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Original Studies |
Is Associated with an Increased Phosphodiesterase Activity in Human Growth Hormone-Secreting Adenomas1
Institute of Endocrine Sciences, Ospedale Maggiore IRCCS (A.L., E.B., S.M., A.S.), Italian Auxological Institute IRCCS (L.P.), and Department of Neurosurgery, Scientific Institute San Raffaele (M.L.), University of Milan, 20122 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 Milano, Italy. E-mail: endosci{at}imiucca
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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gene
that occur in human GH-secreting adenomas are associated with increased
PDE activity. We studied 10 adenomas with wild-type Gs
(gsp-) and 8 with mutant Gs (gsp+).
Although, in the absence of PDE inhibitors, intracellular cAMP levels
were similar in gsp+ e gsp- adenomas,
the PDE blockade with 3-isobutyl-1-methylxanthine induced a marked
increase in cAMP in all but one gsp+ adenoma (%
increase: from 77 to 2900) and a slight rise in only 2
gsp-. Similar results were obtained with the PDE4
selective inhibitor
4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone. In
vitro GH release was significantly higher in
gsp+ than in gsp- adenomas (315 ±
158 vs. 82 ± 53 µg/well; P
< 0.01), and PDE blockade caused a further increase in 3 of 5
gsp+ adenomas but not in gsp- tumors. By
direct measurement, PDE activity was about 7-fold higher in
gsp+ than in gsp- adenomas (320 ±
213 vs. 48 ± 23 pmol/min·mg protein;
P < 0.05) and was largely
4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone sensitive.
This study first demonstrates that activating mutations of the Gs
gene that naturally occur in pituitary adenomas is associated with an
increased PDE activity that might, at least partially, counteract the
constitutive activation of the cAMP-dependent pathway. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, or Gs-coupled receptor genes, in
human hyperfunctioning tissues strongly suggests a proliferative action
of cAMP in selected cell types, such as somatotrophs and thyrocytes
(1, 2, 3, 4, 5, 6, 7, 8). Intracellular cAMP levels depend on the activity of adenylyl
cyclase and cyclic nucleotide phosphodiesterase (PDE), a given cAMP
concentration being the result of a steady state of synthesis and
degradation. Inactivation of cyclic nucleotides is catalyzed by a large
number of different PDE isoenzymes, belonging to at least seven
families (9, 10).
The PDE activity is finely regulated by the modification of
intracellular cAMP levels. In particular, increased cAMP levels induce
a short-term activation of cAMP specific-PDEs (PDE4) via a series of
phosphorylation processes and a long-term activation via gene
expression and protein synthesis regulation (9, 10, 11, 12). Moreover, it has
been recently reported that the feedback mechanism by which cAMP
controls the expression of its own degrading enzymes is overactive in
the presence of the cAMP pathway that is abnormally stimulated (13). In
fact, the PDE-mediated cAMP hydrolysis was demonstrated to be
dramatically increased in FRTL-5 cells expressing a mutated Gs,
conferring constitutive activation to adenylyl cyclase (13).
It is widely accepted that about 30% of human GH-secreting pituitary
adenomas carry gain of function mutations of the Gs gene that cause
constitutive activation of adenylyl cyclase (3, 4, 14, 15, 16, 17). Because the
cAMP pathway is involved in somatotroph replication (18), it has been
proposed that the Gs gene may be converted into an oncogene,
designated gsp (for Gs protein), in selected cell types (4, 6). The in vitro phenotype of somatotrophs that express
these mutations is consistent with the constitutive activation of Gs
protein, because it is characterized by high adenylyl cyclase activity
that is not further stimulated by any agents, resulting in in
vitro GH hypersecretion (3). In contrast, no significant
differences in the in vivo phenotype of acromegalic
patients, bearing adenomas with or without Gs mutations, have been
reported so far, suggesting the existence of regulatory mechanisms able
to counteract the effects of cAMP activation in these tumors (14, 15, 16, 17).
The aim of the study was to investigate whether the activation of the
cAMP-dependent pathway caused by Gs mutations is associated with an
increase in PDE activity in human GH-secreting adenomas.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eighteen tumors, surgically removed by the transsphenoidal route
from patients affected with acromegaly, were included in this study
(Table 1
). 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. Small adenoma fragments were fixed, for light and electron
microscopy, to check the adenomatous nature of the material, as
previously described (19). Part of the tissue was quickly frozen for
Gs gene analysis and PDE activity assay, and part was placed in
sterile culture medium for cell culture. Local ethical approval was
obtained for all studies.
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gene
DNA was obtained from tissue homogenates by acid guanidine
thiocyanate-phenol-chloroform extraction (20). After DNA extraction,
gene amplifications were performed by PCR technique. 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 at 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'.
