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Mutations
Institute of Endocrine Sciences, University of Milan, Istituto Auxologico Italiano IRCCS (L.P., S.B.), Milan 20145 and Ospedale Maggiore IRCCS (A.L., M.F., G.M., A.S.), Milan 20122, Italy; and Division of Reproductive Biology (M.C.), Department of Gynecology and Obstetrics, Stanford University, Stanford, California 94305
Address all correspondence and requests for reprints to: Luca Persani, M.D., Ph.D., Lab. di Ricerche Endocrinologiche, Istituto Auxologico Italiano IRCCS, Via Ariosto 13, 20145 Milano, Italy.
Abstract
Both cAMP production by adenylyl cyclase and cAMP degradation by phosphodiesterases account for intracellular cAMP levels. We previously demonstrated an increased phosphodiesterase activity in GH-secreting adenomas bearing the gsp oncogene. Here we characterize both the activity and the expression of cAMP-specific phosphodiesterase genes in the human pituitary and in gsp+ and gsp- GH-secreting adenomas and analyze the impact of this intracellular feedback mechanism on the levels of cAMP-responsive element-binding protein phosphorylation. Normal pituitary and gsp- GH-secreting adenomas showed similar phosphodiesterase activities, and 7-fold higher levels were observed in gsp+ tumors. In these tumors the increased activity was mainly owing to isobutyl-methyl-xanthine-sensitive phosphodiesterase 4 and to isobutyl-methyl-xanthine-insensitive isoforms. By semiquantitative RT-PCR, all phosphodiesterase 4 transcripts were expressed in the normal and tumoral pituitary. However, the levels of phosphodiesterase 4C and 4D messenger RNAs were significantly higher in gsp+ than in gsp- GH-secreting adenomas and normal pituitary. Expression of the thyroid-specific isobutyl-methyl-xanthine-insensitive phosphodiesterase 8B was absent in the normal pituitary but detectable in almost all GH-secreting adenomas and higher in gsp+ (P < 0.02). Therefore, this study provides a characterization of phosphodiesterase expression in human pituitary and demonstrates a dramatic induction of the cAMP-specific phosphodiesterases 4C and phosphodiesterases 4D and phosphodiesterases 8B in gsp+ GH-secreting adenomas. Similar levels of cAMP-responsive element-binding protein phosphorylation were observed in gsp- and gsp+ GH-secreting adenomas; however, phosphodiesterase blockade caused an increase in cAMP-responsive element-binding protein phosphorylation that was significantly higher in gsp+ than in gsp- adenomas. Because cAMP-responsive element-binding protein represents the principal end point of the cAMP pathway, these results suggest that the enhanced phosphodiesterase activity may have a significant impact on the phenotypic expression of gsp mutations.
IT IS WELL established that somatotrophs represent a cell type in which the activation of the cAMP-dependent pathway leads to cell proliferation and differentiation (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Most of the effects of cAMP on gene expression are mediated by the nuclear transcription factor cAMP-responsive element-binding protein (CREB) that binds to the cAMP- responsive element when phosphorylated by the cAMP- dependent protein kinase A. The actual levels of intracellular cAMP depend on the activity of two classes of enzymes, adenylyl cyclases that account for the cyclic nucleotide production and phosphodiesterases (PDEs) that account for its degradation. PDEs are isoenzymes encoded by at least 22 different genes and organized into 11 families, depending on their biochemical and pharmacological properties, such as substrate affinity (cAMP or 3',5'-cyclic guanosine monophosphate) or sensitivity to specific inhibitors (11, 12, 13, 14, 15, 16, 17). The PDE isoforms with highest affinity for cAMP are defined as cAMP specific and may be either isobutyl-methyl-xanthine (IBMX) sensitive, like the PDE4 isoform (11, 12, 13), or IBMX insensitive, like is the recently cloned PDE8B, which is also characterized by a specific thyroid expression (14).
In different cell systems, increases in cAMP levels have been
demonstrated to up-regulate the activity of some PDE isoforms,
particularly the PDE4 (18, 19, 20). In agreement with this
observation, we recently reported increased PDE activity in pituitary
GH-secreting adenomas and autonomous thyroid adenomas characterized by
the presence of the constitutive activation of adenylyl cyclase owing
to gain of function mutations of Gs
or TSH receptor genes (21, 22). Moreover, in autonomous thyroid adenomas with mutant TSH
receptor or Gs genes, high PDE4 messenger RNA (mRNA) levels were
observed, suggesting that in these adenomas the induction of specific
PDE expression constitutes a mechanism opposing the chronic cAMP
increase (22). This observation prompted us to study the
expression of cAMP-specific PDE genes in the human pituitary and in GH-
secreting adenomas with and without mutant Gs and to investigate
the impact of this intracellular feedback mechanism on CREB
phosphorylation that represents the final target of the cAMP-dependent
signaling pathway.
