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Original Studies |
Gene Are Associated with Low Levels of Gs Protein in Growth Hormone-Secreting Tumors1
Institute of Endocrine Sciences, Ospedale Maggiore IRCCS, Italian Auxologic Center IRCCS (A.M.D.B.), and the Department of Pharmacology, CNR Center of Cytopharmacology, Scientific Institute San Raffaele, University of Milan (L.V.), Milan, Italy
Address all correspondence and requests for reprints to: Anna Spada, M.D., Istituto di Scienze Endocrine Ospedale Maggiore, IRCCS, Via Francesco Sforza 35, 20122 Milan, Italy. E-mail: endosci{at}imiucca.csi unimi.it.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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content and activation of the adenylyl
cyclase system. Data on Gs levels in endocrine tumors
that depend on cAMP for growth, particularly pituitary adenomas, are
still limited. The levels of Gs protein were evaluated
in 11 GH-secreting adenomas with Gs mutations
(gsp+) and 15 without (gsp).
Complementary DNAs from gsp+ tumors
contained very low amounts of wild-type Gs sequences,
indicating a preponderance of the mutant Gs transcripts
in these tumors. Immunoblotting of Gs protein showed
that the two isoforms were present at high levels in all
gsp-, but were undetectable or barely
detectable in gsp+. The low
Gs content in gsp+ tumors was
not due to a reduction in ribonucleic acid synthesis or stability, as
Gs messenger ribonucleic acid levels were similar in
wild-type and mutant tissues. Treatment of
gsp- cells with cholera toxin caused a
marked reduction of Gs levels. As in other cell systems
cholera toxin increases Gs degradation, our data are
consistent with an accelerated removal of mutant Gs.
This may represent an additional mechanism of feedback response to the
constitutive activation of cAMP signaling in pituitary tumors with
mutations in the Gs gene. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit of the stimulatory G protein
(Gs) have reproducibly been found in these
neoplasia (2, 3). Although mutations of the menin gene, recently
localized in 11q13, probably have little role in pituitary
tumorigenesis (4, 5), Gs is a key element for
the activation of the cAMP-dependent pathway that in pituitary cells is
a signal for differentiation and proliferation (6). Point mutations
that have been first identified in GH-secreting adenomas occur at two
specific sites in the Gs gene, codon 201 or
227, the common effect of both being to constitutively activate
adenylyl cyclase by impairing the intrinsic guanosine triphosphatase
activity of the subunit (2, 7). The same gain of function mutations
have been subsequently identified in a subset of toxic thyroid adenomas
and differentiated thyroid adenocarcinomas (8, 9), consistent with the
hypothesis that in selected cell types Gs gene
may be converted into an oncogene, designated gsp for
Gs protein (2, 7, 10, 11, 12, 13).
Expression of Gs can vary over a wide range in
human tissues, and several lines of evidence suggest the existence of a
direct relationship between cellular Gs
content and activation of the adenylyl cyclase system (14). However,
data on Gs expression in endocrine tumors that
depend on cAMP for growth are still limited and controversial.
Increased levels of Gs have been found in
tumoral thyroid samples compared with those in normal tissue; the
overexpression especially involves the mutant
Gs or is independent of the presence of
mutations according to various reports (15, 16, 17). Whereas it has been
recently suggested that high Gs levels may be
sufficient to stimulate phosphorylation of cAMP response
element-binding protein in GH-secreting adenomas (18), the
Gs content in pituitary tumors with and
without activating Gs mutations has been
poorly investigated to date. The present study shows extremely low
amounts, if any, of Gs in GH-secreting
adenomas carrying Gs mutations and
investigates the molecular mechanisms underlying this phenomenon.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The study was carried out on 26 GH-secreting tumors surgically
removed by the transsphenoidal route from patients affected with active
acromegaly. Acromegaly was diagnosed on the basis of clinical features,
elevated insulin-like growth factor I plasma levels and elevated GH
levels not suppressible during oral glucose tolerance test. No patient
had previously undergone pituitary irradiation. Patients did not
receive drugs known to influence G protein expression, in particular
ethanol, opiates, and lithium. Small adenoma fragments were fixed for
light and electron microscopy to check the adenomatous nature of the
material, as previously described (19). The remaining tissue was
quickly frozen for Gs gene analysis, adenylyl
cyclase activity, and immunoblotting. For cell cultures adenomatous
tissues were placed in sterile medium until enzymatic digestion. Local
ethical approval was obtained for all studies.