DNA sequencing was carried out using the thermo sequenase radiolabeled terminator cycle sequencing kit (Amersham, Little Chalfont, UK).
Cell culture
Cells were enzymatically dispersed using trypsin and deoxyribonuclease, as previously described (21). The suspension obtained consisted largely 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 x 105 cells/mL, in 24-well plastic cluster 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 3–4 days, culture medium was removed by aspiration, and the cell monolayers were washed twice and preincubated for 1 h with HBSS supplemented with 0.1% BSA. Cells were then incubated for 1 h in fresh medium, in the absence or presence of either 500 µmol/L 3-isobutyl-1-methylxanthine (IBMX) or 50 µmol/L 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) at 37 C, in triplicate. At the end of the incubation, the medium was removed and stored at -20 C until GH assay, while cell monolayers were treated for intracellular cAMP extraction and measurement. GH was measured in cell culture supernatants, by an immunofluorimetric assay using a commercial kit (Pharmacia, Turku, Finland).
Intracellular cAMP assay
Intracellular cAMP levels were measured as previously described (22). Briefly, after the removal of the incubation medium, cells were washed with HBSS and 0.1% BSA, and then 1 mL ice-cold ethanol (80%) was added. After remaining at -20 C overnight, samples were harvested, transferred to polyethylene tubes, and centrifuged at 180 x g for 5 min at 4 C. The supernatants were then evaporated to dryness and redissolved in cAMP kit buffer (0.5 mL). cAMP was measured without acetylation, using an enzyme immunoassay commercial kit (Amersham). The minimum detection limit was approximately 0.12 pmol/mL for nonacetylated samples; cross-reaction with cGMP was less than 0.001%. The intra- and interassay coefficients of variation were <7% and <10%, respectively.
PDE activity assay
For PDE activity assay, the adenoma tissues were frozen immediately after surgery and stored up to 10 days at -80 C. Tissue homogenization was performed using a glass-Teflon Potter homogenizer in a buffer containing 20 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA, 0.2 mmol/L EGTA, 10 mmol/L sodium pyrophosphate, 50 mmol/L benzamidine, 0.5 µg/mL leupeptin, 0.7 µg/mL pepstatin, 4 µg/mL aprotinin, 10 µg/mL soybean trypsin inhibitor, and 2 mmol/L phenylmethylsulfonylfluoride. PDE assay was carried out on membrane preparations sedimented from the homogenates by centrifugation at 40,000 x g for 30 min and resuspended in the buffer. In some experiments, both the pellet and the supernatant were tested for PDE activity, giving similar results (data not shown). Protein measurement was performed using the bicinchoninic protein assay. PDE assay was carried out using 1 µmol/L cAMP as substrate, according to the method of Thompson and Appleman (23). Samples were assayed in a total vol of 200 µL reaction mixture containing 40 mmol/L Tris-HCl (pH 8.0), 10 mmol/L MgCl2, 1.25 mmol/L 2-mercaptoethanol, 0.15 mg BSA, 1 µmol/L unlabeled cAMP, and 0.1 µCi [3H]cAMP. Triplicate samples were incubated at 34 C for 10 min, and the reaction was stopped by heat denaturation for 1 min at 100 C. To convert AMP to adenosine, 50 µg Crotalus atrox snake venom were added to each sample, and the reaction was carried out for 15 min at 34 C. The formed adenosine was separated by Dowex resin and an aliquot of unbound [3H] adenosine was added to scintillation fluid for counting. The inclusion of ethanol in the resin did not modify PDE activity values. The intra- and interassay coefficients of variation were <7% and <15%, respectively. For the inhibitor studies, samples were incubated in the presence of rolipram or IBMX at the maximal effective concentrations.
Reagents
Culture media, Crotalus atrox snake venom, leupeptin, pepstatin, aprotinin, trypsin, soybean trypsin inhibitor, and IBMX were purchased from Sigma (St. Louis, MO). [2,8-3H] cAMP (20–50 Ci/mmol) was purchased from DuPont NEN (Boston, MA). Dowex 1 x 8 resin was purchased from Fluka (Buchs, Switzerland), and rolipram was purchased from ICN Pharmaceuticals (Costa Mesa, CA). TRI-REAGENT was from Molecular Research Center (Cincinnati, OH). All other chemicals were reagent grade.
Statistical analyses
The results are expressed as the mean ± SD. Paired or unpaired two-tailed Student’s t test was used to detect the significance between two series of data. P < 0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene
Eighteen GH-secreting adenomas were submitted to mutation
analysis. Eight tumors (35.7%) were found to harbor point mutations of
Gs gene as follows: 6 at codon 201 (CGT>TGT/Arg>Cys in tumors no.