Materials and Methods
Pituitary tissue samples
The study included two pools of normal pituitary samples
obtained by autoptic surgery within 8 h after death (kindly
supplied by Prof. F. Basolo, Pisa, Italy) and 12 GH-secreting adenomas
(GH-omas), surgically removed by the transsphenoidal route from
patients affected with acromegaly. Acromegaly was diagnosed on the
basis of clinical features, elevated IGF-I plasma levels, and elevated
GH levels not suppressible during oral glucose tolerance tests. 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
(23). Part of the tissues was quickly frozen for Gs
gene analysis, PDE activity, cAMP-specific PDE mRNA expression studies,
and Western blot analyses of CREB proteins. Tissues from 4 tumors were
placed in sterile medium for cell culture. Local ethical approval was
obtained for all studies.
Analysis of mutations in Gs gene
Genomic DNA was obtained from tissue homogenates by acid
guanidine thiocyanate-phenol-chloroform extraction, as previously
described (24). Sequence analyses of Gs genes were
directly performed by the dideoxy-nucleotide method and by automatic
techniques (ABI Prism 310, Perkin-Elmer Corp., Norwalk,
CT) on PCR products. The hotspots of Gs gene were amplified using
intronic oligonucleotide primers, as previously described
(25). Of the 12 tumors, 4 were found to harbor mutations
of the Gs gene at codon 201 (CGT > TGT/Arg > Cys) and 1
at codon 227 (CAG > CGG/Gln > Arg) and were defined as
gsp+. Both mutations are known to constitutively activate
adenylyl cyclase. Four tumors (3 gsp- and 1
gsp+) had been included in the study of Reference 21.
PDE assay
PDE activity was performed on tissue homogenates in the absence or presence of nonspecific (IBMX, 1 mM) or specific (8 methoxy-IBMX, 50 µM, for CaM-dependent PDE1; rolipram, 10 µM, for cAMP-specific PDE4) pharmacological inhibitors, as previously described (21, 22). After incubation at 34 C for 10 min, the reaction was terminated by heat denaturation, and 50 µg of Crotalus atrox snake venom was added for 15 min at 34 C. The reaction products were separated by anion-exchange chromatography and the amount of radiolabeled adenosine collected was counted. Data were corrected for the protein amount, measured by the bicinchoninic assay (Pierce Chemical Co., Rockford, IL). PDE activity from 4 tumors was reported in Ref. 21 .
cAMP-specific PDE mRNA expression
The mRNA expression of the four PDE4 genes and of the PDE8B gene was studied by a semiquantitative RT-PCR method using the amplification of the GAPDH gene for normalization of the data, as previously described (22). Poly(A+)RNA was prepared from pituitary tissues by using a commercial kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ), and subjected to DNAase digestion. Specific amplification of the four PDE4 transcripts was obtained by means of primers located at the 3' end of each gene; these regions encode the C-terminal portion of the proteins downstream to the catalytic domain and are not affected by the various splicing variants of each gene product (12, 13). For each cDNA, preliminary experiments with different sets of primers (at least two sets for each cDNA) were conducted to determine the PCR cycles corresponding to the exponential phase. The sequences of the selected oligonucleotides for PDE4A, 4B, 4C, 4D, and PDE8B amplification as well as the specific PCR conditions had been previously reported (22). Results from each RNA sample were confirmed from two distinct retrotranscribed materials, with variations of PDE/GAPDH ratios lower than 16%. No product was obtained when the nonretrotranscribed materials were subjected to PCR amplification. The identity of the PCR products was confirmed by direct automatic sequencing analysis. The amount of each amplification product was determined at densitometer analysis (GS-670, Bio-Rad Laboratories, Inc., Hercules, CA) on agarose gel stained with ethidium bromide, and the PDE/GAPDH ratios were calculated for each sample.
