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Original Studies |
Institute of Endocrine Sciences, University of Milan, Istituto Auxologico Italiano IRCCS (L.P., L.A.), and Ospedale Maggiore IRCCS (A.L., R.R., G.M., A.S.), 20145 Milan, Italy; Cattedra di Endocrinologia, Università di Catanzaro (S.F.), 88100 Catanzaro, Italy; and Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University (M.C.), Stanford, California 94305
Address all correspondence and requests for reprints to: Luca Persani, M.D., Ph.D., Laboratorio di Ricerche Endocrinologiche, Istituto Auxologico Italiano IRCCS, Via Ariosto 13, 20145 Milan, Italy. E-mail: persani{at}auxologico.it
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in
autonomous thyroid adenomas (ATAs). Because steady state cAMP results
from production by adenylyl cyclase and degradation by
phosphodiesterases (PDEs), we evaluated PDE activity and expression in
ATAs with wild-type and mutant TSHR and Gs. Activating
mutations of TSHR and Gs were identified in 7 and 1 of
18 ATAs, respectively. No difference was observed in the cAMP content
in ATAs with or without activating mutants. In the surrounding normal
thyroid tissue (NTs), PDE activity was 80% isobutylmethylxanthine
sensitive, with the major contribution by PDE1 and a minor contribution
by PDE4. No differences were observed in PDE activities between NTs and
ATAs with wild-type TSHR and Gs. In contrast, in the
presence of mutant TSHRs or Gs, total PDE activity was
higher. This increase was primarily due to PDE4 induction (917 ±
116% over NTs), associated with a minor PDE1 increase only in ATAs
with mutant TSHR. By RT-PCR, increments of PDE4D and 4C messenger
ribonucleic acids were found in the ATAs with mutant TSHR or
Gs, whereas messenger ribonucleic acids encoding other
cAMP-specific PDEs were not significantly increased. This study
provides a characterization of the PDEs expressed in human thyroid and
demonstrates a dramatic PDE4 induction in the ATAs bearing mutant TSHR
or Gs genes. The increase in cAMP-degrading activity may
represent a marker of constitutive adenylyl cyclase activation and
constitutes an intracellular feedback mechanism with significant impact
on the phenotypic expression of the activating mutations. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies using in vitro experimental systems have provided
evidence that some PDE isoforms are regulated by hormones or other
stimuli via changes in intracellular cyclic nucleotide levels. In
particular, increases in cAMP levels in FRTL-5 cells after TSH
treatment result in short-term activation of cAMP-specific PDEs (such
as PDE4) via protein kinase A-induced phosphorylation and in long-term
activation via gene expression and protein synthesis regulation
(9, 10, 11). An increase in PDE cAMP-degrading activity also has recently
been demonstrated in FRTL-5 cells expressing a constitutively active
Gs protein (12, 13). This finding opens the
possibility that the increased cAMP hydrolysis caused by PDE
activation, if occurring in human thyroid cells, may have a great
impact on the in vivo phenotypic expression of natural
mutations that constitutively activate the cAMP pathway.
In human thyroid tissue, the ligand-independent activation of the
adenylyl cyclase may be caused by mutations affecting two elements of
the cAMP pathway, the TSH receptor, or the Gs
protein itself (14, 15). Indeed, somatic mutations in the TSH receptor
gene are a frequent finding in autonomous thyroid adenomas (ATAs)
(16, 17, 18, 19, 20, 21), whereas somatic mutations of the Gs
gene have been identified in a small subset of these tumors (15, 16, 22). However, no phenotypical differences have yet been described
between ATAs with or without the constitutive activation of the cAMP
cascade.
