Adenovirus-Directed Expression of a Nonphosphorylatable Mutant of CREB (cAMP Response Element-Binding Protein) Adversely Affects the Survival, but Not the Differentiation, of Rat Granulosa Cells
Jeremy P. Somers,
Julie A. DeLoia and
Anthony J. Zeleznik
Departments of Cell Biology and Physiology The University of
Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261
Magee-Womens Research Institute Department of Obstetrics,
Gynecology and Reproductive Sciences University of Pittsburgh
School of Medicine Pittsburgh, Pennsylvania 15213
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ABSTRACT
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Although usually considered to be a
constitutively expressed protein, in the primate ovary the expression
of CREB (cAMP response element-binding protein) is extinguished after
ovulation, and its loss is temporally associated with the cessation of
proliferation of luteal cells and the ultimate commitment of the corpus
luteum to undergo regression. To determine the cellular consequences of
the loss of CREB expression, we expressed a nonphosphorylatable mutant
of CREB (CREB M1) in primary cultures of rat granulosa cells using a
replication-defective adenovirus vector. Expression of CREB M1 did not
block granulosa cell differentiation as assessed by acquisition of the
ability to produce estrogen and progesterone in response to FSH or
forskolin. However, granulosa cells expressing CREB M1, but not
adenovirus-directed ß-galactosidase or enhanced green fluorescent
protein, exhibited a 35% reduction in viability that was further
reduced to 65% after stimulation with 10 µM
forskolin. These results demonstrate that the trophic effects of cAMP
(proliferation and survival) on ovarian granulosa cells are
functionally separate from the effects of cAMP on differentiation and
provide novel evidence that CREB may function as a cell survival factor
in the ovary. The separation of signaling pathways that govern
differentiation and survival in the ovary thereby provides a mechanism
by which progesterone production, which is absolutely essential for the
maintenance of pregnancy, can continue despite the cessation of
proliferation of luteal cells and their commitment to cell death
(luteolysis).
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INTRODUCTION
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The ovarian cycle involves progressive stages of cell
proliferation, differentiation, and cell death (1). As the ovarian
follicle matures under the control of FSH, granulosa cells proliferate
and differentiate into steroid-producing cells to form a mature
Graafian follicle (2). Upon ovulation, the Graafian follicle is
transformed into a corpus luteum whose production of progesterone under
the influence of LH is absolutely required for the establishment and
maintenance of pregnancy (3). An enigma in the understanding of ovarian
function is although both FSH and LH, at least in part, exert their
actions thorough the cAMP intracellular signaling system (4), the
responses of granulosa cells and luteal cells to their respective
trophic hormones differ. In particular, the actions of FSH on the
follicle include both proliferation and stimulation of steroid
production. However, after ovulation, luteal cells no longer
proliferate and become committed to cell death but progesterone
production by these cells remains highly responsive to LH (5, 6).
We demonstrated previously that the expression of the cAMP-dependent
transcription factor CREB (cAMP response element-binding protein) is
extinguished upon the luteinization of granulosa cells in the monkey
ovary and proposed that the loss of CREB signaling might subtract the
expression of a subset of genes involved in the control of cellular
proliferation and survival (7). We reasoned that the consequences of
the loss of CREB in luteal cells could be determined by eliminating the
CREB-signaling pathway in progenitor granulosa cells. To accomplish
this, we used a recombinant adenovirus vector that directs the
expression of a mutant of CREB (CREB M1) in which the activating
phosphorylation site at serine 133 is mutated to alanine (8). Herein we
show that while interfering with CREB signaling in granulosa cells does
not inhibit their differentiation into steroid-producing cells,
expression of CREB-M1 adversely affects their viability. These findings
suggest that CREB may function as a molecular switch that governs cell
survival in the ovary and support the rapidly emerging notion that CREB
may function as a survival factor in a number of different cell
types.
