Transcriptional Regulation of the Human PRL-Releasing Peptide (PrRP) Receptor Gene by a Dopamine 2 Receptor Agonist: Cloning and Characterization of the Human PrRP Receptor Gene and Its Promoter Region
Atsushi Ozawa,
Masanobu Yamada,
Teturou Satoh,
Tsuyoshi Monden,
Koshi Hashimoto,
Hideaki Kohga,
Yasuhiro Hashiba,
Tomio Sasaki and
Masatomo Mori
First Department of Internal Medicine (A.O., M.Y., T.S., T.M., K.H., M.M.), Gunma University School of Medicine, Maebashi 371-8511, Japan; and Department of Neurosurgery (H.K., Y.H., T.S.), Gunma University School of Medicine, Maebashi 371-8511, Japan
Address all correspondence and requests for reprints to: Masanobu Yamada, M.D., Ph.D., First Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: myamada{at}med.gunma-u.ac.jp.
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ABSTRACT
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PRL-releasing peptide receptor (PrRPR) mRNA was expressed in pituitary adenomas but was not detected in patients treated with bromocriptine, a specific agonist of dopamine 2 (D2) receptor. Although medical treatment with bromocriptine is effective for patients with pituitary adenomas, little is known about the molecular mechanisms of gene regulation mediated by D2 receptors. The cloned human PrRPR gene spanned approximately 2.0 kb and contained two exons and one intron. Two functional polyadenylation signals located at 510 and 714 bp downstream from the stop codon. A primer extension analysis demonstrated two major transcriptional start sites at 139 and 140 bp upstream from the translational start site and an additional minor site at -161. The promoter region contained several putative binding sites for transcriptional factors including pituitary-specific transcription factor (Pit 1), activator protein 1 (AP-1), and specificity protein (Sp1), but no typical TATA or CAAT box. This promoter showed the strong activity in the pituitary-derived GH4C1 cells, and the region between -697 and -596 bp was responsible for the stimulation both by forskolin and overexpression of cAMP response element binding protein (CREB). These stimulations were significantly suppressed by incubation with bromocriptine in a dose- and time-dependent manner, and the mutant CREB (S133A) completely abolished the inhibitory events of bromocriptine. However, EMSA studies demonstrated that CREB did not bind to this region, to which an approximately 60-kDa protein was strongly bound, and that antibodies against CREB, c-Fos, and Sp1 did not supershift this complex. Furthermore, the amount of this unknown protein was apparently reduced by treatment with bromocriptine. A series of mutation analyses demonstrated that the specific sequence, 5'-cccacatcat-3', was required for both the binding to the 60-kDa protein and the repression by bromocriptine. Therefore, the transcriptional repression of the PrRPR gene by bromocriptine required CREB but was independent of direct binding of CREB to the gene and that the sequence -663
-672, 5'-cccacatcat-3', bound to the 60-kDa protein appeared to be critical for this event.
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INTRODUCTION
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THE PRL-RELEASING peptide (PrRP) receptor (PrRPR) was originally isolated as an orphan seven transmembrane domain receptor, GPR10 (hGR3), which was expressed extensively in the anterior pituitary (1, 2). Hinuma et al. (3) isolated its specific ligand from bovine hypothalamic tissues and named it PRL-releasing peptide (PrRP). PrRP generated two amidated isoforms PrRP31 and PrRP20, and the later corresponded to the C-terminal 20 residues of the former. Both PrRP 31 and 20 have been demonstrated to have similar abilities to induce arachidonic acid metabolite release and then specifically stimulate the secretion of PRL from the anterior pituitary gland in vivo and in vitro. Although the function of PrRP as a physiological regulator of PRL secretion remains controversial (4, 5, 6), we and other found strong expression of PrRPR mRNA in pituitary adenomas (7).
Pituitary adenomas are benign neoplasms that are monoclonal in origin, suggesting that they arise as clonal expansion of a genomically altered progenitor cell (8, 9). Recently, several genetic aberrations were implicated in the initiation of tumorigenicity, including activation of cellular protooncogenes, loss of function of tumor suppresser genes, or mutations of G protein-coupled receptors that were induced by mutations has been demonstrated in endocrine diseases in man (10, 11, 12, 13). Therefore, abnormal expression of PrRPR may be involved in the tumorigenicity of pituitary adenomas.
PRL release from the anterior pituitary is regulated principally by inhibitory influences imparted by the tuberoinfundibular dopamine system. Medical treatment with long-acting dopamine agonists such as bromocriptine is very effective for reducing PRL levels and restoring gonadal function in patients with prolactinoma (14). In addition, although this effect is most dramatic in macroprolactinoma, bromocriptine has been reported to be beneficial for other pituitary adenomas and has been frequently used for shrinkage of pituitary adenomas before transsphenoidal surgery and to depress hormone levels after insufficient surgery. When tumor shrinkage was first documented in the late 1970s, the mechanism of this dopamine agonist-specific effect was obscure. However, the cloning of the dopamine 2 (D2) receptor revealed that D2 receptor is negatively coupled with adenylate cyclase and was expressed in normal and tumor lactotrophs (15, 16). In fact, in purified intact lactotrophs, a marked decrease of the cellular cAMP level was found within 1 min of dopamine application, correlating with a reduced rate of PRL secretion. These effects could also be induced by dopamine agonists and conversely blocked by D2 receptor antagonists (17, 18). It was, therefore, reported that reduction in intracellular cAMP levels is an important mechanism in which dopamine and bromocriptine inhibit hormone release. However, recent studies demonstrated multiplicity and complexity of the signaling events at the D2 receptor including MAPK and inhibition of phosphatidyl inositol turnover, and intracellular calcium concentrations (19, 20, 22). In addition, Asa et al. (23) reported that D2 receptor knockout mice developed pituitary lactotroph adenomas suggested the direct involvement of the D2 receptor signaling pathway in tumorigenicity of pituitary adenomas. Therefore, regulation of a certain gene by dopamine 2 agonist in the pituitary adenomas may be important for molecular mechanism of treatment of the pituitary adenomas by D2 agonist.
