0021-972X/98/$03.00/0
Journal of Clinical Endocrinology and Metabolism
Copyright © 1998 by The Endocrine Society
Vol. 83, No. 12
Printed in U.S.A.
Growth Hormone Secretagogue Receptor Expression in
Human Pituitary Tumors*
MONICA M. SKINNER, RALF NASS, BEATRIZ LOPES, EDWARD R. LAWS,
MICHAEL O. THORNER
AND
Division of Endocrinology and Metabolism, Department of Medicine (M.M.S., R.N., M.O.T.),
Department of Pathology (B.L.), and Department of Neurosurgery (E.R.L.), University of Virginia
Health Sciences Center, Charlottesville, Virginia 22908
ABSTRACT
The GH secretagogue (GHS) receptor (GHS-R) has been characterized and cloned. It is a member of a family of seven transmembrane
receptors and is closely related to the neurotensin and TRH receptors.
To determine the expression of this receptor in normal anterior pituitary and in 24 human pituitary adenomas, we analyzed GHS-R
messenger ribonucleic acid (mRNA) using a RT-PCR assay. We found
that normal human pituitary was positive for the GHS-R signal. In
addition, all GH-secreting adenomas and the one TSH-secreting adenoma demonstrated the presence of GHS-R mRNA. Three of four
ACTH-secreting tumors and three of nine gonadotroph adenomas
were also positive for the GHS-R mRNA. To determine the amounts
of GHS-R mRNA in normal pituitary and in representative tumors,
T
HE BIOSYNTHESIS and secretion of GH in the anterior
pituitary gland is under complex hormonal regulation.
It is regulated by two hypothalamic hormones, somatostatin
and GHRH, which oppose one another and act through distinct membrane receptors. Somatostatin inhibits GH release
via a family of GTP-binding protein-coupled membrane receptors (1–3), and GHRH activates GH release through a
stimulatory G protein-coupled receptor (4). GH regulates its
own release through negative feedback loops acting at the
level of the hypothalamus via GH receptors in arcuate neurons and the periventricular nucleus (5, 6).
In addition, a class of synthetic molecules, termed GH
secretagogues (GHSs), act at both the pituitary and the hypothalamus to stimulate and amplify pulsatile GH release.
GHSs induce the release of GHRH from arcuate neurons and
act to functionally antagonize somatostatin (7–12). The synthetic hexapeptide GHRP-6 contains d-amino acids and stimulates GH release by a pathway distinct from that of GHRH
(13, 14). The nonpeptide mimetics of GHRP-6, L-692,429 and
MK-0677, have improved pharmacokinetics and oral bioavailability (15). GHSs cause depolarization and inhibition of
potassium channels, induce a transient increase in the concentration of intracellular calcium in somatotrophs, increase
Received June 15, 1998. Revision received August 19, 1998. Accepted
August 31, 1998.
Address all correspondence and requests for reprints to: Dr. Michael
O. Thorner, Department of Medicine, University of Virginia Health
Sciences, Box 466, Charlottesville, Virginia 22908. E-mail: mot@
virginia.edu.
* This work supported in part through a gift from Sal and Francince
Ranieri and a grant from the Deutsche Forschungsgemeinschaft (Na
317/1-1 and Na 317/1-2; to R.N.).
semiquantitative competitive PCR was performed. We determined
that normal pituitary had approximately 750 molecules/L GHS-R
mRNA. The acromegalic tumor had approximately 1.5 3 105 molecules/L, and the TSH-secreting tumor had approximately 7.5 3 103
molecules/L. Other tumor types contained considerably less, with the
ACTH-secreting and gonadotroph tumors expressing 7.5 3 102 and
3 3 102 GHS-R mRNA molecules/L, respectively. These results suggest that GH- and TSH-producing adenomas express GHS-R mRNA
at levels 200 and 10 times higher, respectively, than the normal
pituitary, and that this receptor expression may be involved in the
pathogenesis and growth of these pituitary adenomas. (J Clin Endocrinol Metab 83: 4314 – 4320, 1998)
intracellular concentrations of inositol triphosphate, and increase the activity of protein kinase C (16 –20).
Recently, a receptor was cloned and was shown to be the
target of GHSs (21). Sequence analysis of the GHS receptor
(GHS-R) reveals that it has seven transmembrane domains
and shares only limited sequence homology with other
known G protein-coupled receptors. An initial mapping
study of GHS-R in human tissue revealed its expression in
pituitary, hypothalamus, and hippocampus as well as a weak
signal in pancreas (22). GHS-R is also expressed in human
fetal pituitary tissue (23). Recently, Bennett et al. (24) showed
that hypothalamic GHS-R expression was highly sensitive to
GH, being markedly increased in GH-deficient dw/dw dwarf
rats and decreased in dw/dw rats treated with bovine GH,
suggesting that GHS-R may be involved in feedback regulation of GH.
