Identification of a GonadotropinReleasing Hormone-Responsive Region in the Glycoprotein Hormone Q-Subunit Promoter

Thomas

W. H. Kay* and J. Larry Jameson

Thyroid Unit Massachusetts General Hospital Harvard Medical School Boston, Massachusetts 02114

transcriptional response to GnRH is mediated through one or more elements between -346 and -244 bp and that GnRH-responsive sequences are distinct from those involved in basal and CAMPstimulatedexpression of the (Y promoter. (Molecular Endocrinology 6: 1767-1773, 1992)

Transcriptional regulation of the glycoprotein hormone a-subunit gene has been studied extensively in placental cells, but much less is known about its regulation in the pituitary gland. In this study, transcriptional control of the human a-subunit gene by GnRH was analyzed using transient expression assays in primary cultures of rat pituitary cells using (Y promoter contructs linked to a luciferase reporter gene. Deletion mutants between -846 and -156 basepairs (bp) had little effect on basal expression, but further deletion to -132 bp reduced basal activity by -50%. Deletion of a CAMP response element between -132 and -99 bp caused a marked loss of basal activity, reducing expression to that of background luciferase activity. The same constructs were analyzed for CAMP responsiveness in primary pituitary cells. The degree of stimulation with 1 mM 8-bromo-CAMP (3.6- to 6.0-fold) was relatively unaffected by deletions from -846 to -132 bp, whereas CAMP stimulation was decreased by further deletion to -99 bp, consistent with the presence of previously defined CAMP response elements in this region of the (Y promoter. GnRH stimulation of (Y promoter activity was highly dependent upon the time of hormone addition after transfection, being most effective when added soon after transfection. Under optimal conditions, GnRH stimulated -846aLUC expression by 20-fold. GnRH responsiveness was retained with deletion to -346 bp, but it was decreased by 55% after deletion to -280 bp and by 79% with deletion to -244 bp. A series of DNA sequences between -346 and -180 bp was linked to the truncated -132a promoter to define the GnRH-responsive region in more detail. Sequences -3461-180 and -3461-244 conferred between greater GnRH responsiveness than was seen with a -346/-280 fragment, confirming an important role for the region between -280 and -244 bp for GnRH responsiveness. These studies suggest that the 0888-8809/92/1767-1773$03.00/O Molecular Endocmology Copynght 0 1992 by The Endocrme

INTRODUCTION

GnRH regulates the secretion of the pituitary gonadotropins LH and FSH. Several lines of evidence indicate that GnRH is also necessary for gonadotropin biosynthesis. For example, when endogenous GnRH secretion is suppressed by gonadal steroids (l-5) or diverted from the pituitary (6, 7), administration of exogenous GnRH stimulates gonadotropin mRNA levels and biosynthesis. In addition, nuclear run-on assays indicate that the GnRH-stimulated increase in steady state gonadotropin mRNA levels is mediated in part at the transcriptional level (2, 8). Detailed analyses of the gonadotropin gene promoter elements have been impeded because of the lack of a suitable gonadotropin-producing ceil line. As an alternative model, transient gene expression assays have been performed in transfected primary cultures of pituitary cells (9, 10). In this system, the glycoprotein hormone a-subunit promoter is expressed at relatively high levels, and its expression is targetted primarily to gonadotropes and thyrotropes, as assessed by double labeled immunofluorescence (9). Moreover, N promoter activity is stimulated by treatment with GnRH or TRH, suggesting that transcriptional response elements for these hypothalamic hormones exist within the available 5’-flanking sequence of the (Y gene. Although there is currently little information concerning the regulatory DNA sequences that mediate GnRH responsiveness in the N promoter, there have been extensive analyses of the sequences and transcription factors involved in N gene expression in placental cell lines (11-l 5). Some of the regulatory elements identi-

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fied in placental cells may be shared with those used in the pituitary. For example, the CAMP response elements (CREs), which are located between -142 and -116 basepairs (bp) (12, 13) bind proteins that are present in both placental and pituitary cell lines (14, 16, 17). Thus, (IRE-binding proteins such as CREB or other members of the ATF family of transcription factors may be involved in basal or hormone-stimulated expression in the pituitary as well as placenta (17, 18). On the other hand, it is likely that other DNA sequences will mediate O( gene expression in a tissue-specific manner. There is a trophoblast-specific element (TSE) located up-stream of the CREs (between -180 and -156 bp). The TSE binds a placenta-specific protein that modulates basal expression (14, 15). Promoter sequences involved in thyrotrope-specific expression have been localized in the murine 01 gene up-stream of the TSE (16, 19) supporting the view that distinct response elements may be involved in placental and pituitary regulation of the N gene. The aim of the current study was to localize response elements in the human LYpromoter that mediate GnRH responsiveness. We have optimized conditions for GnRH stimulation in transfected primary cultures of pituitary cells to allow more detailed analyses than were feasible in previous studies (9). We found that GnRH responsiveness is localized up-stream of the CREs and other regulatory elements that control N gene expression in the placenta.

