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Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: ljameson{at}northwestern.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, resulting
in the activation of adenylate cyclase; cAMP can mimic most of the
effects of GHRH, including stimulation of GH synthesis and secretion
and proliferation of somatotrope cells in the pituitary
(1, 2, 3, 4). Inhibition of downstream effects of cAMP leads to
somatotrope depletion and dwarfism in transgenic mice (5).
A recent study revealed that inactivating mutations of the GHRH
receptor gene cause somatotrope hypoplasia and profound dwarfism in the
little mouse (lit/lit) (6). This
mutant receptor fails to bind GHRH (7), precluding
subsequent signaling by the peptide. Mutations in the human (h) GHRH
receptor have recently been identified in families with dwarfism
(8, 9, 10). These mutations generate a severely truncated
receptor or impair receptor processing, leading to hypoplasia of
pituitary somatotropes. Genetic treatment of such patients is
theoretically possible by transfer and expression of a wild-type GHRH
receptor. GH3 cells, a rat pituitary GH- and PRL-producing cell line, have been valuable for studies of hormonal secretory physiology (11) and signal transduction pathways. However, GHRH fails to stimulate GH secretion or GH messenger RNA production in GH3 cells (12), suggesting the absence of GHRH receptors in this cell line. Replication-deficient, recombinant adenovirus vectors represent a highly efficient means for transferring genes in vitro and in vivo and are being used in a wide variety of applications in cell culture, experimental animals, and human gene therapy. We tested the feasibility of GHRH receptor gene transfer using a recombinant adenovirus and analyzed the functional effects of GHRH receptor transfer to GH3 cells, including the effects on signal transduction and cell proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
AdAS, containing the antisense GHRH receptor, was used as a negative
control. AdGHGal, which contains ß-galactosidase driven by the hGH
promoter (-610, +58), was used as a reporter. AdCMVGal
(13) was used to determine the efficiency of gene
transduction. Individual clones of the recombinant adenoviral vectors
were purified and titrated by plaque assays. The sequences of the
expression cassettes in the adenoviral vectors were confirmed by
automated DNA sequencing of viral DNA using specific primers.
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Twenty-four-hour after plating cells, infections were carried out by the addition of viral solutions to cell monolayers and incubation at 37 C for 1 h with brief agitation every 15 min. After the addition of new culture medium, infected cells were returned to the 37 C incubator, and medium was changed 24 h later. Transduction efficiency of adenoviral vectors was tested in cell lines using AdCMVGal. ß-Galactosidase gene expression was detected in 95–100% of GH3 and COS-7 cells at 48 h after infection [5 plaque-forming units (PFU)/cell; data not shown]. Subsequent experiments were performed using the same concentration of the other recombinant adenoviral vectors.
For measurement of cAMP and pituitary hormones and for ligand binding studies, cells were plated in 12-well culture plates at a density of 2 x 105 cells/well. Triplicate wells were infected with adenoviral vectors at 5 PFU/cell. For Western blot analysis, cells were plated in 10-cm culture plates at a density of 5 x 106 cells/well. Cells were analyzed 48 h after infection of adenoviral vectors.
Immunofluorescence of GHRH receptor
Cells were plated on fibronectin-coated chamber slides
(Becton Dickinson and Co., Bedford, MA) and infected with
each virus at 5 PFU/cell. Forty-eight hours after infection, cells were
washed with PBS twice and fixed with 4% paraformaldehyde in PBS (pH
7.4) for 5 min. The slides were incubated with rabbit anti-hGHRH
receptor (1:1000; provided by Dr. Bruce Gaylinn, University of
Virginia, Charlottesville, VA) at room temperature for 1 h. After
washing with Tris-buffered saline/0.025% Tween, staining was performed
using biotinylated secondary antibodies (ABC kit, Vector Laboratories, Inc., Burlingame, CA), and
streptavidin-fluorescein isothiocyanate (1:100; Vector Laboratories, Inc.). The photomicrographs were taken using a
Carl Zeiss microscope (Axioskop, Carl Zeiss,
Oberkochen, Germany) and Fuji Photo Film Co., Ltd. color
film (1600 Super HG, Fuji Photo Film Co., Ltd., Tokyo,
Japan).