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RESULTS
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Recombinant Adenoviruses Direct ß-Galactosidase Expression
with High Efficiency in Primary Cultures of Rat Granulosa Cells
To determine the feasibility of using adenoviruses to direct the
expression of recombinant proteins in granulosa cells, we employed an
adenovirus vector (Ad ß-gal) that directs the expression of a nuclear
targeted ß-galactosidase reporter gene under the control of a
cytomegalovirus (CMV) promoter. Surprisingly, as shown in Fig. 1
, Ad ß-gal-directed ß-galactosidase
activity in primary cultures of immature rat granulosa cells was
hormone responsive. In the absence of hormonal stimulation (panel A),
ß-galactosidase staining was weak. In contrast, incubation of
granulosa cells with either 10 ng/ml human (h)FSH (panel B) or 10
µM forskolin (FSK) (panel D) resulted in a more
intense staining of individual cells. The expression of
ß-galactosidase was ligand-dependent as immature granulosa cells that
lack LH receptors did not respond to the addition of 10 ng/ml hLH
(panel C).

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Figure 1. Effect of FSH, LH, and FSK on ß-Galactosidase
Expression in Primary Cultures of Rat Granulosa Cells Infected with Ad
ß-gal
Granulosa cells were cultured and exposed to Ad ß-gal at a 1:100
dilution. Twenty-four hours after viral infection, the culture medium
was removed and cells were exposed to medium alone (A), 10 ng/ml hFSH
(B), 10 ng/ml hLH (C), or 10 µM FSK (D). After 48 h
of exposure to stimulatory agents, cell cultures were stained for
ß-gal activity for 30 min.
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Functional Characterization of Ad CREB M1
Figure 2
(top
panel) illustrates a Western immunoblot using an antibody directed
against the amino terminus of CREB on whole-cell lysates of granulosa
cells that were infected with increasing amounts of the Ad CREB M1
adenovirus. Exposure of granulosa cells to increasing viral titers
resulted in progressive increases in immunoreactive CREB expression as
compared with noninfected cells. When cell lysates from noninfected
granulosa cells were subjected to immunoprecipitation with an anti-CREB
antibody and the immunoprecipitates exposed to protein kinase A (PKA)
and [32P]-ATP in vitro, intense
phosphorylation of a 43-kDA protein was observed (Fig. 2
, bottom
panel). In contrast, immunoprecipitates of granulosa cells from Ad
CREB M1-infected cells were weakly phosphorylated by PKA in
vitro as would be expected by the elimination of the PKA-dependent
phosphorylation site on ser 133.

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Figure 2. Adenovirus-Mediated Expression of CREB M1 in
Primary Cultures of Rat Granulosa Cells
Top panel, Granulosa cells were cultured and exposed to
increasing concentrations of Ad CREB M1. Ninety-six hours after viral
infection, cell lysates were prepared and assessed for immunoreactive
CREB by immunoblot analysis. Fifty micrograms total cell protein were
applied to each lane. Bottom panel, Fifty micrograms of
whole-cell lysates of granulosa cells that were exposed to control
medium or a 1:100 dilution of Ad CREB M1 for 96 h were
immunoprecipitated with anti-CREB antiserum and the immunoprecipitates
were subjected to in vitro phosphorylation by the
catalytic subunit of PKA and [32P]-ATP. Labeled proteins
were separated by electrophoresis and identified by autoradiography.
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Ad CREB M1 Inhibits the Expression of CMV ß-Galactosidase
To determine whether CMV-ß-gal stimulation is CREB dependent,
primary cultures of rat granulosa cells were infected with increasing
amounts of Ad CREB M1 in the presence of a fixed amount of Ad ß-gal
(1:1000). Figure 3
shows that Ad CREB M1
inhibited FSH-stimulated CMV-ß-galactosidase activity in a
dose-dependent fashion, with the highest Ad CREB M1 concentration
(1:100 dilution) completely inhibiting ß-galactosidase expression.
This concentration of Ad-CREB also inhibited ß-galactosidase
expression in response to 10 µM FSK and 0.5
mM 8-Br-cAMP (data not shown).

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Figure 3. Ad CREB M1 Inhibits FSH-Stimulated CMV
ß-Galactosidase Expression in Cultured Rat Granulosa Cells
Cells were infected with Ad ß-gal (1:1000 dilution) in the presence
of increasing concentrations of Ad CREB M1. Twenty-four hours after
virus infection cells were exposed to medium alone or medium containing
10 ng/ml hFSH. Forty-eight hours after exposure to virus, equal volumes
of granulosa cell lysates were enzymatically assessed for
ß-galactosidase activity. Results show means ± 1
SEM of duplicate independent observations.