In this study we investigate the pattern of activity and expression of
potentially relevant PDEs in the normal thyroid and in ATAs with and
without gain of function mutations of TSH receptors or
Gs. Moreover, we provide biochemical and
molecular evidence for the induction of specific PDE isoforms in the
presence of constitutive activation of the cAMP cascade induced by
these oncogenes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eighteen ATAs and the surrounding normal thyroid tissue (NT) were collected at surgery and included in the study. All ATAs were diagnosed on the basis of the patients’ clinical hyperthyroid features, suppressed TSH secretion, and borderline high or elevated free thyroid hormone levels, in the absence of antithyroid autoantibodies and in the presence of a single capsulated nodule at ultrasound examination. The diagnosis was confirmed when 99Tc uptake confined to the nodular tissue and suppression of the surrounding parenchyma were seen. After adenomectomy, all tissue was quickly frozen for genetic characterization, determination of cAMP content, and studies of PDE activity and expression. NT obtained from four patients who underwent total thyroidectomy for thyroid carcinoma was also included in the study. Local ethical approval was obtained for all studies.
Analysis of mutations in Gs and TSH
receptor genes
Genomic DNA and total ribonucleic acid (RNA) were obtained from
tissue homogenates by acid guanidine thiocyanate-phenol chloroform
extraction, as previously described (23). Sequence analyses of
Gs and TSH receptor genes were directly
performed by the dideoxynucleotide method and by automatic techniques
(ABI Prism 310, Perkin-Elmer Corp., Norwalk, CT) on PCR
products obtained from adenomatous tissue genomic DNA, or cDNA. The hot
spots of the Gs gene were amplified using
intronic oligonucleotide primers, as previously described (24). The 5th
to 10th exons of the TSH receptor gene were amplified either from
genomic DNA or cDNA, using the following set of oligonucleotide
primers: 5–10U, 5'-TGG ACT TAA AAT GTT CCC T-3'; and 5–10L, 5'-AGG
CAT TCA CAG ATT TTC TCT GGC-3' [annealing temperature (Ta), 50 C];
E9U, 5'-AAG CCA CTG CTG TGC CTT TA-3'; and 1264L, 5'-GGG TTG AAC TCA
TCG GAC TT-3' (Ta, 48 C); 1161U, 5'-CCC AGG AAG AGA CTC TAC AAG-3'; and
1535L, 5'-TGC AAA GAC AGT GAA GAA ACC A-3' (Ta, 58 C); 1409U, CTG GCC
TTT GCG GAT TTC TGC-3'; and 1876L, 5'-TTG TCC CCT GGG TTG TAC TGC G-3'
(Ta, 66 C); 1649U, 5'-AGG CAC GCA TGT GCC ATC ATG-3'; and 2084L, 5'-CCT
CTG GAA GGC CTT GGT GAA-3' (Ta, 64 C); 1876U, CGC AGT ACA ACC CAG GGG
ACA A-3'; and 2355L, 5'-TTC CCC TAC CAT TGT GAG-3' (Ta, 59 C). The
intronic oligonucleotide primers for the amplification of 9th and 10th
exons had been previously reported by others (25). PCR products were
obtained in a 50-µL reaction mixture containing 0.1–1 µg DNA,
0.5–1 U Taq polymerase (Perkin-Elmer Corp./Cetus), 10–20 pmol of each primer, 200 mmol/L deoxy-NTPs,
10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, and 1.5–2.0 mmol/L
MgCl2. Each sequence was checked using either the
upstream or downstream primers on two different PCR products.
cAMP content assay
Frozen tissue (
30 mg) was homogenized on ice using a
glass-Teflon Potter homogenizer in 6% (wt/vol) trichloroacetic acid.
The homogenate was centrifuged at 2000 x g for 15 min
at 4 C, and subsequently the pellet was discarded. The supernatant was
washed four times with 5 vol water-saturated diethyl ether, and the
upper ether layer was discarded after each wash. The remaining aqueous
extracts were evaporated and resuspended in cAMP kit buffer (0.25 mL).
cAMP was measured in nonacetylated homogenate samples, using a
commercial RIA kit (NEN Life Science Products, Boston,
MA). The minimum detection limit was 0.1 nmol/L for nonacetylated
samples; cross-reaction with cGMP was less than 0.001%. The intra- and
interassay coefficients of variations were less than 6% and less than
10%, respectively.