Analysis of mutations in Gs gene
DNA and ribonucleic acid (RNA) were obtained from tissue
homogenates by acid guanidine thiocyanate-phenol-chloroform extraction
using commercial kits (Tri-Reagent, Molecular Research Center, Inc., Cincinnati, OH). Analysis of mutations was carried out as
previously described (20). Briefly, 100 ng DNA were amplified in a
50-µL reaction mixture containing 2 U Taq polymerase
(Perkin-Elmer/Cetus, Norwalk, CT), 0.5 µmol/L of each
primer, 0.2 mmol/L 2-deoxy-nucleoside-5'-triphosphate, 10 mmol/L
Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.001%
(wt/vol) gelatin [40 cycles of 94 C for 1 min, 56 C (for codon 201 of
the Gs gene) or 54 C (for codon 227 of the
Gs gene) for 1 min, and 72 C for 1 min]. The
oligonucleotides used to amplify codon 201 of the
Gs gene were 5'-CCAAACTACTCCAGACCTTT-3' and
5'-TGGAAGTTGACTTTGTCCAC-3'; the oligonucleotides used to amplify codon
227 of the Gs gene were
5'-ACAGAGATCATGGTTTCTTG-3' and 5'-TTAACCAAAGAGAGCAAAGC-3'. Amplified
fragments were purified and directly sequenced using the Thermo
Sequenase radiolabeled terminator cycle sequencing kit (Amersham,
Aylesbury, UK).
RT-PCR
Total RNA (500 ng) was reverse transcribed at 42 C for 1 h
in the following reaction conditions: 1 mmol/L of each deoxynucleoside
triphosphate, 1 U RNAsin, 100 pmol random hexamers, and 200 U reverse
transcriptase enzyme. The mixture was then heated at 95 C for 5 min and
quick chilled on ice. PCR was performed on the entire complementary DNA
(cDNA) product with Taq DNA polymerase and specific
oligonucleotide primers. Primer sequences used for amplification of
human Gs cDNA were 5'-CATGGGCTGCCTCGGGAA-3'
and 5'-TTAGAGCAGCTCGTACTGAC-3'. PCR conditions were as follow: 94 C for
1 min, 54 C for 1 min, and 72 C for 2 min for 40 cycles. Amplification
of the housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was performed in all samples studied to verify the integrity of
the ribonucleic acid, as previously described (21). An additional
control was obtained by omitting the RT step to detect the presence of
any contaminating genomic DNA. PCR products were visualized on a 4%
agarose gel stained with ethidium bromide. Amplified fragments were
purified and directly sequenced using the Thermo Sequenase radiolabeled
terminator cycle sequencing kit (Amersham).
Semiquantitative RT-PCR
Levels of Gs RNA transcripts were
evaluated by semiquantitative PCR performed using the GAPDH gene as an
internal standard. For each cDNA, preliminary experiments were
conducted to determine the PCR cycles corresponding to the exponential
phase. Therefore, amplifications of GAPDH and
Gs sequences were performed for 23 and 21
cycles, respectively. Oligonucleotide primers were 5'-end labeled with
[
-32P]ATP. PCR products were visualized on a 10%
polyacrylamide gel. DNA bands were cut off the gel, and radioactivity
was counted in a ß-counter. Ratios of radioactivity detected in
Gs DNA fragments and in their correspondent
GAPDH DNA fragments were calculated.
Immunoblotting of Gs protein
Immunoblotting analysis was performed on pituitary tissues as
previously described (22). Briefly, tissues were homogenized by a
glass-Teflon Potter homogenizer in 10 mmol HEPES (pH 7.3), 150 mmol
NaCl, 1 mmol phenylmethylsulfonylfluoride, 4 µg/mL pepstatin, and 4
µg/mL aprotinin. Total homogenates were centrifuged at 3,000 x
g for 15 min at 4 C, and supernatants were centrifuged at
40,000 x g for 30 min at 4 C. In some experiments the
supernatant fractions were used. Crude membranes were resuspended in
solubilization buffer, and protein concentrations were determined using
the bicinchoninic acid protein assay. Membrane proteins (20 µg) were
separated by 10% SDS-PAGE, electroblotted to nitrocellulose, probed
with specific polyclonal antibodies to the G protein -subunits at
1:250 dilution for 3 h at room temperature, followed by
125I-labeled protein A (4–5 x 105
cpm/mL), and finally subjected to autoradiography with X-Omat x-ray
film (Eastman Kodak Co., Rochester, NY). The regions of
the blot corresponding to the bands and equivalent sized areas not
containing immunoreactive proteins were excised, and radioactivity was
quantitated by -counter (Packard A5550, Downers Grove, IL). In some
experiments detection of Gs was achieved by
using donkey horseradish peroxidase-linked antirabbit IgG followed by
densitometer analysis. Two commercial antibodies were used to detect
Gs: one directed against a peptide
corresponding to residues RMHLRQYELL near the C-terminus of
Gs and the other directed against a peptide
corresponding to amino acids 100–119 mapping within the amino-terminal
domain. Antibodies directed against the C-termini of
Gi1/2, Gi3,
Gq/11, and Gß and the N-terminal sequence of
Go were used to detect the different subunits. The
specificity of the reaction was evaluated as previously described
(22).
