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Molecular Endocrinology, doi:10.1210/me.2007-0116
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Molecular Endocrinology 21 (8): 1969-1983
Copyright © 2007 by The Endocrine Society

Pituitary-Specific Expression and Pit-1 Regulation of the Rat Growth Hormone-Releasing Hormone Receptor Gene

Allison T. McElvaine, Andrew I. Korytko, Signe M. Kilen, Leona Cuttler and Kelly E. Mayo

Department of Biochemistry, Molecular Biology, and Cell Biology (A.T.M., S.M.K., K.E.M.), Northwestern University, Evanston, Illinois 60208; and Department of Pediatrics (A.I.K., L.C.), Case Western Reserve University, Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Kelly E. Mayo, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2205 Tech Drive, Hogan 4-112, Evanston, Illinois 60208. E-mail: k-mayo{at}northwestern.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANTERIOR PITUITARY gland comprises five cell types that are defined by the hormones they secrete. Development of the anterior pituitary is characterized by a series of inductive signals from the neural ectoderm of the ventral diencephalon (1, 2, 3, 4, 5) that stimulates the growth and differentiation of the anterior pituitary primordium, Rathke’s pouch, around embryonic d 8.5 in the mouse (6, 7, 8). Once established, Rathke’s pouch creates an ectodermal boundary that allows subsequent signaling gradients (9), which stimulate the expression of transcription factors in overlapping patterns (9, 10, 11, 12, 13, 14), leading to the differentiation of the various pituitary cell types, which are specified by embryonic d 17.5 (15). An opposing ventrodorsal gradient of BMP2 and FGF8 (9, 16) is required to differentiate the ventral cell phenotypes from the dorsal cell phenotypes, and reciprocal gradients of Pit-1 and GATA-2 are involved in the differentiation of the four most caudal cell lineages, gonadotrophs, thyrotrophs, lactotrophs, and somatotrophs (14).

The pituitary somatotroph cell, which secretes GH, integrates signals from the hypothalamus to regulate GH release. In vertebrates GH promotes proliferation (17, 18, 19, 20, 21), differentiation (22, 23, 24, 25, 26), and metabolism (27, 28, 29, 30) of target tissues throughout the body. GHRH, a hypothalamic peptide hormone, is the primary stimulus for GH synthesis (31, 32) and secretion (32, 33, 34) and acts by binding to its receptor on the surface of pituitary somatotroph cells. The GHRH receptor is a seven-transmembrane G protein-coupled receptor that is closely related to the receptors for the related peptide hormones, vasoactive intestinal polypeptide, secretin, and glucagon (35).

Pituitary somatotroph cells and lactotroph cells, which secrete prolactin, differentiate from a common precursor cell. Terminally differentiated somatotroph cells express the GHRH receptor and respond to GHRH stimulation (36). Therefore, discovery of how the GHRH receptor gene is regulated is expected to lead to a better understanding of what factors are involved in the differentiation of the somatotroph cell lineage from the four other cell types of the anterior pituitary gland. The GHRH receptor gene is known to be regulated by several factors, including glucocorticoids (37, 38, 39, 40, 41), thyroid hormone (41), retinoic acid (41), GHRH (36, 42), and the pituitary-specific transcription factor Pit-1 (38, 43, 44, 45), but the mechanism of regulation has not been elucidated for most of these factors.

The transcription factors Prop-1 (11, 12) and Pit-1 are required for the differentiation of the thyrotroph (46), lactotroph (47), and somatotroph (48) cell lineages from a common pituitary precursor. Pit-1 is a pituitary-specific POU homeodomain transcription factor (49, 50) that is involved in the transcriptional activation of the GH gene in somatotroph cells and of the prolactin gene in lactotroph cells (51, 52, 53). Previous work from our laboratory and others has shown that Pit-1 also leads to activation of the GHRH receptor promoter (38, 43, 44). Further indicative of the important role Pit-1 plays in stimulating GHRH receptor expression, a mutation in a human GHRH receptor Pit-1 binding site results in decreased expression of the receptor in a patient exhibiting GH deficiency (45). Pit-1 has recently been shown to be required for glucocorticoid regulation of the rat (r) GHRH receptor (54), suggesting that cooperation between Pit-1 and other factors may lead to differential cell-specific gene regulation.

Mutations in the GHRH receptor in human (55, 56, 57, 58) and in animal models (59, 60, 61) lead to dwarfism and pituitary hypoplasia due to lack of somatotrophs, indicating an integral role for the receptor in regulation of appropriate GH levels. Given the importance of the GHRH receptor in mediating the neuroendocrine pathway regulating GH levels, understanding regulation of the GHRH receptor gene itself is important.

The current study seeks to determine what promoter elements are required to direct tissue- and cell-specific expression of the GHRH receptor gene. Using transgenic mice and cell culture systems, we show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and that the cell specificity is mimicked in vitro. In further examination of elements within the 1.6-kb promoter, we mapped 10 Pit-1 binding sites and show that five of these contribute to Pit-1-regulated expression of the GHRH receptor. Of these, the greatest contribution to Pit-1-activated expression of the gene is achieved by two well-conserved, high-affinity sites, –888 to –857 and +129 to +164.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary-Specific Expression of the Luciferase Transgene
Two independent lines of transgenic mice were generated in which the 1.6-kb GHRH receptor promoter directs a luciferase reporter. Luciferase assays were performed on pituitaries taken from normal littermate and transgenic mice for each line. Luciferase expression is significant in transgenic pituitaries in each line (Fig. 1AGo). Transgenic mice in line 2 show significantly higher transgene expression than those in line 1 (Fig. 1AGo). Due to the robust transgene expression level in the pituitary of transgenic mice in line 2, this line was used for further analyses and data from line 2 are reported here.


Figure 1
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  		Fig. 1. Luciferase Transgene Expression in Two Independent Lines

A, Transgene expression in normal littermate (NLM) and transgenic (Tx) male mice from two lines detected by luciferase assay performed on homogenized pituitary tissue and normalized to protein. B, Luciferase assay of 15 homogenized tissues from normal littermate and transgenic males, normalized to protein (mbh: medial-basal hypothalamus). Statistical analysis was performed using a two-way ANOVA (*, P < 0.05; ***, P < 0.001; n ≥ 5 for each group). RLU, Relative light units.

