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Original Studies |
Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University (Y.H., M.Y., S.O., H.S.), Furo-cho, Chikusa-ku, Nagoya 464-8601; the Department of Pediatrics, Kamiida Daiichi General Hospital (T.K.), Kita-ku, Nagoya 462-0802; and Ogawa Clinic (M.O.), Nagoya 461-0001, Japan
Address all correspondence and requests for reprints to: Yoshitaka Hayashi, M.D. Ph.D., Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: hayashiy{at}endeavor.riem.nagoya-u.ac.jp
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Abstract |
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A metabolic labeling study in COS-1 cells revealed that a mutant GH with a reduced molecular mass was synthesized from the mutant messenger ribonucleic acid and retained in the cells for at least 6 h. On the other hand, the wild-type GH was rapidly secreted into the medium. Coexpression of mutant and wild-type GH did not result in any inhibition of wild-type GH secretion in COS-1 or HepG2 cells. However, coexpression of mutant GH resulted in significant inhibition of wild-type GH secretion in somatotroph-derived MtT/S cells as well as in adrenocorticotroph-derived AtT-20 cells, without affecting cell viability. We conclude that the dominant negative effect of mutant GH on the secretion of wild-type GH is at least in part responsible for the pathogenesis of IGHD type II. Our results also suggest that neuroendocrine cell type-specific mechanisms, including intracellular storage of the secretory proteins, are involved in the inhibition.
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Introduction |
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We have recently identified two mutations at the donor splice site of
intron 3 in GH-I from Japanese patients with IGHD type II
(see Fig. 1B
; Kamijo, T., manuscript in preparation). One
mutation was a G to A transition of the first nucleotide (mutA), which
was also reported by Cogan et al. (8), and the other was the
same transition of the fifth nucleotide (mutE). In the present report,
we investigated whether coexpression of the wild-type and mutant GH
molecules results in inhibition of wild-type GH secretion. It was also
studied whether expression of the mutant GH-I gene products
affects cell viability.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Informed consent was obtained from all patients and family
members for blood sampling, genomic DNA extraction, and analysis. All
exons and exon-intron junctions of the GH-I gene were
amplified by PCR. The amplified fragments were ligated with pGEM-T
vector (Promega Corp., Madison, MI), followed by sequence
determination using Dye Terminator Cycle Sequencing FS Ready Reaction
Kit (PE Applied Biosystems, Foster City, CA) and DNA
sequencer model 373A-36 (PE Applied Biosystems). Two
different mutations at the donor splice site of exon 3 in the
GH-I gene (see Fig. 1B
), a G
to A substitution at the first nucleotide in intron 3 (referred to as
mutA) and the same substitution at the fifth nucleotide in intron 3
(referred to as mutE), were identified in patients with IGHD as
heterozygotes. A detailed description of the patients and these
procedures will be presented elsewhere (Kamijo, T., manuscript in
preparation).
Cell culture
HepG2, COS-1, and AtT-20 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 5% FBS. A rat pituitary-derived cell line, MtT/S, was obtained from RIKEN cell bank (Tsukuba, Japan) and cultured in a half and half mixture of DMEM and F-12 medium supplemented with 2.5% FBS and 10% horse serum (10, 11). The cells were cultured at 37 C in an atmosphere of 95% room air-5% CO2 and 100% humidity.
Plasmid construction, transfection, and RT-PCR
PCR fragments of the GH-I gene containing mutations,
mutA or mutE, were digested with SacI present in intron 2
and BglII in exon 5. The digested fragments were ligated to
corresponding sites in pTK-GH (Nichols Institute Diagnostics, Los Angeles, CA) (12), in which transcription of
the wild-type GH-I gene is driven by herpes virus thymidine
kinase promoter. The constructs were named pTK-GHmutA and pTK-GHmutE,
respectively. HepG2 cells were cultured in 6-cm dishes (Falcon 3003,
Becton Dickinson and Co., Franklin Lakes, NJ) and
transfected with 2 µg pTK-GH, pTK-GHmutA, or pTK-GHmutE together with
8 µg carrier DNA by a calcium phosphate method as previously
described (13, 14). Seventy-two hours after the transfection, RNA was
extracted from the cells by the acid-guanidium-phenol-chloroform method
as previously described (15). To analyze the transcripts from the
mutant genes, complementary DNA (cDNA) was synthesized using
oligo(deoxythymidine) primer and SuperScript reverse transcriptase
(Life Technologies, Gaithersburg, MD) according to the
manufacturer’s instructions. Human GH cDNAs were amplified by PCR
using a sense primer (5'-GCC CAA CTC CCC GAA CCA CT-3') and an
antisense primer (5'-GAG GCA CTG GGG AGG GGT CAC-3'). The amplified
fragment was ligated to pGEM-T vector for sequence determination. As
will be shown in the results, the transcripts from the two mutant
GH-I genes were devoid of exon 3. To construct a plasmid
expressing GH cDNA without exon 3, the cDNA was inserted into
pcDNAI/Amp (Invitrogen, San Diego, CA) to yield
phGH
Ex3. A plasmid expressing wild-type GH cDNA (phGHwt) was
constructed in a similar manner.