Western blot analysis of CREB proteins
Proteins were obtained by homogenizing the tissues obtained from 12 GH-omas with a glass Teflon Potter homogenizer in 10 mmol HEPES pH 7.3, 150 mmol NaCl, 1 mmol phenyl methyl sulfonyl fluoride, 4 µg/ml pepstatin, and 4 µg/ml aprotinin. The crude nuclear pellets were obtained from total homogenates by centrifugation (1000 rpm for 10 min at 4 C). The whole extraction procedure always lasted less than 15 min. Under these conditions, the possible influence that the addition of phosphatase inhibitors to the extraction buffer may have on the levels of phosphorylated CREB in the nuclear extracts was found to be negligible. Proteins (20 µg) were separated by SDS-PAGE (10%) using a Minigel apparatus (Bio-Rad Laboratories, Inc.) and transferred to nitrocellulose membranes. The filters were then incubated with blocking solution (20 mM Tris, 500 mM NaCl, and 0.2% dry milk) at 4 C overnight (26). The filters were subsequently incubated (at 4 C overnight) in 20 mM Tris-HCl, 150 mM NaCl (pH 7.5), containing the polyclonal antibody recognizing total CREB protein, or the polyclonal antibody specific for the 43-kDa phosphorylated CREB, at 1:300 dilution (Upstate Biotechnologies, Lake Placid, NY). The specificity of the reaction was checked by preincubating the antisera with the specific blocking peptide. The membranes were washed and incubated with goat antirabbit IgG (H+L) antibody conjugated to alkaline phosphatase for 30 min. The membranes were treated with chemiluminescent substrate and enhancer (Immuno-Star chemiluminescent protein detection systems, Bio-Rad Laboratories, Inc.). The bands were determined on x-ray film by scanning densitometry (GS-670, Bio-Rad Laboratories, Inc.).
Cell cultures
Cells, enzymatically dispersed from 4 tumors using trypsin and deoxyribonuclease as previously described (21), were plated at the density of 1 x 106 cells/ml in 6-well plastic cluster dishes and cultured in DMEM supplemented with 10% FCS and antibiotics in an atmosphere of 95% air-5% CO2 in a humidified incubator. After 24 h, culture medium was removed and the cell monolayers were washed twice and preincubated in serum-free medium for 6 h, followed by treatment with or without IBMX (500 µM). After 10 min at 37 C, cells were lysed by the addition of Laemmli’s sample buffer. Equal amounts (20 µg) of whole cell extracts were resolved by SDS-PAGE, followed by Western blotting.
Statistical analyses
The results are expressed as the mean ± SD. A paired or unpaired two-tailed t test was used to detect the significance between two series of data. P < 0.05 was accepted as statistically significant.
Results
PDE activity in normal pituitary and GH-omas with and without gsp oncogene
The total PDE activity measured in normal pituitary samples was
47.2 ± 5.2 pmol/min per milligram protein and was almost
completely blocked by the nonselective PDE inhibitor IBMX. The activity
was equally owing to PDE1 and PDE4 isoforms because 50% of the
activity was inhibited by 8-IBMX, a selective inhibitor of
Ca-Calmodulin-dependent PDE1, and the remaining 50% by rolipram, a
selective inhibitor of the cAMP-specific PDE4 (Fig. 1
).
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gene, the levels
of total PDE activities were similar to those observed in the normal
pituitary (55.5 ± 10 pmol/min per milligram) and were due to both
PDE1 and PDE4 (Fig. 1
). Moreover, in gsp- GH-omas, a
significant fraction of PDE was IBMX insensitive (18.5 ± 11.1
pmol/min per milligram), accounting for 33% of the total activity. In
gsp+ tumors PDE activity, although variable from one tumor
to another, was about 7-fold higher than those measured in
gsp- tumors and in normal pituitary (Fig. 1). No overlap
was seen between PDE activities in this series of gsp- and
gsp+ tumor extracts. The increase in total PDEs was due to
both IBMX-sensitive (265.0 ± 174.7 pmol/min per milligram) and
IBMX-insensitive (100.4 ± 62.8 pmol/min per milligram) PDE
activities. As far as the increase in IBMX-sensitive PDEs is concerned,
this was in large part owing to PDE4 (229.1 ± 138.6 pmol/min per
milligram) because it was almost completely abolished by rolipram (Fig. 1). mRNA expression of cAMP-specific PDEs
The mRNA levels of the cAMP-specific IBMX-sensitive PDE4A, PDE4B,
PDE4C, and PDE4D, and the IBMX-insensitive isoform PDE8B were evaluated
in normal pituitary samples and GH-omas by semiquantitative RT-PCR. A
GAPDH-specific signal was found in all RNA samples indicating that the
failure to detect PDE signals in individual tumors was not due to RNA
degradation (Fig. 2). All transcripts
encoding the four PDE4 isoforms were present in the normal pituitary
(PDE/GAPDH ratios: PDE4A = 125 ± 10; PDE4B = 45 ±
4.7; PDE4C = 114 ± 8.2; and PDE4D = 23 ± 1.8). As
far as PDE expression in GH-omas is concerned, the expression PDE4A and
PDE4B mRNAs was highly variable and independent from the presence of
the gsp oncogene (Fig. 2, Table 1). Conversely, the levels of PDE4C, and
particularly PDE4D, transcripts in gsp+ tumors were
significantly higher than those found in gsp- GH-omas (Fig. 2 and Table 1).
