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Original Studies |
-Subunit Secretion1
Departments of Endocrinology (M.K., A.G., G.M.B., J.P.M.), Clinical Biochemistry (R.H.S., J.M.B.), and Morbid Anatomy (J.F.G.), St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London, United Kingdom E1 2AD
Address all correspondence and requests for reprints to: Dr. R. H. Skelly, Clinical Biochemistry, St. Bartholomews and Royal London School of Medicine and Dentistry, Turner Street, London, United Kingdom E1 2AD. E-mail: r.h.skelly{at}mds.qmw.ac.uk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit gene expression. Its expression
has not been examined previously in human pituitary adenomas
characterized by in vitro hormone secretory profiles. Of
the 34 pituitary adenomas studied, Ptx-1 expression was reduced by more
than 50% compared to that of the housekeeping gene human
glyceraldehyde-3-phosphate dehydrogenase in the 6 corticotroph
adenomas, which also had significantly reduced -subunit production
(all 6 tumors secreting 
0.5 ng/24 h). Mutations of the pituitary
transcription factor Prop-1, which is responsible for the syndrome of
Ames dwarfism in mice, are being increasingly recognized as a cause of
combined pituitary hormone deficiency in humans, although ACTH
deficiency has been described only once. Prop-1 expression was detected
in all 34 pituitary adenomas, including 6 corticotroph adenomas and 5
gonadotroph adenomas. The expression of Prop-1 has not been described
previously in these cell phenotypes. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Constitutively active gene mutations in pituitary transcription factors have not been described to date in pituitary adenomas, and a connection between phenotype expression in pituitary ontogeny and actual hormone hypersecretion remains tenuous. Nevertheless, in light of a previous observation suggesting no difference in Prop-1 expression in a limited variety of pituitary adenomas (11), we sought to examine the expression of this transcription factor in a large series of pituitary adenomas, including corticotroph and gonadotroph adenomas (not previously studied), for which there was full histological and in vitro characterization of tumor phenotype.
The second transcription factor whose role we have studied in pituitary
tumorigenesis, Ptx-1 (pituitary homeobox 1), had first been identified
as being centrally linked to corticotroph expression, exerting
cell-specific transcription of POMC in corticotrophs through
interaction with the corticotroph upstream transcription element (12).
However, it is now clear that several pituitary-specific promoters or
enhancers contain at least one putative Ptx-1-binding site (13, 14),
including those for -subunit (-SU) (13); the ß-subunits of LH,
FSH, TSH, GH, the Pit-1 enhancer (15); as well as the POMC promoter
(12). Ptx-1 had been shown previously to be present at high levels in a
subset of adult mouse anterior pituitary cells that express POMC (12).
However, recent papers associate Ptx-1 expression more closely with
-SU- producing cell subtypes (16).
Studies using Ptx-1 antisense ribonucleic acid (RNA) to generate Ptx-1
knockdown in T3–1 cell lines demonstrated little or no -SU
messenger RNA (mRNA) expression in such cell lines (14). The
predominant expression of Ptx-1 protein in -SU-positive cells (16)
correlates with the higher expression of Ptx-1 protein and mRNA seen in
thyrotroph, gonadotroph, and somatolactotroph cell lines compared to
that in a corticotroph cell line (14).
Recently, it has been shown that Ptx-1 is expressed in pituitary
adenomas as well as normal adult pituitary cells (17). However, in that
study Ptx-1 expression was not correlated with in vitro data
on hormone secretion. The present report concerns the direct comparison
of -SU and pituitary hormone secretion with Ptx-1 and Prop-1 mRNA
expression in secretory and nonfunctioning (so-called null cell)
pituitary adenomas. To this end, we performed studies in a large series
of pituitary tumors for which we had full in vitro and
immunohistochemical data and correlated the expression of these
transcription factors with in vitro hormone release in each
pituitary tumor.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Pituitary tumors were collected from patients at the time of transsphenoidal adenomectomy. Tissues were divided at the time of surgery for diagnostic histological studies and for tissue culture.