PDE assay
ATAs and the corresponding NTs (30 mg) were homogenized using
a glass-Teflon Potter homogenizer in 20 mmol/L Tris-HCl (pH 8)
containing 5 mmol/L 2-mercaptoethanol, 10 mmol/L NaF, 1 mmol/L
ethylenediamine tetraacetate, 0.2 mmol/L
ethyleneglycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, 2 mmol/L phenylmethylsulfonylfluoride, 0.2 µg/mL leupeptin, 0.7
µg/mL pepstatin, 10 µg/mL aprotinin, 10 µg/mL soybean trypsin
inhibitor, and 7.8 mg/mL benzamidine. Tissue and homogenates were
always kept in ice, and an aliquot (5 µL) of the fresh homogenate was
immediately assayed for PDE activity in the absence or presence of
nonspecific (IBMX, 1 mmol/L) or specific [8-methoxy-IBMX (50 µmol/L)
for calmodulin (CaM)-dependent PDE1; rolipram (10 µmol/L) for
cAMP-specific PDE4] pharmacological inhibitors. The PDE assay was
performed as previously described (12, 26) in 200 µL reaction mixture
containing 40 mmol/L Tris-0.1% BSA, 10 mmol/L
MgCl2, 5 mmol/L 2-mercaptoethanol, 1 µmol/L
cAMP, and 1 µmol/L [3H]cAMP (0.1 µCi).
After incubation at 34 C for 10 min, the reaction was terminated by
adding an equal volume of 40 mmol/L Tris-HCl buffer, pH 7.5, containing
10 mmol/L ethylenediamine tetraacetate followed by heat denaturation
for 1 min. To each reaction tube, 50 µg Crotalus atrox
snake venom were added, and the incubation was continued at 34 C for 15
min. The reaction products were separated by anion exchange
chromatography on AG 1-X8 resin (100–200 mesh; Bio-Rad Laboratories, Inc., Hercules, CA), and the amount of
radiolabeled adenosine collected was quantitated by scintillation
counting. In each case, data were corrected for the amount of protein
present in the extracts. Protein content was measured by the
bicinchinonic assay (Pierce Chemical Co., Rockford, IL).
The contributions of the four different PDE4 enzyme isoforms were
assessed by testing the homogenates after immunoprecipitation with
specific antibodies (K112, nonselective polyclonal Ab for all PDE4
enzymes; AC55, polyclonal Ab against PDE4A isoforms; K118, polyclonal
Ab against PDE4B isoforms; M3S1, monoclonal Ab against PDE4D isoforms);
finally, the amount of PDE4C isoforms in one sample was estimated by
the following formula: K112/PDE4 - (AC55/PDE4A + K118/PDE4B +
M3S1/PDE4D) (9, 27). The antibodies were bound to protein A
(polyclonals) or G (monoclonal antibody) Sepharose beads in PBS-0.05%
BSA, pH 7.4; the suspension was incubated at 4 C in rotation for 90
min. Then, the PDE-Ab-beads complex was washed twice with fresh buffer,
and after resuspension in 40 mmol/L Tris-0.1% BSA, an aliquot (5 µL)
of the pellet was immediately tested for PDE activity.