In the present study, we first examined whether PrRPR mRNA was expressed in several pituitary adenomas by Northern blot analysis and found PrRPR mRNA was not detected in patients treated with bromocriptine, a specific agonist of D2 receptor. We hypothesized that the PrRPR gene in the pituitary adenomas may be regulated by D2 receptor agonists. Although the partial structure of the human PrRPR gene was recently reported, its genomic organization, detailed analysis of the transcriptional start sites, and regulation of the promoter region remains to be elucidated (24). Therefore, to directly test our hypothesis we cloned and characterized the entire human PrRPR gene, and gained insight into the molecular mechanisms of regulation of the PrRPR gene by bromocriptine.
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RESULTS
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Expression of PrRP Receptor mRNA in Pituitary Adenomas
The clinical characteristics of all patients whose pituitary specimen was analyzed are summarized in Table 1
. The mean age of patients with each cell type adenoma including prolactinomas, GH-secreting adenomas, and nonfunctioning adenomas was 37.6, 41.6, and 49, respectively. Figure 1
shows representative findings of Northern blot analysis of PrRPR mRNA in PRL and GH-producing pituitary adenomas, which showed a single species, approximately 2.2 kb in length. Significant expression of PrRPR mRNA was observed in all cell types of pituitary adenomas including non-functioning adenomas and an ACTH producing adenoma (Table 1
). There was no significant difference in the expression levels of PrRPR mRNA between different cell types. Furthermore, even in the same cell type of pituitary adenomas, the expression level varied between individual adenomas. However, no expression of PrRPR mRNA was observed in some pituitary adenomas including two of five prolactinomas, two of five GH-secreting adenomas, or one of three nonfunctioning adenomas. Significant signal loss of PrRPR mRNA was observed in four of five samples, who were treated with bromocriptine, a specific D2 receptor agonist. These patients were treated with bromocriptine 2.5
7.5 mg/d for at least 2 wk before surgery. Furthermore, expression levels of PrRPR mRNA were independent of pathological types or gender of the patients. Therefore, these findings suggest the possible regulation of PrRPR gene expression by the dopamine agonist, bromocriptine.

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Figure 1. Northern Blot Analysis of PrRP Receptor mRNA in the Pituitary Adenomas
The black arrow indicates 2.2 kb PrRPR mRNA. Ethidium bromide staining of 18S rRNA confirmed equal application of each total RNA. Representative data from PRL and GH producing pituitary adenomas are shown. PRL1, PRL3, GH2, and GH3 correspond to the patients PRL1, PRL3, GH2, and GH3 in Table 1 , respectively.
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Isolation and Characterization of the Human PrRPR Gene
To determine the mechanism underlying the regulation of the human PrRPR gene, we first cloned a human PrRPR gene from the human genomic DNA library and obtained two overlapped clones encoding the PrRPR gene. Comparison of the genomic sequence with that of the 5'-rapid amplification of cDNA ends (RACE) study established the organization of the PrRPR gene as two exons and one intron (Fig. 2A
). Intron 1 occurred in the 5' untranslated region and was 133 bp in length. Exon 2 started from 6 bp upstream of the ATG initiation codon and contained the nucleotide sequence of the entire coding sequence as well as the 3' untranslated region. The GT-AG sequence was conserved for splice sites in the intron (Fig. 2B
).

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Figure 2. Schematic Representation and Nucleotide Sequence of the Human PrRPR Gene
A, The XhoI and EcoRI restriction sites are indicated. In the human PrRPR gene, black boxes denote exons and the thin line denotes the intron and flanking regions. ATG represents the translational initiation site; Stop, stop codon; poly(A), poly(A) adenylation signals. B, Exons are shown in uppercase letters. Introns are described in lowercase letters. The proposed transcriptional initiation sites are shown with asterisks and the most 3' one is numbered +1. Several potential cis-acting sequences are indicated (AP-1, Sp1, and Pit-1 binding motif). Functional polyadenylation signals (AATAAA and ATTAAA) are represented in boldface.
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Two Functional Polyadenylation Signals of the Human PrRPR Gene
To determine a functional polyadenylation signal in the anterior pituitary, 3'RACE experiments were performed. As shown in Fig. 3A
, a single major amplified product was approximately 300 bp in length and another minor product approximately 150 bp, each of which was subcloned into pGEM-T vector. Sequence analysis indicated that the major functional polyadenylation signal was the typical AATAAA motif located 714 downstream from the stop codon, and thymidine residue 23 bp downstream of the signal was the polyadenylation site. Analysis of another minor 3'RACE fragment indicated an atypical motif ATTAAA 510 bp from the stop codon that was also functional, and the thymidine residue 23 bp downstream of the signal was the polyadenylation site (Fig. 3B
).

