| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Other Original Articles |
Division of Pediatric Endocrinology, University Children’s Hospital, CH-3010 Bern, Switzerland
Address all correspondence and requests for reprints to: Prof. Dr. Primus E. Mullis, Division of Pediatric Endocrinology, University Children’s Hospital, CH-3010 Bern, Switzerland. E-mail: primus.mullis{at}insel.ch
Abstract
G to A transition at position 6664 of the GH-1 gene results in the substitution of Arg183 by His (R183H) in human GH protein and causes a new form of autosomal dominant isolated GH deficiency (type II). Although a weak GH release after standard pharmacological provocation tests is observed in these affected individuals, the dominant inheritance pattern is postulated to be caused by a blockade of the GH-regulated secretion in the somatotrophs. The aim of this study was to analyze the impact of this autosomal dominant mutation not only at a clinical, but also at a cellular, level. The results of the different stimulation tests showed first that the patient possesses a severely impaired, but releasable, GH store, and second that the GH secretion is blocked in a time-dependent and reversible way. To confirm these clinical data, cell culture studies were performed looking at the regulated secretory pathway of GH using AtT-20 cells. Importantly, we were able to show that when the R183H mutant GH was expressed in AtT-20 cells, secretagogue (forskolin) induced a normal R183H GH-regulated secretion, but in AtT-20 cells coexpressing both the R183H mutant GH and the normal GH, forskolin-induced GH secretion was markedly reduced. Together, the experiments seem to support the hypothesis that R183H mutant GH severely impaired the GH-regulated secretion and may, therefore, be the cause of this specific form of isolated GH deficiency type II.
HUMAN GH PLAYS an essential role in postnatal somatic growth. The GH-1 gene is located on the long arm of chromosome 17 (17q22–24) and consists of five exons and four introns (1). Mature human GH, a 191-amino acid (aa) peptide, is characterized by an antiparallel up-up down-down arrangement of four helexes containing two disulfide bonds but no carbohydrates (2). Approximately 75% of circulating human GH is expressed in the anterior pituitary as a major 22-kDa product, whereas 5–10% of the remaining hGH is a minor (20-kDa) product, which is also bioactive at physiological concentrations (3).
To date, autosomal dominant isolated GH deficiency (IGHD type II) was mainly described in patients presenting different patterns of mutations in intron III splice sites that caused skipping of exon III and thus deletion of aa 32–71 (del32–71 GH) (4, 5, 6). In these individuals, even though the normal GH-1 allele is present, the secreted human GH level is severely decreased (6). When both normal and del32–71 GH were coexpressed in different cell lines, the dominant negative expression of the GH-1 gene was observed only in neuroendocrine cell lines (7). Moreover, in neuroendocrine cells coexpressing both normal and del32–71 GH, the mutant GH caused GH deficiency by decreasing the intracellular stability of the normal GH (8).
Recently, we have studied four unrelated families whose members were suffering from a new form of IGHD type II (9). The dwarf phenotype cosegregated clearly with a G6664A transition mutation within exon 5 of the GH-1 gene. This mutation causes an arginine to histidine aa change at position 183 in the human GH protein. This R183H mutation produces a dominant negative expression of the GH-1 gene (9). All affected patients showed delayed growth and impaired, but still present, GH release after standard pharmacological provocation tests (peak GH values, <10 µg/liter) (9). Noteworthy is that the Arg183 is highly conserved in the GH of different species (10, 11). The mechanism by which this G6664A GH-1 gene mutation expresses a dominant negative phenotype remains unknown. Performing GH receptor stimulation assays on human liver cells, R183H mutant GH and normal GH on their own showed equivalent bioactivity (Deladoëy, J., et al., paper submitted), suggesting that the pathophysiological cause must stem from an intracellular event in the somatotrophs that leads to decreased levels of circulating GH.
Other examples of autosomal dominant hormonal deficiencies have been shown to be caused by mutations, for instance, in the AVP gene inducing autosomal dominant familial neurohypophyseal diabetes insipidus and in the signal sequence of PTH. In familial neurohypophyseal diabetes insipidus, mutant AVP precursors accumulate within the endoplasmic reticulum and cause a marked degeneration of neurons responsible for AVP synthesis, resulting in a progressive deficiency of plasma and urinary AVP during childhood (12, 13). In the autosomal dominant C18R mutation in the signal sequence of PTH, accumulation of the mutant PTH in the endoplasmic reticulum is speculated to lead to cellular toxicity (14). However, it is important to stress that the situation might be quite different in patients presenting the R183H GH mutation. As a GH release after either insulin-induced hypoglycemia or GHRH stimulation test was observed in these patients during childhood, we hypothesized that the dominant inheritance pattern of the R183H GH mutation may be caused by a specific blockade within the GH-regulated secretion rather than cell toxicity through mutant GH accumulation in the endoplasmic reticulum.