 
To determine whether the 1.6-kb promoter is sufficient to direct tissue-specific expression of the GHRH receptor, multiple endocrine and nonendocrine tissues from both normal littermate and transgenic mice were examined for luciferase expression. In addition to pituitary the tissues tested include medial-basal hypothalamus, cerebellum, pancreas, cerebral cortex, testes/ovary, heart, lung, adrenal gland, kidney, spleen, muscle, skin, thymus, and liver. Only in the pituitary gland were relative light units significantly higher for the transgenic sample compared with the normal littermate sample (Fig. 1BGo).

Transgene Expression Is Targeted to Somatotroph Cells
To determine whether the transgene is restricted to somatotroph cells, immunocytochemistry was performed on dispersed pituitary cells from transgenic and normal littermate mice. In control samples where the primary antibodies are omitted, no fluorescence is observed (Fig. 2Go, A–H). An antibody to GH was used to localize somatotroph cells. A luciferase antibody was used to examine transgene localization. The GH antibody shows that somatotroph cells in both normal littermate (Fig. 2JGo) and transgenic (Fig. 2NGo) pituitaries occur at equivalent numbers. As expected, the luciferase antibody shows luciferase expression in pituitary cells of only the transgenic mice (Fig. 2OGo).


Figure 2
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  		Fig. 2. Cell Specificity of Transgene Expression

Pituitaries from normal littermate (A–D; I–L) and transgenic (E–H; M–P) male mice, age 21–45 d, were necropsied and cells were dispersed. Immunocytochemistry was performed using primary antibodies to GH and luciferase. Additional samples were processed without primary antibody incubation (normal littermate, A–D; transgenic, E–H). A 4',6-diamidino-2-phenylindole (Dapi) stain identifies cell nuclei (A, E, I, and M). GH-positive cells are visualized on the Texas Red channel (B, F, J, and N), and luciferase-positive cells are visualized on the fluorescein isothiocyanate channel (C, G, K, and O). Overlays show where colocalization of GH and luciferase occurs (D, H, L, and P). Data are representative of at least three independent experiments with n ≥ 6 animals per condition in each experiment. NLM, Normal littermate; Tx, transgenic.

 
Overlays of the channels detecting GH and luciferase antibodies reveal that the two antibodies colocalize in the transgenic pituitary (Fig. 2PGo), indicating that the luciferase transgene is expressed in somatotroph cells of the anterior pituitary gland. In immunocytochemistry experiments, all somatotroph cells express the luciferase transgene (n > 1000 cells). In fewer than 5% of cells expressing the transgene, GH is not detected (n >1000 cells). This small number of cells is not likely significant enough to represent an entire population of an additional pituitary cell type. Thus, we conclude that transgene expression is somatotroph cell specific.

Transgene Expression Is Sexually Dimorphic and Regulated during Postnatal Development
Transgene expression was examined in males compared with females to determine whether sexual dimorphism in transgene expression occurs. Transgene expression levels are strikingly higher in transgenic pituitaries from males than from females, with a difference of more than 5-fold (Fig. 3Go).


Figure 3
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  		Fig. 3. Sexual Dimorphism of Transgene Expression

Pituitaries from male and female normal littermate and transgenic mice were necropsied and homogenized to perform luciferase assays, normalized to protein. Statistical analysis was performed using a two-way ANOVA (*, P < 0.05; ***, P < 0.001; n ≥ 7). NLM, Normal littermate; RLU, relative light units; Tx, transgenic.

 
To determine whether the 1.6-kb promoter regulates changes in receptor expression during postnatal development and aging, luciferase assays were performed on pituitaries from transgenic animals ranging from d 1 postnatal through 1 yr of age (Fig. 4Go). Similar to reports on expression of the endogenous GHRH receptor (62), there is a significant increase in transgene expression in the time leading up to puberty (30–35 d in the male mouse) (Fig. 4Go). In contrast to the endogenous receptor, transgene expression levels do not consistently decrease as the animal ages (Fig. 4Go).


Figure 4
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  		Fig. 4. Transgene Expression during Postnatal Development and Aging

Pituitaries from male transgenic mice age 1 d postnatal through 1 yr were necropsied and homogenized to perform luciferase assays, normalized to protein. Statistical analysis was performed using a one-way ANOVA [data points with different letters are significantly different from one another (P < 0.05); n ≥ 4 for each age group and is indicated at the bottom of each bar]. RLU, Relative light units.

 
Cell-Specific and Pit-1-Activated Expression of the rGHRH Receptor Promoter in Cell Culture
Having established the 1.6-kb promoter as sufficient in vivo to direct tissue- and cell-specific expression, we further examined regulation of this promoter in cell culture experiments. A rGHRH receptor promoter-luciferase reporter construct (rGHRH receptor –1673) is not expressed in a nonpituitary cell line, HeLa T4 cells, but is highly expressed in a GH-secreting pituitary cell line, GH3 cells (Fig. 5Go). In nonpituitary HeLa cells, coexpression of Pit-1 leads to significant activation of the rGHRH receptor –1673 construct. Because Pit-1 is expressed in GH3 cells, Pit-1 coexpression in transfections of these cells leads to little or no further activation of the GHRH receptor promoter reporter (Fig. 5Go). Pit-1-activated levels of this promoter in HeLa cells are 8-fold lower than basal expression levels in GH3 cells, indicating that factors in addition to Pit-1 are required for full activation of the promoter (Fig. 5Go). Given the ability of Pit-1 to stimulate expression of the promoter in a nonpituitary cell line, we focused on identifying functional binding sites for this transcription factor, which appears to be a critical determinant of GHRH receptor gene expression.


Figure 5
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  		Fig. 5. Expression of rGHRH Receptor Promoter Luciferase Reporter in Pituitary and Nonpituitary Cell Lines

The full –1673 rGHRH receptor promoter-luciferase construct was transfected into nonpituitary HeLa cells and pituitary GH3 cells alone or with cotransfected Pit-1 to examine relative expression levels. Data are plotted as relative light units per microgram of protein, as a percentage of Rous sarcoma virus-luciferase values, to normalize for variability in transfection efficiencies of the cell lines. Data represent the average of at least three independent experiments. Error bars represent the SEM. Statistical analysis was performed using a Student’s t test. (***, P < 0.001). RLU, Relative light units.