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Figure 3. 3'RACE and Determination of the Transcriptional Initiation Site of the Human PrRPR Gene
A, Amplification products of the expected sizes are indicated by arrows. The molecular size marker (bp) is indicated. B, The representative sequence data is shown in the right panel. C, 5'RACE study of the human PrRPR gene. Results of Southern blot analysis are shown. The molecular size marker (bp) is indicated on the left. Amplified products were subcloned and sequenced. D, Primer extension using oligonucleotide, PE-1. The end-labeled primers were hybridized to 5 µg of poly(A) RNA (lane poly(A)+) from the human PRL-secreting tumor, and yeast tRNA (lane tRNA), and extended with AMV reverse transcriptase. The primer extended products were separated on an 8 M urea 6% polyacrylamide gel. PE-1 gave positive strong signals at 139 and 140 from the translational initiation site and a weak signal at 161. Marker lanes indicate the sequencing ladder of the human PrRPR gene using the same primer.
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Multiple Transcription Start Sites of the Human PrRPR Gene
To determine the transcriptional start sites in the human PrRPR gene, we first performed 5'RACE of the transcript in intact anterior pituitary. Although Southern blot analysis seems to be a single amplified fragment (Fig. 3C
), sequence analysis revealed several 5' ends of the cDNA of the PrRPR gene. The results of 5'RACE may be due to the premature extension of cDNA or a hairpin loop formation of the priming of the second strand of the cDNA. Therefore, to determine the exact transcriptional start site, we next performed primer extension analysis. The primer extension with 5 µg of poly(A)+ RNA from the human pituitary PRL-secreting adenoma, and the PE-1 primer gave major signals at 139 and 140 bp from the translational initiation site and a minor start site at 161, whereas no significant signals were found in yeast transfer RNA (Fig. 3D
). We confirmed this finding using another primer extension analysis and the primer PE-2 (data not shown). On the basis of these observations, the transcriptional start site of the human PrRPR gene formed a cluster, and the major site was assigned to residues 139 and 140 bp upstream of the translational initiation site. We numbered the 3' site, 139 bp upstream +1 in this study.
Inspection of the sequence of the PrRPR promoter region indicated that there was no typical TATA box, CAAT box, or GC-rich sequence in close proximity to the major transcriptional start site. However, several possible regulatory elements were identified including complete sequence matches for pituitary-specific transcription factor (Pit 1), activator protein 1 (AP-1), and specificity protein (Sp1) (Fig. 2B
).
Promoter Activity of the Human PrRPR Gene
To determine whether the putative promoter region was functional, the 697-bp promoter region was subcloned into a luciferase reporter plasmid. As illustrated in Fig. 4A
, the strongest expression was observed in rat pituitary tumor-derived GH4C1 cells with 214 ± 12.9% [
2,000,000 arbitrary light units (ALU)/10 sec/100 µg protein] of that obtained from the thymidine kinase (TK) promoter. In mouse neuroblastoma-derived NB41A3 cells, the same construct showed 113.7 ± 0.1% of that of the TK promoter, 81.2 ± 8.02% in the human neuroepithelioma-derived HTB-10 cells, 19.3 ± 1.83% in human adenocarcinoma of the cervix-derived HeLa cells, and almost no activity with 6.15 ± 0.40% in monkey kidney-derived CV-1 cells.

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Figure 4. The Promoter of the Human PrRPR Gene Was Most Active in the Pituitary-Derived GH4C1 Cells
A, Constructs containing the PrRPR gene upstream fragments were subcloned in front of the luciferase reporter gene, and transiently transfected into GH4C1, NB41A3, HTB-10, HeLa, and CV-1 cells. The values are expressed as relative luciferase activity (arbitrary light units of the PrRPR promoter/that of the TK promoter). Values represent means ± SEM of triplicate determinations. At least three independent experiments were performed. B, GH4C1 cells were transiently transfected with the indicated plasmids, and luciferase activity was measured. The value of the TK promoter activity was set as 100%, and other values are presented as means ± SE of triplicate determinations. The pGL3-Basic was the promoterless luciferase plasmid, the typical activity of which measured 500 ALU/10 sec/100 µg protein.
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Deletion analysis of the promoter region of the human PrRPR gene in GH4C1 cells revealed no significant changes in the promoter activity among the plasmids -697+133Luc, -697+76Luc, -431+76Luc, and -219+76Luc, indicating that the regions between -697 and -219, and +76 and +133 from the major transcription start site (TSS) were not essential for the basal promoter activity (Fig. 4B
). In contrast, deletion of the region between -23 and +76 significantly reduced its activity, and this result was compatible with that of the above primer extension study showing that the major transcription start site was in this region.
