Involvement of a cAMP-responsive DNA element in mediating TRH responsiveness of the human thyrotropin α-subunit gene

TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH α-subunit gene, we have used transient transfection of GH₃ rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone α-subunit gene linked to luciferase. Analysis of 5'- deletions of the α-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to - 135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinase-luciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a 5-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12- myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone. The calcium channel blocker, verapamil, and the calcium chelator, EGTA, completely blocked induction by TRH of the α- subunit reporter gene. The -151 to -100 region of the α-subunit gene contains two copies of a consensus CRE. Deletion of one copy of the CREs resulted in a loss of TRH and calcium responsiveness. Overall, these results indicate that the TRH responsive element of the α-subunit gene likely involves a previously characterized CRE. At least a portion of the mechanism of TRH effects on the α-subunit gene likely involves changes in intracellular calcium levels leading to activation of calcium-responsive protein kinases and phosphorylation of CRE-binding proteins.