Acromegaly was diagnosed on the basis of persistently measurable GH and
inadequate suppression on administration of an oral glucose load.
Patients with corticotroph adenomas presented with typical clinical and
biochemical features of cortisol excess. A pituitary source of ACTH
hypersecretion was confirmed by inferior petrosal venous sampling.
Those patients with presumed lactotroph macroadenomas represented a
subset of patients with hyperprolactinemia that had not responded to
treatment with dopamine agonist therapy and required debulking surgery
before external beam radiotherapy. Patients who presented due to mass
effect, without clinical features of pituitary hormone secretion, and
with LH/FSH levels inappropriately low for the level of their gonadal
steroids were classified as clinically nonfunctioning pituitary
adenomas (NFPAs). In NFPAs with PRL hypersecretion, all had PRL easily
suppressed to below assay detection limits with low dose dopamine
agonist therapy. The diagnosis of prolactinoma was on the basis of
clinical features of PRL hypersecretion with raised serum PRL above
1000 mU/L that was not easily suppressed to below the hormone assay
detection limit by low dose dopamine agonist therapy. Furthermore, all
clinical prolactinomas subsequently had diagnosis confirmed on tumor
removal (all tumors stained strongly positive on immunostaining with
PRL antibody and secreted elevated in vitro levels of PRL).
After surgery, all tumors were classified according to their
morphological and immunocytochemical characteristics by light
microscopy. Tumors were excluded from the series if microscopy
suggested an alternative histological diagnosis, if significant normal
pituitary tissue was present on microscopy, or if the tissue appeared
necrotic. We also excluded tumors in which ACTH was present in the
culture medium in the absence of a clinical diagnosis of corticotroph
adenoma, suggesting contamination with normal tissue. NFPAs all stained
negative for PRL and were subsequently classified as being gonadotroph
adenomas if they demonstrated strong immunocytochemical staining for
LHß and/or FSHß. The remainder of NFPAs were classified as being
null cell adenomas, including those that demonstrated positivity for
-SU alone.
Morphology and immunocytochemistry
All tumors were examined by standard hematoxylin and eosin,
reticulin, and periodic acid-Schiff stains, and routine immunostaining
was performed for GH, PRL, ACTH, TSH, LH, FSH (antibodies against the
whole molecule obtained from BioGenex Laboratories, Inc.,
Berks, UK), and -SU (rabbit polyclonal, UCB Bioproducts,
Braine-L’Alleud, Belgium) using a standard
streptavidin-biotin-horseradish peroxidase method. The distinction
between normal pituitary gland and tumor was confirmed by reticulin
staining in all cases. The amount of positivity in all tumors was
initially assessed and was quantified by means of a three-point scale
(-, negative or scattered cells representing <10%; +, 10–50%
positive; ++, >50%; Table 1
) (24).
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Pituitary adenoma tissue was transported to the laboratory in DMEM containing 10% (vol/vol) heat-inactivated FCS, 0.06 g/L penicillin, 0.1 g/L streptomycin, and 2.5g/L fungizone (Life Technologies, Inc., Paisley, U.K.) and buffered with HEPES (0.02 mol/L), hereafter referred to as culture medium. Tumor tissue was dispersed enzymatically with trypsin as described previously (18). Dispersed cells were harvested by centrifugation, washed once, and subsequently resuspended in culture medium. Cell viability was assessed using trypan blue exclusion and was more than 90% in all of the tumors studied after cell dispersion. Cell yield from each tumor varied from 1–15 x 106 cells. The cells were plated in six-well plates at approximately 1 x 106 cells/well in 4 mL medium. Cultures were incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2 for 48 h to allow cell attachment to occur, after which time the medium was collected and assayed for basal hormone secretion as described below. Adherent cells were lysed with a buffered guanidinium thiocyanate solution and stored at -70 C before mRNA extraction (see below).