cAMP-specific PDE messenger RNA (mRNA) expression
The mRNA expression of the four PDE4 genes and the PDE8B gene was studied by means of a semiquantitative RT-PCR method, using amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for the normalization of the data, as previously described (28). Poly(A+) RNA was prepared from ATAs and surrounding NTs using a commercial kit (Amersham Pharmacia Biotech, Piscataway, NJ) and was subjected to deoxyribonuclease 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 from the catalytic domain and are not affected by the various splicing variants of each gene product (3, 4). 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 were: PDE4A: CT0s, 5'-ATC AAT GGC CCA GAT ACC-3'; and CT0as, 5'-ACA GGG ACA GAG GTC TG-3' (Ta, 54 C; PCR product, 518 bp); PDE4B: CT1s, 5'-ATT CTG AAG GAC CTG AGA-3'; and CT2as, 5'-GTG TCT TGA TCA GAA TCG-3' (Ta, 54 C; 404 bp); PDE4C: CT2s, 5'-GCC TGA CAG ATT CCA GTT-3'; and CT2as, 5'-ATT CCT AAG TCC TCT GGT T-3' (Ta, 52 C; 197 bp); PDE4D: CT0s, 5'-TGG TGA GTC AGA CAC GGA-3'; and CT1as, 5'-AGT TTT TGC ACT GTT ACG TG-3' (Ta, 54 C; 215 bp); and PDE8B: S3, 5'-AAC AGC CCA GTC ACA GTA GC-3'; and AS3, 5'-ACC TTT AAG CCC AGA TAA ACC A-3' (Ta, 62 C, 344 bp). Each reaction was preceded by 3 min of denaturation at 94 C, followed by a hot start; amplifications of PDE4A, -4B, and -4C were run for 30 cycles, those of PDE4D, PDE8B, and GAPDH were run for 25, 28, and 23 cycles, respectively (each cycle: 93 C for 1 min, Ta for 45 s; 72 C for 1 min). No product was obtained when the nonretrotranscribed materials were subjected to PCR amplification. The identities of the PCR products were confirmed by direct automatic sequencing analysis. The amount of each amplification product was determined at densitometer analysis (Multianalyst software, Bio-Rad Laboratories, Inc.) on agarose gel stained with ethidium bromide, and the PDE/GAPDH ratios were calculated for each sample.
Statistical analyses
The results are expressed as the mean ± SD. Paired or unpaired two-tailed Student’s t test was used to detect significance between two series of data. P < 0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TSH
receptor genes
Mutations known to cause the constitutive activation of
cAMP-dependent pathway were found in 8 of 18 cases. Heterozygote
mutations in the TSH receptor gene were found in 7 adenomas (cases
1–7): 6 ATAs had previously reported mutations [the single nucleotide
substitution G to A at codon 281 (S281N) in ATA 4; the substitution T
to C at codon 453 (M453T) in ATAs 1, 2, and 6; the substitution A to T
at codon 486 (I486F) in ATAs 3 and 5], whereas 1 (ATA 7) showed a
novel T to A substitution at codon 631 (F631I) in the VI transmembrane
domain (Fig. 1
). In 3 different
experiments, the F631I mutant exhibited a cAMP accumulation 2.0 ±
0.1-fold higher than the wild-type TSH receptor when transfected in
COS7 cells (data not shown). One adenoma was found to harbor the
Gs mutant (ATA 8), with the substitution A to
G at codon 227 (Q227R) in the heterozygote state.
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The cAMP concentrations in the homogenates from normal thyroid
tissue obtained at thyroidectomy for thyroid carcinoma and ATAs were
largely overlapping. Moreover, no significant differences were found
among the cAMP levels in NTs and ATAs regardless of whether they had
mutant TSH receptors or Gs, although slightly
higher cAMP concentrations were seen in the group of ATAs bearing a
mutant TSH receptor [NTs, 3.7 ± 1.2 pmol/mg protein (n =
4); range, 2.3–4.7; ATAs with wild-type TSH receptor or
Gs, 3.7 ± 1.1 (n = 4); range,
2.7–5.1; ATAs with mutant TSH receptor, 5.0 ± 1.6 (n = 4);
range, 3.3–6.9].
