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Department of Physiology and Biophysics, University of Alabama, Birmingham, Alabama 35294-0005
Address all correspondence and requests for reprints to: Dr. Jimmy D. Neill, Department of Physiology and Biophysics, 812 McCallum Building, 1918 University Boulevard, University of Alabama, Birmingham, Alabama 35294-0005. E-mail: neill{at}uab.edu
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Introduction |
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The mechanisms giving rise to these alterations in GnRH receptor
signaling are unknown, but they do not appear to be due to changes in
GnRH receptor number (4, 5, 6) or to alterations in LH stores in the
pituitary gland (4, 5, 6). Two other molecular loci, however, have been
implicated recently as potential modulators of GnRH receptor signaling:
one is the receptor itself (7), and the other is the G protein that
transduces the signal generated by the receptor binding to its ligand
(8). With respect to the latter mechanism, regulators of G protein
signaling (RGSs) have been identified (9, 10, 11) that antagonize the
interaction of the G
protein with its effector, such as
phospholipase Cß (12, 13), or that accelerate the hydrolysis of GTP
by the G protein, thereby prematurely terminating signaling due to
reassociation of the G protein with Gß
-subunit complex (9).
With respect to action at the receptor, G protein-coupled receptor
kinases (GRKs) act in an agonist-specific manner to induce
phosphorylation of intracellular regions of the receptor; this permits
ß-arrestins to bind, thereby preventing G protein association with
the receptor (14, 15).
In earlier studies that suggested a role for GRKs and RGSs in modulating GnRH receptor signaling, we used a heterologous cell system (COS kidney cell line) in which the complementary DNAs (cDNAs) encoding the GnRH receptor and GRK (7) or RGS (8) were cotransfected before testing for GnRH-stimulated inositol trisphosphate (IP3) production. A heterologous rather than a homologous cell system was used in these studies because heterologous cells can be transfected, whereas pituitary cells cannot; about 30% of COS cells (a homogeneous cell line) can be transfected and hence express the protein encoded by the cDNA, whereas pituitary cells that are composed of only 10–15% gonadotropes (16) can be transfected at an efficiency of less than 1% using even the most effective methods available (our unpublished findings). In the studies described here, we adopted a gene transfer approach (17) using adenoviruses for expression of GRK2 in pituitary cells that have an efficiency of infection near 100%. The gene transfer approach has the important advantage that a secretory product of gonadotropes, LH, is the parameter measured to determine the effect of GRK2 on GnRH receptor signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HEK293 cells (ATCC CRL-1573, human embryonic kidney, adenovirus type 5 transformed) were obtained from the American Type Culture Collection (Manassas, VA) and cotransfected with the pACCMVpLpa and pJM27 plasmids using lipofectamine (Life Technologies, Grand Island, NY). Lipofectamine (16.7 µl) was diluted in 300 µl OptiMEM I, as was 0.83 µg of each of the two plasmids (pJM17 and GRK2-pACCMVpLpa); the two 300-µl aliquots were combined and allowed to stand at room temperature for 15 min. Then the lipofectamine/plasmid solution was added, and the dish was placed in an incubator (37 C, 8% CO2) for 5 h. Next, DMEM/2 x Pen-Strep/20% FBS was added to each dish, which was returned to the incubator. When the medium in a dish turned yellow (usually 3–4 days), 80% of it was removed, and 4 ml fresh DMEM/10% FBS containing penicillin, streptomycin, and fungizone were added. Two to 3 weeks after cotransfection when most of the 293 cells had detached, the cells and medium were frozen (-70 C) and thawed three times to release virus from the cells (lysate), and cellular debris was removed by centrifugation at 2500 x g for 10 min.
PCR was performed on viral DNA extracted from each lysate to confirm the presence of the GRK cDNA insert. In brief, viral DNA was extracted from each dish by combining its lysate 1:1 with Hirt buffer (40 mM EDTA, 0.1% SDS, and 0.5 mg/ml proteinase K) followed by incubation at 56 C for 1 h. Samples were then treated with phenol/chloroform twice and precipitated with ethanol. The DNA from each sample was resuspended in 50 µl TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0), and PCR was performed on each sample using primers directed at vector sequences on either side of the multiple cloning site in pACCMVpLpa (5'-CTCTGTAGGTAGTTTGTCCA-3' directed at the simian virus 40 sequence and 5'-GAGGTCTATATAAGCAGAGC-3' directed at the cytomegalovirus promoter; see map of the pACCMVpLpa plasmid in Ref. 17). Conditions for PCR were standard (20), and 0.75 µM of each primer was used. The cycle was 95 C for 1 min, 56 C for 1 min, and 72 C for 2 min. The presence of an insert on an electrophoretic agarose gel having the size appropriate for GRK2 cDNA was accepted as evidence of the presence of adenoviruses bearing the GRK2 cDNA insert.