Characterization of the GHS receptor provides evidence
for the presence of an endocrine pathway distinct from that
described for GHRH and somatostatin that contributes to the
control of GH release. Thus, it is important to determine its
expression in different pituitary cell types. X-Linked inactivation studies have demonstrated that the vast majority of
pituitary tumors are monoclonal and are comprised solely of
one cell type (25–28), thus offering a unique opportunity to
determine cell type-specific expression of GHS-R as well as
to study its possible effect on tumor proliferation. It is unknown which, if any, factor selectively stimulates the clonal
proliferation of pituitary tumor cells. Alterations in hormone
biosynthesis and secretion as well as abnormal cell surface
receptor gene expression by pituitary adenomas may provide clues to the underlying cellular mechanisms that modulate pituitary cell proliferation.
4314
GH SECRETAGOGUE RECEPTOR
Therefore, we used a semiquantitative approach to determine the amount of GHS-R messenger ribonucleic acid
(mRNA) in different pituitary tumor cell types and in nonadenomatous postmortem pituitary. As the ligands for the
GHS-R behave as amplifiers of GHRH activity and functional
antagonists of somatostatin, the expression of GHRH receptor (GHRH-R) and the somatostatin receptor (SSTR2) was
comparatively analyzed by RT-PCR in these same tumors.
Subjects and Methods
Patients
Pituitary tumors were collected from 24 patients at the time of transsphenoidal resection and were snap-frozen at 270 C until the time of
RNA preparation. These tumors were classified according to their immunohistochemical staining for ACTH, GH, PRL, FSH, LH, and TSH.
Ten patients had tumors positive for GH (AT-1 to AT-10), nine patients
had tumors positive for FSH or LH (NT-1 to NT-9), four patients had
tumors positive for ACTH (CT-1 through CT-4), and one patient had a
TSH-positive tumor (TT-1). The clinical and pathological characteristics
of the patients and the tumors are summarized in Tables 1 and 2. Normal
human postmortem pituitary tissue was obtained frozen from the National Hormone Pituitary Program.
RNA preparation
Total RNA was extracted from the GH- and TSH-secreting tumors
and normal pituitary tissue using the product TriReagent (Molecular
Research Center, Inc., Cincinnati, OH). Polyadenylated [poly(A)1] RNA
was extracted from the nine gonadotroph adenomas and the four
ACTH-secreting tumors using the QuickPrep Micro mRNA purification
kit from Pharmacia Biotech (Piscataway, NJ). Quantitation of the RNA
was performed by spectrophotometry using an optical density of 260
nm. The integrity of the RNA samples was tested using PCR with
primers for the human glyceraldehyde 3-phosphate dehydrogenase
gene (Stratagene, La Jolla, CA). Each sample produced the expected
4315
600-bp fragment when analyzed by agarose gel electrophoresis (data not
shown).
RT-PCR analysis of GHRH-R, GHS-R, and SSTR2 mRNA
For each RT reaction, either 1.25 g total RNA or 62.5 ng poly(A)1 RNA
from each specimen were heated to 65 C for 5 min and then cooled on
ice. The RNA was then incubated with 0.625 g oligo(deoxythymidine)15
primer (Promega Corp., Madison, WI), 2.5 L 10 mmol/L of each of the
four deoxy (d)-NTPs (Promega Corp.), 5 L 100 mmol/L dithiothreitol,
10 L 5 3 reaction buffer (375 mmol/L KCl, 250 mmol/L Tris-HCl, and
15 mmol/L MgCl2), and 500 U Moloney murine leukemia virus
(MMuLV) reverse transcriptase (Life Technologies, Gaithersburg, MD;
total reaction volume, 50 L). RT reactions were carried out at 25 C for
10 min [oligo(deoxythymidine) annealing], followed by a 60-min elongation step at 37 C and MMuLV-RT heat inactivation at 99 C for 5 min.
PCR oligonucleotide primer sets for GHRH-R, GHS-R, and SSTR2
were designed to amplify 350-, 429-, and 892-bp products, respectively.