Metabolic labeling and GH assay
COS-1 cells or AtT-20 cells were cultured in six-well plates
(Falcon 3043, Becton Dickinson and Co.) and transfected by
the calcium phosphate or Lipofectamine (Life Technologies)
method, respectively. Each well received 2 µg plasmid in total, which
contains 100 ng pSV-ßGal (Promega Corp.); 900 ng phGHwt,
phGHEx3, and/or pcDNAI/Amp without any cDNA inserts; and 1 µg
pBluescript (Stratagene, La Jolla, CA). MtT/S cells
cultured in six-well plates were transfected with SuperFect
Transfection Reagent (QIAGEN, Valencia, CA) according to
the manufacturer’s instruction; each well received 2 µg plasmids
containing 1800 ng GH-expressing plasmids and 200 ng pSV-ßGal
together with 4 µL SuperFect reagent.
For metabolic labeling, cells were cultured in the corresponding medium for 48 h after transfection. After 2-h preincubation in a medium devoid of methionine and serum, cells were labeled for 2 h in the same medium containing [35S]methionine (1000 Ci/mmol; Amersham International, Aylesbury, UK) at a concentration of 10 µCi/mL. After labeling, cells were immediately harvested or were left for further incubation in the complete medium for the chase. Cells were lysed in buffer consisting of 150 mmol/L NaCl, 10 mmol/L Tris (pH 8.3), 1 mmol/L ethylenediamine tetraacetate, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and 0.1% L-methionine. Human GH in cell lysate or medium was immunoprecipitated with anti-GH antibody obtained from NIH (Bethesda, MD); detailed procedures were described previously (16, 17). The immunoprecipitate was fractionated through 12% or 15% SDS-PAGE and visualized by autoradiography.
To determine the level of human GH secreted into the medium, 200-µL aliquots of medium were collected at intervals after a medium change following the transfection procedure. The medium was diluted in 10 mmol/L sodium phosphate buffer (pH 7.0) containing 0.1 mol/L NaCl and 0.1% sodium azide by more than 50-fold and directly subjected to an enzyme immunoassay using the PICOIA HGH PLATE kindly provided by Sumitomo Pharmaceutical, Inc. (Tokyo, Japan). The lower detection limit of the assay is 2 pg/mL (18). To monitor the transfection efficiency, cells were harvested after final collection of the medium and subjected to a ß-galactosidase assay using a Luminescent ß-galactosidase detection kit (CLONTECH Laboratories, Inc., Palo Alto, CA) as previously described (15). All the data were analyzed by Student’s t tests, and differences with P < 0.05 were considered statistically significant.
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Results |
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. Two mutations at the donor splice site of intron 3
(bold line in Fig. 1A), mutA and mutE, are depicted in Fig. 1B along with the consensus sequence for the donor splice site. At
first, the structure of mRNAs transcribed from GH-I genes
with these mutations was analyzed. As shown in Fig. 1C, a fragment 625
bp in length was amplified by RT-PCR using the RNA extracted from HepG2
cells transfected with pTK-GHmutA (lane 1), in agreement with exon 3
skipping as previously reported (6, 7, 8). A fragment with the same size
was also amplified from cells transfected with pTK-GHmutE (lane 2),
indicating that the mutation of the guanine residue in mutE affects the
splicing in the same manner as mutA. Sequencing verified that the
transcripts of both mutant GH-I genes lacked exon 3. It is
of note that the guanine at the first and fifth nucleotides of introns
are conserved 100% and 84%, respectively (19). The lack of exon 3 in
the transcripts of mutE indicated the significance of the fifth residue
for the splicing. Similar results were reported by Missarelli et
al. (20) recently. As exon 3 of GH-I gene consists of
120 nucleic acid residues, a mutant GH protein with a deduced molecular
mass of 17.5 kDa, which lacks 40 amino acid residues in-frame, was
predicted to be synthesized from the two mutant GH-I genes.