 
Pit-1 Binding to the 1.6-kb rGHRH Receptor Promoter
To determine whether Pit-1 binds to the rGHRH receptor promoter and where it binds, EMSAs were performed using overlapping probes spanning the proximal promoter. A Western blot shows Pit-1 expression in nuclear extracts from cells transfected with the Pit-1 construct, which were used in the binding reactions, and no expression in nuclear extracts from mock-transfected cells, which were used as a negative control (Fig. 6AGo). DNA probes are shown schematically in Fig. 6CGo. In the rat, four transcriptional start sites for the GHRH receptor gene have been identified (43); these are depicted as arrows in the schematic (Fig. 6CGo). We refer to the 5'-most of these start sites as "+1." Only the 3'-most probe was negative for Pit-1 binding (Fig. 6BGo). Pit-1 binds to each of the other seven probes, as nuclear extracts from cells overexpressing Pit-1 shift the mobility of the promoter probes (Fig. 6BGo, lanes 3). An unlabeled oligonucleotide composed of a consensus Pit-1 binding site competes away Pit-1 binding (Fig. 6BGo, lanes 4), but an unlabeled oligonucleotide with a mutated Pit-1 binding site has no effect on binding (Fig. 6BGo, lanes 5). Incubation of the nuclear extracts with an antibody to Pit-1 supershifts the Pit-1 binding band (Fig. 6BGo, lanes 6), whereas normal serum does not affect binding (Fig. 6BGo, lanes 7). Pit-1 binding to the –1673 to –1323 region is less definitive than for the other six regions, because normal serum, in addition to the Pit-1 antibody, supershifted the Pit-1 band, although this band has a distinct mobility from the Pit-1 antibody shift.


Figure 6
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  		Fig. 6. Expression of Pit-1 in Nuclear Extracts and Pit-1 Binding across the 1.6-kb rGHRH Receptor Promoter

A, Nuclear extracts from either Pit-1 overexpressing HeLa T4 cells (lane 1) or mock-transfected HeLa T4 cells (lane 2) were separated by SDS-PAGE on a 10% gel and transferred to nitrocellulose. The membrane was incubated with an antibody to Pit-1, and antibody-antigen complexes were detected by chemiluminescence. B, EMSAs were run by incubating nuclear extracts with radiolabeled DNA probes spanning the promoter. Lane 1 in each panel is probe alone. Lane 2 is probe incubated with nuclear extracts from mock-transfected cells. Lanes 3–7 are probes incubated with nuclear extracts from Pit-1-overexpressing cells. Lane 4 includes an unlabeled oligonucleotide including a Pit-1 consensus binding sequence. Lane 5 includes an unlabeled mutated Pit-1 oligonucleotide binding sequence. Lane 6 includes an antibody to Pit-1, and lane 7 includes normal rabbit serum. C, A schematic represents the overlapping DNA promoter probes and the four transcriptional start sites in the rGHRH receptor promoter. Data are representative of at least three independent experiments.

 
Pit-1 Specifically Binds 10 Sites in the 1.6-kb rGHRH Receptor Promoter
To determine short DNA regions that bind Pit-1 specifically, sequential sets of overlapping smaller oligonucleotide probes were generated to each region mapped in Fig. 6Go and used in EMSAs. In a previous report, four putative Pit-1 binding sites in the 1.6-kb promoter were identified computationally (43). The binding experiments show that, in fact, 10 short regions of the 1.6-kb rGHRH receptor promoter bind Pit-1 specifically (Fig. 7Go, lanes 3). The binding of Pit-1 to these probes is competed by an unlabeled Pit-1 site oligonucleotide (Fig. 7Go, lanes 4), not competed by a mutated Pit-1 site oligonucleotide (Fig. 7Go, lanes 5), supershifted by incubation with an antibody to Pit-1 (Fig. 7Go, lanes 6), and unaffected by incubation with normal rabbit serum (Fig. 7Go, lanes 7). Not surprisingly, several of these promoter probes also bind nuclear proteins from mock-transfected cells (Fig. 7Go, lanes 2). For most of these, the band representing binding of other (non-Pit-1) proteins migrates at a different size than Pit-1. For others (–348 to –313 and –81 to –46) the band migrates at a similar size, but the band intensity is much greater in the Pit-1 nuclear extract lane. Pit-1-specific binding was determined by competition with a cold Pit-1 consensus oligonucleotide (but not with a mutated version of it) and supershift with Pit-1 antibody. No defined binding sites were detectable within the –1673 to –1323 region, possibly indicative of the existence of multiple weak-affinity sites within this region, or lack of specificity of binding, as discussed for Fig. 6Go.


Figure 7
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  		Fig. 7. Identification of 10 Pit-1 Binding Sites in the 1.6-kb rGHRH Receptor Promoter

EMSAs were performed using nuclear extracts from Pit-1-overexpressing HeLa T4 cells and radiolabeled oligonucleotide probes. Shown are the 10 short probes that specifically bind Pit-1. Lane 1 in each panel is probe alone. Lane 2 is probe incubated with nuclear extracts from mock-transfected cells. Lanes 3–7 are probes incubated with nuclear extracts from cells overexpressing Pit-1. Lane 4 includes an unlabeled oligonucleotide including a Pit-1 consensus binding sequence. Lane 5 includes an unlabeled mutated Pit-1 oligonucleotide binding sequence. Lane 6 includes an antibody to Pit-1, and lane 7 includes normal rabbit serum. An arrow marks the Pit-1 band. The second gel in each panel is an identical experiment with a mutated probe. Data are representative of at least three independent experiments. Mut, Mutation.

 
When key nucleotides are mutated from A/T to G (Table 1Go), Pit-1 binding to these sequences is completely abolished (Fig. 7Go, Mut panels). Because no strong consensus Pit-1 binding site exists, it was not possible to predetermine a single target for effective mutation in these probes. Instead, several nucleotides were mutated within the presumed core sequence of each Pit-1 probe (Table 1Go).