The following GHRH-R primers were used (59-39): MS7 TCTGAGCCCTTTCCACCTTACCCTGTG and MS8 GCTGAAGTTGGTCATGGTGGCGAAATG. Oligonucleotide primers for human GHS-R were (59-39)
MS11 CTCTGCATGCCCCTGGACCTCGTTCGC and MS12 CTGCCGATGAGACTGTAGAGGACCGTGAGAC. Oligonucleotide primers
for human SSTR2 were (59-39) MS15 GATGATCACCATGGCTGTG and
MS16 CAGGCATGATCCCTCTTC. All amplifications were carried out
by the following method. Ten liters of each reverse transcribed product
were incubated at 95 C for 5 min with 20 pmol each of specific 59- and
39-oligonucleotide primers, water, and a wax bead (Perkin Elmer,
Branchburg, NJ; total reaction volume, 40 L). After cooling to room
temperature for 10 min, 8 L 10 3 reaction buffer (100 mmol/L Tris-HCl,
15 mmol/L MgCl2, and 500 mmol/L KCl, pH 8.3), 2 L 10 mmol/L
deoxy-NTPs, 2.5 U Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), and water to a final volume of 80 L were layered on top
of the wax barrier. The reactions were carried out in a DNA thermal
cycler 480 (Perkin Elmer) for 40 cycles (1 min at 94 C, 1 min at 45 C, and
90 s at 72 C). The PCR products were electrophoresed on 1.0% agarose
gel and visualized under UV light. Control reactions without MMuLV
reverse transcriptase were also performed to exclude genomic DNA
contamination as a source of amplified signal (data not shown).
TABLE 1. Immunospecific staining of surgically excised pituitary macroadenomas
Patient no.
Sex
Age (yr)
FSHb
LHb
a—
Subunit
TSHb
GH
PRL
ACTH
AT-1
AT-2
AT-3
AT-4
AT-5
AT-6
AT-7
AT-8
AT-9
AT-10
M
F
M
F
F
M
F
M
F
M
38
66
46
22
46
37
58
40
34
39
—
—
—
—
—
—
—
—
—
NA
—
—
—
—
—
—
—
—
—
NA
—
111
111
—
—
1
—
1
111
NA
—
—
—
—
—
—
—
—
—
NA
111
111
111
1
1
111
1
1
111
NA
—
11
1
—
—
—
1
—
—
NA
—
—
—
—
—
—
—
—
—
NA
NT-1
NT-2
NT-3
NT-4
NT-5
NT-6
NT-7
NT-8
NT-9
F
F
M
M
M
M
F
M
M
47
21
55
77
NA
50
38
70
75
NA
1
—
11
1
11
111
111
1
NA
1
—
11
11
111
—
—
1
NA
—
—
11
11
11
11
111
11
NA
—
—
—
—
11
—
—
—
NA
—
—
—
—
—
—
—
—
NA
—
—
—
—
—
—
—
—
NA
—
—
—
—
—
—
—
—
CT-1
CT-2
CT-3
CT-4
F
M
F
F
57
61
55
57
—
—
—
—
1
—
—
—
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
111
111
111
111
TT-1
F
58
—
—
1
1
—
—
—
Staining was graded as — (none), 1 (weak and/or focal), 11 (moderate strength and/or a majority of cells), or 111 (strong and/or most of
cells). NA, Not available.
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JCE & M • 1998
Vol 83 • No 12
SKINNER ET AL.
TABLE 2. Serum hormone data of patients with pituitary adenomas
Patient no.