For isolation by cloning of adeno-GRK2, we diluted viral lysates by 103-, 104-, 105-, 106-, 107-, 108-, and 109-fold in DMEM/10% FBS; 2 ml diluted virus were placed in a dish of HEK293 cells, and incubated for 1 h at 37 C. Agarose (1.3%; PharMingen, San Diego, CA) in DMEM, penicillin/streptomycin, fungizone, and 2% FBS were mixed at 40 C and added to each dish. After solidification of the agarose, the dishes were kept in the incubator upside down until viral plaque formation, which required 4–7 days. At that time a dish was chosen with plaques that were well separated so that individual plaques could be retrieved using large orifice pipette tips (200 µl; USA Scientific, Ocala, FL); the resulting core of agarose containing the viral plaque was then frozen and thawed three times as described above to release the viruses. For small scale amplification of the virus, we added the lysate containing the cloned adeno-GRK2 to dishes of subconfluent 293 cells and incubated them for 1 h at 37 C. Four milliliters of DMEM/10% FBS containing fungizone were added. Cells and medium were harvested as before when most of the cells had detached from the petri dish (typically 2–3 days). Cells and medium were frozen and thawed as before to release the virions, and the lysate was collected after centrifugation as described above. PCR analysis to confirm the presence of the GRK2 cDNA insert was performed as detailed above.
For large scale amplification and titrating of adeno-GRK2, we added viral lysate to cultures of 293 cells in T175 flasks. After incubation at 37 C for 30 min in the CO2 incubator, 27 ml DMEM/5% FBS were added, and the flasks were returned to the CO2 incubator. When the cells were mostly detached (24–48 h), the remaining adherent cells were dislodged by gentle pipetting of the incubation medium. The cell suspension was pelleted by centrifugation at 350 x g for 10 min. Ninety percent of the supernatant fluid was aspirated and discarded; the pelleted cells were gently put into suspension using a Pasteur pipette. The cells were frozen and thawed three times using a -70 C freezer and cool water for thawing. Tubes were spun at 2500 x g for 10 min to pellet the cellular debris. The lysates from the tubes were collected, pooled, and stored at -70 C. This lysate contains the adeno-GRK2 for infection of rat pituitary cells. It was titrated by the viral plaque assay described earlier using triplicate determinations at 106-, 107-, 108-, and 109-fold dilutions of the lysate. Viral yields were in the range of 109 plaque-forming units/ml lysate.
Infection of rat pituitary cells with adenovirus
Sprague Dawley CD rats (Charles River Laboratory, Inc., Wilmington, MA) were maintained in accordance with the NIH
Guide for the Care and Use of Laboratory Animals, and the experimental
protocol was approved by the institutional animal care and use
committee. Anterior pituitary glands from rats undergoing normal
estrous cycles were dispersed with trypsin as described previously
(21). The cells were plated into poly-L-lysine-coated petri
dishes in DMEM/10% horse serum containing 10-9
M estradiol to optimize the GnRH responsiveness of the
gonadotropes (22) and were incubated overnight at 37 C in a
CO2 incubator.
For infection of pituitary cells, a volume of adeno-GRK2 or
adeno-ß-galactosidase (adeno-ßgal) necessary to achieve the chosen
multiplicity of infection (MOI; number of viral particles per pituitary
cell) diluted to 0.3 ml was further diluted in 1.2 ml TS buffer (8
g/liter NaCl, 0.38 g/liter KCl, 0.1 g/liter
Na2HPO4, 3.0 g/liter Tris, 200 mg/liter
CaCl2·2H2O, and 100 mg/liter
MgCl2·6H2O). The medium from cell cultures
was aspirated and replaced with the virus solution, and the cells were
then incubated at 37 C for 1.5 h. The medium in the dishes was
replaced next by DMEM/10% horse serum containing 10-9
M estradiol. The cells were then incubated at 37 C in a
CO2 incubator.