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  		Table 1. rGHRH Receptor Promoter Pit-1 Binding Sequences and Mutations

 
The rGHRH Receptor Promoter Pit-1 Binding Sites Exhibit Varying Affinities for Pit-1
The 10 Pit-1 binding sites were examined more closely to determine which site or sites might be important to Pit-1 activation of the GHRH receptor gene. Because the observation was made in initial experiments that different sites seem to bind Pit-1 with different affinities, we examined relative binding affinities of the sites. EMSAs were performed by incubating increasing amounts of Pit-1 nuclear extracts, ranging from 0 µM to 2000 µM, with each of the labeled probes (Fig. 8Go, A and B). Average relative affinities were compared with the affinity of a known Pit-1 binding sequence (63), which is referred to as the Pit-1 consensus sequence (Fig. 8Go). Relative affinities for the 10 sites are highly variable, with EC50 values ranging from 300 µM for the +129 to +164 site to greater than 2000 µM for six of the sites (Fig. 8C). Affinity ranks are listed for comparison (Fig. 8CGo).


Figure 8
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  		Fig. 8. Varying Relative Affinities for Pit-1 among the 10 Binding Sites in the rGHRH Receptor Promoter

Relative affinities were deduced from EMSAs using increasing amounts of Pit-1 nuclear extracts. A, In each panel, the first lane, marked "P," is probe alone. The second lane, marked "M," is probe incubated with mock-transfected nuclear extract. The last six lanes are probe incubated with increasing amounts of nuclear extract from cells overexpressing Pit-1 in the following order: 1 µg (40 µM), 5 µg (200 µM), 10 µg (400 µM), 20 µg (800 µM), 30 µg (1200 µM), and 50 µg (2000 µM). Shown is a representative of three independent experiments. B, Arbitrary densitometric units were calculated for each probe using ImageQuant software. Data are representative of three independent experiments. C, Relative affinities of Pit-1 binding sites were determined by calculation of the EC50 values for each probe, using the GraphPad Prism program (N.E., nuclear extract). A relative affinity rank is also provided, with the rank of first having the highest affinity and the rank of tenth having the lowest. Conservation of Pit-1 binding probe sequences was determined across multiple species, and the degree of conservation was translated to a rank, shown here. The rank of first is most highly conserved, and tenth is least highly conserved.

 
Conservation of Pit-1 Binding Sequences across Multiple Species
To examine evolutionary conservation of the Pit-1 binding sequences, the rat promoter sequence was aligned to the genomic sequences of other species (data not shown). As might be expected, there was strong homology between rat and mouse that was less robust in other species (human, chimpanzee, dog, and rhesus monkey). A percent conservation and conservation raking was calculated for each of the sites, and the ranks are shown (Fig. 8CGo). There is some correlation between relative affinities and amount of conservation of the sequences. For example, the most highly conserved sequence, +129 to +164, is the site with the highest relative affinity for Pit-1, and the least well-conserved sequence, –348 to –313, is among the lowest affinity sites (Fig. 8CGo).

Two human GHRH receptor promoter Pit-1 binding sites have been identified (44). The more 5' of these human sites, which contributes most to Pit-1 activation of the human GHRH receptor, is highly conserved and corresponds to the high-affinity +129 to +164 rat site, indicating a possible functional importance for this site in regulating the rGHRH receptor gene.

Functional Analysis of Pit-1 Binding Sites
Because Pit-1 does not further activate expression of the 1.6-kb rGHRH receptor construct in GH3 cells, we performed experiments in HeLa cells to examine the contribution of Pit-1 to regulation of the receptor gene and compared these data to basal reporter expression in GH3 cells.

In HeLa cells, Pit-1 significantly activates a series of 5'-deletion constructs up to the rGHRH receptor –131 construct, which still contains the high-affinity, highly conserved +129 to +164 site (Fig. 9AGo). When this site is removed, in the rGHRH receptor +148 construct, Pit-1 activation is lost (Fig. 9AGo). Because basal expression of the 5'-deletion constructs varied, we also examined fold increase in reporter expression with Pit-1 cotransfection for each of the promoter deletion constructs. This analysis shows that Pit-1 significantly increases promoter activity for each construct except the shortest, which lacks Pit-1 binding sites (Fig. 9BGo). Pit-1 coexpression has the greatest effect on the full-length –1673 construct, suggesting that multiple binding sites are necessary for full Pit-1 responsiveness of this gene (Fig. 9BGo).


Figure 9
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  		Fig. 9. Pit-1 Activation of rGHRH Receptor Promoter Luciferase Reporter Deletion and Mutation Constructs

A, HeLa T4 cells were transfected with 5'-deletion rGHRH receptor promoter constructs alone or with a cotransfected Pit-1 expression construct and analyzed for luciferase expression. Data are representative of at least three independent experiments performed in triplicate. Error bars represent the SEM. Statistical analysis was performed using a Student’s t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001). B, The fold increase in reporter activity with Pit-1 coexpression is plotted for each of the 5'-promoter-deletion constructs. Data represent the average of at least three independent experiments. Error bars represent the SEM. Statistical analysis was performed using a one-way ANOVA (ns, P > 0.05; *, P < 0.05; ***, P < 0.001). C, Individual mutations of the 10 Pit-1 binding sites were examined by transfection and luciferase assay in HeLa cells. Data are representative of at least three independent experiments performed in triplicate. Error bars represent the SEM. Statistical analysis was performed using a Student’s t test (ns, P > 0.05; *, P < 0.05). D, The fold increase in reporter activity with Pit-1 coexpression is plotted for each of the 5'-promoter deletion constructs. Data represent the average of at least three independent experiments. Error bars represent the SEM. Statistical analysis was performed using a one-way ANOVA (ns, P > 0.05; *, P < 0.05; **, P < 0.01). Mut, Mutation; ns, nonsignificant; RLU, relative light units.

 
To further examine the individual contributions of the identified Pit-1 binding sites, functional analysis of the Pit-1 binding site mutations was performed. Five of the Pit-1 binding sites exhibit significantly reduced Pit-1-activated expression when mutated (Fig. 9CGo). The most dramatic loss of Pit-1 activation is observed when either the –888 to –857 or +129 to +164 site is mutated. Because changes in basal expression were apparent in these experiments, fold increase in reporter expression with Pit-1 coexpression was examined for these mutation constructs. The fold increase is significant for each of the mutations except the +129 to +164 site (Fig. 9DGo), supporting that Pit-1 binding to this site is essential for activating the rGHRH receptor promoter.