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Effect of Ad CREB M1 on Granulosa Cell Steroidogenesis
To determine the effects of the CREB M1 mutant on granulosa
cell function and the specificity of the Ad CREB M1 vector, we compared
the steroidogenic responses and CMV-ß-galactosidase expression of
primary cultures of rat granulosa cells not infected by adenoviruses
with those infected with Ad CREB M1 as well as those exposed to
identical concentrations of an adenovirus that directs the expression
of enhanced green fluorescent protein under the control of the CMV
promoter (Ad EGFP). Consistent with previous results, both FSH
and FSK stimulated ß-galactosidase activity in noninfected cells and
cells infected with the control Ad EGFP adenovirus. In contrast,
ß-galactosidase expression was completely abrogated in cells infected
with the Ad CREB M1 virus (Fig. 4A
). The
observation that ß-galactosidase activity was somewhat lower in Ad
EGFP-infected cells in comparison to noninfected cells may be due to
competition of the CMV promoters for endogenous transcriptional
regulators, as both basal and FSK-stimulated ß-galactosidase
activities were reduced proportionately. The relative increases in
ß-galactosidase activity in response to FSK were comparable in cells
not exposed to virus and those exposed to Ad EGFP (18-fold
vs. 13-fold, respectively).

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Figure 4. Effects of Ad CREB M1 and Ad EGFP on Granulosa Cell
Steroidogenesis
Granulosa cells were cultured and exposed to either Ad EGFP or Ad
CREB M1 at 1:100 dilutions in the presence of a 1:1000 dilution of Ad
ß-gal. Twenty-four hours after virus infection, cells were exposed to
medium containing 10 ng/ml testosterone (as a substrate for aromatase),
testosterone plus 10 ng/ml hFSH, or testosterone plus 10
µM FSK. Medium was collected at 24 and 48 h after
addition of FSH or FSK for estrogen and progesterone analysis by RIA.
After 48 h exposure to FSH or FSK, granulosa cells (floating and
attached) were collected, and equivalent amounts of cell lysates were
assessed for ß-galactosidase activity. Panel A illustrates
ß-galactosidase activity; panel B, estradiol; and panel C,
progesterone concentrations in culture medium. Results show means
± 1 SEM of quadruplicate independent incubations.
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Figure 4B
illustrates that estrogen production after 48 h exposure
to FSH or FSK was comparable by cells not exposed to adenovirus and
cells exposed to Ad EGFP. The responses to FSK were statistically
significant (P < 0.05, n = 4 independent
observations) when compared with untreated cells. Although cells
exposed to Ad CREB M1 produced lower amounts of estrogen when compared
with noninfected cells or Ad EGFP-infected cells, they retained their
responsiveness to FSH and FSK (P < 0.05). For all
groups, there was a net increase in estrogen concentration between
samples collected at 24 and 48 h after exposure to FSH or FSK
(data not shown). Identical results were observed with progesterone
production (Fig. 4C
).
Effect of Ad CREB M1 on Granulosa Cell Survival
Figure 5
illustrates the morphology
of the granulosa cells from the study shown in Fig. 4
. The top
panel presents fluorescent microscopic analysis in the granulosa
cells infected with Ad EGFP. Like that of the CMV-ß-gal, the
expression of the CMV promoter-directed EGFP was FSH and FSK
responsive. The center panel illustrates the morphology of
Ad EGFP-infected granulosa cells. Well defined monolayers were present
in cells not exposed to stimuli as well as cells exposed to FSH or FSK.
The lower panel illustrates the morphology of granulosa
cells exposed to Ad CREB M1. In the absence of hormone stimulation or
in the presence of FSH, granulosa cells were present in well defined
monolayers. In marked contrast, Ad CREB M1-infected granulosa cells
that were exposed to 10 µM FSK displayed pronounced
morphological alterations that included profound cell clumping, which
was associated with the detachment of the majority of the cells from
the culture dish.