Cortisol
(mg/dL)
AT-1
AT-2
AT-3
AT-4
AT-5
AT-6
AT-7
AT-8
AT-9
AT-10
11.0
14.2
21.0
NT-1
NT-2
NT-3
NT-4
NT-5
NT-6
NT-7
NT-8
NT-9
5.4
7.0
,3.0
39.2
,10.0b
6.0
15.4
TSH
(mIU/mL)
T4
(mg/dL)
FSH
(mIU/mL)
LH
(mIU/mL)
GH
(ng/mL)
IGF-I
(ng/mL)
PRL
(ng/mL)
23.0
28.7
24.0
5.3
4.6
2.9
3.8
0.94
4.2
25.0
13.6
3.2
2.5a
6.7
8.0a
3.4
1.8a
8.3a
27.7
759
685
986
1100
457
977
1023
3.2
17.2
10.0a
34.5
7.0
10.0
5.3
43.0
2.9
2.6
26.3
3.0
737
812
13.7
54.0
3.2
,0.2
29.0
25.0
8.4
2.4
,0.5
0.8
26.0
20.6
,0.5
1.7
194
44
79
11.9
49.5a
46.0
19.0a
7.4
3.0
0.8
0.2
1.8
26.0
1.2
28.0
1.6
10.3
0.6
2.6
19.2
0.5
9.0
1.36
2.73
1.91
1.1
3.6
14.0
0.69
8.4
9.0
8.4
4.7
6.9
5.9
8.2
9.0
1.6
0.64
5.3
6.5
2.6
0.81
1.37
5.5
7.8
8.8
3.7
8.8
3.65
1.3
0.44
3.1
7.0
7.2
7.1
3.9
1.38
0.35
5.6
7.2
4.9
3.8
30.0
29.0
32.0
1.25
0.5
,0.5
1.7
0.3
0.7
11.58
11.5
26.0
47.2
20.4
14.3
CT-1
CT-2
CT-3
CT-4
43.0
34.4b
TT-1
22
a-Subunit
(ng/mL)
T3 uptake
(%)
24.0
,0.3
47
105
99
,0.3
3.4
,0.3
57
210
191
0.4
,0.2
,0.2
9.8
22.1
22.3
60.9
ACTH
(pg/mL)
0.3
1.1
1103
294
109
361
35.4
Cortisol (mg/dl): morning, 5–18; evening, 2–9; TSH (mIU/mL): 0.35–5.50; T4 (mg/dL): 4.5–10.9; T3 uptake (%): 25–35; FSH (mIU/mL): follicular
phase, 2.5–10.2; midcycle peak, 3.4 –33.4; luteal phase, 1.5–9.1; postmenopausal phase, 23–116.3; males, 1.4 –18.1; LH (mIU/mL): follicular
phase, 1.9 –12.5; midcycle peak, 8.7–76.3; luteal phase, 0.5–16.9; postmenopausal, 5–52.3; males, 20 –70 yr, 1.5–9.3; .70 yr, 3.1–34.6; IGF-I
(ng/mL): male, female, 25–39 yr, 114 – 492; 40 –54 yr, 90 –360; .54 yr, 71–290; PRL (ng/mL): males, 2.1–17.7; females, nonpregnant, 2.8 –29.2;
postmenopausal, 1.8 –20.3; a-subunit (ng/mL): ,0.1; postmenopausal, ,3.6; ACTH (pg/mL): 7–51. Note that the diagnosis of acromegaly was
made clinically and by elevated IGF-I levels when GH results during an OGTT were not available.
a
GH (ng/mL) random levels: minimum GH during an oral glucose tolerance test (OGTT).
b
Outside hospital.
Quantitation of the GHS-R using MIMIC PCR
A competitive PCR approach using a nonhomologous internal standard called a PCR MIMIC (Clontech, Palo Alto, CA) was used to analyze
the level of GHS-R mRNA in normal human pituitary tissue and in
human pituitary tumors. A PCR MIMIC consists of a heterologous DNA
fragment with primer templates that are recognized by a pair of genespecific primers. The template mimics the target and is amplified during
PCR.
To construct the 559-bp GHS-R PCR MIMIC, two rounds of PCR
amplification were performed according to the manufacturer’s protocol (Clontech). In the first PCR reaction, two composite primers
were used (59-39): MS13 CTCTGCATGCCCCTGGACCTCGTTCGCCGCAAGTGAAATCTCCTCCG and MS14 CTGCCGATGAGACTGTAGAGGACCGTGAGACATTTGATTCTGGACCATGGC. Each composite primer has the GHS-R gene primer sequence attached to a 20nucleotide stretch of sequences (underlined) designed to hybridize to
opposite strands of the MIMIC DNA fragment (574-bp BamHI/EcoRI
fragment of v-erbB). A dilution of the first PCR reaction was then amplified again using the GHS-R gene-specific primers MS11 and MS12 (see
above). The GHS-R PCR MIMIC was purified using a CHROMA
SPIN1TE-100 column (Clontech), and the yield was calculated according to manufacturer’s instructions. A portion of the concentrated PCR
MIMIC was diluted to 100 attomol/L (equal to 6 3 107 molecules/L)
from which 10-fold serial dilution stock solutions were made. The 10221027 10-fold serial dilutions (equal to 6 3 105 to 6 3 100 molecules/L,
respectively) were used to titrate a constant amount of target complementary DNA (cDNA).
RT reactions were performed using tumor samples that were positive
for GHS-R by RT-PCR. The reactions were performed as described
above, except that 37.5 ng poly(A)1 or 0.75 g total RNA were used in a
scaled reaction with a final volume of 30 L. Two liters of each serial
dilution of the GHS-R PCR MIMIC were added to PCR amplification
reactions containing constant amounts (2 L) of cDNA from normal
pituitary or tumor samples, 5 L 10 3 PCR reaction buffer [100 mmol/L
Tris-HCl (pH 8.3), 500 mmol/L KCl, and 20 mmol/L MgCl2], 1 L 10
mmol/L dNTPs, 1 L 20 mol/L MS11, 1 L 20 mol/L MS12, 2 U Taq DNA
polymerase (Boehringer Mannheim), and water to a final volume of 50
L. Reactions were carried out for 40 cycles (45 s at 94 C, 45 s at 50 C, 90 s
at 72 C). The PCR products were electrophoresed on 1.6% agarose gel,
stained with ethidium bromide, and visualized under UV light.