GHRH receptor promoter deletion and mutation constructs were also examined in pituitary GH3 cells. Interestingly, the pattern of basal reporter expression for the promoter 5'-deletion constructs follows the pattern of Pit-1-activated reporter expression seen in HeLa cells (Fig. 10AGo). Mutation of seven of the 10 Pit-1 binding sites results in significantly reduced basal expression in GH3 cells (Fig. 10BGo). These sites include those that exhibit reduced Pit-1-activated expression when mutated in HeLa cells, in addition to two others. As in HeLa cells, no activation of the reporter occurs when Pit-1 is unable to bind the critical +129 to +164 site (Fig. 10BGo).


Figure 10
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  		Fig. 10. Expression of rGHRH Receptor Promoter Luciferase Reporter Deletion and Mutation Constructs in Pituitary Cells

A, GH3 cells were transfected with 5'-deletion rGHRH receptor promoter constructs and analyzed for luciferase expression. Data are representative of at least three independent experiments performed in triplicate. Error bars represent the SEM. Statistical analysis was performed using a one-way ANOVA (ns, P > 0.05; *, P < 0.05; ***, P < 0.001). B, Mutation of the +129 to +164 Pit-1 binding site was examined by transfection and luciferase assay in GH3 cells. Data are representative of six independent experiments performed in triplicate. Error bars represent the SEM. Statistical analysis was performed using Student’s t test (ns, P > 0.05; *, P < 0.05). mut, Mutation; ns, nonsignificant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The primary goal of this study was to expand our understanding of pituitary- and somatotroph cell-specific expression of the GHRH receptor gene. Examination of transcriptional regulation of this gene, which is expressed early in terminally differentiated somatotroph cells, will lead to a better understanding of the events leading to cell specification of the somatotroph lineage. The approach we took was to characterize regulation of the rGHRH receptor promoter in vivo, in an attempt to define elements required to direct tissue- and cell-specific expression, and to further study regulation of the defined promoter in a cell culture system.

Previous cell culture experiments have shown that the 1.6-kb rGHRH receptor promoter was able to direct expression of reporter constructs in a pituitary cell line (43). To determine whether this part of the promoter was sufficient in vivo to direct tissue- and cell-specific expression of the receptor, transgenic mice were generated. The transgene contains the –1673 to +289 fragment cloned upstream of a luciferase reporter. The data show that the transgene is specifically expressed in the pituitary, is targeted to somatotroph cells, and is expressed in a sexually dimorphic and age-dependent manner.

Just as endogenous GHRH receptor expression is largely restricted to the pituitary (35), transgene expression is specific to the pituitary in transgenic mice, suggesting that tissue specificity of expression is driven by elements within the 1.6-kb promoter. Interestingly, no transgene expression is observed in the kidney, where an alternative GHRH receptor mRNA is expressed in the rat (35). Studies in our group (Zheng, J., and K. Mayo, unpublished) suggest that this mRNA is transcribed from a different promoter. A report has suggested that endogenous GHRH receptor expression is restricted to somatotroph cells of the anterior pituitary gland (64) in experiments in which cell type was morphologically determined. Although a small percentage of nonsomatotroph cells express the GHRH receptor promoter transgene, all somatotroph cells, defined by GH-positive immunocytochemistry, express the transgene. The results support the finding that endogenous GHRH receptor expression is somatotroph specific and suggest that the cell-specific expression is determined by elements in the 1.6-kb proximal promoter.

Conflicting data have been reported on whether GHRH receptor expression is dependent upon sex. Some reports suggest that males express the receptor mRNA at much higher levels than females (36, 65). Other studies have reported no difference between receptor expression in males and females (66, 67). The explanation for the conflict in the literature is not clear. One possibility is that female cycle status should be taken into account, considering that the GHRH receptor is known to be regulated by estrogen (38, 68). Expression of the GHRH receptor promoter transgene is consistently higher in males than in age-matched females, with an approximate 5-fold difference. The data indicate that sexual dimorphism of GHRH receptor expression is controlled by elements in the 1.6-kb receptor promoter. Sexually dimorphic GHRH receptor gene expression may contribute to the differing pulsatile pattern of GH release in males and females and the resulting average size difference between the sexes.

A previous study in the rat using ribonuclease protection assays has shown that expression of the endogenous GHRH receptor transcript is regulated during early postnatal development and aging. Endogenous GHRH receptor expression is high in newborns and declines until puberty, when expression increases, before again declining throughout aging (62). Subsequent reports have further substantiated that GHRH receptor mRNA expression dramatically decreases in the aged animal (66, 67). In the transgenic mouse, the transgene expression pattern during postnatal development shows an increase in expression as the animals approach puberty, although transgene levels do not steadily decrease during aging, as endogenous expression levels do. The data suggest that the regulatory elements for the pubertal surge in GHRH receptor expression are contained within the 1.6-kb promoter, but other upstream elements might be involved in the decreased expression during the aging of the animal. Alternatively, regulation of GHRH receptor protein levels may differ from mRNA regulation, possibly explaining the differences reported here. Another possibility is that this aspect of regulation of the GHRH receptor gene differs between rat and mouse species.

The data show that 1.6 kb of the rGHRH receptor promoter is sufficient to target tissue- and cell-specific expression of the receptor in vivo. In addition, the data suggest that the increase in GHRH receptor expression coincident with the pubertal increase in GH levels and sex-dependent expression of the receptor are regulated by elements within this 1.6-kb proximal promoter. Having established this part of the promoter as sufficient in vivo for appropriate expression, we further examined its regulation in cell culture experiments.

We showed that the cell specificity of expression driven by the 1.6-kb promoter is mimicked in cell culture. Because coexpression of Pit-1 led to activation of this promoter reporter construct in nonpituitary HeLa cells, we focused on Pit-1 regulation of the 1.6-kb rGHRH receptor promoter with a goal of identifying functional Pit-1 binding sites.

We were able to effectively narrow down specific Pit-1 binding sites to ten 25- to 35-bp regions. Only two of the 10 sites correspond to computationally predicted Pit-1 binding sites (43), illuminating the lack of consensus for Pit-1 binding sequences. To determine whether a single or few sites contributes most to Pit-1 activation of the rGHRH receptor promoter, we examined relative binding affinities and sequence conservation to zero in on candidate regions. Combined with functional analysis of promoter 5'-deletion and Pit-1 binding mutation constructs, the data revealed that multiple binding regions contribute to Pit-1 activation of the rGHRH receptor promoter. Of particular importance are two well-conserved sites with high relative affinities for Pit-1, –888 to –857 and +129 to +164.