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Figure 5. Morphology of Granulosa Cell Monolayers
Before removal of culture medium and collecting granulosa cells for
assessment of ß-galactosidase activity, cell cultures from the study
presented in Fig. 3 were photographed. The top panel
shows granulosa cells exposed to Ad EGFP and photographed with
fluorescence microscopy (x200). The center and lower
panels show bright field photographs of granulosa cells exposed
to Ad EGFP and Ad CREB M1, respectively (x100). Identical results were
observed in four independent experiments.
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To determine whether the morphological changes observed in Ad CREB
M1-infected cells were associated with a reduction in cell viability,
granulosa cells were exposed to either medium alone, Ad EGFP, or Ad
CREB M1 followed by incubation in the presence and absence of 10
µM FSK; cells were then assessed for viability at
the indicated times after the addition of FSK as shown in Fig. 6
. There were no significant differences
in the viability of cells (P > 0.05) exposed to Ad
EGFP compared with cells not exposed to virus either in the presence or
absence of FSK. Although not apparent by visual examination, granulosa
cells infected with Ad CREB M1 and not exposed to FSK exhibited a
significant (P < 0.01) decline in viability at 48 and
72 h when compared with cells not infected with virus or cells
infected with Ad EGFP. At both 48 h and 72 h, incubation of
Ad CREB M1-infected cells with 10 µM FSK resulted in a
further decrease in viability when compared with Ad CREB M1-infected
cells not exposed to FSK (P < 0.01). The reduced
production of estrogen and progesterone in Ad CREB M1-infected
granulosa cells in response to FSK (Fig. 4
, B and C) is thus likely due
to a diminished number of viable cells in the culture.

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Figure 6. Effect of Ad CREB M1 and Ad EGFP on Granulosa Cell
Viability
Granulosa cells were cultured and exposed to either no virus, Ad EGFP,
or Ad CREB M1 at 1:100 dilutions. Twenty-four hours after virus
infection, cells were exposed to M199 alone or M199 containing 10
µM FSK. Cell cultures were assessed for viability by the
CellTiter assay at 48 and 72 h after the addition of FSK. Results
show means ± 1 SEM of four independent experiments.
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Although expression of CREB M1 adversely affected survival of granulosa
cells in primary culture, a comparable effect was not observed in
established ovarian cancer cell lines. No alteration of cell morphology
in response to 10 µM FSK was observed in Ad CREB
M1-infected human ovarian cancer cell lines SKOV-3 or OV 1063.
MTT viability assay (-FSK vs. +FSK) were 0.968
vs. 1.008 for SKOV-3 cells and 1.650 vs. 1.699
A490 units for OV 1063 cells (means of duplicate
observations). In addition, no cytopathic effects in response to 10
µM FSK were evident in Ad-CREB M1-infected primary
cultures of rat Sertoli cells under conditions in which Ad CREB M1
completely blocked the FSK stimulation of a c-fos-CRE
reporter gene (9).
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DISCUSSION
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Our results demonstrate that expression of a CREB M1 in ovarian
granulosa cells adversely affects their survival but does not abolish
their ability to differentiate into steroid-producing cells in response
to agents that stimulate cAMP production. The separation of signaling
pathways that govern differentiation (steroidogenesis) and survival in
the ovary thereby provides a mechanism by which progesterone
production, which is absolutely essential for the maintenance of
pregnancy and the survival of the species, can continue despite the
cessation of proliferation of luteal cells and their commitment to cell
death (luteolysis). The observations that CREB may participate in a
proliferation/survival pathway in the ovary is consistent with findings
from a number of other cell systems. Expression of CREB M1 in
transgenic animals results in proliferative defects of pituitary
somatotrophs, T lymphocytes, and cardiac myocytes (8, 10, 11), and
transfection of FTRL-5 thyroid cells with a different CREB mutant
reduced thymidine incorporation in response to TSH (12). In addition,
human somatotroph adenomas resulting from activating mutations of G
s
are associated with elevated levels of phosphorylated CREB (13).
CREB was first identified as a nuclear protein that is phosphorylated
by PKA and binds to the cAMP response element (CRE) on the
somatostatin gene promoter and stimulates transcription (14). Since its
identification as a PKA-dependent transcriptional activator, it has
become apparent that CREB is a nuclear target of a number of
intracellular signaling pathways including mitogen-activated
protein kinase (15), p90RSK (16), and calmodulin
kinases (17, 18) as well as the antiapoptotic Akt/PKB pathway (19).