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).
Furthermore, the mRNA levels detected in gsp+ adenomas were
significantly higher than those found in gsp- tumors
(P < 0.02) (Table 1). The PDE8B expression observed in
gsp+ GH-omas was similar to that found in the normal thyroid
(Fig. 3), the only normal tissue known to express this gene
(14).
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CREB proteins were detected by either an anti-CREB serum
recognizing CREB regardless of the phosphorylation state of Ser 133, or
an antibody specific for the phosphorylated form of CREB (P-CREB) on
nuclear extracts from gsp+ and gsp- GH-omas. The
absolute levels of total and phosphorylated CREB were estimated by
densitometer analysis and they did not differ significantly between
gsp+ and gsp- adenomas (total CREB:
gsp+ = 1.67 ± 0.68; gsp- = 1.64 ±
0.81; P-CREB: gsp+ = 1.85 ± 1.18; gsp- =
1.38 ± 1.4). To determine the relative activation of CREB, the
intensity of P-CREB bands were normalized to those of total CREB. Also
in this case, the relative levels of P-CREB in gsp+ tumors
did not differ appreciably from those found in gsp- tumors.
In fact, the ratio of P-CREB to total CREB was 1.11 ± 0.83 (range
0.34–2.38) in gsp+ and 0.84 ± 0.47 (range 0.23–1.48)
in gsp- tumors (P = 0.48) (Fig. 4).
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).
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This study describes the activity and the expression of several cAMP-specific PDE isoforms in normal human pituitary and in GH-secreting adenomas. In the normal human gland, PDE activity was almost completely inhibited by IBMX treatment, with major and similar contributions by the Ca-Calmodulin-dependent PDE1 and the cAMP-specific PDE4. The relative amount of rolipram-sensitive PDE4 in human pituitaries was similar to that reported in mouse pituitaries (26) or pituitary cell lines (27, 28). The pattern of activities observed in normal pituitary was only partially maintained in GH-omas because in both gsp- and gsp+ tumors, a significant component of PDE activity was IBMX insensitive owing to the induced transcription of normally unexpressed PDE isoforms, particularly PDE8B. This study confirms and extends our previous observations of a dramatic increase in the PDE activities, particularly rolipram-sensitive PDE4 and IBMX-insensitive PDEs, in gsp+ GH-omas (21). The increase observed in gsp+ tumors was more sustained than that found in autonomous thyroid adenomas (29).
The increase in cAMP-specific PDE activities may be the result of a
series of phosphorylation processes and/or of the induction of gene
transcription. To determine the expression of genes encoding the
cAMP-specific PDEs, we used a semiquantitative RT-PCR technique. In
principle, other RT-PCR applications, such as competitive PCR, would
give higher accuracy in the measurement of mRNA. However, in the case
of PDEs, as well as in the case of genes with similar characteristics,
the coexistence of various isoforms represents a major problem for
constructing specific competitors. Northern blot analysis would not be
an alternative possible choice owing to the small amount of tumoral
tissue available. By semiquantitative RT-PCR, we demonstrated that the
four PDE4 transcripts were present in the normal pituitary and that
their relative abundance was different in GH-omas, depending on the
presence or absence of gsp mutations. In fact, although
PDE4A and PDE4B mRNAs did not significantly differ between the two
types of tumors, the steady-state of PDE4C and PDE4D transcripts was
significantly enhanced in gsp+ adenomas, thus indicating
that the high PDE4 activity observed in these tumors resulted, at least
in part, from increased expression of these two isoforms. This
different pattern of mRNA expression is consistent with previous
studies carried out in other cell systems indicating a marked cAMP
inducibility of PDE4D and a modest cAMP effect on the promoter activity
of PDE4A and 4B genes (29, 30, 31). Moreover, these data are
in accordance with previous data showing overexpression of PDE4D
transcript in FRTL5 cells expressing gsp mutations and in
human autonomous thyroid adenomas with mutant TSH receptor or Gs
(22, 32).