The required +129 to +164 site has previously been identified as a Pit-1 binding site that synergizes with glucocorticoid receptor to activate transcription of the rGHRH receptor promoter (54). In addition, this site corresponds to one of the human sites identified by Iguchi et al. (44), which contributes most to Pit-1 activation of the human GHRH receptor promoter. Interestingly, a mutation in a different human Pit-1 site, which corresponds to a region in the rat that does not bind Pit-1, has been discovered in a human familial isolated GH deficiency type IB case (45). Our data confirm that the +129 to +164 site is essential to Pit-1 activation of the GHRH receptor gene. Furthermore, we have identified four additional functional Pit-1 binding sites that contribute to activation of the rat promoter. Of these, the –888 to –857 site contributes most to Pit-1 activation. Interestingly, there is a predicted glucocorticoid receptor binding site within this region, suggesting that Pit-1 might synergize with GR at this site, as well.

The current study identifies the 1.6-kb promoter as sufficient for tissue- and cell-specific expression of the rGHRH receptor gene in vivo. Further analysis of the promoter shows that cell specificity of expression is maintained in cell culture. Pit-1 is an important activator of the GHRH receptor promoter, and an in-depth characterization of Pit-1 regulation of this gene reveals at least 10 short regions that specifically bind Pit-1. The identification of several Pit-1 binding sites within the promoter of a single gene is not unexpected. Multiple binding sites for Pit-1 have been identified in the promoters of other known Pit-1 targets (48, 63, 69, 70), as well, which seems to be indicative of the importance of this factor in regulating cell-specific genes in the thyrotroph, lactotroph, and somatotroph lineages.

A report on estrogen regulation of the prolactin promoter shows a requirement for multiple Pit-1 binding sites to facilitate estrogen responsiveness (70), which confers cell-specific expression of this gene. It is likely that the Pit-1 binding sites identified in the rGHRH receptor promoter similarly synergize with cell-specific factors to facilitate somatotroph-specific expression of the gene. Future work will focus on examining the factors that work in addition to Pit-1, or in synergy with it, to regulate the cell-specific expression of the GHRH receptor gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation and Maintenance of Transgenic Mice
Transgenic mice were generated by the Case Western Reserve University (CWRU) transgenic facility according to the standard technique described in Ref. 71 and following protocols approved by the CWRU institutional animal care and use committee. The transgene was created by cloning the 1.6-kb GHRH receptor proximal promoter into the pA3 luciferase vector. NcoI digestion linearized the transgene, which was purified and diluted in T10E1 buffer. Transgene DNA was microinjected into the pronucleus of zygotes at 2–4 ng/µl. Injected embryos were surgically transferred to the oviducts of pseudopregnant recipient females. Animals used to generate founders were F1 hybrids of C57/Bl6 x SJL. Two founder male mice were identified and used to establish two separate transgenic lines. The transgenic lines were maintained by crossing transgenic offspring to C57/Bl6 mice. Genotyping was performed on genomic DNA extracted from tail biopsies by PCR using primers to amplify the luciferase reporter (5'-GATTCTAAAACGGATTACCAG-3' and 5'-GTGTTGTAACAATATCGATTC-3'). Mice were housed and bred in a controlled barrier facility at Northwestern University’s Center of Comparative Medicine. Temperature, humidity, and photoperiod (12-h light, 12-h dark) were kept constant. Mice were fed a phytoestrogen-free diet (Harlan Teklad Global 2019 or Harlan Teklad Breeder diet 2919). All animal protocols were approved by the animal care and use committees of CWRU (Cleveland, OH) and Northwestern University (Evanston, IL), and animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Luciferase Assays on Pituitary Protein Extracts
Pituitary glands were removed after euthanasia by CO2 and decapitation. Pituitaries were placed in 100 µl 1x Reporter Lysis Buffer (Promega Corp., Madison, WI) and homogenized using a hand-held motorized grinder. Samples were stored on ice for 15 min, cleared by centrifugation, and frozen at –80 C for at least 1 h to facilitate efficient cell lysis. Thawed samples were recentrifuged, and 25 µl of the supernatant was used in a luciferase assay by injecting 100 µl of Luciferase Assay Reagent (Promega Corp.) with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Protein assays were performed using the Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Inc., Richmond, CA). Relative light units were normalized to protein concentration for each sample. Statistical analysis was performed using GraphPad PRISM 4.0 (GraphPad Software, Inc., San Diego, CA).

Pituitary Dispersions
Pituitaries were removed after euthanasia by CO2 and decapitation. Tissues were immediately transferred to Wash/Collection Media [3 g/liter BSA; 25 mM HEPES (pH 7.4), in low-glucose DMEM (Invitrogen Life Technologies, Inc., Gaithersburg, MD), with penicillin/streptomycin and fungizone] on ice. Pituitaries were quartered with a scalpel on a siliconized glass slide and washed twice with wash/collection media. Pituitary quarters were rinsed in HEPES Dissociation Buffer (HDB) [1x Hanks’ Balanced Salt Solution (Invitrogen Life Technologies, Inc., Gaithersburg, MD) and 25 mM HEPES, pH 7.4] in a 15-ml conical tube. HDB was removed, and pituitaries were dissociated in 5 ml Collagenase II solution [0.4% Collagenase, type II (Invitrogen Life Technologies, Inc.)] 0.4% BSA Fraction V (Sigma, St. Louis, MO), 0.2% glucose, 80 U/ml DNase II (Sigma) in HDB; filter-sterilized aliquots were stored at –20 C before use in a 15-ml conical tube by shaking at 100 rpm in a 37 C water bath for 2 h. During the last half-hour of dispersion, the cells were triturated with a siliconized Pasteur pipette. Dispersed cells were centrifuged at 1000 rpm, and the supernatant was discarded. Freshly prepared 1x pancreatin (Sigma) (2.5 mg/ml in HDB) was added to the cells, and the cells were incubated at 37 C, 100 rpm for an additional 7 min. Charcoal-stripped fetal bovine serum (2 ml) (Invitrogen Life Technologies, Inc.) was added to stop the action of pancreatin, and cells were centrifuged at 1000 rpm for 10 min. Cells were washed twice with 420 µl culture media (low-glucose DMEM with 10% horse serum and 2.5% fetal bovine serum with penicillin/streptomycin and fungizone). Cells were resuspended in culture media, filtered through nylon mesh, and viable cells were counted on a hemocytometer using Trypan Blue staining. Cells were plated on collagen I-coated eight-well chamber slides (BD Biosciences, San Jose, CA) at approximately 1.5 x 105 cells per well.