Given the wide diversity of inputs that may converge on CREB, it is not
unreasonable to suggest that CREB may be involved in basic processes of
cellular homeostasis including proliferation and cell death. Our
current finding that expression of CREB M1 results in diminished
viability of granulosa cells is consistent with other recent studies
indicating that CREB may function as a survival factor. Thus, in
addition to causing cell cycle arrest in T cells, the expression of a
dominant-negative mutant of CREB in transgenic animals also resulted in
apoptosis of these cells in response to agents that would normally
stimulate activation (10). Likewise, expression of a dominant-negative
mutant of CREB in human melanoma cells led to reduced tumorigenesis as
well as an enhanced sensitivity of these cells to apoptosis (20).
Whether the diminished viability of granulosa cells as assessed by the
MTT assay seen in the current study is the result of apoptotic
cell death is not known and will be the subject of future research.
During the course of our studies, we observed that the expression
of ß-galactosidase under the control of a CMV promoter was highly
responsive to FSH, FSK, and cAMP stimulation. In retrospect, the
responsiveness of the CMV promoter to cAMP is not surprising as it has
been shown by others that the CMV promoter contains three cAMP response
elements (CREs) and is stimulated by FSK (21). Using the hormonal
dependence of CMV-ß-galactosidase as a reporter for documenting the
effectiveness of CREB M1, we demonstrated that FSH- and FSK-stimulated
ß-galactosidase expression was completely blocked by overexpression
of the mutant CREB. Assuming that there are no differences between the
ability of CREB M1 to inhibit the transcription of the extrachromosomal
CMV-ß-galactosidase gene and endogenous genes, we would conclude that
CREB-mediated expression of endogenous genes was dramatically
compromised in these cells. Despite the apparent inhibition of
CREB-dependent transcription, estradiol and progesterone production by
CREB M1-expressing granulosa cells remained highly responsive to
FSH and FSK. Although we have not measured mRNA levels for
steroidogenic enzymes in the present study, it is well recognized that
the acquisition of the ability of granulosa cells from immature rats to
produce estrogen and progesterone is due to cAMP-mediated increases in
mRNAs for aromatase (P450arom), cholesterol side chain cleavage
(P450scc), and 3ß-hydroxysteroid dehydrogenase, 54 isomerase
(3ß-HSD) (22). We would therefore infer from our studies that those
genes involved in progesterone and estrogen production are not CREB
dependent. This is consistent with the findings of others that the
promoter regions of P450scc and 3ß-HSD do not appear to contain CREs
(23, 24) and that P450arom, which does contain a CRE-like sequence,
also appears to be responsive to steroidogenic factor-1 (25). The
separation of cAMP-regulated signaling pathways that control survival
from those that control differentiated phenotype (steroidogenesis) in
the ovary would serve to ensure that progesterone production, which is
essential for the maintenance of pregnancy, continues even though
trophic actions of cAMP are lost after ovulation.
A number of potential mechanisms exist by which the elimination of
CREB-mediated signaling in granulosa cells may adversely affect cell
proliferation and survival. CREB may act as a transcription factor that
directly regulates genes involved in these processes. One candidate
gene is proliferating cell nuclear antigen (PCNA), an auxiliary factor
for DNA polymerase
, which is required for both replicative and
repair DNA synthesis, as both the murine and the human PCNA gene
promoters contain obligatory CREs (26, 27). Indeed, in the primate
ovary, the expression of both PCNA and CREB cease upon luteinization
(7). A second possible candidate gene is cyclin D2, which has been
shown to be FSH and FSK responsive in rat granulosa cells (28, 29).
Interestingly, ovarian function in the cyclin D2 knockout mouse is
similar to that seen in our current study as granulosa cell
proliferation, but not granulosa cell differentiation
(steroidogenesis), is disrupted (28). It is not yet known, however,
whether expression of the cyclin D2 gene is CREB dependent.