On the other hand, a 745-bp fragment encoding wild-type GH with a
molecular mass of 22 kDa was amplified from RNA extracted from HepG2
cells transfected with pTK-GH (lane 3). Very little, if any, mRNA
encoding the 20-kDa variant GH (21, 22) or other splicing variants (7)
could be detected.
The synthesis and secretion of mutant and wild-type GH were then
studied. It was also examined whether the mutant GH can exert dominant
negative effects on the secretion of wild-type GH. COS-1 cells were
transfected with simian virus 40-derived expression vectors carrying
mutant or wild-type GH cDNAs for metabolic labeling. As shown in Fig. 2A, a 22-kDa protein was detected in both
medium and cell lysate from the cells transfected with phGHwt after
labeling for 2 h. In contrast, a protein with a smaller molecular
mass, which is in agreement with a deletion of 40 amino acids encoded
by exon 3, was detected in the cell lysate, but not in medium, when
transfected with phGHEx3. In cells transfected with both phGHwt and
phGHEx3, both peptides were detected in cell lysate, but only the
22-kDa wild-type GH was secreted into the medium. As shown in Fig. 2B, the 17-kDa mutant GH was retained in the cells, even 6 h after the
chase, without significant intracellular degradation, whereas the
wild-type GH was secreted into the medium with minimum retention after
1 h of chase. Secretion of wild-type GH was not affected by
coexpression of mutant GH.
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Ex3 to phGHwt
could result in inhibition of wild-type GH secretion. As shown in Fig. 3A, COS-1 cells transfected with phGHwt
secreted GH, with the concentration reaching 150 ng/mL 5 days after
transfection. Naturally, no GH was detected in the medium when
phGHEx3 was transfected. Cotransfection of a 9-fold excess of
phGHEx3 with phGHwt did not inhibit GH secretion. The activity of
ß-galactosidase, which was coexpressed to monitor the transfection
efficiency, was not significantly different among the transfected cells
(Fig. 3B). This result also suggests that expression of the mutant GH
is not toxic for cell survival (23). Similar experiments were carried
out in HepG2 cells using plasmids expressing GH cDNA (wt combined with
Ex3) or the GH-I gene (pTK-GHwt combined with mutA or
mutE). No inhibition of wild-type GH secretion by coexpression of the
mutant GH-I genes or cDNA was observed (data not shown).
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Ex3 or pcDNAI/Amp with no
insert, indicating that PICOIA HGH PLATE, the enzyme immunoassay
employed in this study, does not detect rGH (Fig. 4A). On the other hand, cells transfected
with phGHwt secreted GH into the medium, with concentrations reaching 3
ng/mL after 6 days of incubation. Although cotransfection of equal
amounts of phGHEx3 and phGHwt did not result in any inhibition of GH
secretion, cotransfection of a 5-fold excess of phGHEx3 resulted in
a significant decrease in GH secretion (Fig. 4A). Similar results were
produced in the experiments using pTK-GH and pTK-GH mutants rather than
GH cDNA-expressing plasmids (data not shown). This is the first
demonstration of a dominant negative effect of the mutant GH on
wild-type GH secretion in cultured cells. We then tested the effect of
dexamethasone treatment on the secretion of GH and the dominant
negative effect by the mutant GH, as this treatment is known to
increase the content of secretory granules in MtT/S cells (24). As
shown in Fig. 4B, the concentration of GH in medium 6 days after
transfection reached 15 ng/mL in cells transfected with phGHwt. The
magnitude of inhibition by cotransfection of a 5-fold excess of
phGHEx3 was 67% (Fig. 4B), which was greater than 50% inhibition
in the absence of dexamethasone (Fig. 4A). As shown in Fig. 4C, the
efficiency of transfection was consistent.
