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Interactions of Thyrotropin-Releasing Hormone, Phorbol Ester, and Forskolin-Sensitive Regions of the Rat Thyrotropin-β Gene

Division of Endocrinology Metabolism, Department of Medicine, University of Virginia Health Sciences Center Charlottesville, Virginia 22903
Brigham and Women's Hospital, Howard Hughes Medical Institute, Harvard Medical School Boston, Massachusetts 02115

Address requests for reprints to: Dr. M. A. Shupnik, Box 511, Division of Endocrinology, University of Virginia Medical Center, Charlottesville, Virginia 22908.

Abstract

Our previous studies demonstrated TRH stimulation of TSHβ gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between –1.3 kilobases and –204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSHβ gene transcription. In cultured pituitary cells, TSHβ gene transcription was stimulated by 2 h of 10^–9 M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 µM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSHβ gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH₃ cell line to delineate DNA sequences conferring this regulation. Transfected TSHβCAT constructs containing TSHβ gene sequences from –2100/+27I150, –1295/+27I150, and –520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSHβ gene sequences from –703 to –85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin. For each construct, stimulation by treatment with TRH plus PMA, TRH plus forskolin, or all three agents was additive compared to that with each treatment alone. Stimulated half-maximal effective concentrations for TRH (10^–10 M), PMA (10 nM), and forskolin (200 nM) were identical for all constructs. In contrast, a TSHβCAT construct containing TSHβ gene sequences from –204/+27I150 was not stimulated by TRH or PMA, but retained some (1.5- to 2-fold) stimulation by forskolin. Therefore, TRH-, PMA-, and some forskolin-responsive regions of the rat TSHβ gene lie in the region between –520 and –204 bp. The additivity of the responses suggests that the three stimulators may have independent modes of action. Synthetic oligonucleotides corresponding to two regions of Pit-1 homology in this gene region were inserted into tkCAT vectors and tested for TRH, PMA, and forskolin responses. One region located at –274 to –262 bp can confer a TRH and a PMA response to tkCAT. While a second sequence from –333 to –325 bp can confer a forskolin stimulatory response. Thus, there are at least two separate transcriptional response elements in this area of the TSHβ gene that respond distinctly to TRH, PMA, and forskolin.

FOOTNOTES

This work was supported by funding (to M.A.S.) from the University of Virginia (NIH Grant 5-S07-RR-05431–28) and the University of Virginia Cancer Center (NCI Grant P30-CA-44579–03).Some of these data was presented at the 64th Annual Meeting of the American Thyroid Association, San Francisco, CA, 1989, p T55 (Abstract).

Received for publication January 12, 1990. Revision received March 15, 1990. Accepted for publication March 19, 1990.

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