Immunocytochemistry
Immunocytochemistry was performed on dispersed pituitary cells after culturing for 16–24 h at 37 C in culture media. Cells were fixed in 1% paraformaldehyde for 30 min at 4 C and permeabilized in 0.02% Triton X-100 for 5 min at room temperature, and blocking was performed by incubation in 6% BSA for 30 min at room temperature. Primary antibodies were diluted in 1% BSA in 1x PBS. Mouse antiluciferase (Zymed Laboratories, South San Francisco, CA; Invitrogen) was used at 30 µg/ml, and guinea pig antirat GH (National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA) was used at a 1:50 dilution. Primary antibody incubations were performed at 4 C for 16 h. A two-step secondary antibody incubation was performed using the Alexa Fluor Signal Amplification Kit (Molecular Probes, Invitrogen). For the first step, cells were incubated for 30 min at room temperature in a 1:200 dilution of rabbit antimouse conjugated to Alexa Fluor 488 and a 1:200 dilution of goat antiguinea pig conjugated to Alexa Fluor 546 (Molecular Probes, Eugene, OR) in 1% BSA in 1x PBS. To amplify the signal of the luciferase primary antibody, a second step was performed. Cells were incubated for 30 min at room temperature in a 1:200 dilution of goat antirabbit conjugated to Alexa Fluor 488 and a 1:200 dilution of goat antiguinea pig conjugated to Alexa Fluor 546. Vectashield mounting media with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) was applied to the samples and the slides were coverslipped. Fluorescence was examined using a Leica DM5000B fluorescence microscope and OpenLab 4.0 software (Improvision, Lexington, MA). Images were captured at x400.

Vaccinia Transfection System
HeLa T4 cells, maintained in DMEM with 4.5 g/liter glucose and L-glutamine (Mediatech, Inc., Herndon, VA) and 5% fetal bovine serum (Mediatech, Inc.), were transfected with constructs using the vaccinia virus-T7 polymerase expression system (obtained under license from Dr. Bernard Moss, National Institutes of Health, Bethesda, MD), as described elsewhere (72). For transfection, the cells were incubated with vaccinia at a multiplicity of infection of 10 in PBS/0.1% BSA for 30 min. Cells were transfected either with a Pit-1 expression construct (a gift from Dr. Holly A. Ingraham, University of California at San Francisco) or with empty vector. Plasmid DNAs to be transfected were incubated with liposomes (73, 74) at 5 µg lipid/µg DNA in OptiMEM media (Life Technologies, Inc., Gaithersburg, MD) for 15–20 min at room temperature. After infection, the virus was aspirated, and the DNA/transfectAce was added. Cells were transfected for 6 h at 37 C in 5% CO2 and allowed to recover overnight in DMEM.

Extraction of Nuclear Proteins
After transfection, nuclear extracts were collected, as described previously (75). HeLa cells were washed twice with cold 1x PBS and scrape collected in 1 ml 1x PBS with protease inhibitors (1 µg/ml aprotinin, leupeptin, antipain, and pepstatin) in microcentrifuge tubes. Cells were pelleted by spinning for 5 min at 500 x g at 4 C. The cell pellet was resuspended in 5x volume of Buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride with 1 µg/ml aprotinin, leupeptin, antipain and pepstatin] and incubated on ice for 15 min, followed by homogenization by 10 strokes with a glass-glass Dounce homogenizer. Samples were spun at 1,500 x g for 2 min at 4 C. The nuclear pellet was resuspended in two thirds volume Buffer C (20 mM HEPES, pH 7.9; 1.5 mM MgCl2; 420 mM NaCl; 0.2 mM EDTA; 25% glycerol; 0.5 mM DTT; 0.5 mM phenylmethylsulfonylfluoride with 1 µg/ml aprotinin, leupeptin, antipain, and pepstatin) while constantly stirring with a micro stir bar on ice. After stirring for 30 min, samples were spun at 13,000 x g for 10 min. The supernatant was aliquoted and stored at –80 C. Protein concentration was determined by Bradford Protein Assay using Bio-Rad dye reagent (Bio-Rad Laboratories, Hercules, CA).

Western Blot Analysis
Nuclear extracts (10 µg) were boiled for 5 min in sample buffer and size separated by SDS-PAGE on a 10% acrylamide gel. Proteins were transferred to a nitrocellulose membrane (BA-85; Schleicher & Schuell, Keene, NH) by electrophoresis. The membrane was blocked with 3% nonfat dry milk in PBS (blocking buffer) for 20 min at room temperature with shaking, followed by incubation with a 1:100 dilution of Pit-1 antibody (PRB-230C; Covance Laboratories, Inc., Berkeley, CA) in blocking buffer overnight at 4 C with shaking. The blot was washed twice with water and incubated with a 1:3000 dilution of secondary antibody [goat-antirabbit conjugated to horseradish peroxidase (Promega)] in blocking buffer for 90 min. The blot was washed twice with water, then in PBS/0.05% Tween for 5 min, and finally washed in five changes of water. The antibody-antigen complexes were detected by enhanced chemiluminescence (ECL-Plus kit, Amersham, Little Chalfont, Buckinghamshire, UK).