After about 72 h, the cells were collected by brief trypsinization and counted. Then they were handled as follows 1) without further manipulations, some cells were tested for viability and others were used for receptor radioassay; and 2) cells were plated for 2 h at 37 C, and then subjected to X-Gal staining, to enzyme-linked immunosorbent assay (ELISA) for GRK2, to measurement of LH content, or to stimulation by D-Ala6-desGly10-GnRH ethylamide (GnRH-A) for measurement of LH release or intracellular IP3 content. Cell counts and viabilities were determined using trypan blue solution (0.4% in saline) and a hemocytometer. Recoveries of cells at this point averaged about 50% of the number plated before the control or adenovirus treatments were applied. Differing numbers of cells were plated depending on their intended use; for GnRH stimulation of LH secretion, we plated 100,000 cells/well in a 24-well plate; 50,000 cells/well were plated in a 24-well plate for X-Gal staining, 10,000 cells/well were plated in 96-well plates for ELISA, 100,000 cells/well were plated in a 6-well plate for measurement of LH content, and 3 x 106 cells/60-mm dish were used for measurement of IP3.
Detection of ß-galactosidase expression in cells infected with
adeno-ßgal
Cells were fixed with 0.05% glutaraldehyde (Sigma Chemical Co., St. Louis, MO) in PBS. The X-Gal stain
(Fisher Scientific, Fairlawn, NJ) was first dissolved as a
2.0% solution in dimethylformamide, which was then diluted to 0.2% in
buffer (2 mM MgCl2, 5 mM
K4Fe(CN)6·3H2O, and 5
mM K3Fe(CN)6 dissolved in PBS). The
solution was then filtered using a 0.22-µm syringe filter to remove
potentially undissolved X-Gal crystals. The X-Gal solution was then
incubated with the cells for 3 h at 37 C. Stained cells were
stored in PBS. The percentage of stained pituitary cells was determined
microscopically by counting 500 cells/well.
Measurement of GRK2 expression by ELISA (23) in cells infected with
adeno-GRK2
The cells were fixed in freshly prepared 3.5% paraformaldehyde
in PBS for 8 min at room temperature. Then, 0.1% Nonidet P-40
(Sigma Chemical Co.) was added for a 15 min incubation at
room temperature to achieve cell permeabilization. The wells were then
rinsed three times with PBS before a blocking solution (3% BSA in PBS)
was added and incubated for 2 h at room temperature. Next, GRK
antiserum diluted 1:1000 in 3% BSA was incubated with the cells
overnight at 4 C. Then, second antibody (goat antirabbit IgG conjugated
to alkaline phosphatase obtained from Sigma Chemical Co.)
diluted in 3% BSA was incubated with the cells for 3 h at room
temperature. Substrate (Sigma Chemical Co. 104–0)
dissolved at 1 mg/ml in diethanolamine solution (210 mg diethanolamine
and 20.3 mg MgCl2 · 6H2O in 200 ml of
H2O, pH 9.5) was added to each well. Plates were incubated
at room temperature and analyzed spectrophotometrically at
OD405 in a Molecular Devices UV Max Microplate
reader.
The antibody used to detect GRK2 was provided by Dr. Robert J. Lefkowitz. It is a polyclonal antibody generated in rabbits against the carboxyl-terminal 220 amino acids of rat GRK3 fused to glutathione-S-transferase (rabbit 7428) (24). This antiserum binds both GRK3 and GRK2, as there is 76% homology between the amino acid sequences of the C-termini of these two proteins (24); indeed, it binds GRK2 much more strongly than GRK3 (24).
Assay of GnRH-stimulated LH secretion in cells infected with
adeno-GRK2
For LH secretion assays, medium was aspirated after the 2-h
plating period (25) and was replaced with DMEM/0.1% BSA containing
10-7 M GnRH-A) for incubation at 37 C for 90
min. The medium was then collected and centrifuged at 200 x
g for 10 min to pellet any detached cells. The supernatant
was carefully removed and stored at -20 C before LH RIA.
LH RIAs were performed using reagents and instructions provided by Dr. A. F. Parlow on behalf of the National Hormone and Pituitary Program, NIDDK, NIH. Rat LH (NIDDK-rLH-I-9) was used for radioiodination, the LH antiserum was NIDDK-anti-rLH-S-11 prepared in rabbits, and the reference preparation was NIDDK-rLH-RP3.