Measurement of ligand binding
GH3 or COS-7 cells infected with adenoviral vectors were
rinsed with 1.0 ml binding buffer: 50 mM HEPES, 100
mM sucrose, 5 mM
CaCl2·6H2O, 0.1%
(wt/vol) ovalbumin. 125I-labeled (50,000 dpm)
hGHRH-(1–44) amide (Amersham Pharmacia Biotech,
Piscataway, NJ) was added to each well with various amounts of
unlabeled hGHRH-(1–44) amide (Sigma, St. Louis,
MO) in binding buffer at the indicated concentration. Incubation was
continued at room temperature for 3 h, and cells were rinsed twice
with 1.0 ml binding buffer followed by the addition of 0.5 ml 1.0
N NaOH. After 1 h, the wells were scraped, and the
contents were transferred to a glass tube and washed twice with 0.25 ml
1.0 N NaOH. 125I radioactivity was
measured in a 
-counter (United Technologies Packard, Downers Grove,
IL), and binding curves were determined using Prism analysis software
(GraphPad Software, Inc., San Diego, CA).
Measurement of intracellular cAMP levels
GH3 and COS-7 cells infected with adenoviral vectors were
treated with 0.1 mM isobutylmethylxanthine for 20 min at 37
C. The hGHRH (1–44) amide was added in fresh warmed medium, and
incubation was continued for 30 min at 37 C. The medium was removed,
and 0.5 ml cold 0.1 M HCl was added to each well and
harvested. Cell lysates were centrifuged for 10 min at 4 C to remove
protein, and the supernatants were neutralized with an equal volume of
150 mM Tris-HCl (pH 8.6) containing 4 mM EDTA.
The cAMP levels were measured using a RIA kit (Biomedical Technologies, Stoughton, MA) according to the manufacturer’s
instructions.
Measurement of inositol phosphate accumulation
Twenty-four hours after infection, triplicate wells of cells
were labeled with tritiated myo-inositol (NEM Life Science Products,
Inc., Boston, MA) for 24 h. The medium was replaced by
Krebs-Ringer-bicarbonate buffer (pH 7.4) containing 20 mM
lithium chloride and hGHRH-(1–44) amide (10 nM) and
incubated for 1 h at 37 C. Ice-cold methanol (0.75 ml) was added
to each well, and cells were scraped into a tube containing 0.66 ml
chloroform and diluted by the addition of 0.75 ml water. The tubes were
sonicated for 10 sec and centrifuged at 10,000 rpm at 4 C. The
tritiated inositol phosphates (IP) in the aqueous layer were isolated
by ion exchange chromatography. The organic phase (0.2 ml), containing
nonhydrolyzed phosphatidyl inositol (PI), was evaporated in a
scintillation vial. Tritium was measured in a liquid scintillation
counter (Beckman Coulter, Inc., Fullerton, CA). Inositol
phosphate accumulation is expressed as a percentage tritiated IP
divided by the sum of tritiated IP and PI.
Measurement of GH and PRL in cell culture medium
GH3 cells infected with adenoviral vectors were washed
twice with prewarmed DMEM/Ham’s F-12 without serum, and the same
medium containing hGHRH-(1–44) amide (1 nM) was added.
Aliquots were collected at 5, 15, 30, and 60 min after the addition of
ligand. The samples were frozen at -20 C until assayed for GH and PRL.
The levels of GH and PRL were measured with a RIA kit provided by the
National Hormone and Pituitary Program (NIDDK, NIH).
Western blot analysis
GH3 cells and COS-7 cells were grown in a 10-cm plate and
infected with adenoviral vectors at 5 PFU/cell. Forty-eight hours after
infection, cells were treated with hGHRH-(1–44) amide (1
nM) for various time intervals. Cells were washed with
ice-cold PBS and harvested with 5 ml PBS+ (PBS, 1
mM EDTA, 1 mM phenylmethysulfonylfluoride and 1
mM dithiothreitol). Nuclear extracts were prepared by the
Shapiro method (14) modified by the addition of protease
inhibitor cocktail tablets, Complete (Roche Molecular Biochemicals, Indianapolis, IN), and 25 mM NaF. In
the experiments that involved GHRH receptor and
mitogen-activated protein kinase (MAPK), whole cell lysis was prepared
with lysis buffer [25% glycerol, 0.5 M NaCl, 1.5
mM MgCl2, 20 mM HEPES (pH
7.9), 1 mM phenylmethysulfonylfluoride, 0.2 mM
EDTA, 25 mM NaF, and protease inhibitor cocktail tablets,
Complete]. Proteins were solubilized in 1% Nonidet P-40. Equal
amounts of proteins were resolved by 10% SDS-PAGE and transferred onto
nitrocellulose filters. The membranes were blocked with 3% nonfat milk
in PBS for 1.5 h and then incubated overnight at 4 C with rabbit
polyclonal antibodies against either total cAMP response
element-binding protein (CREB) or
Ser133-phosphorylated CREB (Upstate Biotechnology, Inc., Lake Placid, NY). To detect Pit-1, c-Fos,
MAPK, or GHRH receptor, membranes were incubated with rabbit anti-Pit-1
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit
anti-c-Fos (Santa Cruz Biotechnology, Inc.), rabbit
anti-phospho-p44/p42 MAPK (New England Biolabs, Inc.,
Beverly, MA), or rabbit anti-hGHRH receptor antibody (provided by Dr.