Cell culture
Tumor fragments were enzymatically dissociated using trypsin and deoxyribonuclease as previously described (23). The cell suspensions obtained largely consisted of single cells with a viability, as assessed by trypan blue exclusion, greater than 90%. The dispersed cells were plated at a density of 2.5 x 105 cell/mL in plastic dishes and cultured in DMEM supplemented with 10% FCS and antibiotics at 37 C in an atmosphere of 95% air-5% CO2 in a humidified incubator. After 24 h, medium was removed by aspiration, and the cell monolayers were washed twice and incubated with cholera toxin (1 µg/mL for 8 h) or forskolin (10 µmol/L for 1 h) in Hanks’ Balanced Salt Solution, 500 µmol/L 3-isobutyl-1-methylxanthine (IBMX) and 0.1% BSA. At the end of incubation, cells were lysed in solubilization buffer and homogenized for immunoblotting as described above.
Adenylyl cyclase assay
The adenylyl cyclase assay was carried out as previously described on crude membrane preparations sedimented from tumor homogenates by centrifugation at 40,000 x g for 30 min. The assay mixture contained 25 mmol/L Tris-HCl (pH 7.4), 500 µmol/L IBMX, 1 µmol/L GTP, 0.2 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 2 mmol/L ATP, 7 mmol/L phosphocreatine, and creatine phosphokinase (20 U/mL). The reaction was initiated by the addition of membranes (0.5 mg protein/mL), and incubation proceeded at 30 C for 8 min. The estimation of the amount of cAMP formed was previously described (24).
Materials
Culture medium, leupeptin, pepstatin, aprotinin,
phenylmethylsulfonylfluoride, trypsin, soybean trypsin inhibitor,
donkey horseradish peroxidase-linked antirabbit IgG, IBMX, forskolin,
and cholera toxin were purchased from Sigma Chemical Co.
(St. Louis, MO). The bicinchoninic acid protein assay was purchased
from Pierce (Rockford, IL). Nitrocellulose membrane and
mol wt standards were obtained from Bio-Rad Laboratories
(Hercules, CA). Polyclonal rabbit anti-G protein -subunit antibodies
and [125I]protein A were purchased from New England
Nuclear-DuPont (Boston, MA). An additional
anti-Gs serum (amino acids 100–119) and
control Gs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). X-Omat x-ray film was
obtained from Eastman Kodak Co. Antirabbit Ig.
biotinylated species-specific whole antibody, and streptavidin Texas
Red were obtained from Amersham. All other chemicals were reagent
grade.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene
Genomic DNAs from 26 GH-secreting adenomas were analyzed for
mutations in codons 201 and 227. Eleven tumors (42%) were found to
harbor point mutations of the Gs gene as
follows: nine at codon 201 (CGT>TGT/R201C in eight and
CGT>AGT/R201S in one) and two at codon 227 (CAG>CGG/Q227R) and were
defined as gsp+. All mutations reported here are
known to constitutively activate adenylyl cyclase. Indeed, adenylyl
cyclase activity was 102 ± 19 pmol cAMP/mg protein·min in
gsp+ vs. 10.5 ± 2.0 in
gsp- tumors (P < 0.001).
Although genomic DNAs from gsp+ tumors showed
both mutant and wild-type alleles, cDNAs obtained by reverse
transcribing RNA from the four tumors tested contained very low
amounts, if any, of wild-type Gs sequences,
indicating a preponderance of the mutant Gs
transcripts in these tumors (Fig. 1
and
data not shown).
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in
wild-type and mutant GH-secreting adenomas
Figure 2 summarizes the data of
immunoblots obtained with antiserum to Gs in
gsp- and gsp+ adenomas.
