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Original Studies |
Department of Endocrinology and Metabolism, Magdeburg University Hospital (K.D.D., H.L.), 39120 Magdeburg; Institute for Neurobiology (E.D.G.), 39118 Magdeburg; and Department of Neurosurgery, University Hospital Eppendorf (D.K.L.), 20246 Hamburg, Germany
Address all correspondence and requests for reprints to: Klaus Dieterich, Department of Endocrinology and Metabolism, Magdeburg University Hospital, Leipzigerstrasse 44, 39120 Magdeburg, Germany. E-mail: dieteric{at}hermes.med.uni-magdeburg.de
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-R and CRF2ß-R), encodes proteins of
411 and 431 amino acids, respectively (9, 10, 11, 12, 13). In the anterior
pituitary, the CRF1-receptor is the predominant form,
whereas in various brain areas, as well as in nonneuronal tissues, both
subtypes are present (3, 9, 14). Cushing’s disease is a disorder caused by autonomous ACTH secretion of a pituitary adenoma. ACTH-secreting pituitary adenomas are rare and account for approximately 80% of cases of Cushing’s syndrome (15). Pituitary adenomas may be functional (ACTH-secreting tumor, active tumor) or clinically silent (nonfunctioning, inactive tumor). Functioning ACTH cell adenomas have an unusually high female preponderance, whereas silent ACTH cell adenomas occur somewhat more frequently in men (16). Most Cushing’s disease patients with ACTH-secreting pituitary tumors exhibit an increased ACTH release after exogenous CRF application compared with normal individuals (17, 18). However, the mechanism underlying this phenomenon is not yet understood.
Moreover, it is still unresolved whether Cushing’s disease is primarily a disorder of the hypothalamus (pituitary responding to excess of CRF), the pituitary, or a phenomenon implying both or other factors. However, recent data suggest that most ACTH-secreting pituitary adenomas, as well as nonfunctioning pituitary tumors, are monoclonal, suggesting a genetic defect at the pituitary level (19, 20, 21, 22, 23, 24, 25, 26). To test the hypothesis that a possible mutation of the CRF-R might be involved in the pathogenesis of Cushing’s disease, we analyzed ACTH-producing pituitary adenomas, as well as inactive adenomas and normal anterior pituitaries as control tissues, by PCR and direct cycle sequencing. In addition, we assessed expression levels of CRF-R transcripts by PCR.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Fifteen ACTH-secreting pituitary adenomas, 4 inactive pituitary
tumors, and 11 normal pituitaries were analyzed.
Corticotropin-secreting as well as inactive pituitary tumors were
obtained by transsphenoidal surgery at the University Hospital
Eppendorf, Hamburg. As control tissue we used normal anterior
pituitaries, which were obtained postmortem and kindly provided by the
Department of Neuropathology, Prof. Dietzman, Otto-von-Guericke
University Hospital Magdeburg. The anterior lobes had been dissected
from all postmortem pituitaries. The diagnosis of Cushing’s disease
was established by standard diagnostic tests and controlled
morphologically and immunohistochemically. Clinical details are shown
in Table 1
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Total RNA was extracted by a method based on the combination of guanidinium thiocyanate, lithium chloride, and cesium trifluoroacetate using the Quick Prep Total RNA Extraction Kit from Pharmacia (Freiburg, Germany).
RT-PCR and sequence analysis
For complementary DNA (cDNA) synthesis and following
amplification, the Ready-To-Go You-Prime First-Strand Beads kit from
Pharmacia was used. RNA was reverse transcribed into cDNA using the
Moloney murine leukemia virus (Mo-MLV) reverse transcriptase and 0.2
µg hexamer random primer (Pharmacia). Two micrograms total RNA was
heated at 65 C for 10 min and chilled on ice for 2 min. The RNA was
then transferred to the reaction mixture consisting of 2.4
mM each deoxynucleotide triphosphate (dNTP), 50
mM Tris (pH 8.3), 75 mM KCl, 7.5 mM
dithiothreitol (DTT), 10 mM MgCl2, and 0.08
mg/mL BSA. The reaction mixture was incubated for 60 min at 37 C. The
resulting cDNA was used as a template for the following PCR. For
amplification of the CRF1-R cDNA two primers (Nos. 1 and 2;
see Table 2; 20 pmol each) encompassing
the entire coding region of the CRF1-R cDNA were used,
according to the published sequence (GenBank accession no. L23332).