Bruce Gaylinn). Immunoreactive proteins were detected using an
antirabbit horseradish peroxidase-conjugated antibody (1:5000) and the
enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL). Bands were detected with
Kodak (Rochester, NY) X-Omat film and quantitated using a
GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc.,
Hercules, CA).
GH promoter activation by GHRH in AdGHRH-R-infected GH3 cells
AdGHGal, an adenoviral vector containing the ß-galactosidase
gene driven by 0.67 kb of the hGH promoter, was used as a reporter to
evaluate the GHRH activation of GH promoter. Twenty-four hours after
coinfection (5 PFU/cell of each virus) of AdGHGal with AdGHRH-R or
AdAS, hGHRH-(1–44) amide was added, and the cells were incubated
overnight. Culture medium was aspirated, and cell lysis solution was
added. Triplicate wells of infected cells were used to measure
ß-galactosidase activity using O-nitrophenyl
ß-D-galactopyranoside as a substrate
(Sigma, St. Louis, MO). Cell lysates were mixed with the
O-nitrophenyl ß-D-galactopyranoside
substrate solution and incubated in 37 C for 1 h. The reaction was
stopped with 100 µl 1 M
Na2CO3. Absorption was
measured at 405 nm, and ß-galactosidase activity was calculated using
a standard curve.
Cell proliferation assays of GH3 cells infected with AdGHRH-R
Cell proliferation was measured using a nonradioactive cell
proliferation assay according to the manufacturer’s protocol
(Cell-Titer 96 Aqueous NonRadioactive Cell Proliferation Assay,
Promega Corp., Madison, WI). GH3 cell cultures were
depleted of estrogen for 4 days using phenol red-free DMEM/Ham’s F-12
containing 10% dextran/charcoal-stripped FBS. One day after plating
1 x 104 cells in quadruplicate wells of
96-well plates, adenoviral vectors were infected at 5 PFU/cell. Fresh
medium containing 1 nM hGHRH-(1–44) amide was added every
day for 4 days, at which time cell proliferation was measured. The
growth of GH3 cells was expressed relative to the growth
(mean ± SD) of cells infected with AdGHRH-R without
addition of GHRH.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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). A GHRH receptor-specific band of approximately 52
kDa was detected in GH3 and COS-7 cells infected with AdGHRH-R.
This band was not observed in cells infected with AdAS.
AdGHRH-R-infected GH3 cells also showed a higher molecular mass
band, presumably corresponding to different form of glycosylated GHRH
receptor.
GHRH binding to receptors was analyzed in cells infected with
adenoviral vectors (Fig. 3). GHRH
bound to AdGHRH-R-infected GH3 cells with a
Kd of 3.46 nM. The number of
expressed receptors expressed ranged from 186,000–277,000/cell. No
GHRH binding was detected in cells infected with AdAS. COS-7 cells
infected with AdAS exhibited a low amount of specific GHRH binding,
suggesting that these cells may have a small number of endogenous
GHRH-binding sites.
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). GH3 cells infected with AdAS
showed no cAMP increase in response to GHRH, whereas AdAS-infected
COS-7 cells showed a slight increase (24.5 ± 0.9
pM/well) in cAMP at high concentrations of GHRH
(10-7 M).
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). This suggests that the
cellular capacity to generate cAMP may be saturated by these high
levels of GHRH receptor expression.
To investigate the possibility of involvement of protein kinase C (PKC)
pathway in GHRH receptor signal transduction, inositol phosphate
accumulation was measured in cells expressing the GHRH receptor. TRH
was used as a positive control because the endogenous TRH receptor in
GH3 cells is linked to the PKC pathway. GHRH did not stimulate
inositol phosphate accumulation in GH3 or COS-7 cells infected
with AdGHRH-R (Fig. 5). In contrast, TRH
stimulated high levels of inositol phosphate accumulation in GH3
cells. This suggests that GHRH does not activate the PKC pathway in
somatotrope cells.
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, A and B).
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). There was no significant change in
the pCREB level in cells infected with AdAS. The total amount of CREB
was unchanged throughout this time course (Fig. 7).
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, Pit-1 was abundantly expressed in the
absence of GHRH in both GH3 cell groups infected with AdAS or
AdGHRH-R. GHRH did not alter Pit-1 expression. In contrast, GHRH
induced MAPK activation within 5 min and dramatically increased the
level of c-Fos by 2 h in GH3 cells infected with AdGHRH-R
(Fig. 8C).