11, 12, 14, 17, and 18; and CGT>AGT/Arg>Ser in no. 13) and 2 at codon
227 (CAG>CGG/Gln>Arg in tumors no. 15 and 16) and were defined as
gsp+. All mutations reported here are known to
constitutively activate adenylyl cyclase. No significant difference in
tumor size and serum GH levels (13. 6 ± 9.2 µg/L in gsp-
vs. 16.7 ± 13.5 in gsp+; P, not
significant) between patients, with or without Gs mutations, was
observed.
Effect of PDE inhibitors on intracellular cAMP levels
Despite the presence of activating mutations of the Gs gene,
cells obtained from seven of eight gsp+ tumors showed basal
intracellular cAMP levels similar to those obtained from tumors with
wild-type Gs (gsp-) (6.5 ± 7.3 pmol/well in
gsp+ vs. 3.5 ± 3.7 pmol/well in
gsp-; P, not significant), whereas only 1
gsp+ adenoma (no. 16) had very high basal cAMP levels
(22.1 ± 2.0 pmol/well) (Fig. 1).
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). In contrast, in
gsp+ tumors, the blockade of PDE activity resulted in a
marked stimulation of intracellular cAMP accumulation, the increase
induced by IBMX ranging from 77–2900%. Similar results were obtained
by incubating cells with rolipram (Fig. 1b). Interestingly, the only
gsp+ tumor with very high cAMP levels in basal conditions
showed a poor response to PDE inhibition (22% increase by IBMX and
17% by rolipram) (Fig. 1b). Effect of PDE inhibitors on in vitro GH release
The effect of PDE inhibitors on in vitro GH release was
evaluated in 11 adenomas (6 gsp- and 5 gsp+).
Contrary to results observed when measuring intracellular cAMP levels,
in vitro GH release from cells expressing mutant Gs was
markedly higher than that from cells with wild-type Gs (315 ±
158 vs. 82 ± 53 µg/well; P < 0.01),
in the absence of PDE inhibitors. As far as the PDE blockade was
concerned, neither IBMX nor rolipram was effective in increasing GH
release in gsp- tumors (Fig. 2, and data not shown). In contrast, in 3
of 5 gsp+ tumors, the incubation with both PDE inhibitors
induced a significant increase in GH levels (Fig. 2).
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PDE activity was measured in 6 gsp- and 5
gsp+ tumors. The enzyme activity, though variable from one
tumor to another, was about 7-fold higher in gsp+ than in
gsp- GH-secreting adenomas (320 ± 213 vs.
48 ± 23 pmol/min·mg protein; P < 0.05) (Fig. 3). Because of the small size of
pituitary adenomatous tissues, it was not possible to measure PDE
activity in all tumors (in particular, tumor no. 16, which was the only
gsp+ tumor with cAMP levels poorly affected by IBMX). To
further characterize the properties of the PDE activated in cells
expressing Gs mutations, the PDE activity was measured in the
presence of the nonselective PDE inhibitor (IBMX) and the
PDE4-selective inhibitor (rolipram). The increase in basal PDE activity
that was observed in gsp+ tumors was caused, in large part,
by rolipram-sensitive PDE isoforms, given that 64% of the activity was
abolished by rolipram (Fig. 4). In
contrast, in gsp- cells, only 28% of the PDE activity was
inhibited by the PDE4-selective inhibitor (Fig. 4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene, naturally occurring in human GH-secreting pituitary
adenomas, are associated with increased PDE activity. Both the
modifications of cAMP levels induced by PDE inhibitors in intact cells
and the direct measurement of the enzyme activity in tumoral tissues
point to a marked difference of tumors, with and without Gs
mutations, in the rate of cAMP degradation. In fact, inhibition of PDE
activity resulted in a marked increase in cAMP accumulation in cells
from gsp+ tumors, whereas the effect was little, if any, in
gsp- cells. These data were consistent with the existence
of high PDE activity in cells with constitutive activation of the
cAMP-dependent pathway, that was confirmed by direct measurement.
Although the small size of pituitary adenomatous tissues made PDE assay
feasible only in a limited number of tumors, the PDE activity measured
in gsp+ tumors was about 7-fold higher than that observed in
wild-type tumors. Because the difference between these tumor subtypes
is the presence of constitutive activation of adenylyl cyclase, it is
most likely that increased PDE activity in gsp+ tumors is
dependent on increased cAMP production, suggesting that, in these
tumors, the feedback loop by which the activation of the cAMP-dependent
pathway increases the rate of cAMP hydrolysis is operating. The lack of
normal human pituitary as control makes it impossible to determine
whether the low PDE activity observed in tumors with wild-type Gs is
related to the tumoral nature of the tissues or is characteristic of
the somatotroph cell type. Moreover, data on the expression of PDE
isoforms in rat pituitary and pituitary cell lines are scarce and
contradictory (24, 25).