In the two groups of tumors the levels of Gs
expression were markedly different. In particular, the two isoforms of
Gs arising from alternative splicing of
messenger RNA (mRNA) transcripts were expressed at high levels in all
gsp- adenomas (Fig. 3). In contrast,
Gs was undetectable in blots obtained from
eight gsp+ tumors and barely detectable in the
remaining three; the reduction was independent from the location of the
mutation (Fig. 3). A similar reduction in Gs
was observed in three gsp+ tumors using a
different antiserum raised against a peptide corresponding to amino
acids 100–119 mapping within the amino-terminal domain (data not
shown). With the exception of two microadenomas, all tumors were tested
at least twice, giving similar results. Data were confirmed by
visualizing the reaction with peroxidase-linked antirabbit IgG followed
by densitometer analysis (data not shown). Moreover,
Gs was undetectable or detectable at very low
levels in the supernatant fractions resulting from the second
centrifugation of gsp+ tumors (data not
shown).
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. In fact, probing with the antiserum
specific for the common carboxyl-terminal sequence of
Gq and G11 detected a single
band of approximately 44 kDa that was expressed at high levels in both
gsp- and gsp+ tumors
(Fig. 4). Similarly, using antiserum
specific for the ß-subunit common to G proteins, all tumors showed
high amounts of this protein (Fig. 4). As far as the levels of the G
proteins of the Gi family are concerned,
Gi1/2 and Gi3 were detected at very low
levels and Go was found in significant amounts in both
gsp- and gsp+ tumors
(Fig. 4 and data not shown).
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mRNA levels in
wild-type and mutant GH-secreting adenomas
As the amounts of tumoral tissue available were too limited to
perform Northern blot analysis, we employed semiquantitative RT-PCR to
determine levels of Gs mRNA in GH-secreting
adenomas (seven gsp- and six
gsp+ tumors). Despite the large individual
variations, the mean Gs mRNA levels were
similar in wild-type and mutant tissues (target/standard ratio,
0.202 ± 0.150 in gsp- vs.
0.144 ± 0.094 in gsp+; P =
NS), indicating that the low levels of Gs
protein in gsp+ tumors were not due to a
reduction in Gs transcripts.
Modification of Gs levels in cultured
cells from GH-secreting adenomas
Cells obtained from GH-secreting adenomas were treated with
cholera toxin, a toxin known to mimic gsp mutations by
covalently modifying Arg201. Treatment of cells obtained
from three gsp- tumors with cholera toxin (1
µg/mL) for 8 h caused a marked reduction
inGs levels, as assessed by immunoblotting
analysis (Fig. 5).
Gs levels observed after the treatment were
similar to those observed in untreated cells obtained from
gsp+ tumors. In contrast, incubation with
forskolin (1 µmol/L) did not reduce Gs
levels, thus ruling out cAMP as a mediator of the toxin-induced
decrease in immunoreactive Gs (data not
shown). None of these treatments modified Gs
levels in cells obtained from two gsp+ tumors
(data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene are
characterized by extremely low levels of the mutant protein, thus
confirming our previous preliminary data obtained in individual tumors
(13). In the present series heterozygous gain of function mutations of
the Gs gene were detected in about a third of
GH-secreting adenomas, as reported in previous studies (2, 7, 25, 26);
the substitution CGT
TGT at codon 201 was the most frequently
observed. By analogy with most dominant oncogenes and in agreement with
previous reports (2), the large majority of the
Gs transcript originated from the mutant
allele. In fact, although the analysis of tumor genomic DNA revealed
equal amounts of mutant and wild-type Gs
sequences, cDNA amplified by PCR from the same tumors almost
exclusively showed mutant Gs.
Tumors with and without Gs mutations were
markedly different in their content of Gs
protein. All gsp- tumors showed high levels of
the two Gs isoforms arising from alternative
mRNA splicing that, although variable among the different tumors, were
similar to those observed in prolactinomas and nonfunctioning adenomas
and in the normal pituitary (22) (our unpublished observations).
Conversely, in gsp+ tumors
Gs protein was undetectable or, in a minority
of tumors, barely detectable; this pattern was in contrast with the
overexpression that characterizes the common oncogenes. The reduction
in Gs content was a general phenomenon, being
present in all gsp+ tumors independently from
the location of the mutation, and a selective event, as the other G
proteins were present in similar amounts in gsp+
and gsp- tumors. The reduction in
Gs levels obtained with an antiserum directed
against a sequence in the C-terminus of Gs was
confirmed using a different antiserum recognizing amino acids within
the amino-terminal domain. The parallel results with two antibodies
directed against spatially separated portions of
Gs polypeptide make it unlikely that mutations
at codons 201 and 227 somehow prevented the antibodies from detecting
the mutant protein. Although these antibodies did not discriminate
between wild-type and mutant proteins, the reduction of
Gs in gsp+ tumors
probably affected the mutant protein because these tumors mainly
contained mutant Gs transcripts.