PDE activity
The total PDE activity in unfractionated homogenate of the normal thyroid tissue surrounding the ATAs was 43.2 ± 10.2 (±SD) pmol/min·mg protein (range, 27.8–62.0). This activity was mostly IBMX sensitive (31.3 ± 7.6 pmol/min·mg protein; range, 19.2–47.4); in all cases, however, a significant fraction of total PDE activity was IBMX insensitive (28 ± 10%; range, 14–44%). The activity sensitive to 8-methoxy-IBMX (calcium-CaM-dependent PDE1) was 11.4 ± 4.8 pmol/min·mg protein (range, 3.8–18.2), whereas 7.8 ± 3.3 pmol/min·mg protein (range, 4.9–14.8) were rolipram sensitive (cAMP-specific PDE4); PDE1 accounted for 40 ± 22%, and PDE4 accounted for 26 ± 10% of IBMX-sensitive activity, respectively. A similar pattern of PDE activities was found in the normal tissue removed at surgery for thyroid cancer (data not shown).
ATAs lacking activating mutations of cAMP pathway had PDE activities
similar to those of their surrounding NTs (Fig. 2). In the ATAs bearing constitutively
active mutant TSH receptors, the total PDE activities were always
higher than those of surrounding NTs (248 ± 26% over NTs; range,
209–289%). This increase in total PDEs was due to the induction of
both IBMX-sensitive (264 ± 37%; range, 214–327%) and
insensitive PDE activities (205 ± 74%; range, 116–325%). As
far as IBMX-sensitive activity is concerned, in these adenomas we
observed a dramatic induction of rolipram-sensitive PDE4 (917 ±
116% over NTs; range, 745-1085%) that was associated with a
significant increase in PDE1 activity (238 ± 30%; range,
211–292%; Fig. 2). All of these PDE activities were higher than those
observed in the other adenomas without mutant TSH receptors and
Gs. Contrary to observations of the adenomas
bearing the mutant TSH receptors in the homogenate of the ATA bearing
the mutant Gs, the high increase in
IBMX-sensitive PDE activity (293% over NT) was exclusively due to
PDE4; the level of PDE1 was similar to that in the homogenate of the
normal surrounding tissue (ATA vs. NT, 7.5 ± 1.9
vs. 8.7 ± 2.0 pmol/min·mg protein; P
= NS; Fig. 2).
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). The PDE4 activity recovered after
immunoprecipitation with K112, an antibody recognizing all PDE4
isoforms, confirmed the dramatic increase observed in ATAs bearing the
ligand-independent activation of adenylyl cyclase (850% and 466% over
NTs, in cases 1 and 7, respectively). The main contribution of PDE4D
was documented in both cases by immunoprecipitation with a
PDE4D-specific antibody (M3S1; 976% and 523% over NTs), whereas a
residual PDE4C activity could be demonstrated only in the adenomatous
tissue. In these two ATAs, minor increases in PDE4A and PDE4B
activities were also observed (Fig. 3).
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cAMP-specific PDE4 mRNA levels were evaluated by means of
densitometric analysis in five cases with mutant TSH receptors (no.
1–5) and in three cases (no. 9, 12, and 15) with wild-type TSH
receptors and Gs (Fig. 4). The data are expressed
as the ratio (x1000) between the PDE fragments and GADPH. In the case
of the ATAs with normal TSH receptors and Gs,
no significant difference (P = NS) was observed between
NTs and ATAs for all PDE4 mRNA levels (Table 1). In the cases with mutant TSH
receptors, significant differences were observed for 4C and 4D mRNA
levels (Table 1). When the statistical analysis was performed between
the ATAs with or without mutant TSH receptors, significant differences
were found only in the case of PDE4D (P = 0.008).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pattern of PDE activity in the normal thyroid was maintained in the
ATAs expressing wild-type TSH receptors and
Gs, although it was dramatically modified in
the presence of mutations in these genes. Consistent with the high
prevalence of mutations in the TSH receptor gene and with the rarity of
those located in the two hot spots of the Gs
gene (16, 22), 7 of 18 ATAs of the present series expressed mutant TSH
receptors and only 1 mutant Gs. All
substitutions had been previously demonstrated to constitutively
activate the cAMP cascade (16, 17, 18, 19, 20, 21), with the exception of the F631I
mutation, which is a new substitution (Ile for Phe) at a codon already
reported to be affected by other substitutions (Leu or Cys for Phe)