A 2-fold serial dilution series was made for each sample after determining which 10-fold MIMIC dilution produced PCR MIMIC and target
cDNA template bands of equal intensity. The MIMIC dilution 10-fold
less dilute than the dilution that gave bands of equal intensity was used
to start the 2-fold serial dilutions. PCR amplification reactions were
carried out as described above, except with 2 L of each 2-fold serial
dilution of the GHS-R PCR MIMIC added to constant amounts (2 L) of
cDNA from normal pituitary or tumor samples. By knowing the amount
of GHS-R PCR MIMIC added to the reactions, the amount of GHS-R
target template was determined in each sample.
Results
Expression of GHS-R in normal and neoplastic human
pituitary tissues
The results of the RT-PCR using primers MS11 and MS12
for the GHS-R are shown in Fig. 1. All 10 acromegalic tumors
were positive for the 429-bp GHS-R fragment (Fig. 1A), as
were three of nine gonadotroph adenomas (Fig. 1B, lanes 2,
5, and 10), three of four of the ACTH-secreting tumors (Fig.
1C, lanes 2, 4, and 5), and the TSH-secreting tumor (Fig. 1D).
The normal human pituitary sample was also positive for the
GHS-R signal (Fig. 1E).
GH SECRETAGOGUE RECEPTOR
FIG. 1. GHS-R expression in pituitary tumors. Total RNA or poly(A)1
RNA extracted from pituitary tumors and normal pituitary tissue was
reverse transcribed, and the resulting products were amplified by
PCR with specific oligonucleotide primers for GHS-R. The PCR products were electrophoresed on an agarose gel and stained with
ethidium bromide, as shown above. A, Acromegalic tumors AT-1 to
AT-10 (lanes 2–11) exhibited a RT-PCR product of the anticipated size
(429 bp). B, Gonadotroph tumors NT-1, NT-4, and NT-9 were positive
for the GHS-R product (lanes 2, 5, and 10, respectively). C, ACTHsecreting tumors CT-1, CT-3, and CT-4 were positive for GHS-R (lanes
2, 4, and 5, respectively). D, TSH-secreting tumor TT-1 produced the
429-bp GHS-R PCR product (lane 2). E, Normal pituitary sample
produced the GHS-R product (lane 2). The DNA mol wt markers are,
from top to bottom, 517, 396, and 344 bp in lane 1 of all panels.
Expression of GHRH-R in normal and neoplastic human
pituitary tissues
RT-PCR results for the GHRH-R are shown in Fig. 2. Analysis of the GHRH-R mRNAs with primers MS7 and MS8
demonstrated that the expected 350-bp PCR fragment was
detectable in all 10 acromegalic tumors (Fig. 2A), all 9 gonadotroph adenomas (Fig. 2B), all 4 ACTH-secreting tumors
(Fig. 2C), the TSH-secreting tumor (Fig. 2D), and the normal
pituitary sample (Fig. 2E).
Expression of SSTR2 in normal and neoplastic human
pituitary tissue
Results from the RT-PCR using primers MS15 and MS16
for SSTR2 are shown in Fig. 3. The 10 acromegalic tumor
samples were positive for the 892-bp fragment of SSTR2 (Fig.
3A). Seven of nine gonadotroph adenomas (Fig. 3B, lanes 2,
3, 5–7, 9, and 10), all four ACTH-secreting tumors (Fig. 3C),
and the TSH-secreting tumor (Fig. 3D) were also positive for
SSTR2. The normal pituitary sample was positive for the
892-bp fragment (Fig. 3E).
Quantitation of the GHS-R in normal and neoplastic
human pituitary tissues
Results from the GHS-R competitive PCR using 10-fold
serial dilutions of the GHS-R MIMIC are shown in Fig. 4.
Normal pituitary tissue produced MIMIC and target cDNA
template bands of equal intensity at 6 3 102 GHS-R molecules/L (Fig. 4A, lane 4).
