Thyroid Hormone-Responsive Elements of the Prolactin Gene: Evidence for Both Positive and Negative Regulation

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Abstract
To analyze the regulation of PRL gene expression by thyroid hormone (T3), fusion gene constructs containing various lengths of the rat PRL gene 5''-flanking sequence linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected into the GH3 cell line. Thyroid hormone had no effect on basal or cAMP-stimulated CAT expression in constructs containing more than 1.7 kilobasepairs of the 5''-sequence. However, deletion to 1.5 or 0.6 kilobasepairs resulted in an inhibition of both basal and cAMP-stimulated expression by T3. A construct containing the proximal enhancer region (positions -292 to -38 basepairs) linked to the herpes simplex thymidine kinase promoter (TK) and the CAT reporter gene also responded to T3 with inhibition of basal and cAMP-induced CAT expression. The distal enhancer region (positions -1714 to -1495) linked to thymidine kinase promoter CAT responded to T3 with a stimulation of CAT expression, and the response was additive with the stimulatory response to cAMP. Deletion analysis of the distal enhancer revealed that the sequence between positions -1530 and -1565 was required for the stimulatory response to T3. The stimulatory response to T3 was additive with the response to estradiol, suggesting distinct elements, but deletion to position -1565 abolished the response to estradiol and permitted an inhibitory response to T3. Mutation of the estrogen response element prevents the response to estradiol, but only blunted the response to T3. Mutation of the sequence GGTCA and positions -1555 to -1551 resulted in an inhibitory response to T3, implicating this sequence in the stimulatory response. The interaction of [125I]T3-labeled receptor with specific DNA sequences demonstrated that the distal enhancer binds T3 receptor specifically and that sequences downstream of position -1565 are required. The proximal enhancer region did not bind T3 receptor. The results support the role of both stimulatory and inhibitory T3-responsive elements in regulation of the expression of the PRL gene in GH3 cells.

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