binding site identified previously in the α-promoter adjacent to the TATA box, potentially reflecting a locus for thyrotrope-specific factors involved in T3 repression.