The induction of PDE isoforms by gsp mutations was not limited to PDE4 but also involved the cAMP-specific PDE8B. In agreement with a previous report (14), no message for PDE8B was found in the normal pituitary; by contrast, this isoform was expressed in almost all GH-omas. The differential expression of PDE8B was consistent with the pattern of IBMX-insensitive PDE activities that was undetectable in the normal pituitary but relevant in GH-omas. Moreover, in gsp+ GH-omas, the expression of PDE8B was increased when compared with those found in gsp- tumors. The present study describes for the first time that PDE8B, whose expression was shown to be confined to thyroid tissue (14), is induced in tumoral somatotrophs. Although it is likely that PDE8B overexpression in gsp+ tumors results from the constitutive activation of cAMP formation, other mechanisms are probably involved because PDE8B was also present in gsp- tumors.
The overexpression of several cAMP-specific PDEs is likely to have a
significant impact on the phenotype of cells with gsp
mutations. This view is strongly supported by the results we obtained
in GH-omas by evaluating the phosphorylation of CREB, the final target
of the cAMP-dependent signaling pathway. By quantifying the relative
levels of Ser-133 phosphorylated CREB, no correlation between CREB
phosphorylation and gsp mutations was found, although it
should be considered that Ser-133 phosphorylated CREB does not
unequivocally represent activated CREB (33). Our results
are in agreement with a previous study in which GH-omas were
demonstrated to contain levels of phosphorylated CREB that, although
higher than those detected in nonfunctioning pituitary adenomas, did
not correlate with the presence of mutant Gs (34). In
that study, the lack of difference in Ser-133 phosphorylated CREB
between gsp+ and gsp- tumors was attributed to
the overexpression of wild-type Gs protein in gsp-
tumors, mimicking the activated phenotype caused by mutant Gs in
gsp+ tumors. Although we were unable to confirm Gs
protein overexpression in gsp- tumors (35),
the present study provides evidence for a different mechanism
responsible for the large overlap of P-CREB values among
gsp+ and gsp- tumors. In fact, the observation
that the blockade of endogenous PDEs in gsp+ tumors resulted
in a dramatic increase in P-CREB strongly supports the view that
up-regulation of PDEs is effective in limiting the cAMP-induced
activation of this nuclear regulatory protein in tumors with
gsp mutations. Indeed, gsp- tumors may harbor
somatic alterations of other elements of this pathway downstream to
cAMP intracellular concentrations, such as protein kinase A R1A
mutations in Carney Complex (36), that could also lead to
increased P-CREB values.
To the best of our knowledge, this is the first report describing the
pattern of expression of cAMP-specific PDE genes in normal and tumoral
human pituitary. Overexpression of PDE4 and PDE8 isoforms in GH-omas
with gsp mutations as well as CREB phosphorylation induced
by PDE blockade in these tumors support the view that up-regulation of
PDEs is a mechanism counteracting the constitutive activation of cAMP
production with a significant impact on the phenotypic expression of
gsp mutations. In particular, the manifestations that may be
influenced by the PDE up- regulation include the rates of GH
secretion or tumor growth. Because a similar overexpression of
cAMP-specific PDEs occurs in autonomous thyroid adenomas with mutations
of TSH receptor or Gs (22), PDE overexpression is
likely to constitute a general feedback mechanism opposing the chronic
increase of cAMP in endocrine disorders.
Acknowledgments
We acknowledge Miss E. Giammona for her skillful technical support. We are indebted to Prof. F. Basolo (Pisa, Italy) for the supply of autoptic pituitary samples.
Footnotes
This work was supported in part by NIH Grant HD-20788 (to M.C.) and by the Ricerca Corrente Funds of Istituto Auxologico Italiano IRCCS, Milano, Italy (to L.P.) and by MURST (Rome, Italy) Grant 9806261488.004.
Abbreviations: CREB, cAMP-responsive element-binding protein; GH-oma, GH-secreting adenoma; IBMX, isobutyl-methyl-xanthine; mRNA, messenger RNA; PDE, phosphodiesterase.
Received December 27, 2000.
Accepted April 26, 2001.
References
is associated with an increased phosphodiesterase activity in human
growth hormone secreting adenomas. J Clin Endocrinol Metab 83:1624–1628 gene are associated with low levels of
Gs protein in growth hormone-secreting tumors. J Clin Endocrinol Metab 83:1624–1628
. Mol Endocrinol 9:1279–1287[Abstract]
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