The biological mechanisms responsible for the low amount of mutant
Gs protein in gsp+
tumors were further investigated. Using RT-PCR analysis to monitor
endogenous Gs steady state mRNA levels, no
significant difference in Gs mRNA levels
between gsp+ and gsp-
tumors was detected, thus ruling out that reduced mRNA synthesis or
stability might be responsible for the low expression of
Gs in gsp+ tumors.
Experiments were carried out by treating gsp-
tumors with cholera toxin, a toxin known to mimic gsp
mutations by covalently modifying Arg201 and to
constitutively activate adenylyl cyclase. The toxin was able to
dramatically reduce Gs protein levels; the
resulting phenotype was superimposable to that characterizing
gsp+ tumors. These data are in agreement with
previous studies showing that several hours of exposure to cholera
toxin leads to a progressive Gs disappearance
from GH3 pituitary cells and S49 lymphoma cells (27, 28).
Similarly, low levels of Gs were observed in
S49 cyc- cells expressing mutant
Gs (R201C) (28). In these experimental models
a conformational change that loosens Gs
attachment to membranes and increases its degradation rate has been
proposed to occur. Accelerated degradation of mutant
Gs in pituitary adenomas is consistent with
the lack of difference in Gs mRNA levels
between gsp- and gsp+
tumors and with the absence of detectable Gs
in the cytosolic fractions from gsp+ tumors.
Taking into account that both cholera toxin and gsp
mutations activate adenylyl cyclase by inhibiting
Gs guanosine triphosphatase and thereby
preventing the formation of the inactive ß complex, it is
conceivable that in the activated free state, -subunit is highly
susceptible to degradation (27, 28).
The low amounts of Gs found in pituitary tumors is
in contrast with previous reports showing that thyroid toxic adenomas
and papillary and follicular carcinomas bearing
Gs mutation at codon 201 contain high amounts
of the corresponding protein (15, 16). As it seems unlikely that the
rates of degradation of mutant Gs protein may
be different in thyroid and pituitary tissues, it is possible to
hypothesize that thyroid tumors contain both wild-type and mutant
transcripts, and that the immunoreactive protein found in these tumors
is the stable wild-type Gs. However, this
hypothesis needs to be supported by experimental evidence.
As observed in cells treated with cholera toxin,
gsp+ tumors showed high adenylyl cyclase
activity, suggesting that the concentrations of
Gs are substantially higher than that required
for activation of adenylyl cyclase. However, several lines of evidence
support the idea that the amount of available Gs
is important for stimulation of cAMP synthesis. In fact, in cells
transfected with cDNA for Gs there is a close
relationship between Gs levels and adenylyl
cyclase activation (14). Moreover, it is well known that heterozygous
loss of function mutations of Gs causes the
resistance to PTH and other hormones that characterizes
pseudohypoparathyroidism type I, indicating that
Gs limits the maximal activity of adenylyl
cyclase (29).
Several mechanisms able to contrast the consequence of the constitutive
activation of the cAMP-dependent pathway have been recently identified
in gsp+ GH-secreting adenomas. In particular, it
has been proposed that the high sensitivity to the inhibitory action of
somatostatin and the increased degradation of cAMP caused by
phosphodiesterase overactivity may be involved in determining the
in vivo phenotype of gsp mutations (12, 20, 26).
The low content of mutant Gs, probably due to
its accelerated removal, may represent an additional mechanism of
feedback response to the constitutive activation of cAMP signaling in
pituitary tumors carrying gain of function mutations of
Gs gene.
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Acknowledgments |
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Footnotes |
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Received July 29, 1998.
Revised September 2, 1998.
Accepted September 12, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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enhances activation of the adenylyl cyclase
signal transduction cascade. Mol Endocrinol. 11:1053–1061. subunit
in tumors bearing a mutated Gs gene. J Cancer Res Clin
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protein-adenylate
cyclase pathway may not be sufficient to generate toxic thyroid
adenomas. J Clin Endocrinol Metab. 81:1898–1904.[Abstract]
is
associated with an increased phosphodiesterase activity in human growth
hormone-secreting adenomas. J Clin Endocrinol Metab. 83:1624–1628.1-Adrenergic stimulation of in vitro growth
hormone release and cytosolic free Ca2+ in rat
somatotrophs. Endocrinology. 122:1419–1425.[Abstract]
mutations in somatotroph adenomas. J Clin Endocrinol Metab. 83:1604–1610.
subunit of Gs in intact cells alters its abundance,
rate of degradation and membrane avidity. J Cell Biol. 119:1297–1305.This article has been cited by other articles:
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