mRNA analysis
Cell extract from cultured cells was thawed on ice and centrifuged to remove cell debris. mRNA was extracted according to a standard technique (Pharmacia Biotech, Uppsala, Sweden) using oligo(deoxythymidine)-cellulose and washed first with 0.5 mol/L NaCl (high salt) and then with 0.1 mol/L NaCl (low salt), each containing 10 mmol/L Tris-HCl (pH 7.5) and buffered with 1 mmol/L ethylenediamine tetraacetate. Bound polyadenylated RNA was further washed with low salt buffer in a MicroSpin column (Pharmacia Biotech) and then eluted in 10 mmol/L Tris-HCl (pH 7.5) with 1 mmol/L ethylenediamine tetraacetate. mRNA was recovered by precipitation with glycogen (0.25 mg/mL) and 0.25 mol/L potassium acetate (pH 5.0) and centrifugation in 95% ethanol at -20 C. Quantitation was performed by measurement of absorbance at OD260; approximately 0.5–1.0 µg mRNA was extracted/106 cells.
After denaturation at 65 C, first strand complementary DNA (cDNA)
synthesis was catalyzed by Moloney murine leukemia virus reverse
transcriptase using a
NotI-(deoxythymidine)18 bifunctional
primer with a first strand cDNA biosynthesis kit (Pharmacia Biotech). PCR was carried out using the equivalent of 0.1 µg
mRNA in a volume of 50 µL using 200 nmol/L deoxy-NTPs. Primers were
designed from GenBank sequences [for Ptx-1, NM 002653; for Prop-1, AF
076215; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NM
002046; for primers and PCR conditions, see Table 1
]. Primers were
designed to span introns. For Prop-1 an initial PCR amplification
series was performed, and a subsequent set of amplification reactions
using a second set of primers was used to amplify the first Prop-1 PCR
amplification product (diluted 1:1000). This was necessary because of
the very low level of expression of Prop-1 in all of the pituitary
adenomas. After optimization of PCR conditions for each primer pair,
comparative kinetic analyses (19, 20) were performed to determine the
phase during which there was an exponential increase in PCR product
before a plateau was reached. It was at this point that the PCR was
terminated, allowing comparative data to be obtained.
Negative controls included mock amplification of cDNA in the absence of
template with the same PCR reaction mix. To ensure that were was no
genomic contamination in the subsequent PCR reactions, a control
reverse transcriptase reaction of tumor mRNA was performed in the
absence of Moloney murine leukemia virus reverse transcriptase. This
failed to amplify Ptx-1 or Prop-1 subsequently. The integrity of RNA
from each specimen was verified in each reaction by PCR using GAPDH
primers (Table 1).
The size of the predicted product was visualized by 1.6% agarose gel electrophoresis with ethidium bromide staining. By performing serial dilutions of known quantity starting mRNA it was possible to determine that there was a linear relationship between starting mRNA and the optical density of the PCR band generated. Confirmation of Prop-1 and Ptx-1 products was confirmed by direct DNA sequencing.
Hormone assays
GH, PRL, LH, FSH, and TSH were measured using two-site
chemiluminescent enzyme immunometric assays on the Immulite
autoanalyzer (Euro/DPC Ltd., Gwenedd, UK). The intra- and interassay
coefficients of variation for all of these assays are less than 6% and
10%, respectively. ACTH was measured by a specific double antibody RIA
(Euro/DPC Ltd.) with inter- and intraassay coefficients of variation of
less than 10%. -SU concentrations were measured by a direct double
antibody RIA using antibodies purchased from UCB Bioproducts (Brussels,
Belgium) and chloramine-T-iodinated antigen (National Institute for
Biological Standards and Control reagent 76/508, Potters Bar, UK) and
were calibrated against First International Reference Preparation
75/569 (National Institute for Biological Standards and Controls).