Regulation of the PrRPR Gene by Forskolin, cAMP Response Element Binding Protein (CREB), and Bromocriptine
Bromocriptine is a specific agonist for D2 receptor, and mainly stimulates an intracellular molecule of Gi to decrease the cAMP level (25, 26, 27). cAMP is known to mediate the hormonal stimulation of a number of eukaryotic genes through a transcription factor, CREB. Therefore, we examined the effects of forskolin and overexpression of CREB on the promoter activity of the PrRPR gene in GH4C1 cells. An addition of 10 µM forskolin showed no effects on the promoter activity of TK or those of -595+76, -556+76, -499+76, -431+76 and -219+76Luc (139 ± 15.5%, 100 ± 3.4, 112 ± 3.5, 100 ± 4.6, 103 ± 5.5% and 116 ± 12.3% of the control, respectively). In contrast, significant stimulation of the promoter activity (approximately 2.5-fold) (237 ± 9.8% of the control, P < 0.01, n = 3) was observed specifically in the plasmid -697 + 76Luc (Fig. 5A
). Similarly, as shown in Fig. 5B
, overexpression of CREB caused significant stimulation only in the plasmid -697 + 76Luc (317 ± 10.5% of the control, P < 0.001, n = 3).

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Figure 5. Stimulation by Forskolin and Overexpression of CREB Specifically on the Promoter -697 -596 bp Region of the PrRPR Gene
A, The indicated plasmids were transfected into GH4C1 cells in the presence or absence of 1 µM of forskolin. B, The same plasmids were transfected into GH4C1 cells with the expression vector of CREB driven by CMV promoter. The values (A and B) are presented as the n-fold activation of the promoter activity in the presence of 1 µM forskolin or CREB, and are the means of at least three separate experiments ± SE.
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To evaluate the possibility of regulation of the promoter activity of the PrRPR gene by the dopamine agonist, we next examined the effects of bromocriptine in each deletion mutant of the promoter region. As pituitary derived GH4C1 cells have been reported to have no expression of dopamine receptors (19), human neuroblastoma-derived NB41A3 cells possessing functional D2 receptors were used for the subsequent experiment with bromocriptine (28, 29). As shown in Fig. 6A
, incubation of 1 µM of bromocriptine for 48 h alone did not alter the promoter activity of TK or constructs containing the promoter region of the PrRPR gene. However, when cells were incubated with 10 µM of forskolin simultaneously, incubation of bromocriptine significantly reduced the promoter activity specifically in plasmid -697+76Luc showing 47.5 ± 4.7% (P < 0.001, n = 3) of the control without treatment of bromocriptine. In contrast, incubation with the same dose of bromocriptine did not alter the promoter activity of TK and the constructs -595 + 76, -556 + 76, -499 + 76, -431 + 76 or -219+76Luc. These data indicated that the regulatory region by bromocriptine was located in the region between -697 and -596 from the major TSS. Furthermore, as shown in Fig. 6
, C and D, the reduction of the PrRPR gene promoter activity by bromocriptine was dose and time dependent. The effect of bromocriptine reached a plateau within 24 h and was observed from as little as 0.01 µM of bromocriptine and reached a plateau at 1 µM.

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Figure 6. Bromocriptine Reduced the Promoter Activity of the PrRPR Gene in NB41A3 Cells Possessing Functional D2 Receptors
A, The indicated plasmids were transfected into NB41A3 cells in the presence or absence of 1 µM bromocriptine. B, The same transfection was performed except for adding 10 µM of forskolin simultaneously. All values are presented as the % of the control in the presence of 1 µM bromocriptine, and are the means of at least three separate experiments ± SE. C and D, The effect of bromocriptine on the forskolin-stimulated promoter activity was time dependent (C) and dose dependent (D). The indicated time (hours) was incubated with 1 µM bromocriptine and 10 µM of forskolin, and measured promoter activities (C). The indicated concentration of bromocriptine was incubated with 10 µM forskolin (D).
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Involvement of the Residue Ser 133 of the CREB in the Regulation of the PrRPR Gene by Bromocriptine
It was reported that cAMP activated the cAMP-dependent PKA, which phosphorylates CREB at Ser-133 located in the kinase-inducible domain, and that the phosphorylation of Ser-133 allows CREB to associate with CREB binding protein (30, 31, 32). Therefore, we examined whether the Ser-133 residue of CREB was involved in the regulation of the PrRPR gene by bromocriptine using a mutant which exchanged serine with alanine at 133 (S133A) of the CREB. Similar to the inhibitory effect of bromocriptine on the forskolin stimulated PrRPR gene, treatment with 1 µM of bromocriptine induced a significant reduction of the promoter activity stimulated by overexpression of CREB specifically in the plasmid -697+76Luc (64.2 ± 3.6% of the control, P < 0.01, n = 3), but no significant effect in the TK or other shorter constructs were observed (Fig. 7A
). Furthermore, although the mutant CREB (S133A) less effectively stimulated the basal promoter activity of the PrRPR gene compared with the wild-type (211 ± 8.5%, P < 0.01), the inhibitory effect of bromocriptine was completely abolished, suggesting the essential involvement of the Ser 133 residue of CREB to produce the inhibitory effect of bromocriptine (Fig. 7B
).

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Figure 7. Involvement of Residue Ser 133 of CREB in the Regulation of the PrRPR Gene by Bromocriptine
A and B, The indicated plasmids and the same amount of CREB (black bars) or mutant CREB (S133A) (hatched bars) expression vector were cotransfected into NB41A3 cells in the presence or absence of 1 µM bromocriptine. All values are presented as the percentage of the control in the presence of 1 µM bromocriptine, and are the means of at least three separate experiments ± SE. C and D, Incubation with 10 µM nimodipine and 50 µM PD98059 did not affect the inhibitory effect of the PrRPR gene by bromocriptine.