Alternately, CREB could also act by controlling the expression of other
transcription factors that regulate genes involved in proliferation and
survival such as c-fos and C/EBPß, both of which are
CREB-dependent (30, 31). In this regard it is noteworthy that the
C/EBPß knockout mouse is infertile due to defects in the
luteinization process (32).
In our current study, pronounced morphological disruptions and reduced
granulosa cell viability were most apparent when Ad CREB M1-infected
cells were stimulated by FSK. This observation is analogous to those
obtained with other cells in which the expression of inactive CREB
mutants resulted in diminished cell viability. Thus, Jean et
al. (20) did not observe marked cytopathic effects of a CREB
mutant on melanoma cells unless the cells were treated with
thapsigargin, which elevates intracellular free Ca++
concentrations. Likewise, Barton et al. (10) did not observe
pronounced cell death in T cells that express a nonphosphorylatable
CREB mutant unless the cells were presented with activating stimuli. As
FSK has been shown to elevate intracellular free Ca++ in
granulosa cells (33), it is possible that the elimination of
CREB-mediated signaling allows other pathways that are antagonistic to
cell proliferation and survival to exert dominance, possibly by
increasing their ability to interact with limited amounts of nuclear
coactivators (34). In this regard, previous studies by Aharoni
et al. (35) demonstrated that treatment of granulosa cells
from rat preovulatory follicles with very high concentrations of FSK
(50 µM) promoted apoptosis but, like that seen in our
study, did not inhibit steroid production. As we did not observe any
cytotoxic effects of a lower concentration (10 µM) of FSK
in noninfected cells or cells infected with either Ad ßgal or Ad EGFP
(Figs. 1D
, 5
and 6), it appears that the loss of CREB signaling
rendered granulosa cells more sensitive to adverse effects of FSK, as
would be expected if competition exists between CREB and other
antagonistic signaling pathways.
A caveat to the interpretation of our current results is the assumption
that the cytopathic effect of CREB M1 on granulosa cells is due
specifically to the competition of this mutant protein with endogenous
CREB for binding to CREs within the regulatory regions of
CREB-responsive genes. However, it remains a possibility that
overexpression of CREB M1 in granulosa cells may squelch the activity
of other signaling pathways. For example, in addition to forming
homodimers, CREB is also able to form heterodimers with other members
of the bZip family of transcription factors, such as ATF-1 (36). Thus,
an alternate explanation for our findings could be that the effects of
CREB M1 could indirectly be mediated through sequestration of ATF 1 or
other transcription factors. Although we cannot rule this out, our
previous studies with the primate ovary failed to detect, by
Southwestern analysis, any CRE-binding proteins other than CREB in
luteal cells (7). In addition, CREB has been shown to interact in a
phosphorylation-independent manner with components of the TFIID complex
(37). However, such an interaction with the general transcription
factor apparatus would be expected to globally suppress transcription,
which would be inconsistent with our findings that CREB M1 did not
block granulosa cell differentiation. Finally, it is important to note
that cytopathic effects of CREB M1 expression were not observed in
either ovarian epithelial cancer cells (as presented in
Results) or primary cultures of rat Sertoli cells (9). The
latter is significant in view of the fact that Sertoli cells, which are
the testicular homologs of granulosa cells, do not require FSH-mediated
cAMP signaling for their survival in vivo (38).
In summary, our current results suggest that CREB may function as a
molecular switch that governs cell proliferation and survival in the
ovary, results that are in keeping with other recent observations that
CREB may function as a general regulator of cell proliferation and
survival (8, 9, 10, 11, 12, 13, 19, 20). Extrapolation of these observations to the
in vivo state suggests that the loss of CREB expression that
occurs in the primate corpus luteum could be directly causal to the
cessation of proliferation of luteal cells and, ultimately, the cell
death that occurs during luteal regression.
The recent report that phosphorylated CREB is absent from nuclei of rat
luteal cells indicates that an impairment in CREB signaling also occurs
in rodents (39). Although the downstream targets of CREB that may
participate in cell proliferation and survival are not known, the
ability to rapidly and effectively interfere with CREB-mediated
signaling in primary cell cultures with replication-defective
adenovirus vectors provides a novel approach to address this important
problem.