In gsp+ tumors, the high PDE activity was caused largely by
increased activity of the cAMP-specific, rolipram-sensitive, cAMP-PDE
(PDE4). In fact, rolipram, a selective PDE4 inhibitor, was nearly as
effective as IBMX, a nonselective PDE inhibitor, in stimulating cAMP
accumulation in intact cells and blocking the enzyme activity in
membrane preparations. The involvement of isoenzymes of the PDE4 family
in the feedback mechanism activated by the cAMP pathway in
gsp+ GH-secreting adenomas is consistent with the pattern of
PDE4 regulation. In fact, the activation of Gs-coupled receptors, as
well as the increase of cAMP levels induced by cholera toxin or
forskolin, seems to selectively increase PDE4 activity (11, 12). From
our study, it is not possible to distinguish whether the increase in
PDE4 activity is caused by an activation or an increased expression of
the enzyme. However, it is reasonable to hypothesize that the long-term
adaptation of somatotrophs that express mutant Gs to the
constitutively activated cAMP-dependent pathway might require both
enzyme activation and increased gene expression.
The increased PDE activity had a major impact on the in
vitro phenotype of somatotrophs expressing mutant Gs. In fact,
although substitutions at either codon 201 or 227 in Gs gene are
known to constitutively stimulate adenylyl cyclase by inhibiting the
intrinsic GTPase activity (17), no difference in cAMP levels was
observed in adenomatous somatotrophs, with and without Gs mutations,
unless PDE inhibitors were added to the cells. In this respect, it is
worth noting that the only gsp+ tumor in which PDE blockade
was ineffective in increasing cAMP accumulation was characterized by
extremely high cAMP levels in basal conditions. Therefore, it is most
likely that the high cAMP levels in gsp+ tumors previously
reported might be caused by the routine inclusion of PDE inhibitors in
the incubation buffer used to study cAMP production (3).
Although the activation of the PDE feedback loop seems to counteract
the ability of Gs mutations to constitutively stimulate cAMP levels,
the phenotype of mutant Gs was characterized by an in
vitro GH release that was definitely higher than that observed in
wild-type tumors. It is possible to hypothesize that small differences
in cAMP levels might result in significant differences in biological
responses, such as GH secretion. These data suggest that mutant Gs
phenotype may be reverted only in part by the increased PDE activity,
and they further strengthen the view that multiple processes contribute
to determine the oncogenic potential of activating mutations of Gs
in the different cell types. Moreover, the observation that (contrary
to the results observed in vitro) serum GH levels in
patients with and without gsp oncogene did not differ
significantly, points to an involvement of central inhibitory
influences in determining the in vivo phenotype.
The data obtained in tumoral somatotrophs are consistent with those
obtained in mammalian cells transfected with mutant Gs (13, 26, 27).
In particular, it has been reported that FRTL-5 thyroid cells
expressing the Gln227Leu mutation of Gs are characterized by an
increased PDE activity, the blockade of PDE4 resulting in a further
stimulation of cAMP levels and cell proliferation (13). Moreover, it
has been reported that Swiss 3T3 fibroblasts expressing mutated Gs
had mitogenic activity only in the presence of PDE4 inhibitors (26). In
contrast, GH3 cells expressing the same mutation showed a high
proliferation rate, even in the absence of PDE blockade (27).
To the best of our knowledge, this is the first demonstration that
mutations of the Gs gene that naturally occur in pituitary tumors
are followed by an increase in PDE activity in vivo.
Although other mechanisms may be involved in determining the in
vivo phenotype of patients bearing gsp+ tumors, such as
the high sensitivity of these patients to the inhibitory action of
somatostatin (6, 14), it is most likely that the increased degradation
of cAMP caused by PDE overactivity may, at least in part, contrast the
consequence of the constitutive activation of the cAMP-dependent
pathway. It remains to be elucidated whether the PDE feedback loop is
also activated in other endocrine disorders caused by mutations
in Gs, such as thyroid toxic adenomas and the McCune-Albright
syndrome (8, 28).
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Acknowledgments |
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Footnotes |
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Received December 23, 1997.
Revised January 23, 1998.
Accepted January 30, 1998.
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References |
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genes as a cause of toxic thyroid adenomas. J
Clin Endocrinol Metab. 82:2695–2701.. Mol
Endocrinol. 9:1279–1287.[Abstract]
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