In the present study to test this hypothesis we have performed repeated GHRH stimulation tests in a patient presenting with a G6664A mutation to investigate his regulated GH secretion in vivo. Further, we have expressed the human R183H mutant GH (alone or coexpressed with the normal human GH) in mouse pituitary cell line AtT-20 to analyze its phenotype at the cellular level, especially its regulated mode of secretion. AtT-20 cells were used, as by many groups, to study specifically the regulated secretory pathway (15). These cells have been shown first to package the endogenous ACTH and the exogenous (transfected) normal human GH in secretory granules with the same efficiency and second to release them when the cells are stimulated with a secretagogue (16, 17). These properties allow comparison of the secretion of endogenous ACTH (internal control) with secretion of exogenous normal and R183H mutant GH.
Subjects and Methods
Patient’s history
The patient, of Turkish (Kurd) ancestry (9),
immigrated to Switzerland at the age of 11 yr. He presented at this
time with short stature [-4.8 SD score for age and sex
(18)] caused by insufficient GH secretion (peak GH value
of 4.1 µg/liter in an insulin tolerance test). The pedigree of his
nonconsanguineous family is presented in Fig. 1
. The criteria for IGHD
(19) included no demonstrable causes of IGHD, stature
-2.5 SD score or less for chronological age
(18), delayed bone age (>2 yr), peak GH level less than
10 µg/liter after standard pharmacological stimulation test
(insulin-induced hypoglycemia), height velocity SD score of
less than -2 for chronological age and sex (18), and
otherwise normal endocrinology. Standard auxological assessment was
performed (20). He had no evidence of an organic disease,
psychological deprivation, or any eating disorder, and renal and
hepatic functions were normal. At the molecular level, a familial
G6664A heterozygous mutation of the GH-1 gene was found, and
therefore, IGHD (type II) was diagnosed (19). Recombinant
human GH (Novo-Nordisk, Gentofte, Denmark) treatment was started, and a
dose of 25 µg/kg was administered sc in the evening on a daily basis.
The growth velocity increased up to 12.1 cm (+8.5
SD score) during the first year of treatment
(pretreatment growth velocity, 2.2 cm/yr; -4 SD
score). Thereafter, doses were continuously adapted and were in the
range of 25–45 µg/kg·d over the years. Before the clinical study,
a dose of 43 µg/kg·d was given, which is well in the normal range
of the suggested dosing (21). When the clinical study was
performed the patient was 17 yr of age, 163.7 cm tall (-1.9
SD score), and weighed 46.7 kg (-2.3
SD score), and the pubertal stages according
Tanner were P5 and G5 with testicular volumes of 12 ml (left) and 15 ml
(right) (18, 22). He was in good health and normal in
school performance.
|
Informed consent was obtained from the patient and his parents. In addition, the study was approved by the ethical committee of the University of Bern. GH responses to GHRH injections are of a high intra- and interindividual variability (23). This variability might represent the effect of a variable endogenous somatostatin secretion (23). To inhibit the somatostatin tone, we have given pyridostigmine, a potent cholinergic agonist. To test the reversibility of GH production and/or secretion in this patient with IGHD type II, a first test was conducted after 1 d of recombinant human GH therapy withdrawal. Pyridostigmine (60 mg; Novartis, Basel, Switzerland) was given orally to the patient 1 h before the beginning of GHRH test, defined as iv injection of 1 µg/kg GHRH (Ferring Pharmaceuticals Ltd., Duebendorf, Switzerland). Blood samples were collected serially 1 h and 15 min before the beginning of the test and every 15 min thereafter for 2 h. A second and third identical tests were performed 2 wk and 4 months after interruption of recombinant human GH treatment, respectively. Glucose, GH, cortisol, free testosterone, IGF-I, TSH, free T3, and free T4 were measured in samples collected 1 h before GHRH injection. At the injection time, glucose, GH, and cortisol were also measured. Thereafter, GH was analyzed in each sample.
Hormonal analysis
Human GH was measured by an immunoradiometric assay using a commercial kit (Biochem Immunosystems, Freiburg, Germany). The coefficients of variation were 2% (intrassay) and 2.4% (interassay). ACTH was measured by chemiluminescence immunoassay using a commercial kit (Nichols Institute Diagnostics, San Juan Capistrano, CA) with intra- and interassay coefficients of variation of 6.9% and 4.2%, respectively.