(19). The ligand-independent activity of the F631I mutant was similar
to that reported for previous missense substitutions (29). In ATAs with
mutant Gs or TSH receptors, a 2- to 3-fold
increase in total PDE activity was observed. This increase was due
largely to the dramatic induction of PDE4 activity, with a 10-fold
higher activity than that measured in the homogenates of the
surrounding normal tissue. The increase in PDE activities is in
agreement with data obtained in human GH-secreting pituitary adenomas
(24) or FRTL-5 cells (12) expressing mutant
Gs. Moreover, PDE up-regulation is likely to
have a significant impact on the phenotype of cells expressing these
activating mutations, as the increased cAMP degradation may account for
the large overlap of cAMP content between normal thyroid samples and
the ATAs bearing, or not, the mutant TSH receptor or
Gs. The cAMP-dependent stimulation of PDE4 may
be the result of a series of phosphorylation processes and/or of the
induction of gene transcription (9, 10, 11). Semiquantitative RT-PCR
experiments suggest that the stimulation of cAMPspecific PDE4 is
the consequence of an increased mRNA steady state and protein
synthesis, at least in the case of PDE isoforms encoded by the 4C and
4D genes. The PDE4C mRNAs were almost undetectable in the normal
tissue and in the ATAs with wild-type TSH receptors and
Gs, although the PDE4D transcripts were
constantly abundant in the ATAs with mutant TSH receptors or
Gs and were low in the adenomas not expressing
these mutants. These results are consistent with data obtained by
studying the different isoforms at the protein level. Indeed, in the
ATAs with the constitutive activation of adenylyl cyclase, about 60%
of the PDE4 cAMP-degrading activity was immunoprecipitated with the Ab
specific for the PDE4D isoforms, although the activity attributable to
PDE4C isoforms reached about 20%; minor changes were seen for the
activities immunoprecipitated with the Abs specific for the PDE4A and
-4B isoforms. These results are consistent with the data showing 1) a
marked cAMP inducibility of PDE4D expression and the presence of
cAMP-responsive elements in the promoter region of PDE4D gene (10, 30),
and 2) a modest cAMP effect on the promoter activity of PDE4A and -4B
genes in different cell systems (30, 31, 32). Furthermore, our data
indicate a marked cAMP-dependent inducibility of PDE4C expression in
human thyroid cells.
In addition to the induction of PDE4, our data show that ATAs
expressing mutant TSH receptors are associated with a significant
stimulation of 8-methoxy-IBMX-inhibited, CaM-dependent PDE1 activity,
consistent with the constitutive activation of phospholipase C caused
by the mutant receptor. Interestingly, in the homogenate of the ATA
carrying mutant Gs, no induction of PDE1
activity was observed, consistent with a poor phospholipase Cß
stimulation by the mutant Gs. Although not
detected in the functional studies of M453T or S281N TSH receptors
expressed in COS-7 cells (20, 21), the ability to stimulate the
inositol phosphate cascade has been observed in the case of other
mutant TSH receptors (19), including I486F (16). In the present study
the stimulation of CaM-dependent PDE1 was similar among the ATAs
bearing these different mutant receptors (data not shown). Recently,
the possibility has been put forth that the constitutive activation of
different intracellular pathways may contribute to the superior
oncogenic potential of a mutant TSH receptor (33).
This is the first study evaluating the expression of the cAMP-specific PDE8B in human normal and adenomatous tissue. This PDE isozyme has been recently described to be IBMX insensitive and to have a thyroid-specific expression (5). Our results indicate that this new PDE may account for about 20% of the total activity present in the homogenates of normal tissue. In the presence of the constitutive activation of the cAMP pathway, a significant increase in IBMX-insensitive PDE activity is observed. As no enhancement of the PDE8B mRNA steady state is detected, we can hypothesize that the PDE8B expression is already maximal in normal thyroid tissue and is only marginally regulated at the transcriptional level by cAMP, whereas the reported increase in IBMX-insensitive PDE activity in the ATAs may be secondary to cAMP-dependent phosphorylation.