4317
FIG. 2. GHRH-R expression in pituitary adenomas. Total RNA or
poly(A)1 RNA extracted from pituitary tumors and normal pituitary
was reverse transcribed, and the resulting products were amplified by
PCR with specific oligonucleotide primers for GHRH-R. The PCR
products were electrophoresed on an agarose gel and stained with
ethidium bromide, as shown above. A, Acromegalic tumors AT-1 to
AT-10 (lanes 2–11) exhibited a RT-PCR product of the anticipated size
(350 bp). B, Gonadotroph adenomas NT-1 to NT-9 displayed the expected 350-bp product (lanes 2–10). C, ACTH-secreting tumors CT-1
to CT-4 produced the GHRH-R PCR product (lanes 2–5). D, TSHsecreting tumor TT-1 exhibited the expected 350-bp product (lane 2).
E, Normal pituitary produced the 350-bp GHRH-R product (lane 2).
The DNA mol wt markers are, from top to bottom, 396, 344, and 298
bp in lane 1 of all panels.
The acromegalic tumor AT-4 produced MIMIC and target
cDNA template bands of equal intensity at 6 3 104 GHS-R
molecules/L (Fig. 4B, lane 3). The gonadotroph tumor NT-1
had 60 GHS-R molecules/L (Fig. 4C, lane 6). The ACTHsecreting tumor CT-4 produced a band that was of equal
intensity to 6 3 102 GHS-R MIMIC molecules/L (Fig. 4D, lane
5), whereas the TSH-secreting tumor TT-1 had 6 3 103 GHS-R
molecules/L (Fig. 4E, lane 3).
A 2-fold serial dilution series was made for each sample
starting with the MIMIC dilution 10-fold less dilute than the
dilution that gave MIMIC and target cDNA bands of equal
intensity. After running the PCR samples on an agarose gel,
it was determined that 750 MIMIC molecules/L produced a
band equal in intensity to the normal pituitary sample (Fig.
5A, lane 4).
The acromegalic tumor AT-4 had a band of equal intensity
at 1.5 3 105 mol/LIMIC moleculeszL (Fig. 5B, lane 3), and the
gonadotroph tumor NT-1 was equal at 3 3 102 mol/LIMIC
moleculeszL (Fig. 5C, lane 2); 7.5 3 102 molecules/L MIMIC
produced a band equal in intensity to the target cDNA band
for the ACTH-secreting tumor CT-4 (Fig. 5D, lane 4). The
TSH-secreting tumor TT-1 had a band equal to 7.5 3 103
molecules/L (Fig. 5E, lane 4).
Discussion
The majority of human pituitary adenomas are monoclonal neoplasms and originate from a single adenohypophy-
4318
SKINNER ET AL.
FIG. 3. SSTR2 expression in pituitary tumors. Total RNA or poly(A)1
RNA extracted from pituitary tumors and normal pituitary was reverse transcribed, and the resulting products were amplified by PCR
with specific oligonucleotide primers for SSTR2. The PCR products
were electrophoresed on an agarose gel and stained with ethidium
bromide, as shown above. A, Acromegalic tumors AT-1 to AT-10 (lanes
2–11) exhibited a RT-PCR product of the anticipated size (892 bp). B,
Gonadotroph adenomas NT-1, NT-2, NT-4 to NT-6, NT-8, and NT-9
displayed the SSTR2 PCR product (lanes 2, 3, 5–7, 9, and 10, respectively). C, ACTH-secreting tumors CT-1 through CT-4 produced the
expected 892-bp PCR product (lanes 2–5). D, TSH-secreting tumor
TT-1 produced the SSTR2 product (lane 2). E, The normal pituitary
sample displayed the SSTR2 product (lane 2). The DNA mol wt
marker is 1018 bp and is shown in lane 1 in A–E.
seal cell (29). The incidence of pituitary tumors is high, but
only seldom do they become clinically evident or malignant.
There is evidence that many hypothalamic factors exist that
may control the growth of pituitary adenomas (30). For example, tumor shrinkage after administration of dopamine
agonists to prolactinomas (31) or of somatostatin analogs to
somatotroph cell adenomas (32) supports the concept that
hypothalamic hormones and their receptors play a role in
pituitary tumor progression.