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As involvement of intracellular calcium concentrations and MAPK in D2 receptor action have been recently reported, we performed the same experiment with a Ca++ ion channel blocker and a specific MAPKK inhibitor. As shown in Fig. 7
, C and D, 10 µM nimodipine and 50 µM PD98059 did not affect the inhibitory effect of the PrRPR gene by bromocriptine.
An Unknown Factor That Was Not CREB Bound the Region -596
-697 of the PrRPR Gene, Whose Amount Was Reduced by Bromocriptine Treatment
Inspection of the -596
-697 sequence of the PrRPR gene indicated no typical consensus sequence for the reported CRE (TGACGTCA), but a sequence homologous to the half-site of CRE (CGTCA) was observed at -689
-692 and -616
-619. Therefore, to clarify whether CREB binds to the -596
-697 region, we performed an EMSA study with nuclear extract from NB41A3 cells. As shown in Fig. 8A
, the consensus CRE clearly bound to CREB in nuclear extract that was supershifted by a specific antibody against CREB. Although a similar factor strongly bound to the -633
-697 region, it was not supershifted with the CREB antibody, and the same results were observed with nuclear extract from the cell extracted with CREB expression vector (data not shown). Furthermore, no significant binding of any factor was observed in other fragments, -578
-657, -539
-602, -482
-563, and -415
-506. These findings demonstrated that CREB did not bind the responsible region of the PrRPR gene for the bromocriptine effect.
Homologous sequences for Sp1 and AP-1 were also observed in the region -633
-697; therefore, we next attempted to identify the above unknown factor using specific antibodies against c-Fos and Sp1 (Fig. 8B
). As shown in Fig. 8B
, after incubation with nuclear extracts, the consensus sequence of AP-1 and Sp1 clearly made a single complex, and those were supershifted by antibodies against Sp1 and c-Fos. However, alteration in the migration rate of the complex of the unknown factor and the -697
-633 fragment did not occur in the presence of antibodies for c-Fos or Sp1. Importantly, treatment with bromocriptine led to a reduction in the amount of unknown factor binding to the -633
-697 fragment (Fig. 8C
).
The Specific Region -663
-672 Was Critical for Both Binding to the Unknown Factor and for the Inhibitory Event by Bromocriptine
To further characterize the responsible region of the PrRPR gene for bromocriptine effect, we made a series of mutants in the -697
-633 region and first performed an EMSA study. We first identified that the 5' half-site of this region -678
-697 did not bind to the unknown factor. Within the 3' region -658
-677, changing 5 bases of the 5' end to 5 thymidine residues (mutant 1, Mut. 1) induced no significant change, and the same change of the 3' end (Mut. 4) slightly reduced the amount of the binding of the unknown factor. In contrast, mutation of the middle region (Mut. 2 and 3) led to a complete abolishment of binding of the unknown factor, demonstrating that the region -663
-672 was essential for binding to the unknown factor (Fig. 9A
).
To confirm whether this region is also important for the inhibition of the PrRPR promoter activity by bromocriptine, we performed functional analysis with the same mutations (Mut. 2 and 3). As shown in Fig. 9B
, the mutations 3 or 2 caused loss of the inhibition of the PrRPR promoter activity by bromocriptine.
The Unknown Protein that Bound to the Responsible Region of the PrRPR Gene for the Bromocriptine Effect Was Approximately 60 kDa in Size
To further characterize the unknown protein that bound to the responsible region for bromocriptine effect, we attempted to determine the molecular weight of this protein using the biotin/streptavidin affinity system and SDS-PAGE. As shown in Fig. 10
, whereas the fragment -678
-697 did not coprecipitate any protein, the -658
-677 fragment coprecipitated a single protein stained strongly with approximately 60 kDa in size.

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Figure 10. The Unknown Protein Bound to the -663 -672 Was Approximately 60 kDa in Size
After coprecipitation of the unknown factor with 500 pmol of biotinlated oligonucleotides encoding the indicated region of the PrRPR gene using the biotin/streptavidin affinity system, it was resolved on an 8% SDS-polyacrylamide gel and stained with coomassie brilliant blue. A protein of approximately 60 kDa was coprecipitated with the -677 -658 oligonucleotide.
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DISCUSSION
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In the present study, we first demonstrated expression of PrRP receptor mRNA in all cell types of pituitary adenomas examined using Northern blot analysis. Expression levels of PrRPR mRNA among cell types of pituitary adenomas varied, and even in the same cell type of pituitary adenoma, its level differed between individual adenomas. These findings are consistent with those reported by Zhang et al. (7) showing PrRPR mRNA expression in pituitary adenomas using RT-PCR. Although they found expression of PrRPR mRNA in all pituitary adenomas, the present Northern blot analysis revealed undetectable expression in some pituitary adenomas including two of five prolactinomas, two of five GH-secreting adenomas, and one of three nonfunctioning adenomas. This discrepancy may be due to the differences in the sensitivity between PCR and Northern blot analysis, the different doses and/or periods of bromocriptine treatment, or discrete species. All patients with prolactinoma or GH producing adenoma who showed no detectable expression of PrRPR mRNA by Northern blot analysis were treated with bromocriptine, a D2 agonist. This observation raised the possibility of the involvement of dopamine in the regulation of the PrRPR gene expression in pituitary adenomas. To directly test this hypothesis, we isolated and characterized the human PrRPR gene and investigated the regulation of its promoter activity by the dopamine agonist in vitro.