Taken all together, the data indicate that the constitutive activation
of the cAMP pathway in autonomous thyroid adenomas is constantly
associated with the up-regulation of rolipram-sensitive PDE activity
(PDE4). This finding would be in accordance with the studies of
GH-secreting adenomas and FRTL-5 cells expressing the mutant
Gs (12, 15). Because the ATAs lacking any
mutation in the TSH receptor or in the hot spots of the
Gs gene are associated with a normal
cAMP-specific PDE4 activity, the biochemical finding of an increased
PDE4 activity may represent a marker of a constitutively active cAMP
pathway.
Furthermore, the enhanced cAMP-degrading PDE activity constitutes a
compensatory mechanism opposing the chronic increase in cAMP production
induced by the activating mutation, thus explaining the large overlap
of values of cAMP content among the normal and adenomatous samples.
There are two possible interpretations of this phenomenon. The
increased cAMP degradation by PDEs could represent an intracellular
mechanism counteracting the phenotypic expression of these two types of
oncogene. This interpretation supports the hypothesis that in addition
to TSH receptor or Gs mutations, other events
might be required for tumor progression. Alternatively, some
experiments have shown that increases in intracellular cAMP may have
biphasic effect on cell growth, with prolonged and sustained increases
being associated with the arrest of the cell cycle at the
G1-S border (34). It is conceivable that the
enhanced PDE cAMP-degrading activity in the tumors characterized by a
constitutively active adenylyl cyclase may be a necessary event for
tumor progression by determining intracellular cAMP concentrations that
are compatible with the initiation of cell replication.
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Acknowledgments |
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Footnotes |
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Received November 29, 1999.
Revised January 25, 2000.
Accepted February 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Mol Endocrinol. 9:1279–1287.[Abstract]
genes as a cause of toxic thyroid adenomas. J Clin Endocrinol Metab. 82:2695–2701.
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.
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H. Shinozaki, V. Butnev, Y.-X. Tao, K. L. Ang, M. Conti, and D. L. Segaloff Desensitization of Gs-Coupled Receptor Signaling by Constitutively Active Mutants of the Human Lutropin/Choriogonadotropin Receptor J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1194 - 1204. [Abstract] [Full Text] [PDF]
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 M. Conti, W. Richter, C. Mehats, G. Livera, J.-Y. Park, and C. Jin Cyclic AMP-specific PDE4 Phosphodiesterases as Critical Components of Cyclic AMP Signaling J. Biol. Chem., February 14, 2003; 278(8): 5493 - 5496. [Full Text] [PDF]
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L. Alberti, M. C. Proverbio, S. Costagliola, R. Romoli, B. Boldrighini, M. C. Vigone, G. Weber, G. Chiumello, P. Beck-Peccoz, and L. Persani Germline Mutations of TSH Receptor Gene as Cause of Nonautoimmune Subclinical Hypothyroidism J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2549 - 2555. [Abstract] [Full Text] [PDF]
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L. S. Weinstein, S. Yu, D. R. Warner, and J. Liu Endocrine Manifestations of Stimulatory G Protein {alpha}-Subunit Mutations and the Role of Genomic Imprinting Endocr. Rev., October 1, 2001; 22(5): 675 - 705. [Abstract] [Full Text] [PDF]
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L. Persani, S. Borgato, A. Lania, M. Filopanti, G. Mantovani, M. Conti, and A. Spada Relevant cAMP-Specific Phosphodiesterase Isoforms in Human Pituitary: Effect of Gs{alpha} Mutations J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3795 - 3800. [Abstract] [Full Text] [PDF]
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K. Krohn and R. Paschke Progress in Understanding the Etiology of Thyroid Autonomy J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3336 - 3345. [Full Text] [PDF]
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