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), presumably because these cells lack
the specific transcription factors required for GH promoter
function.
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GHRH receptor is an N-linked glycoprotein, and photoaffinity cross-linking studies have revealed three distinct receptor glycosylation forms, two complex and one core-glycosylated (high mannose) form (7, 17). In Western blot analyses, two GHRH receptor bands were seen in GH3 cells, whereas only one band was detected in COS-7 cells, suggesting that different forms of glycosylation may occur in various cell types.
It is notable that COS-7 cells, which are derived from kidney, exhibit low levels of GHRH binding and respond to high doses of GHRH. Mayo et al. (5) reported GHRH receptor messenger RNA expression in rat kidney using a RT-PCR assay. The biological role of the GHRH receptor in kidney, if any, is unknown.
The efficiency of infection combined with the strong activity of the CMV promoter presumably resulted in supraphysiological receptor expression in excess of available G protein. These conditions may result in uncoupled, low affinity receptors, as suggested by the relatively high Kd for GHRH. In contrast, cAMP production, which requires the coupled receptor, showed the expected higher affinity EC50.
An established function of the GHRH receptor in pituitary somatotrope cells is to mediate the release of GH in response to GHRH. Increased cAMP levels mediate the phosphorylation of ion channels, leading to the opening of a Na+-permeable ion channel, depolarization of the cell membrane, and influx of Ca2+ through the L-type Ca2+ channels. The increase in intracellular Ca2+ levels promotes GH release through the process of exocytosis (18). We have shown that adenovirus-mediated expression of GHRH receptor in GH3 cells confers GH secretion in response to GHRH. The ability to reconstitute the GHRH receptor signaling system using the adenovirus system will allow further investigation of GH secretory pathways.
GHRH stimulates transcription of the GH gene in pituitary somatotrope cells (19, 20). Stimulation of the cAMP pathway and activation of protein kinase A lead to phosphorylation and activation of the transcription factor CREB. It has been suggested that CREB induced synthesis of Pit-1 or perhaps other action of phospho-CREB lead to a subsequent increase in GH gene expression. In this study, we observed rapid CREB phosphorylation and GH gene stimulation after the addition of GHRH to GH3 cells expressing the GHRH receptor. However, the amount of Pit-1 protein did not increase significantly during the time of GHRH treatment. In agreement with previous studies (21, 22), basal expression of Pit-1 (in the absence of GHRH stimulation) is relatively high in these cells. These observations may explain the high basal activity of the GH promoter in GH3 cells, but the basis for further activation of the GH promoter by GHRH remains to be determined. Possible explanations include 1) phosphorylation-mediated activation of Pit-1 protein; 2) cAMP responsiveness through nonclassical cAMP response element motifs in the hGH promoter; 3) CREB-independent regulation by CREB binding protein; or 4) MAPK activation.
It has been demonstrated that GHRH induces c-Fos expression (23, 24), a factor that may be involved in the proliferation of somatotrope cells. Somatostatin inhibits the proliferation of GH3 cells, and it inhibits GHRH induced c-fos expression (25). The mitogenic effect of GHRH has been known to be mediated by the activation of the cAMP pathway; increased cAMP stimulates c-Fos expression through its cAMP response element. In GH3 cells, Pit-1 also enhances c-Fos promoter activity by binding to the serum response element in the c-Fos promoter (26). In this study we observed that GHRH stimulated the proliferation of GH3 cells expressing GHRH receptor. We also demonstrated activation of MAPK and an increase in c-Fos expression after treatment with GHRH. Recently, Pombo et al. (27) demonstrated MAPK activation by GHRH in CHO cells stably expressing GHRH receptor. It has been demonstrated that cAMP analogs and TRH activate MAPK, leading to cellular proliferation of GH3 cells (28, 29). MAPK is instrumental in several signal transduction pathways involved in cell. Once activated, MAPK translocates to the nucleus, where it induces transcription factors, including c-Fos and c-Jun. It is likely that MAPK activation plays a role in mediating the mitogenic action of GHRH.
In summary, we demonstrated restoration of GHRH responsiveness of pituitary GH3 cells in response to GHRH after expression of functional hGHRH receptor using recombinant adenoviral vectors. These results indicate that adenoviral vectors carrying the hGHRH receptor gene are useful for in vitro studies of GHRH receptor biology and represent a first step toward the long-term goal of gene therapy for dwarfism caused by GHRH receptor mutations.
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Acknowledgments |
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Footnotes |
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Received August 1, 2000.
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