EMSAs
DNA probes for EMSAs were generated either by PCR incorporating 32P-dCTP (GE Healthcare, Piscataway, NJ) for probes longer than 100 bp or by end labeling annealed complementary oligonucleotides in a T4 kinase reaction using 32P-{gamma}ATP (MP Biomedicals, Solon, OH), for shorter probes. Labeled probes were gel purified on 5–12% acrylamide/TBE (Tris-borate, EDTA) gels and eluted from the gel in 0.5 M NH4OAc and 1 mM EDTA. Probe eluates were spun through nylon wool, ethanol precipitated twice, and resuspended in 50 µl double distilled H2O. Each probe (1 µl) was counted on a scintillation counter, and probes were diluted for use at 1.0 x 104 cpm per reaction. In each reaction, 5 µg nuclear extract was used, unless otherwise indicated. Nuclear extracts were incubated with 1x EMSA buffer (5x EMSA buffer: 5 mM DTT; 5 mM MgCl2; 50 mM Tris, pH 7.5), 0.2 µg/µl polydeoxyinosinic deoxycytidylic acid double strand (Amersham Biosciences, Piscataway, NJ), and radiolabeled probe in a final volume of 10–15 µl for 20 min at room temperature. For supershift experiments, 2 µl Pit-1 antibody (200 µg/ml) (sc-16289; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was incubated with nuclear extract for 30 min on ice before the addition of the probes. TBE loading dye (30% glycerol/70% TBE with 16 mg/ml bromophenol blue and 16 mg/ml xylene cyanole) was added to each sample, and the samples were run on a 5% acrylamide/TBE gel at 4 C. Gels were exposed to Kodak X-OMAT AR film (Rochester, NY) overnight, and exposed to PhosphorImager screens (Molecular Dynamics, Inc., Sunnyvale, CA) for quantification using a STORM 860 phosphor imager (GE Healthcare) and ImageQuant 5.0 software (Molecular Dynamics).

Generation of 5'-Deletion and Mutation Constructs, Transient Transfections, and Luciferase Assays
rGHRH receptor promoter 5'-deletion constructs were generated by PCR using 5'-primers with a KpnI site and a 3'-primer with either an NheI site (–1673, –1355, –1115) or a HindIII site (–840, –601, –396, –131) using the –1673 rGHRH receptor promoter construct as a template. Primers were generated by Integrated DNA Technologies (Coralville, IA). Primer sequences follow: –1673 5', 5'-GCGGTACCCCATGGCCTC TGCATCAACTTCTG-3'; –1355 5', 5'-GCGGTACCGGTTGTGGGAGCTCCCTACTGCAT-3'; –1115 5', 5'-GCGGTAC CGGGTGTGGCAGCCTCTGTCCCCTTTTA-3'; –840 5', 5'-GCGGTACCTGGCACCCCCC AGCACAACAGCTCTGTAA-3'; –601 5', 5'-GCGGTACCAGCCCCAGAGATCCCTCTGAG AGTCCT-3'; –396 5', 5'-GCGGTACCTGTGGTGCCAGGGATTTG AACCTGGAGCC-3'; –131 5', 5'-GCGGTACCCGTGCAGGTATAGGAGGCCTCTCTGAG-3'; +148 5', 5'-GCGGTA CCTGTCCCTCCTGTTGGCCCTGCCTACACAA-3'; +264 NheI 3', 5'-GGTGCTAGCCAAA GAGCCGTGGCCCTTCCCTCCCA-3' and +264 HindIII 3', 5'-GGAAGCTTCCAAAGAGCC GTGGCCCTTCCCTCCCA-3'. PCR products were digested with the appropriate enzymes and ligated into the pLuc IAV Link V.4 luciferase vector (a gift from Dr. Richard Day, University of Virginia). The –1673 luciferase clone was used as a template for generation of Pit-1 binding site mutation clones. Mutations are shown in Table 1Go and were generated using the QuikChange (Stratagene, La Jolla, CA) protocol by PCR with complementary oligonucleotides incorporating the desired mutations. The parental DNA was digested by DpnI for 1 h at 37 C, and the mutant DNA was transformed by electroporation into Top 10 competent cells.

Clones were sequence verified and used in transient transfections of GH3 and HeLa T4 cells. Cells were plated into 12-well plates and cotransfected while subconfluent with mutation or deletion constructs and either a Pit-1 expression vector (a gift from Dr. Holly Ingraham, University of California at San Francisco) or empty vector with 1 µg each plasmid (2 µg total DNA per well) in Opti-MEM containing lipofectamine 2000 (2 µl/µg DNA) (Invitrogen, Carlsbad, CA) for 4–6 h. Cells transfected with an Rous sarcoma virus-luciferase construct served as a positive control, to which individual transfection experiments were normalized. Transfected cells were allowed to recover overnight in complete media. Cells were washed with 1x PBS and lysed in 150 µl 1x Reporter Lysis buffer (Promega) for 20 min on ice. Cells were scrape collected, and lysis was completed by freezing at –80 C for at least 1 h. For the luciferase assay, 100 µl of each lysate was aliquoted into cuvettes, which were injected with luciferase assay substrate (Promega), and light emission was measured using a Monolight 2010 luminometer (Analytical Luminescense Laboratory, San Diego, CA). Relative light units were normalized to protein concentration, which was determined by Bradford Protein Assay using Bio-Rad dye reagent (Bio-Rad Laboratories).


   ACKNOWLEDGMENTS
 
We thank Dr. A. F. Parlow (Harbor-UCLA Research and Education Institute), the National Hormone and Peptide Program, and National Institute of Diabetes and Digestive and Kidney Diseases for the antibody to GH. We thank Dr. Bernard Moss (National Institutes of Health) for the use of the Vaccinia-T7 polymerase system, Dr. Holly Ingraham (University of California at San Francisco) for the Pit-1 expression construct, and Dr. Richard Day (University of Virginia, Charlottesville, VA) for the pLuc IAV Link luciferase vector. We also thank Dr. Ishwar Radhakrishnan (Northwestern University, Evanston, IL) for guidance on affinity experiments. We thank the staff of Northwestern University’s Center for Comparative Medicine for care and maintenance of the animals used in this study.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK-48071 (to K.E.M.).

Present address for A.I.K.: Applied Molecular Evolution, Eli Lilly, San Diego, California 92110.

Disclosure of Potential Conflicts of Interest: A.T.M, A.I.K., S.M.K., and L.C. have nothing to disclose. K.E.M. has equity interests in Ligand Pharmaceuticals.

First Published Online May 29, 2007

Abbreviations: DDT, Dithiothreitol; HDB, HEPES dissociation buffer; TBE, Tris-borate, EDTA.

Received for publication March 1, 2007. Accepted for publication May 22, 2007.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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