Reverse hemolytic plaque assay for detection of LH secretion from
individual gonadotropes
This assay was performed as described previously by us (26). In
brief, ovine red blood cells (oRBC) were covalently coupled to protein
A, and the pituitary cells in culture were trypsinized and mixed with
oRBC for infusion into poly-L-lysine-coated Cunningham
chambers. The chamber was then filled with DMEM/0.1% BSA containing LH
antiserum and 10-7 M GnRH-A. The slides were
incubated in a CO2 chamber for 2 h at 37 C, and guinea
pig complement was added and incubated for 30 min. LH secretion results
in the complement-mediated lysis of LH antibody-coated oRBC around the
gonadotropes so that clear areas of lysis (plaques) surrounds them. The
presence of a plaque around a cell identifies it as a secretory
gonadotrope.
Intracellular IP3 measurements
These measurements were made with a RRA described by us
previously in detail (7, 27). In brief, IP3 receptors in
calf cerebellar membranes were mixed with
[3H]IP3 and a sample comprised either of
unlabeled IP3 standard (0.3–48.0 pmol) or pituitary cell
extract. Bound and free [3H]IP3 were
separated by centrifugation, and the precipitate was dissolved in NaOH
solution before liquid scintillation spectroscopy. In preparation for
measurements of IP3 concentrations, 3 x
106 anterior pituitary cells infected with adeno-GRK2 (6
MOI) or adeno-ßgal (6 MOI) 72 h previously were preincubated for
2 h and then treated with 10-7 M GnRH-A
in DMEM-0.1% BSA for 5 min. IP3 was extracted by removing
the incubation medium and adding cold 16.6% trichloroacetic acid. The
cells were scraped from the wells and centrifuged, and the supernatant
was extracted with diethyl ether to remove the trichloroacetic acid.
The samples were then heated to evaporate the residual ether before
being subjected to IP3 assay.
Assay of LH contents in pituitary cells
For LH RIA measurements of cell contents, 1 x
105 anterior pituitary cells infected, or not, 72 h
previously with adeno-GRK2 (60 MOI) or adeno-ßgal (60 MOI) were
trypsinized and plated for 2 h. LH was extracted from the cells
(28) by removing and discarding the medium and adding 1 ml extraction
buffer (150 mM NaCl, 50 mM Tris, 5
mM EDTA, and 1 mM bacitracin, pH 7.4) followed
by freeze-thawing twice. After centrifugation, the supernatant was
removed and subjected to LH RIA as described above.
RRA measurements of GnRH receptors
About 3 x 106 pituitary cells infected, or
not, 72 h previously with adeno-ßgal (6 MOI) or adeno-GRK2 (6
MOI) were trypsinized, and cell membranes containing the GnRH receptors
were prepared by placing the cells in a Dounce homogenizer (Kontes Co.,
Vineland, NJ) and disrupting them with 30 strokes of the pestle. The
cell membranes were pelleted by centrifugation at 46,000 x
g and then resuspended in 10 mM Tris buffer. The
GnRH RRA on the membranes was performed as described previously (7, 27). Cell membranes and 1 x 105 cpm
[125I]GnRH-A were incubated at 0 C for 90 min.
Nonspecific binding was determined as counts per min bound in the
presence of 10-6 M unlabeled GnRH-A. Bound and
free [125I]GnRH-A were separated by filtration. The
filters were counted by -scintillation spectrometry.
Data analysis
The results are presented as the mean ± SEM of
at least three independent experiments unless indicated otherwise. The
statistical tests used were one-way ANOVA with significant differences
(P < 0.05) identified by Bonferroni’s method.
Statistical analysis was performed using SigmaStat Statistical Software
for Windows (Jandel Scientific, San Rafael, CA).
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Results |
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Our initial approach to the feasibility of adenovirus-mediated gene
transfer in cultured anterior pituitary cells was to determine the
fraction of cells that were infected by the virus and that subsequently
expressed a marker protein. Therefore, we infected pituitary cells with
an adenovirus encoding ß-galactosidase, a protein not normally
expressed by pituitary cells, and one that can be detected with a
simple and sensitive assay. Figure 1
illustrates that about 60 MOI of adeno-ßgal infected nearly all of
the pituitary cells as indicated by the expression of ß-galactosidase
in the infected group vs. the control group. In 10
independent experiments, 97.4 ± 1.7% (mean ±
SD) of all pituitary cells were infected with 60 MOI of
adeno-ßgal (range, 94.6–100.0%).