Cell culture and treatment
Mouse pituitary (AtT-20/D16v-F2) cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM (4.5 g/liter glucose) supplemented with 10% heat-inactivated horse serum and 100 U/liter penicillin/streptomycin. Chinese hamster ovary (CHO-K1) cells were a gift from Prof. U. Wiesmann (Inselspital, Bern, Switzerland) and were cultured in Ham’s F-12 medium supplemented with 10% heat-inactivated FCS and 100 U/liter penicillin/streptomycin. Both cell lines were incubated at 37 C in 5% CO2.
Antibodies
Polyclonal rabbit anti-ACTH and polyclonal rabbit antihuman GH antibodies were purchased from Chemicon International, Inc., through Juro AG (Luzern, Switzerland) and from ICN Pharmaceuticals, Inc. (Eschwege, Germany) respectively. Monoclonal rat antihemagglutinin (anti-HA)- and monoclonal mouse anti-Myc indocarbocyanine 3 (Cy3)-conjugated antibodies were purchased from Roche (Rotkreuz, Switzerland). Polyclonal anti-Myc antibody was purchased from MBL through LabForce AG (Nunningen, Switzerland). All conjugated antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc., through Milan Analytica AG (La Roche, Switzerland).
Construction of expression vectors
For molecular tagging by PCR, the pAT 153 plasmid containing human GH cDNA cloned into the PstI restriction site was used as a template to generate the following fragments: GH cDNA (wild-type, wt) without tag (with sense primer 5'-AGTGGATCCACCATGGCTACAGGCTCCCGGA-3' and antisense primer 5'-CTTAAGCTTCTAGAAGCCACAGCTGCCCTCCTCCACAGA-3'), GH cDNA with HA as a tag (wt.HA; sense primer, see above, and, antisense primer 5'-CTTAAGCTTCTAGGCGTAGTCGGGCACGTCGGGCACGTCGTAGGGGTAGAAGCCAC-AGCTGCCCTCCACAGA-3'), and GH cDNA with Myc as a tag (wt.Myc; sense primer, see above, and antisense primer 5'-CTTAAGCTTCTACAGGTCCTCCTCGCTGATCAGCTT-CTGCTCGAAGCCACAGCTGCCCTCCACAGA-3'). The reaction consisted of 6 min at 94 C, followed by 40 cycles of 1 min at 94 C, 1 min at 55 C, and 30 sec at 72 C. Thereafter, PCR products were purified from a gel, digested with BamHI and HindIII (sites underlined on primer sequences), and ligated to the BamHI and HindIII restriction sites of pcDNA3.1(-)neo (Invitrogen, La Jolla, CA). Thereafter, the two pcDNA3.1.GH (with and without, ± Myc tag) were used as templates for site-directed mutagenesis (ExSite PCR-Based Site-Directed Mutagenesis Kit, Stratagene) and construction of the two new plasmids: R183H without tag (R183H) and R183H with the Myc tag (R183H.myc), sense primer 5'-pCAGTGCCACTCTGTGGAGGGCA-GCTGT-3' and antisense primer 5'-pCACGATGCGCAGGAATGTCTCGACCTT-3'. The reaction consisted of 1 cycle (4 min at 94 C, 2 min at 50 C, and 2 min at 72 C) followed by 8 cycles of 1 min at 94 C, 2 min at 56 C, and 1 min at 72 C, and a final extension of 5 min at 72 C. After PCR, template DNA was digested with DpnI (30 min at 37 C), and fragments were polished with Pfu DNA polymerase (30 min at 72 C). Additionally, gel-purified R183H.myc and wt.HA inserts from the pcDNA3.1(-)neo vector were introduced in the pcDNA3.1(-)hygro vector as described above. All constructs were confirmed by sequence analysis.
Stable expression of the recombinant proteins
AtT-20 cells and CHO cells were transfected with 1.5 µg DNA of each vector using the FuGENE6 transfection reagent (Roche), followed by selection either with 300 µg/ml geneticin (G418, Promega Corp., Madison, WI) for the AtT-20 cells or with 600 µg/ml G418 for the CHO cells. Antibiotic-resistant clones were picked and screened for expression by measuring GH in the supernatant by ELISA (Roche). Selected clones were further maintained under the same concentration of G418. AtT-20 clones expressing R183H (with or without the Myc tag) mutant GH were confirmed by DNA sequencing. Thereafter, R183H.myc AtT-20 mutant clone was stably transfected either with 1.5 µg DNA of wt.HA or with 1.5 µg DNA of R183H.myc, both inserted in the pcDNA3.1(-)hygro. These clones were selected and maintained with 300 µg/ml G418 and 100 µg/ml hygromycin B (Roche). For further studies, clones with the highest GH expression were selected. To obtain the mock-transfected cells, AtT-20 cells were transfected with pcDNA3.1(-)neo according to the protocol described above.