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, AtT-20 cells transfected with phGHwt secreted GH, and the concentration
of GH reached 30 ng/mL after 3 days of incubation. As observed in other
cell lines, no GH in the medium could be detected when phGHEx3 was
transfected. A metabolic labeling experiment in this cell line
confirmed that mutant GH is not secreted into the medium as it is in
COS-1 cells (data not shown). Cotransfection of phGHEx3 resulted in
significant inhibition of wild-type GH secretion. The magnitude of
inhibition was increased by increasing the amount of phGHEx3. Again,
the efficiency of transfection was consistent (among the wells)
(Fig. 5B).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the present study, we showed by metabolic labeling experiments using COS-1 cells that mutant GH is not secreted into the medium, but is retained in the cells. We also demonstrated that mutant GH can inhibit the secretion of wild-type GH in two neuroendocrine cell lines containing abundant secretory granules, namely AtT-20 and MtT/S (10, 25). As the inhibition was not observed in COS-1 or HepG2 cells, it was suggested that neuroendocrine cell-specific mechanisms, probably involving intracellular storage of secretory proteins, are required for the inhibition of wild-type GH secretion by mutant GH. The magnitude of inhibition in MtT/S cells was enhanced by dexamethasone treatment, which increases the content of secretory granules (24). This finding supports the hypothesis that intracellular storage is involved in the inhibition. This is the first experimental demonstration of the inhibition of wild-type GH secretion by mutant GH, which accounts at least in part for the pathogenesis of IGHD type II.
It should be noted that the inhibition of wild-type GH secretion observed in the present study was partial and required an excess of mutant GH-expressing plasmids. In contrast, serum GH levels remains as low as the lower limit of detection even after the administration of GHRH in patients with IGHD type II (6, 8). These discrepancies may be due to the relatively low efficiency of GH production in AtT-20 or MtT/S in this study. Pituitary somatotrophs produce huge amounts of GH, with the contents of GH reaching nearly 3% of the weight of the pituitary gland (26). We speculate that expression of large amounts of mutant GH could result in more prominent inhibition of wild-type GH secretion due to an increased chance of intermolecular interaction.
The normal 22-kDa GH molecule contains four cysteine residues, and these residues form two pairs of intramolecular disulfide bonds. As the mutant GH lacks 40 amino acid residues, including 1 cysteine residue, it has been previously speculated that uncoupled cysteine residues in the mutant GH molecule lead to the formation of intermolecular disulfide bonds, resulting in multimeric GH aggregates containing both mutant and wild-type molecules (6). Recently, a biologically inactive mutant GH that competes with wild-type GH for its receptor binding was reported (27). Interestingly, this mutant GH also has an uncoupled cysteine residue resulting from substitution of an arginine residue at codon 77 to cysteine. Yet, the heterozygous patient has high serum GH levels, indicating that the presence of an uncoupled cysteine residue is not sufficient to inhibit the secretion of wild-type GH.
Beside inhibition of wild-type GH secretion, cell toxicity by the accumulation of mutant GH should be considered as a mechanism of IGHD type II. The present study also provides an opportunity to test this possibility by monitoring ß-galactosidase activity, as it has been demonstrated that the reduction of reporter gene activity is linked to cell loss in transient transfection studies (23). The activity of ß-galactosidase in cells transfected with the mutant GH-expressing plasmid was not significantly lower than that in cells transfected with the wild-type GH-expressing plasmid. Thus, it is unlikely that the accumulation of mutant GH causes cell loss. However, we cannot exclude the possibility that the expression of mutant GH may lead to different consequences in premature somatotrophs during development. In familial central diabetes inspidus, the secretion of arginine vasopressin is not impaired in early life. The affected patients who are heterozygous for point mutations in the arginine vasopressin precursor manifest symptoms long after birth (28). In such a disease with delayed onset, a reduction of the number of cells due to the accumulation of the mutant protein is more likely to be involved in the pathogenesis. Indeed, cell loss by accumulation of mutant vasopressin precursors was recently demonstrated by experiments using cultured cells (29).
There are few dominantly inherited diseases caused by mutations in the genes encoding secretory proteins. Moreover, as far as we know, almost complete inhibition of secretion of the wild-type molecule by the mutant molecule is unique to IGHD type II. The present study for the first time demonstrated that mutant GH synthesized from IGHD type II mutant GH-I genes can inhibit wild-type GH secretion, and a neuroendocrine cell-specific mechanism and/or cosegregation of wild-type and mutant GH in the secretory granules were suggested to be involved in this mechanism.
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Acknowledgments |
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Footnotes |
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Received December 2, 1998.
Revised February 17, 1999.
Accepted February 24, 1999.
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References |
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X174 molecular mass marker. Lane
1, pTK-GHmutA; lane 2, pTK-GHmutE; lane 3, pTK-GHwt.