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illustrates our findings from
two independent experiments that all LH plaque-forming cells stained
for ß-galactosidase, and that there was no difference in the size or
number of LH plaques between the control and adeno-ßgal-infected
groups.
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(upper panel) illustrates our finding from two independent
experiments that 60 MOI of adeno-ßgal infected 97.2% of the
pituitary cells, as indicated by their expression of ß-galactosidase;
92.7% of the cells were viable, as determined by trypan blue
exclusion. A higher dose (600 MOI) of virus (data not shown) was toxic;
although 98.2% of the cells expressed ß-galactosidase, viability was
only 65%. Lower doses of viruses (Fig. 3) were uniformly nontoxic,
with viabilities ranging from 98.7–99.5%; however, the fraction of
cells infected by adeno-ßgal fell progressively with reduction in
dose of virus so that 80% of the cells were infected at 6 MOI, 50% at
0.6 MOI, and 10% at 0.06 MOI. Clearly, 60 MOI is the near-optimal dose
of adenovirus.
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, bottom panel). The two lowest doses of adeno-GRK2
(0.06 and 0.6 MOI) did not increase GRK expression above that observed
in the control group; all three groups exhibited OD405
values in the range of 0.6–0.7, probably reflecting endogenous
expression of GRK2, because substitution of normal rabbit IgG for the
GRK antiserum produced an OD405 of less than 0.1. The two
highest doses of adeno-GRK2 (6 and 60 MOI) elevated expression of GRK2
to approximately 1.4 and 2.6 OD405 units, illustrating that
adeno-GRK2 infection of rat pituitary cells induces synthesis of GRK2.
The approximately 5-fold increase in OD405 values derived
from intracellular GRK2 translates into an approximately 12.5-fold
increase in GRK2 expression; this conclusion rests on a comparison of
the cell ELISA with a standard ELISA where recombinantly produced GRK2
was attached directly to the wells of a 96-multiwell plate before
reaction with GRK2 antiserum using the same conditions as those for the
cell ELISA (data not shown). Measurement of GnRH-stimulated LH
secretion in the groups expressing GRK2 yielded a significant negative
correlation between GRK2 concentration and LH secretion (r =
-0.84; P < 0.05); LH concentrations fell
progressively from 9.8 ng/ml in the control group to 2.8 ng/ml in the
group infected with 60 MOI of adeno-GRK2. Collectively, these findings
demonstrate that 60 MOI of adenovirus is the optimum infective dose,
that infection of rat pituitary cells in vitro with
adeno-GRK2 induces high level expression of GRK2, and that the level of
GRK2 expression is related inversely to the amount of LH secretion
evoked by GnRH treatment.
Additional experiments using only the two highest doses of adeno-GRK2
and adeno-ßgal (60 and 6 MOI) for infection were performed to
increase the data to a level sufficient for statistical analysis (Fig. 4). Sixty MOI of adeno-ßgal infected
96.3 ± 0.52% (mean ± SEM) of the anterior
pituitary cells, whereas 6 MOI infected 79.8 ± 2.39%. These
values are similar to those shown in Fig. 3. Some 94.9 ± 1.3% of
the pituitary cells were viable when infected with 60 MOI of adenovirus
(adeno-GRK2 and adeno-ß-gal), whereas 98.3 ± 0.6% were viable
after infection with 6 MOI. Figure 4 illustrates that GRK2
immunoreactivity was expressed at high levels in the adeno-GRK2 groups
relative to those in the adeno-ßgal and control groups
(P < 0.05). Sixty MOI induced a greater increase in
GRK2 expression compared with 6 MOI (P < 0.05). The
normal rabbit serum group (Fig. 4) was comprised of cells that were not
infected with adenovirus and in which normal rabbit serum was
substituted for the GRK3 antibody; the results demonstrate that the
immunoreactivity observed in the control (uninfected) and adeno-ßgal
groups was probably due to endogenously expressed GRK2 in the rat
anterior pituitary cells. Confirming findings presented earlier (Fig. 3), an approximately 5-fold increase (P < 0.05) in the
GRK2 OD405 value was observed in the 60 MOI adeno-GRK2
group relative to those in the adeno-ßgal and control groups (Fig. 4); as noted before, this translates into a 12.5-fold increase in GRK2
expression. GnRH-stimulated LH secretion, on the average, varied from
14.8–17.7 ng/ml in the control and adeno-ßgal groups (Fig. 4),
differences that were not statistically significant (P
> 0.05). Both doses of adeno-GRK2 (60 and 6 MOI) inhibited
GnRH-stimulated LH secretion about the same (P > 0.05)
despite 60 MOI inducing a higher level of GRK2 expression
(P < 0.05). These inhibitions were on the order of
80–85% (Fig. 4). Therefore, 60 MOI adeno-GRK2 infection induced a
12.5-fold increase in GRK2 expression, which was associated with an
85% decrease in GnRH-stimulated LH secretion.