After an initial denaturing step at 95 C for 5 min, 36 cycles were
performed at 95 C for 1 min, 66 C for 1 min, and 72 C for 3 min,
followed by a final extension at 72 C for 5 min. A few normal pituitary
cDNAs showed a very faint band. In this case 40 PCR cycles were applied
to get appropriate amounts of cDNA. The reaction mixture consisted of
0.8 mM each dNTP, 15 mM Tris (pH 8.3), 25
mM KCl, 2.5 mM DTT, 3 mM
MgCl2, 0.025 mg/mL BSA, and 2.5 U Taq
polymerase.
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) encompassing the entire
coding region of the CRF1-R cDNA. PCR products were
sequenced using fluorescent dideoxynucleotides and the ABI PRISM Dye
Terminator sequencing kit (Perkin-Elmer, Norwalk, CT). Cycle
sequencing was carried out on an Applied Biosystem (Foster City,
CA) model 373 automated DNA sequencer. Expression studies
The cDNAs from all tissues were used for PCR. A 297-bp fragment
of the CRF1-R coding region was coamplified with the
housekeeping gene glyceraldehyde-phosphate dehydrogenase (GAPDH;
according to the sequence published under GenBank accession no.
M33197). The concentrations used for CRF primers (nos. 6 and 7; Table 2) and GAPDH primers (see Table 2) were 30 pmol and 10 pmol,
respectively. The reaction mixture consisted of 1 mM each
dNTP, 15 mM Tris (pH 8.3), 25 mM KCl, 2.5
mM DTT, 5 mM MgCl2, 0.025 mg/mL
BSA, and 5 U Taq polymerase. PCR amplification for
CRF1-R and GAPDH was carried out as follows: 95 C for 5
min, 36 cycles (at 95 C for 1 min, 55 C for 1 min, 72 C for 3 min), and
72 C for 5 min. The number of cycles used for both CRF1-R
and GAPDH were in the linear range of product amplification. PCR
products were run on a 2.5% agarose gel at 100 V for 40 min and
visualized by ethidium bromide staining. The absorbance values for each
band were measured by densitometry with a video documentation system
for image analysis (Herolab, Wieslach, Germany). For each
individual sample a ratio was calculated between CRF1-R and
GAPDH bands.
ACTH-secreting pituitary tumors are confined to the corticotrophic cell portion of the pituitary, which constitutes approximately 15–20% of the anterior pituitary (27, 28). Thus, when comparing active tumor samples with normal anterior pituitaries, we assumed a 7-fold dilution of the CRF-R message in normal tissue, taking into consideration the lower proportion of corticotroph cells in the normal pituitary. Therefore all CRF1-R/GAPDH ratio data of the active tumor samples have been corrected by dividing the ratios by this diluting factor.
Statistical analyses
The values of the CRF1-R/GAPDH ratios have been compared by an ANOVA based on the significance level of 5% between the three groups of two pituitary tumors and normal pituitaries.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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As shown in Table 1, 15 active and 2 inactive pituitary tumors, as well
as 2 normal anterior pituitaries, were analyzed. The resulting
sequences in 16 of the 17 tumors and normal tissues appeared to be
100% identical to the published CRF1-R sequence, detecting
no mutations in the entire coding region of the CRF1-R
gene. However, in one ACTH-secreting tumor from a 12-yr-old patient
(Table 1), we found two single base substitutions in the coding region.
The point mutations appeared in codon 223, where thymidine was
substituted by cytosine (ACT 
ACC, both codons coding for threonine)
and in codon 258, where cytosine was substituted by thymidine (TGC
TGT, both codons coding for cysteine). It is unlikely that the base
substitutions were created by the Taq polymerase, because
two independent experiments yielded identical results. Both point
mutations do not affect the amino acid sequence of the
CRF1-R protein and thus may not be functionally involved in
tumorgenesis.