Intra- and interassay coefficients of variation were less than 6% and
less than 11%, respectively. Cross-reactivities (nanograms per
nanogram) with purified LH, FSH, and TSH were 3.6%, 1.9%, and 1.3%,
respectively. The detection limits of the above assays, defined as the
concentration 2 SD above the response at zero dose, were as
follows: GH, 0.5 mU/L; PRL, 10 mU/L; LH, 0.4 IU/L; FSH, 0.6 IU/L; TSH,
0.008 mU/L; ACTH, 4 pmol/L; and -SU, 0.1 µg/L. All samples from
each individual tumor were analyzed in the same assay. Hormone data
were initially obtained as concentrations, but were then corrected for
cell number and incubation time. The data presented (Table 2) are therefore expressed as the amount
of hormone secreted per 24 h/106 cells. The
reported detection limits were as follows: GH, 2.0 µU; PRL, 50 µU;
LH, 2.0 mIU; FSH, 3.0 mIU; TSH, 0.1 µU; ACTH, 20 fmol; and -SU,
0.5 ng.
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All data are expressed as the mean ± SD. Data
for -SU secretion comparison were compared by the Kruskall-Wallis
test. Ptx-1 expression was compared by ANOVA followed by Tukey’s
multiple comparison test. Spearman rank correlation coefficients (
)
were calculated to examine the correlations between the secretion of
-SU and Ptx-1 expression. For all tests, P < 0.05
was considered statistically significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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shows a representative gel for Prop-1 expression). Due to the low level
of Prop-1 expression, a seminested approach with a second reverse
primer was used to analyze Prop-1 expression; it was not possible,
therefore, to quantitate Prop-1 gene expression compared to that of a
housekeeping gene as was done with Ptx-1. Nevertheless, we determined
that Prop-1 was expressed in all pituitary adenomas including
gonadotroph adenomas and all 6 corticotroph adenomas.
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; P < 0.001). Ptx-1
expression was highest in the nonfunctioning and gonadotroph tumors,
(2.2 ± 0.3 and 2.3 ± 0.3, respectively; Fig. 1;
P < 0.05 for both compared to all other tumors).
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-SU secretion for all
tumors and then correlated with -SU expression for each tumor type.
There was no relation between Ptx-1 expression and overall tumor -SU
secretion (Spearman correlation coefficient = 0.2490; two-tailed
P = 0.1556). When each tumor type was examined
separately for -SU secretion, a different picture emerged, in that
Ptx-1 expression was lowest in corticotrophs (P <
0.001), which also had the lowest -SU secretion (P
< 0.05), as confirmed by immunohistochemistry (Table 3). There was, however, no significant
difference in -SU secretion in the other tumor groups despite the
significantly higher Ptx-1 expression in gonadotrophs and
nonfunctioning tumors (Table 3 and Fig. 2). Ptx-1 expression did not correlate
with the absence or presence of any other pituitary hormone in the
tumors studied (data not shown). Representative gels of Prop-1 and
Ptx-1 expression in human pituitary adenomas are shown (Figs. 3 and 4,
respectively). In summary, therefore, for the two transcription
factors, Prop-1 and Ptx-1, Ptx-1 has a lower level of expression in
corticotroph adenomas (associated with a lower level of -SU
secretion), whereas Prop-1 is ubiquitously expressed regardless of
tumor phenotype, albeit at a low level of expression.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-SU expression, as evidenced by studies showing absent -SU
expression in Ptx-1-deficient cell lines (14) and stimulation of -SU
by Ptx-1 (13). Studies in fetal mouse pituitary show Ptx-1 expression
to be highest in -SU-expressing tissue and to colocalize with -SU
(16). In this study we show that Ptx-1 expression is diminished in
corticotrophs compared to that in other tumor cell types, similar to
-SU secretion. However, there was no correlation between overall
Ptx-1 expression and -SU secretion in all pituitary tumors studied,
making a specific effect of Ptx-1 on general -SU gene transcription
less likely. Nonetheless, it is of interest that both -SU
secretion and Ptx-1 expression were significantly diminished in
corticotrophs.