Indirect immunofluorescence staining
AtT-20 cells expressing normal and mutant GH (±tag) were grown on a Lab-Tek two-chamber slide (Falcon; Nalge Nunc International, Naperville, IL), fixed with 4% paraformaldehyde, and permeabilized with 0.3% Igepal CA-630 (Sigma, St. Louis, MO). The cells were incubated with rabbit anti-ACTH antibody (diluted 1:40). After washing in PBS, cells were incubated with goat antirabbit fluorescein isothiocyanate (FITC)-conjugated antibody (diluted 1:100) and mouse anti-Myc (diluted 1:20). For double indirect immunofluorescence, AtT-20 cells were incubated with rat anti-HA high affinity antibody (2 µg/ml) and rabbit anti-Myc antibody (1:250). After the washing procedure, cells were incubated with donkey antirat Cy3- conjugated antibody and goat antirabbit FITC-conjugated antibody (both diluted 1:100). The incubation time was 30 min at 37 C, and photographs were taken using a Leitz fluorescence microscope (Rockleigh, NJ).
Immunoprecipitation, SDS-PAGE, and immunoblotting
After culture in 60 x 15-mm tissue culture dishes, subconfluent stable cells expressing normal or R183H mutant GH (with or without the tag) were harvested with a rubber policeman, lysed with 1% Igepal CA-630 in 25 mM Tris, pH 8.0 containing 1% deoxycholate, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml apoprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 3.5 µg/ml benzamidine. Lysates and media were precleared by treatment with 50 µl protein A-Sepharose beads and were transferred into tubes containing 5 µl undiluted polyclonal rabbit antihuman GH antibody. After incubation overnight at 4 C, 50 µl protein A-Sepharose beads were added for 4 h at 4 C. After washing the beads (24), immunoprecipitates were solubilized with 50 µl electrophoresis sample buffer, containing 50 mM dithiothreitol, and analyzed on a 15% SDS-PAGE gel. The gel was transferred to a polyvinylidene difluoride membrane and probed with polyclonal rabbit antihuman GH (1:200), polyclonal rabbit anti-Myc (1:600) or monoclonal rat anti-HA (0.5 µg/ml). After washing, membranes were incubated with goat antirabbit phosphatase-conjugated antibody (1:5000) or goat antirat phosphatase-conjugated antibody (1:5000), respectively. Subsequently, proteins were detected using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate solution (Roche).
Additionally, 50–75 µg AtT-20 cells total protein lysates were directly mixed with 50 µl electrophoresis sample buffer containing 50 mM dithiothreitol and further analyzed on a 15% SDS-PAGE gel as described above.
Forskolin stimulation assays
The amount of human GH in the supernatant of AtT-20 and CHO stably transfected cells was determined by human GH an immunoradiometric assay with and without a secretagogue (50 µM forskolin) to test a possible regulated secretory pattern of the R183H mutant GH. We have modified the method of Fleischer et al. (25). Briefly, clones were seeded at a concentration of 1 x 105 cells on 60 x 15-mm tissue culture dishes. Neither G418 nor hygromycin B was added to the medium. After 3 d, incubation medium was removed, and cells were washed twice with 3 ml PBS. To measure basal and regulated secretion, cells were incubated with either DMEM with dimethylsulfoxide as vehicle (Fluka, Buchs, Switzerland) or with 50 µM forskolin (Sigma). After 2-h incubation, media were sampled and filtered (0.2-µm pore size filter, Ministart, Sartorius, Gottingen, Germany) to remove cellular debris before measuring human GH. The ACTH concentration also was determined in media from AtT-20 clones.