Whereas SSTR2 and GHRH-R gene expression have been
demonstrated in pituitary adenomas (33–35), little is known
about the occurrence of the GHS-R in these tumors. As the
natural ligand of this receptor has not yet been found, the
measurement of its receptor provides a mechanism to gather
information about this system. Using RT-PCR and semiquantitative RT-PCR analysis, we present data which show
that GHS-R, GHRH-R, and SSTR2 mRNA are expressed in
normal human pituitary tissue and in a variety of pituitary
tumors, including GH-, TSH-, and ACTH-secreting tumors,
as well as nonfunctioning pituitary tumors. Specifically, a
GHS-R PCR product was detected in all acromegalic tumors,
in three of four ACTH-secreting tumors, and in one third of
the nonfunctioning tumors. As there was only one TSHsecreting tumor, it is not possible to assess the prevalence of
GHS-R mRNA in this tissue type. GHS-R mRNA expression
was 200-fold higher in the somatotroph tumor and 10-fold
higher in the TSH-secreting tumor than that in normal pituitary tissue. The other pituitary tumors (ACTH secreting
JCE & M • 1998
Vol 83 • No 12
FIG. 4. Semiquantitative competitive PCR of GHS-R expression in
normal pituitary and pituitary tumors. A portion of the concentrated
GHS-R MIMIC was diluted to 100 attomol/L (equal to 6 3 107 molecules/L), from which 10-fold serial dilution stock solutions were made
and used to titrate a constant amount of target cDNA. PCR was
performed as indicated in Materials and Methods. The GHS-R product
is 429 bp, and the GHS-R MIMIC product is 559 bp. A, Competitive
PCR using 10-fold serial dilutions of the GHS-R MIMIC sequence
added to cDNA from normal pituitary tissue. B, Competitive PCR of
10-fold serial dilutions of the GHS-R MIMIC added to cDNA from the
acromegalic tumor AT-4. C, Competitive PCR of 10-fold serial dilutions of the GHS-R MIMIC added to cDNA from the gonadotroph
adenoma NT-1. D, Competitive PCR of 10-fold serial dilutions of the
GHS-R MIMIC added to cDNA from the ACTH-secreting tumor CT-4.
E, Competitive PCR of 10-fold serial dilutions of the GHS-R MIMIC
sequence added to cDNA from the TSH-secreting tumor TT-1. Lanes
1– 6 represent GHS-R MIMIC dilutions 100-1025, respectively, in A
and E, whereas lanes 2–7 represent GHS-R MIMIC dilutions 1001025, respectively, in B–D. The DNA mol wt markers are, from top to
bottom, 517, 396, and 344 bp in lane 7 in A and E and in lane 1 of B–D.
and nonfunctioning) showed levels in the same range or
lower than normal pituitary tissue. These results suggest that
the somatotroph cell is the main target cell at the pituitary
level for the natural ligand of the GHS-R, which plays a less
important role in the development of nonsecreting tumors.
The presence of GHS-R mRNA in somatotroph cells suggests that GHSs can act directly on pituitary cells to stimulate
GH release. This result concurs with earlier demonstrations
that the GHSs are able to directly stimulate GH release from
rat primary pituitary cells in vitro (36) and in acromegalic
subjects (37). In addition, Renner et al. (38) showed that GH
secretion was stimulated by the secretagogue GHRP-6 in
100% of 12 different somatotroph adenoma cell cultures,
whereas only a subset of the adenomas responded to GHRH,
TRH, or octreotide. This suggests that GHS-R and its signaling pathway are expressed more consistently in somatotroph adenoma cells than are receptors and signaling pathways for GHRH, TRH, and somatostatin. In a recent report
by Adams et al. (39), GHS-R mRNA was detected in 6 human
pituitary somatotropinomas by RT-PCR, 4 of which hydrolyzed phosphatidylinositol and secreted GH in response to
GHRP-2 in vitro. In addition, 3 prolactinomas expressed
GHS-R, with 2 of the tumors showing significant stimulation
of PRL secretion and phosphatidylinositol hydrolysis in cul-
GH SECRETAGOGUE RECEPTOR
FIG. 5. Two-fold serial dilutions of GHS-R MIMIC in normal pituitary tissue and pituitary adenomas. A, Two-fold serial dilutions were
made from the 1022 10-fold dilution (equal to 6 3 103 molecules/L) of
the GHS-R MIMIC and used in competitive PCR with cDNA from the
normal pituitary sample. B, Competitive PCR of 2-fold serial dilutions
started from the 100 10-fold dilution (equal to 6 3 105 molecules/L) of
the GHS-R MIMIC added to constant amounts of cDNA from the
acromegalic tumor AT-4. C, Competitive PCR of 2-fold serial dilutions
started from the 1023 10-fold dilution (equal to 600 molecules/L) of the
GHS-R MIMIC added to constant amounts of cDNA from the gonadotroph tumor NT-1. D, Competitive PCR of 2-fold serial dilutions
started from the 1022 10-fold dilution (equal to 6 3 103 molecules/L)
of the GHS-R MIMIC added to constant amounts of cDNA from the
ACTH-secreting tumor CT-4. E, Competitive PCR of 2-fold serial
dilutions started from the 1021 10-fold dilution (equal to 6 3 104
molecules/L) of the GHS-R MIMIC added to constant amounts of
cDNA from the TSH-secreting tumor TT-1. Lanes 2–7 represent the
GHS-R MIMIC serial 2-fold dilutions in A–E. The DNA mol wt markers are, from top to bottom, 517, 396, and 344 bp in lane 1 in A–E.