Another parameter that may be affected in pituitary tumor cells is the
level of functional CRF-Rs. Therefore, the possibility of a
differential expression of the CRF1-R gene in adenomatous
corticotrophs has been investigated. For expression studies we analyzed
9 active tumors, 4 inactive pituitary tumors, and 11 normal anterior
pituitaries (Table 1). As an internal control, the housekeeping gene
GAPDH was coamplified. As seen in Fig. 1
and Table 3 significant differences were
detected in the expression level of the CRF1-R message
(297-bp band) between active tumors and normal anterior pituitaries or
nonfunctioning tumors.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-chain (29),
it was of considerable interest to search for comparable mutations in
the CRF1-R gene in corticotroph adenomas. We therefore
screened Cushing’s disease patients for possible mutations in the
coding region of the CRF1-R gene. Only in one case were two
sites of silent point mutations in a 12-yr-old patient detected. Thus,
without control DNA, a polymorphism cannot be excluded. Because
mutations that affect the amino acid sequence have not been observed in
all tissues analyzed, we suggest that mutations of the
CRF1-R are unlikely to be involved in the pathogenesis of
ACTH-secreting pituitary adenomas. Although excluding activating
mutations as a possible genetic defect in Cushing’s disease, we
further focused on possible regulatory defects at the level of
CRF1-R expression. We demonstrated an abnormal expression of the CRF1-R gene in ACTH-secreting pituitary adenomas as indicated by a significant up-regulation of the CRF1-R mRNA. Similar results have been reported for the vasopressin receptor in corticotropin-secreting pituitary tumors (30, 31). Besides CRF, vasopressin is known as an important regulator of the HPA axis. Because both CRF and vasopressin are acting synergistically (32), it is conceivable that their receptor capacities change accordingly. The increased expression of the CRF1-R gene may not necessarily contribute to the disease but rather could be the consequence of a disturbed feedback regulation and sensitivity of the HPA axis, as has been shown in Cushing’s disease patients (33).
Several findings are in line with this hypothesis. 1) In contrast to the receptor down-regulation observed in normal corticotrophs (34, 35, 36, 37, 38, 39), corticotroph adenoma cells lack any CRF-R desensitization after prolonged exposure to CRF (40). 2) CRF up-regulates CRF1-R mRNA levels in human corticotrophic adenoma cells in vitro, whereas in normal rat anterior pituitary cells, CRF down-regulates CRF1-R transcript levels (41). 3) In the mouse pituitary tumor cell line AtT-20, CRF-R1 mRNA levels increased after CRF exposure (42). Thus, a possible explanation would be that in Cushing’s disease patients CRF enhances CRF-R expression, indicated by increased transcript levels of the CRF1-R, corroborating a disturbed receptor regulation in corticotroph adenoma cells.
However, the underlying mechanism may be more complex. Apparently conflicting data were presented by a recent study (43), showing that in adenomatous corticotrophs CRF-R expression is disturbed with respect to both number and distribution. Accordingly, the authors demonstrated a decrease in the number of CRF binding sites in adenomatous pituitaries that was associated with a redistribution of binding sites to the cell periphery. Whereas high levels of intracellular binding sites were observed in normal pituitaries, in adenomatous corticotrophs, CRF binding sites were found exclusively at the cell periphery. A disturbed internalization process, as suggested by the authors, together with a defective processing of the CRF-R in adenomatous corticotrophs could explain this finding. Thus, the increase in CRF-R transcript levels we observed in this study may represent a counterregulation to compensate this defect.
In summary, we conclude that mutations of the CRF-R are unlikely to be
involved in the pathogenesis of Cushing’s disease. However, an
increase in CRF1-R transcript levels was detected. As shown
in Table 1, most Cushing’s disease patients respond to exogenous
application of CRF with increased ACTH and cortisol levels as compared
with normal individuals (17, 18). This indicates a higher CRF
sensitivity of adenomatous pituitaries and may be reflected by an
increase of CRF-R numbers at the cell surface. It remains to be
clarified whether the increase in transcript levels directly leads to
enhanced receptor numbers or reflects a cellular response to defective
CRF-R processing in adenomatous corticotrophs.
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Footnotes |
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Received January 8, 1998.
Revised May 1, 1998.
Accepted June 5, 1998.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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