RESULTS Parameters That Maximize and GnRH Stimulation

Transfection

Efficiency

A number of variables were tested to maximize expression and GnRH stimulation of aLUC constructs in transfected primary pituitary cells. Increased sensitivity of the transient expression system was achieved with the use of a pA3-based plasmid that contains three copies of simian virus-40 (SV40) transcription termination signals that decrease “read-through” transcription from cryptic initiation sites within the plasmid and thereby reduce background luciferase activity (20). The luciferase activity of primary pituitary cells transfected with the promoterless pA3 vector was no greater than that in untransfected cells, allowing more extensive deletion analyses of the CYpromoter than was feasible with the LUC or CAT expression plasmids used previously (9). The method of transfection was varied to include electroporation, lipofectin, DEAE-dextran procedures, as well as the CaP04 technique, which was used in previous studies (9) (Table 1). When compared in a paradigm in which GnRH was added 24 h after transfection, electroporation resulted in a very high level of basal expression, but the degree of GnRH stimulation (2.3-fold) was less than that seen with calcium phosphate transfection (5.7-fold). Electroporation also resulted in a variable amount of cell death when per-

Table 1. Effects

of Various Parameters on 846r~LUC Transfection Efficiency and GnRH Stimulation in Primary Cultures of Rat Pituitary Cells Lucrferase Activity Fold Condition tested” Stimulation -GnRH +GnRH Transfection method Lipofectin Electroporation CaP04 DEAE GnRH analog” Native GnRH o-Ala-GnRH o-His-GnRH

1,299 17,630 5,140 225

1,216 40,489 29,371 197

0.9 2.3 5.7 0.9

8,418

33,899 67,824 49,072

4.0 8.1 5.8

a In the experiments comparing GnRH and its analogues and different transfection techniques, GnRH was added 24 h after transfection, rather than 4 h after transfection. In this paradigm, there is increased basal activity, but diminished GnRH responsiveness (see Fig. 1). b Luciferase activity is expressed in arbitrary light units and is the mean of three transfections. ‘The GnRH analogs are: o-Ala GnRH, des-Gly”‘-[o-Ala’]GnRH ethylamide; and o-His-GnRH, des-Gly”-[o-His(Bzl)G]GnRH ethylamide. Native GnRH and both analogs were used at maximal doses (1 O-’ M).

formed under conditions that resulted in efficient transfection (data not shown). DEAE-dextran did not result in significant levels of basal expression, and the degree of GnRH stimulation was reduced with the lipofectin technique. Overall, the CaP04 method of transfection was the most reliable, in that it resulted in consistently effective basal and GnRH-stimulated expression. The activities of maximal doses (1 Om7M) of native GnRH and two of its long-acting agonists were also compared (Table 1). When added 24 h after transfection, both the o-Ala and the D-His analogs were more effective than native GnRH, perhaps reflecting greater stability of the analogs in tissue culture. For the remainder of the experiments, the GnRH analog Des-Gly’“-[o-Ala6]GnRH ethylamide was used routinely and is referred to as GnRH. The timing of GnRH addition after transfection proved to be a critical parameter for maximal stimulation of the N promoter. Maximal stimulation (typically 20-fold) occurred when GnRH was added 4 h after transfection rather than after 24-48 h, as in the original transfection protocol (9) (Fig. 1). The decreased degree of GnRH stimulation was due to an increase in basal luciferase expression as well as a decrease in GnRH-stimulated luciferase activity. When GnRH was added immediately after the final 4-h wash in the transfection protocol, GnRH responsiveness in separate experiments varied between lo- and 25fold. However, within a given experiment, the variability in GnRH responsiveness in separately transfected plates was rarely greater than 15%. Consequently, different constructs and parameters were always compared within a single experiment. Because continuous exposure to GnRH desensitizes gonadotropin secretion (21), the length of exposure to

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Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter.

Transcriptional regulation of the glycoprotein hormone alpha-subunit gene has been studied extensively in placental cells, but much less is known abou...
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