ture. The rat pituitary tumor cell line GH3 was also found to
express GHS-R mRNA, but these cells did not respond to
GHRPs. This group was unable to detect GHS-R mRNA in
8 functionless human pituitary tumors. The RT-PCR results
of the 6 somatotropinomas are consistent with our RT-PCR
data that all somatotroph adenomas are positive for GHS-R.
Our semiquantitative data also demonstrate that the receptor
is expressed at a much higher level in somatotroph adenomas
than in normal pituitary, which consists of a mixture of cell
types. We were, however, able to detect GHS-R in 1 of 3
gonadotroph tumors by RT-PCR. When examined by semiquantitative competitive PCR, the gonadotroph tumor NT-1
had only 300 GHS-R molecules/mL. This is 2.5-fold fewer
than the 750 GHS-R molecules/mL observed in normal pituitary tissue. Perhaps it is the low level of GHS-R in gonadotroph tumors that caused this discrepancy in results. We
also detected GHS-R in 75% of ACTH-secreting tumors and
in 1 TSH-secreting tumor. The ACTH-secreting tumor CT-4
had 750 GHS-R molecules/mL, equal to that in normal pituitary, whereas the TSH-secreting tumor expressed GHS-R
at levels 10-fold higher than normal. These results are consistent with our findings that not only are all somatotroph
adenomas positive for GHS-R, but the receptor is expressed
at a much higher level than in normal pituitary, which consists of a mixture of cell types.
However, the other pituitary tumors (ACTH secreting and
4319
nonfunctioning) showed GHS-R levels in the same range or
lower than those in normal pituitary tissue. The existence of
increased levels of GHS-R in somatotroph adenomas might
explain why patients with acromegaly exhibit major GH
discharges to GHRP despite the fact that high GH levels
decrease GHS-R in the hypothalamus (24), but only some
patients respond similarly to GHRH (40). This suggests a lack
of GHS-R down-regulation in the neoplastic somatotroph
that has been previously described for the GHRH-R (41, 42).
Thapar et al. (42) also demonstrated that overexpression of
the GHRH gene in the pituitary is associated with neoplastic
progression and clinical aggressiveness of somatotroph adenomas. Whether such a potential role exists for the natural
ligand of the GHS-R in the development of somatotroph
adenomas requires further study.
Our observation that GHS-R mRNA is present in 75% of
the ACTH-secreting pituitary tumors is consistent with the
findings of animal and human studies, which showed that
the administration of GH secretagogues increased ACTH
and cortisol levels (43, 44). Ghigo and colleagues (44) have
speculated that 1) the ACTH-releasing activity of GHRP
might be used in differentiating pituitary from ectopic
ACTH-dependent Cushing’s syndrome; and 2) GHRP stimulates at least in part ACTH in Cushing’s disease, independent of CRH-mediated mechanisms. Our results support the
latter conclusion. However, as not all ACTH-secreting adenomas expressed GHS-R mRNA, the utility of GHRP as a
diagnostic tool to differentiate between ectopic and pituitary
tumors is questionable.
Measurement of GHS-R and the assignment of this receptor to different tumor types may lead to the selective use of
a receptor-antagonist as seen with the competitive antagonist
of GHRH, (N-Ac-Tyr1,d-Arg2)GHRH-(1–29)NH2, which suppressed the GH concentration in a patient with acromegaly
and ectopic GHRH secretion (45), and the GHRH antagonists
MZ-4-71 and MZ-5-156, which inhibited elevated GH levels
caused by overproduction of human GHRH in transgenic
mice (46). Therefore, the demonstration of the GHS-R and its
quantification might provide valuable information for future
therapeutic approaches in the medical treatment of somatotroph pituitary adenomas.
The quantitative screening of a large number of pituitary
tumors and correlation with clinical outcome will provide
further information about the involvement of the GHS/
GHS-R system in the development and neoplastic progression of pituitary adenomas.
Acknowledgments
We thank Dr. Susan Kirk for assistance in collecting data and for
discussion of the significance of the observations, and Drs. Alan Dalkin
and Richard Day for advice regarding PCR methodologies.
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