Comparison of the genomic sequence with the cDNAs obtained by 5'RACE established the complete structure of the human PrRPR gene, which contained two exons and one intron and spanned a region of approximately 2.0 kb. The first exon contained only the 5' untranslated region, and the second exon began from 6 bp upstream of the translational initiation site, and the second exon contained the entire coding sequence and the 3' untranslated region. Primer extension and 5'RACE studies revealed multiple transcriptional start sites around 140 bp upstream of the translational start site. Inspection of the sequence of the promoter region of the human PrRPR gene revealed no consensus TATA or CAAT boxes at the appropriate positions, but several possible cis-acting regulatory sequences in the promoter region. Of special interest was the identification of a putative Pit 1 binding site at -87
-82. Pit 1, an anterior pituitary-specific transcription factor, was reported to be involved in the regulation of anterior pituitary hormones, for example, in the activation of the GH, TSH, and PRL genes (33, 34). As PrRPR mRNA was identified in PRL and GH-producing tumors, Pit 1 may contribute to the regulation of anterior pituitary PrRP receptors through activation of the PrRPR gene. In fact, our preliminary experiment showed coexpression of Pit 1 led to a significant increase of the PrRPR promoter activity.
The fragment encoding the 697-bp promoter region of the human PrRPR gene was transcriptionally most active with approximately 200% of the control TK in GH4C1 cells, which were derived from rat pituitary tumor cells. The lower activity was observed in neuroblastoma-derived NB41A3 and neuroepithelioma-derived HTB-10 cells. As PrRPR mRNA has been reported to be expressed extensively in the anterior pituitary and moderately in the rat brain, the cloned promoter region of (
-697 from the TSS) may be responsible for the tissue specific expression of the PrRPR gene (35, 36).
Transcriptional regulation on stimulation of the adenylate cyclase signaling pathway is mediated by a family of cAMP-responsive nuclear factors such as CREB, CREM, and ATF-1 (31). These factors contain the basic domain/leucine zipper motifs and bind as dimers to the CRE of the target genes. The function of CREB is modulated by phosphorylation at Ser 133 by cAMP-dependent protein kinase, which promotes recruitment of the coactivator CREB binding protein and p300, histone acetyltransferases that have been proposed to mediate target gene activation, in part, by destabilizing promoter bound nucleosomes and thereby allowing assembly of the transcriptional apparatus (30, 31, 32). Although recent studies have provided these findings regarding the mechanism underlying stimulation of genes by the adenylate cyclase signaling pathway, the detailed mechanism by which D2 agonists, such as bromocriptine, inhibit expression of the responsive genes remains to be clarified.
The present transfection study first demonstrated that the region -697
-595 was responsible for the stimulation by forskolin and CREB. Although bromocriptine alone did not alter the basal promoter activity of the PrRPR gene, it significantly reduced the forskolin and CREB-stimulated promoter activity specifically in the same promoter region. These findings suggested that the basal cAMP level in NB41A3 cells may not be sufficient to stimulate the PrRPR gene, and once stimulated by cAMP, the promoter activity of the PrRPR gene was reversed to the basal level by bromocriptine. The findings with mutant CREB (S133A) suggested that the residue 133 serine of CREB was partially important for the basal activation of the PrRPR gene but was essential for the inhibitory event of bromocriptine. In addition, Ca++ ion channel blocker and MAPKK inhibitor did not affect bromocriptine action. Therefore, although other signal transduction pathways such as MAPK and inhibition of phosphatidyl inositol turnover, and intracellular calcium concentrations have also been reported to be involved in the D2 receptor action (19, 20, 21, 22, 37, 38), the present findings strongly suggested the direct involvement of the cAMP signal transduction pathway in the inhibition of the PrRPR gene by bromocriptine.
From these findings, it was expected that CREB directly bound the region -697 and -596 bp of the PrRPR gene. However, unexpectedly, the EMSA study revealed no binding of CREB to this region, indicating that the transcriptional regulation of the PrRPR gene by bromocriptine appeared to require CREB, but was independent of direct binding of CREB to the gene. The absence of direct binding of CREB to the functionally responsible region raised the possibility that 1) bromocriptine indirectly regulated the PrRPR gene through other cAMP and CREB responsive genes or 2) the protein and protein interaction of CREB and an unknown factor bound to the -697
-633 region. The subsequent detailed EMSA and biotin/streptavidin affinity studies demonstrated that an unknown factor, approximately 60 kDa in size, extracted from NB41A3 cells significantly bound this region. Importantly, the treatment of bromocriptine led to a significant reduction in the amount of this factor bound to the above region. Further mutation analyses demonstrated that the specific sequence -663
-672 was important both for binding to this unknown protein and for the inhibition of the promoter activity by bromocriptine. Cloning of this unknown protein is currently in progress using a yeast-one hybrid system in our laboratory.