Results
Combined pyridostigmine and GHRH tests
The GH levels after the GHRH test procedures in an euglycemic
nonobese IGHD (type II) patient with a normal thyroid, corticosteroid,
and sex steroid hormone profile are shown and summarized in Fig. 2 and Table 1. The idea behind the testing was to
analyze GH secretion in this disorder at different time points (1 d, 2
wk, and 4 months) after withdrawal of GH replacement therapy (43
µg/kg·d). In the first test, extremely high levels of IGF-I (+5.52
SD score) confirmed the patient’s compliance with the GH
replacement therapy. The GH peak value of 8.2 µg/liter after the iv
bolus of 1 µg/kg GHRH represents a subnormal, but substantial,
release (Fig. 2 and Table 1). Two weeks after GH therapy withdrawal, a
second test was performed. The very low IGF-I level (-3.45
SD score) indicated that the patient was off therapy and
that his pituitary gland secreted an extremely low basal amount of GH
during that time. Compared with the first test, GH release after GHRH
stimulation was increased (21.1 vs. 8.2 µg/liter; Table 1). However, this GH peak value is still subnormal when compared with
values obtained in normal children and adolescents using combined
pyridostigmine and GHRH tests (normal range, 22.6–90.0 µg/liter;
n = 94; results not influenced by pubertal stage)
(26). Under these circumstances, the withdrawal of
exogenous GH may cause an increase in endogenous GHRH and consequently
might result in an increase in GH synthesis. However, as the direct
feedback loops are more complicated, this relatively high GH peak may
be further explained first through the absence of the negative IGF-I
feedback and second through an acute release of the blocked GH store
obtained by a supraphysiological GHRH stimulation. Nevertheless, the
findings of the second test imply that at the age of 17 yr, the
patient’s somatotroph cells are still able to synthesize and release
GH. This is of importance, as 17 yr of production of the mutant GH was
not toxic to the somatotrophs. Finally, a third test was performed 4
months after therapy withdrawal, and a blunted GH peak was revealed.
All GH values remained at the basal level (<3.5 µg/liter; Fig. 2 and
Table 1). It is important to stress that in this patient, presenting
with a GH-1 gene alteration, insulin-induced hypoglycemia
performed at the age of 11 yr demonstrated isolated GH deficiency, this
probably after years of obvious endogenous GHRH stimulation (feedback
mechanism).
|
|
Individual AtT-20 cell lines were characterized for their specific
protein expression. After immunoprecipitation of GH, R183H mutant GH
(Fig. 3D, lanes 3, 4, 7, and 8) and
normal GH (Fig. 3D, lanes 1, 2, 5, and 6) were detected in cell lysate
as well as in medium. Moreover, no lower mol wt bands were observed on
the immunoblot of total cell protein lysate of R183H.myc compared with
the total protein lysate of the AtT-20 cells expressing wt.HA (Fig. 3E, lane 2 vs. lane 3). This finding indicated that R183H GH was
not significantly degraded within AtT-20 cells. Therefore, measurement
of the secreted concentration of GH was used for quantification and
estimation of the efficacy of GH production in our different clones.
Using immunofluorescence, ACTH (Fig. 3A) and R183H.myc (Fig. 3B) were
localized as a punctate pattern at the tips of the cell processes,
strongly suggesting that both are directed into secretory granules. A
similar pattern of localization was shown with the wt.HA clone (data
not shown). As described in previous work (17), no
morphological or functional differences were seen between recombinants
with tag added to the carboxyl-terminus of human GH and those without
(data not shown). Thereafter, we determined whether AtT-20 cells
synthesized and secreted the R183H mutant GH to the same extent as
normal GH and as well as their endogenous ACTH. The addition of 50
µM forskolin, a potent secretagogue, to the
medium of the R183H.myc clone increased GH release by up to 19 ±
3.5% (expressed as a percentage of basal secretion), which is similar
to the GH release of 21.3 ± 3.2% observed in the wt.HA clone
(Table 2). As an internal control for the
function of the regulated secretory pathway, ACTH was determined in the
supernatants in parallel with GH measurements. Thereafter, to quantify
the efficiency of GH targeting to the regulated secretory pathway, we
calculated a secretion index corresponding to the ratio of fold
stimulation of GH secretion over that of the endogenous ACTH
(27). The secretion indexes for R183H.myc and wt.HA were
very close to 1 (1.04 ± 0.04 and 0.95 ± 0.05,
respectively), indicating that the R183H.myc, wt.HA, and endogenous
ACTH were targeted to the regulated secretory pathway with equal
efficiency (Table 2), which is of importance as transfectants are
artificial constructs, possibly with different efficacies of hormone
production. Because differences in the intracellular GH pool due to the
artificial construct themselves may have a specific impact on the
regulated GH secretion, R183H mutant GH was coexpressed with itself
(R183H.myc x R183H.myc clone) to assess the effect of an higher
construct production on its regulated secretion. However, the secretion
index remained close to 1 (1.03 ± 0.03) even when the basal level
of GH was doubled (Table 2). It implied first that an increased
expression of R183H mutant GH did not disturb its own regulated
secretion, and second that a high level of exogenous GH expression did
not affect the regulated secretion in AtT-20 cells. When expressed as a
percentage of basal secretion, R183H mutant GH release (19 ±
3.5% for R183H.myc and 25.5 ± 4.9% for R183H.myc x
R183H.myc) was statistically not different from ACTH release (15.2
± 6.3% and 21.4 ± 1.2%, respectively). In contrast, GH release
by the R183H mutant GH and the normal GH individually transfected into
CHO cells, which do not have a regulated pathway of secretion, did not
change with forskolin (data not shown). Mock-transfected AtT-20 cells
presented a GH level below 0.4 µg/liter (data not shown).