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). Adeno-ßgal was viewed as being a more appropriate control than
adenovirus without an insert because it induced high level protein
synthesis, as did adeno-GRK2.
Several additional experiments were performed to initiate a search for
the molecular site of GRK2 action. In the first (Fig. 5A), we measured the LH content of rat
pituitary cells. Figure 5A demonstrates that depletion of LH stores is
an unlikely explanation for GRK2-induced inhibition of GnRH-stimulated
LH secretion because adeno-GRK2 infection significantly
(P < 0.05) increased LH stores in the cells relative
to the control group; the LH contents did not differ significantly
between the adeno-ßgal and adeno-GRK2 groups (P >
0.05). These results suggest the existence of a small, but significant,
change in pituitary gonadotrope LH concentrations. The fact that it was
an increase and not a decrease suggests that it is not related to the
decrease in GnRH-stimulated LH secretion; indeed, the inhibition of LH
release by GRK2 might have been greater except for this apparent
nonspecific effect.
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, decreases in GnRH receptor binding do not account for the suppression
of GnRH-stimulated LH secretion by GRK2. In fact, infection with 60 MOI
of either adeno-ßgal or adeno-GRK2 increased GnRH receptor binding by
50–100% (P < 0.05), whereas the two adenoviral
groups were not different from each other (P > 0.05;
Fig. 5B). These results, like those in Fig. 5A, suggest a specific
change induced by the adenovirus. The fact that a significant increase
in GnRH-stimulated LH release (Fig. 4) was not observed in the
adeno-ßgal group vs. the control group suggests that GnRH
receptor binding is poorly correlated with responsiveness of
GnRH-stimulated LH release (Fig. 5B), an observation that has become
classic (4, 5, 6).
A third experiment on the potential molecular site of the GRK2
inhibitory action was measurement of GnRH-stimulated intracellular
concentrations of IP3. Adeno-GRK2 infection significantly
(P < 0.05) suppressed GnRH-stimulated IP3
production compared with that observed in cells infected with
adeno-ßgal (Fig. 6). The results of
this experiment suggest that the site of GRK2 action is phospholipase
Cß or earlier in the signal transduction process
(Gq protein or the GnRH receptor).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Some success has been reported using transient transfections of primary
pituitary cells using the glycoprotein gene promoter together with
a reporter gene (34); however, these studies require that only a small
fraction of the gonadotropes express this construct. Insofar as we are
aware, there are no studies in which most of the gonadotropes have
undergone DNA-mediated transfection or otherwise were loaded with the
protein of interest. We have conducted an extensive series of
experiments attempting to transfect all or a significant fraction of
pituitary cells in culture (unpublished); we used a eukaryotic green
fluorescent protein vector (pEGFP-N1, CLONTECH Laboratories, Inc., Palo Alto, CA) as a reporter together with numerous
transfecting reagents: 1) CaCl2 (18), 2) diethylaminoethyl
dextran (18); 3) lipofectin and lipofectamine, 4) SuperFect
(Qiagen), and 5) diethylaminoethyl dextran together with
adenovirus (35). In all cases, a significant fraction of COS-1 cells
was transfected (10–30%), but in no case did we observe more than 1%
of the pituitary cells to be transfected. We also tried two methods
that are dependent on permeabilizing cells in the presence of a protein
or antibody: 1) scrape-loading (36) of pituitary cells in the presence
of goat antimouse IgG coupled to fluorescein resulted in loading fewer
than 1% of the cells; and 2) streptolysin 0 permeabilization (37)
completely inhibited GnRH responsiveness of gonadotropes as measured in
the reverse hemolytic plaque assay even though lactotropes remained
secretory. Clearly, methods of transfection or permeabilization that
are effective in many cells and cell lines do not work with pituitary
cells, including gonadotropes. Finally, we considered but rejected
microinjection (38) into gonadotropes preidentified by reverse
hemolytic plaque assay because of the laboriousness of the injection
procedure as well as the subsequent measurement of secretion in
individual gonadotropes.