In conclusion, we demonstrated, for the first time, not only the complete structure and the promoter activity of the human PrRPR gene, but also a novel function of bromocriptine on the gene. We also indicated the importance of the specific sequence 5'-cccacatcat-3' located -663
-672 on the PrRPR gene for the bromocriptine action and possible involvement of the 60 kDa unknown protein with this event.
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MATERIALS AND METHODS
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Subjects and Materials
As shown in Table 1
, samples included five prolactinomas, five cases of GH-secreting adenomas, three nonfunctioning tumors, and one ACTH-secreting pituitary adenoma. Pituitary adenoma tissues were obtained from patients at the time of transsphenoidal surgery. Informed consent was obtained from each family, and this study was approved by the ethical committee on human research of Gunma University. Poly(A)+ RNAs and total RNA were prepared from each adenoma using the modified acid-phenol methods as described previously (39).
Northern Blot Analysis
Total RNA (10 µg) was extracted from individual pituitary adenomas by a modified acid-phenol method, resolved through a 1.2% formaldehyde agarose gel and transferred onto nylon membranes (GeneScreen Plus, NEN Life Science Products, Boston, MA) as reported previously (39). The membranes were hybridized under high stringency with the human PrRPR cRNA probe. Human PrRPR cDNA was generated by PCR with sets of primers; a sense primer, 5-tttctgacttattttctgggctgccgcc-3 and an antisense primer, 5-aacaccgacacatagacggtgacc-3. This amplified cDNA (PrRPR1) encompassed the region between nucleotides 32 and 589 of the PrRPR cDNA from the translational start site. The PCR product was subcloned into the pGEMT vector (Promega Corp., Madison, WI) and used for generating the cRNA probe labeled with [32P]UTP with digestion of endonuclease and SP6 RNA polymerase. After overnight hybridization, the membrane was washed twice in 2x SSC and 1% SDS at room temperature for 15 min and twice in 0.1x SSC and 1% SDS at 68 C for 60 min, then exposed to x-ray film ( XAR-5, Kodak, Rochester, NY) at -70 C for 16 h. The hybridization bands were quantitatively measured using Adobe Photoshop 4.0 (Adobe Systems Corp., San Jose, CA) and NIH Image (Scion Corp., Frederick, MD). To compare the expression level of PrRPR mRNAs, the level of a prolactinoma with the strongest expression of PrRPR mRNA was set as 100% and the relative expression level was indicated in each patient. A sample from an individual pituitary adenoma was used for each hybridization, and the experiment was repeated at least twice.
Isolation of Human PrRPR Genomic Clones
A human genomic DNA library derived from placental DNA in EMBL3 SP6/T7 (CLONTECH Laboratories, Inc., Palo Alto, CA) was used in this study. Approximately 1 x 106 recombinants were screened using a 32P-labeled human PrRPR1 cDNA. Filter hybridization was performed at 42 C using the previously described method (40, 41). Filters were washed twice at room temperature for 15 min in 2x SSC, twice at 50 C for 15 min in 2x SSC, and 1% SDS. Two genomic clones were isolated and characterized by restriction endonuclease mapping. The restriction digests were subjected to electrophoresis, transferred onto a nylon membrane, and hybridized with the PrRPR1 probe. All hybridized genomic fragments were subcloned into pGEM4Z or 3Z for further restriction analysis and sequenced using a PRISM model 310 autosequencer (PE Applied Biosystems, Tokyo, Japan).
Analysis of the 3' Untranslated Region by 3'RACE
To detect functional polyadenylation signals in the 3' untranslated region of the human PrRPR gene, 3'RACE was performed as described previously (40). Briefly, the first strand was synthesized with 5 µg of total RNA from the PRL producing adenoma and an oligo(deoxythymidine)17+adapter (5'-gactcctgcagacatcgattttttttttttttttt-3'). The first amplification was performed under the conditions described above between the adapter (5'-gactcctgcagacatcga-3', 25 pmol) and a sequence-specific primer (3R1, 5'-ggtgagagtcttgctctttgcttgg-3'). The second round of PCR was performed using 1 µl of the first PCR product with an adapter and a downstream internal primer (3R2, 5'-agtaccatgatattgcttagtccattt-3'). The PCR products were analyzed electrophoretically using 1% agarose gels. Two amplified fragments were subcloned and sequenced as described above.
5'RACE
To obtain the 5' portion of the human PrRPR cDNA, 5'RACE was performed using a human pituitary Marathon Ready cDNA (CLONTECH Laboratories, Inc.). Amplification products were subjected to Southern blot analysis with a fragment containing exon 1 of the gene as a probe, and an amplified fragment was gel purified, subcloned into the pGEMT-Easy plasmid (Promega Corp.) and sequenced as described above.
Primer Extension Analysis
Primer extension was carried out using two synthetic oligonucleotides, PE-1, 5'-tcgcgggaagaaggggcagaattt-3' and PE-2, 5'-ggccctcggtagtcct ctgcccacg-3'. The oligonucleotides were end-labeled with [
-32P]ATP, hybridized to 5 µg of the poly(A)+ RNA extracted from the human pituitary prolactinoma and extended using AMV reverse transcriptase. Forty micrograms of yeast transfer RNA were used as a negative control. The primer-extended products were separated on an 8 M urea 6% polyacrylamide gel. The gel was then dried and exposed to Kodak XAR-5 film. The sizes of the resulting labeled primer-extended products were inferred from their comigration with a sequencing ladder, which was obtained using the same primer with an exon 1 containing clone.