|
|
Protein expression of clones coexpressing both R183H.myc and wt.HA
were assessed with double indirect immunofluorescence (IF) and
immunoprecipitation. For double indirect IF, the first antibodies used,
rat anti-HA and rabbit anti-Myc, did not shown any cross-reactivity
either on Western blots of cell lysates (Fig. 3E
) or in our IF-negative
controls (data not shown). Using this method, the wt.HA and R183H.myc
showed a similar distribution perinuclearly as well as at the tips of
the cell processes (Fig. 4A). However,
the apparent similarity in distribution of wt.HA and R183H.myc does not
unequivocally demonstrate that normal and mutant GH are in the same
secretion granules at an equimolar ratio. Furthermore, under standard
conditions, no coimmunoprecipitation between wt.HA and R183H.myc was
observed (Fig. 4B). In the cell lysate, when R183H.myc was
immunoprecipitated with an anti-Myc antibody, no band was detected on
membranes blotted with an anti-HA antibody (Fig. 4B, lower
panel). In addition, when wt.HA was immunoprecipitated with an
anti-HA antibody, a nonspecific band was obtained on membranes blotted
with an anti-Myc antibody (Fig. 4B, upper panel). When
analyzing these data, we have to bear in mind that a strong interaction
between R183H.myc and wt.HA is not excluded under conditions normally
occurring in secretory granules, such as low pH and high calcium and
zinc concentrations. Further, in the wt.HA x R183H.myc clone
founded from the R183H.myc clone, the secretion index of 0.74 ±
0.04 revealed a significantly reduced regulated secretion
(P < 0.05) compared with the secretion indexes of
wt.HA, R183H.myc, and R183H.myc x R183H.myc clones (Table 2).
When expressed as a percentage of basal secretion, a reduction of the
GH release after forskolin stimulation (9.9 ± 4.5%) is observed,
which is dramatic compared with endogenous ACTH release (49.2 ±
9.7%; Table 2). These results support the hypothesis that normal and
mutant GH block their own regulated secretion whenever they are
coexpressed.
|
Based on this newly described GH-1 gene mutation (R183H), which causes an autosomal dominant form of IGHD, the aims of this study were to define clinically the reserve of GH secretion in these patients and to investigate the in vitro effect of this mutation on the regulated secretory pathway.
The patient was 11 yr of age when he was referred to the hospital. At that time he was short and presented a very low height velocity, and hypoglycemia-induced GH stimulation revealed GH deficiency (4.1 µg/liter). After 6 yr of successful GH replacement therapy his GH reserve was retested by a pyridostygmine-GHRH test. Interestingly, GH release could be stimulated, but remained subnormal (26). Moreover, after 4 months of GH therapy withdrawal, GH secretion did not increase after GHRH stimulation. These data imply that the R183H mutant form of GH might not have a fully cytotoxic effect on the somatotroph cells as has been suggested in other autosomal dominant endocrine disorders (12, 14). Rather, the mutant form might have a major impact on the regulated secretory pathway, blocking its secretion over time, which, however, seems to be fully reversible. It is suggested that the somatotroph cells might recover from the constant endogenous GHRH stimulation on exogenous GH replacement. To challenge these hypotheses, in vitro studies were performed.
First, by analyzing the in vitro experiments the relatively
small amount of regulated GH and ACTH release observed in transfected
AtT-20 cells is not surprising, as only a vestige of the regulated
pathway of secretion remains in this tumor cell line (15).
Moreover, the ACTH and GH basal values presented in Table 2 showed a
wide variation between these different clones. However, the relation
between (transfected) human GH release and the endogenous murine ACTH
release allowed us to compare results by correcting the difference in
the basal constitutive secretion between these clones. When R183H.myc
and wt.HA were individually transfected into AtT-20 cells, R183H mutant
GH as well as the normal GH were secreted after forskolin stimulation
in a clear regulated pattern similar to its own endogenous ACTH
secretion. Moreover, when R183H mutant GH was highly expressed in the
R183H.myc x R183H.myc clone, R183H mutant GH release was still
equal to its own ACTH release, implying that AtT-20 cells were able to
secrete correctly even a large amount of exogenous mutant GH. Together,
these results show that R183H mutant GH does not impair its own
regulated secretion. In contrast, AtT-20 cells expressing both R183H
mutant GH and normal GH presented a dramatically decreased, but still
present, GH-regulated secretion. Thus, the in vitro results
indicate that R183H mutant GH may severely impair the regulated
secretion of GH whenever both mutant and normal GH are coexpressed,
which correlated to the in vivo results.