Ptx-1 has previously been reported to have a strong stimulatory effect
on -SU promoter activity (13), where putative Ptx-1-binding sites
have been located at bases -389 to 384 (13) (which may mediate most of
the Ptx-1 activity) (14) and at -399 to -375 of the -SU promoter
(13). This along with cell transfection work involving knockout of
Ptx-1 expression in -T3–1 cells, where -SU gene expression was
found to be absent, would seem to argue for a central role for Ptx-1 in
-SU expression. We examined the expression of both Ptx-1 and Prop-1
by RT-PCR analysis because of the difficulty in obtaining adequate
amounts of tumor tissue by transsphenoidal surgery to quantitate by
Northern blotting. The finding that Ptx-1 expression is significantly
decreased in the corticotroph adenomas studied, which also have
significantly lower -SU secretion than all other pituitary tumor
groups, is in agreement with a previous study showing diminished Ptx-1
expression in murine embryonic and adult pituitary cells that are
-SU negative on immunostaining (16). Although -SU synthesis in
corticotroph tumors is not as uncommon as previously thought, being
present on histological immunostaining in as many as 60% of true
corticotroph adenomas (18), -SU immunostaining was negative in all 6
corticotroph adenomas studied, and we speculate that the diminished
Ptx-1 expression may be responsible for this decreased -SU
expression. Consistent with this hypothesis is the observation that in
transgenic Ptx-1 knockout mice there is a marked reduction in -SU
expression (22). There is no clear correlation between Ptx-1 expression
and -SU secretion, however, in pituitary tumor cells; the cell types
with highest Ptx-1 expression (gonadotrophs and null cell tumors)
having no higher -SU secretion than other pituitary tumors as a
whole. Nevertheless, it is possible that Ptx-1 expression above a
threshold level is sufficient for -SU synthesis in adult pituitary
cells.
Nakamura et al. recently described (11) the expression of Prop-1 in normal adult pituitary and in 14 pituitary adenomas. Their study was limited by the relatively small number of subjects examined (n = 17), the lack of in vitro biochemical data, and the fact that only one corticotroph adenoma and no gonadotropin-secreting tumors were examined. These pituitary adenomas were not classified according to in vitro hormone secretion, thus making it difficult to ascribe with certainty a role for Prop-1 as a panhormonal transcription factor similar to Ptx-1. In the present study the finding that Prop-1 is expressed in all pituitary tumors examined (n = 34), including all 6 corticotrophs and 6 gonadotroph adenomas (data not previously described) is consistent with previous literature documenting decreased ACTH (10) and gonadotroph reserve in patients suffering from homozygous mutations of the Prop-1 gene (7, 8, 9) and suggests that, at least in the adult pituitary, this transcription factor continues to be expressed in all pituitary cell types and may play a role in ACTH and gonadotroph expression as distinct from that of Pit-1. It may therefore activate transcriptional factors other than Pit-1. However, further studies are needed to elucidate this. In summary, we report that Prop-1 is expressed at low levels in all pituitary tumors, distinct from Ptx-1, which is specifically decreased in corticotroph tumors.
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Acknowledgments |
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Footnotes |
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Received September 2, 1999.
Revised January 20, 2000.
Accepted March 15, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cys mutation at codon 120 (R120C). J Clin
Endocrinol Metab. 83:3727–3734.-subunit gene
promoter in thyrotropes. Mol Cell Endocrinol. 140:31–36.[CrossRef][Medline]
-subunit production and plurihormonality in
human pituitary corticotroph tumours an in vitro and
immunohistochemical study. Eur J Endocrinol. 133:25–32.[Abstract]
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x174 DNA digested
with HincII); lane 1, H2O; lane 2,
thyrotroph adenoma; lane 3, lactotroph adenoma; lane 4, gonadotroph
adenoma; lane 5, somatotroph adenoma; lane 6 nonfunctioning adenoma;
lane 7, corticotroph adenoma.