Cell Culture and Transfection
GH4C1, CV-1, HTB-10, HeLa, and NB41A3 cells were cultured in DMEM supplemented with 10% (vol/vol) FBS, penicillin (100 U/ml), streptomycin (100 µg/ml) (Life Technologies, Inc.), and Amphotericin (0.25 µg/ml) (Sigma, St. Louis, MO). Cells were plated 24 h before transfection into 30 mm six-well plates at subconfluent density. Transient transfection was performed by the calcium phosphate precipitation method with 3 µg of reporter construct. Glycerol shock was performed 16 h after transfection (except for 4 h for HTB-10 cells) for 2 min with 15% glycerol in PBS. The cells were then harvested after a further 24 h. For estimation of the effect of bromocriptine and forskolin, 1 µM bromocriptine diluted in ethanol and 10 µM forskolin in ethanol were added after glycerol shock.
Plasmid Construction
The pGL3 basic vector (Promega Corp.) is a promoterless luciferase expression vector. The pGLTK vector contains the herpes simplex virus TK promoter sequence linked to pGL3 basic and was used to monitor transfection efficiency in each cell line and as an internal standard between different experiments. The human PrRPR gene fragment containing 697 bp of the promoter region, 133 bp of exon 1 and 2 bp of intron 1 was subcloned into the pGL3 basic vector and named -697 + 133Luc. The other constructs and mutants of the PrRPR promoter encoded the region between the indicated numbers (i.e. -218 + 76Luc) were generated by deletion of the 5' and 3' fragments from the plasmid -697 + 133Luc or by PCR with the same plasmid as a template. These constructs were transfected into above cell lines. The same amount of expression vector (CREB or CREB S133A) was cotransfected with reporter plasmid. The human CREB or the mutant S133A, which exchanged serine at 133 to alanine, was generated by PCR and pKCR2 expression vector (42).
Luciferase Assay
To determine the luciferase (Luc) activity, cell monolayers were rinsed twice with PBS, then lysed with 400 µl 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol, and 1% vol/vol Triton X-100 (39). Cells were scraped from the dishes and centrifuged at 12,000 x g for 5 min at 4 C. Assays for Luc activity were performed using 100 µl aliquots of cell lysate and 350 µl of 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO4, 4 mM EGTA, 16 mM KPO4, 1 mM dithiothreitol, and 2 mM ATP. The reaction was initiated by addition of 200 µl of 0.2 mM d-luciferin and light emission was measured for 10 sec using a luminometer. Luc activity was expressed as ALU per microgram of cellular protein.
EMSA
The EMSA study was performed on nuclear extracts from NB41A3 cells and radiolabeled CRE, AP-1, or SP1 fragments or a series of mutants of the PrRPR gene, as described previously (43, 44). The consensus sequences used as CRE, AP-1 and SP1, were 5'-agagattgcctgacgtcagagagctag-3'(SC-2504), 5'-cgcttgatgactcagccggaa-3'(SC-2501) and 5'-attcgatcggggcggggcgagc-3'(SC-2502), respectively (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The preparation of nuclear extracts from NB41A3 cells was performed as described previously (45). Double-stranded oligonucleotides were labeled with [
32P]deoxy-CTP by a fill-in reaction using a klenow fragment of DNA polymerase I or by PCR. The binding reaction, gel electrophoreses and autoradiographies were performed under conditions described previously (45). Gel supershift studies were performed with specific antibodies against c-Fos (sc-253), CREB-1 (C-21. sc-186) or Sp1 (PEP2, sc-59) (Santa Cruz Biotechnology, Inc.).
Biotin/Streptavidin Affinity System and SDS-PAGE
Nuclear extracts (300 µg) from NB41A3 cells were mixed with 500 pmol of biotinlated oligonucleotides encoding the indicated region of the PrRPR gene in binding buffer [50 mM Tris-Cl (pH 7.9), 12.5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 20% Glycerol and 10 µg poly(deoxyinosine-deoxycytidine)] at room temperature for 1 h. After this incubation, 100 µl of streptavidin-agarose (Life Technologies, Inc.) was added and incubated at 4 C for an additional hour. Streptavidin-agarose resin was precipitated by centrifugation, washed intensively with binding buffer, resolved on an 8% SDS-polyacrylamide gel and was stained with coomassie brilliant blue.
Statistical Analysis
Statistical analysis was performed by ANOVA and t test or the Duncans multiple range test.
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ACKNOWLEDGMENTS
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FOOTNOTES
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The nucleotides sequences reported in this study will appear in the DDBJ, and GenBank/EMBL Data Bank with accession numbers AB048946.
Abbreviations: ALU, Arbitrary light units; AP-1, activator protein 1; CREB, cAMP response element binding protein; D2, dopamine 2; Pit 1, pituitary-specific transcription factor; PRrP, PRL-releasing peptide; PRrPR, PRrP receptor; RACE, rapid amplification of cDNA ends; Sp1, specificity protein; TSS, transcription start site.
Received for publication September 13, 2001.
Accepted for publication December 7, 2001.
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