Sorting of secretory proteins into secretory granules from the trans-Golgi network remains a controversial debate (for reviews, see Refs. 15 and 28). Two different, but not exclusive, models have been proposed to explain the biogenesis of secretory granules. The active sorting model postulates that secretory proteins bind to one or more sorting receptors concentrated in the trans-Golgi network and are consequently delivered to the immature secretory granules (ISGs). Alternatively, according to the passive sorting model, entry into forming secretory granules is not selective and does not require any receptor. However, only the selectively aggregated proteins are retained in forming secretory granules; thus, they will be secreted in a regulated fashion. The nonaggregated soluble proteins are progressively removed and directed to the constitutive secretory pathway.
Dittié et al. (29) hypothesized that when the amount of newly synthesized regulated secretory protein is large, such as GH in the somatotroph cells, the second model requiring a selective aggregation could be more appropriate. Furthermore, since other histidine residues in the GH protein (His18 and His21, together with Glu174) play an important role in the zinc-dependent GH dimerization (30), we could expect that the new His183 aa in the R183H mutant GH might disturb the aggregation process. Two alternative hypotheses may be proposed. On the one hand, the dimerization between normal GH and R183H mutant GH is impaired; a significant part of GH remains nonaggregated and is excluded from the ISGs. On the other hand, the dimerization remains unaffected or is increased, but the newly formed aggregate blocks the maturation of the ISGs.
Besides these hypotheses about disturbed GH aggregation, it has been suggested that His183 might interfere with the correct Cys182-Cys189 bonding, and hence folding of the R183H GH (5). However, as R183H GH does not accumulate within the endoplasmic reticulum, which is normally the case with misfolded proteins (12, 31), this hypothesis becomes less attractive.
In conclusion, we studied an autosomal dominant form of IGHD (type II) caused by an R183H altered GH peptide at the clinical as well as the cellular level. The results present evidence that this specific form of IGHD type II is caused by a time-dependent, but nevertheless reversible, alteration of the regulated secretory pathway that is induced by the mutant GH form, rather than by a direct cell toxic effect on the somatotrophs. These observations indicate a potential reason why some patients with IGHD type II still produce and secrete GH even at a reduced level. Furthermore, these data may act as a model to explain and challenge the possible underlying mechanisms that cause the autosomal dominant endocrine disorders at the level of the secretory granules.
Acknowledgments
We thank Prof. Melvin Grumbach for his help and valuable advice while reviewing the manuscript, Drs. Elizabeth Bürgi and Jean-Marc Vuissoz for advice and stimulating discussions, and Prof. Jürg Girard for technical assistance.
Footnotes
This work was supported by the Swiss National Science Foundation (32-53714.98; to P.E.M.) and a M.D.-Ph.D. grant from the Swiss Academy of Medical Sciences (31-54879.98; to J.D.).
Abbreviations: aa, Amino acid; Cy3, indocarbocyanine 3; FITC, fluorescein isothiocyanate; HA, hemagglutinin; IF, immunofluorescence; IGHD, isolated GH deficiency; ISG, immature secretory granule; R183H, arginine to histidine substitution at codon 183.
Received November 9, 2000.
Accepted April 25, 2001.