Another method for expression of proteins in cultured cells is adenovirus-mediated gene transfer (17). Adenovirus is a DNA virus commonly used for gene therapy (39, 40, 41). Replication deficient, but infectious, adenovirus vectors have been generated by replacing the E1 gene (which is essential for replication) with the gene of interest and an enhancer-promoter element; the recombinant virus vectors are then replicated in cells such as HEK293 that express the E1 gene (41). This approach has been used to infect and express a gene of interest in 70–100% of normal cells from liver and endocrine pancreas (17), brain (40), and cardiac muscle (42), among many others. Newgard and colleagues in particular have used recombinant adenovirus to advantage in studies of metabolic regulation and endocrine mechanisms (17, 43). Anterior pituitary cells do not seem to have been tested for their infectivity by adenovirus and their subsequent expression of genes of interest.
We report here that pituitary cells are remarkably efficiently infected by the recombinant adenovirus. Indeed, they rival the near 100% infectivity of hepatocytes, the cell type among all others that is most efficiently infected by adenovirus (17). As long as the dose of adenovirus is kept at 60 MOI or less there do not appear to be untoward effects, including cell death. In fact, a change in LH content and in GnRH receptors was induced by the adenovirus, but it was an increase; however, this did not complicate the interpretation of the pivotal result, which was a decrease in LH secretion. Therefore, we seem to have successfully applied a powerful new approach to the study of GnRH receptor signaling in gonadotropes. Of course, this approach should be applicable as well to the study of lactotropes, corticotropes, somatotropes, thyrotropes, and folliculo-stellate cells, as nearly all pituitary cells are infected by the adenovirus. It will be interesting to determine whether this approach can be used to ablate gene products of interest using antisense cDNA constructs in adenovirus or their neutralization using dominant negative mutant constructs. Also of interest will be the determination of whether this approach can be extended to infection of pituitary cells in the whole organism, as has been done for hepatocytes and pancreatic ß-cells (17).
Inhibition of LH secretion from gonadotropes adenovirally infected with
and expressing GRK2 confirms our earlier studies using transfection and
expression of GRK2 in heterologous cells, where decreases in the second
messenger, IP3, were used to indicate inhibition of GnRH
receptor signaling (7). GRK2 was shown here to also suppress
GnRH-stimulated IP3 increases in gonadotropes; this
suggests that GRK2 is acting at one of three loci: phospholipase Cß,
the G protein that transduces GnRH effects
(Gq), or the GnRH receptor. The classic site
of action of GRKs is the receptor where they phosphorylate its
intracellular regions, thereby permitting ß-arrestins to bind that
prevent G protein association with the receptor (14, 15). However, we
have been unable to detect phosphorylation of the epitope-tagged GnRH
receptor expressed in COS-1 cells undergoing GnRH-induced
desensitization (27) or in similar cells transfected with and
expressing GRK2 (7). The failure to detect phosphorylation of the GnRH
receptor in our previous studies may be due simply to unrecognized
technical problems in phosphorylating or immunoprecipitating the
receptor. This possibility is under investigation using a different
epitope for tagging the GnRH receptor. Alternatively, this failure may
reflect the fact that GRKs do not phosphorylate the GnRH receptor but,
instead, inhibit the function of the receptor by other mechanisms
(44).
In conclusion, the significance of our findings is 2-fold: 1) adenovirus-mediated gene transfer permits investigation of the regulatory role of gene products in the cell of interest, the gonadotrope, rather than in heterologous cell systems; and 2) additional, stronger evidence is provided that supports a role for GRKs in setting the responsiveness of GnRH receptor signaling (7).
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Acknowledgments |
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Footnotes |
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Received September 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits. Cell 86:445–452[CrossRef][Medline]
and block
activation of phospholipase Cß by -thio-GTP-Gq.
Proc Soc Natl Acad Sci USA 94:428–432T3–1 and HEK-293 cells expressing the GnRH
receptor. Endocrinology 136:5228–5231[Abstract]
3 and
Gs, in dopamine and thyrotropin-releasing hormone signal
transduction: evidence for competition and commonality. J Endocrinol 148:455
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,
P < 0.05 vs. the 60 MOI adeno-GRK2
group, both adeno-ßgal groups, and the control group. 