References
This article has been cited by other articles:
 |
|
 |
|
  K. S. Alatzoglou, J. P. Turton, D. Kelberman, P. E. Clayton, A. Mehta, C. Buchanan, S. Aylwin, E. C. Crowne, H. T. Christesen, N. T. Hertel, et al. Expanding the Spectrum of Mutations in GH1 and GHRHR: Genetic Screening in a Large Cohort of Patients with Congenital Isolated Growth Hormone Deficiency J. Clin. Endocrinol. Metab., September 1, 2009; 94(9): 3191 - 3199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dateki, K. Hizukuri, T. Tanaka, N. Katsumata, P. Katavetin, and T. Ogata An immunologically anomalous but considerably bioactive GH produced by a novel GH1 mutation (p.D116E) Eur. J. Endocrinol., August 1, 2009; 161(2): 301 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Shariat, R. C. C. Ryther, J. A. Phillips III, I. C. A. F. Robinson, and J. G. Patton Rescue of Pituitary Function in a Mouse Model of Isolated Growth Hormone Deficiency Type II by RNA Interference Endocrinology, February 1, 2008; 149(2): 580 - 586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Petkovic, D. Lochmatter, J. Turton, P. E. Clayton, P. J. Trainer, M. T. Dattani, A. Eble, I. C. Robinson, C. E. Fluck, and P. E. Mullis Exon Splice Enhancer Mutation (GH-E32A) Causes Autosomal Dominant Growth Hormone Deficiency J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4427 - 4435. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Hess, Y. Hujeirat, M. P. Wajnrajch, S. Allon-Shalev, Z. Zadik, I. Lavi, and Y. Tenenbaum-Rakover Variable Phenotypes in Familial Isolated Growth Hormone Deficiency Caused by a G6664A Mutation in the GH-1 Gene J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4387 - 4393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. I Iliev, N. E Wittekindt, M. B Ranke, and G. Binder In vitro analysis of hGH secretion in the presence of mutations of amino acids involved in zinc binding J. Mol. Endocrinol., August 1, 2007; 39(2): 163 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Petkovic, A. Besson, M. Thevis, D. Lochmatter, A. Eble, C. E. Fluck, and P. E. Mullis Evaluation of the Biological Activity of a Growth Hormone (GH) Mutant (R77C) and Its Impact on GH Responsiveness and Stature J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 2893 - 2901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Petkovic, M. Thevis, D. Lochmatter, A. Besson, A. Eble, C. E Fluck, and P. E Mullis GH mutant (R77C) in a pedigree presenting with the delay of growth and pubertal development: structural analysis of the mutant and evaluation of the biological activity Eur. J. Endocrinol., August 1, 2007; 157(suppl_1): S67 - S74. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Salemi, S. Yousefi, D. Lochmatter, A. Eble, J. Deladoey, I. C. A. F. Robinson, H.-U. Simon, and P. E. Mullis Isolated Autosomal Dominant Growth Hormone Deficiency: Stimulating Mutant GH-1 Gene Expression Drives GH-1 Splice-Site Selection, Cell Proliferation, and Apoptosis Endocrinology, January 1, 2007; 148(1): 45 - 53. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Vivenza, L. Guazzarotti, M. Godi, D. Frasca, B. di Natale, P. Momigliano-Richiardi, G. Bona, and M. Giordano A Novel Deletion in the GH1 Gene Including the IVS3 Branch Site Responsible for Autosomal Dominant Isolated Growth Hormone Deficiency J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 980 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Salemi, S. Yousefi, K. Baltensperger, I. C A F Robinson, A. Eble, D. Simon, P. Czernichow, G. Binder, E. Sonnet, and P. E Mullis Variability of isolated autosomal dominant GH deficiency (IGHD II): impact of the P89L GH mutation on clinical follow-up and GH secretion Eur. J. Endocrinol., December 1, 2005; 153(6): 791 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Besson, S. Salemi, J. Deladoey, J.-M. Vuissoz, A. Eble, M. Bidlingmaier, S. Burgi, U. Honegger, C. Fluck, and P. E. Mullis Short Stature Caused by a Biologically Inactive Mutant Growth Hormone (GH-C53S) J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2493 - 2499. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. E. Mullis, I. C. A. F. Robinson, S. Salemi, A. Eble, A. Besson, J.-M. Vuissoz, J. Deladoey, D. Simon, P. Czernichow, and G. Binder Isolated Autosomal Dominant Growth Hormone Deficiency: An Evolving Pituitary Deficit? A Multicenter Follow-Up Study J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2089 - 2096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. E Mullis Genetic control of growth Eur. J. Endocrinol., January 1, 2005; 152(1): 11 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Marino, E. Chaler, M. Warman, M. Ciaccio, E. Berensztein, M. A. Rivarola, and A. Belgorosky The Serum Growth Hormone (GH) Response to Provocative Tests Is Dependent on Type of Assay in Autosomal Dominant Isolated GH Deficiency because of an ARG183HIS (R183H) GH-I Gene Mutation Clin. Chem., June 1, 2003; 49(6): 1002 - 1005. [Full Text] [PDF]
|
|
|
|
|
|
L. McGuinness, C. Magoulas, A. K. Sesay, K. Mathers, D. Carmignac, J.-B. Manneville, H. Christian, J. A. Phillips III, and I. C. A. F. Robinson Autosomal Dominant Growth Hormone Deficiency Disrupts Secretory Vesicles in Vitro and in Vivo in Transgenic Mice Endocrinology, February 1, 2003; 144(2): 720 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. L. Zhu, B. Conway-Campbell, M. J. Waters, and P. S. Dannies Prolonged Retention after Aggregation into Secretory Granules of Human R183H-Growth Hormone (GH), a Mutant that Causes Autosomal Dominant GH Deficiency Type II Endocrinology, November 1, 2002; 143(11): 4243 - 4248. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