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MATERIALS AND METHODS
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Construction of the CREB M1 Recombinant Adenovirus
The adenovirus shuttle vector pACsk.2CMV CREB M1 was
constructed by excising a 2.2-kb fragment containing the CREB M1 coding
region and the SV40 polyadenylation signal from the plasmid pRSVCREB M1
(8) by digestion with EcoRI. The CREB M1 insert was ligated
in frame into the multiple cloning site of EcoRI-digested
pET-23a (Novagen, Madison, WI) to create a T7-tag CREB M1 fusion gene.
The T7-CREB M1 coding region was excised from the pET vector by
digestion with SalI and BglII and then subcloned
downstream from the CMV5 promoter into the SalI and
BglII sites of the multiple cloning site of the adenovirus
shuttle plasmid pACsk.2CMV5 (gift of Dr. Joseph
Alcorn).
The methods for generating and propagating recombinant adenoviruses are
described in detail elsewhere (40). In brief, 10 µg of the plasmid
pACsk.2CMV CREB M1 were cotransfected with 10 µg of the
plasmid pJM17, a plasmid containing a circularized adenovirus type 5
(variant dl309) genome (40) into the human embryonic kidney cell line
293 (41) using a calcium phosphate transfection system according to the
manufacturers instructions (Life Technologies, Inc.,
Gaithersburg, MD). After transfection the cells were maintained in DMEM
containing 4.5 g/liter glucose (Life Technologies, Inc.)
and 10% FBS at 37 C in 5% CO2 for 13 days at which time
the cells exhibited viral cytopathic effect. Cells and tissue culture
supernatants were collected, frozen on dry ice and thawed three times,
and then centrifuged (1000 x g, 4 C, 10 min) to remove
cellular debris. Aliquots of virus stocks were diluted 50- and 100-fold
in lysis solution [0.1% SDS, 10 mM Tris-Cl (pH 7.4), 1
mM EDTA] and incubated for 10 min at 56 C in a shaking
water bath. The absorbance of the samples was measured at 260 nm, and
the value obtained was used to calculate virus content using the
equation 1.0 absorbance units = 1.1 x 1012 virus
particles/ml (42). Adenoviruses were propagated by infecting 293 cells
with approximately 108 particles/ml in tissue culture
medium without serum. Infected cells were incubated until they
exhibited nearly complete cytopathic effect and processed as above.
Virus stocks were prepared to a concentration of 6 x
1012 particles per ml as described above and diluted for
use as indicated in Results.
Granulosa Cell Culture and Adenovirus Infection
All procedures were approved by the Magee-Womens Research
Institute Institutional Animal Use and Care Committee. Immature female
rats (20 or 25 days old) were purchased from Taconic Farms, Inc. (Germantown, NY) and were housed under standard husbandry
conditions. For studies shown in Fig. 2
, animals received a
subcutaneously placed 1-cm SILASTIC capsule containing crystalline
diethylstilbestrol (Sigma Chemical Co., St. Louis,
MO) on day 20 of age and were killed on day 25 of age. The remainder of
the studies used granulosa cells from untreated 22- to 25-day-old rats.
Granulosa cells were collected from the ovaries by puncturing follicles
with a 25-gauge hypodermic needle, and cells were expressed into Medium
199 (M199; Life Technologies, Inc.) containing 10% FBS.
Granulosa cells were seeded into 6-well (
106 cells per
well) or 24-well (
2 x 105 cells per well) tissue
culture plates and allowed to attach overnight. The next morning,
medium and unattached cells were removed and the granulosa cell
monolayers were exposed to adenoviruses in M199 without protein
supplements for 2 h at 37 C with occasional rocking. Medium was
replaced with fresh M199 containing 1 mg/ml BSA (Sigma Chemical Co.). Twenty-four hours after exposure to adenoviruses, medium
was removed and replaced with M199 plus BSA also containing 10 ng/ml
testosterone (Sigma Chemical Co.) alone or with addition
of human FSH (AFP 4161B; 3205 IU FSH/mg < 225 IU LH/mg) or FSK
(Sigma Chemical Co.).
Immunoblot Analysis of Lysates from AdCREB M1-Infected Primary
Rat Granulosa Cell Cultures
Granulosa cells were harvested by scraping into ice-cold PBS
followed by centrifugation (16,000 x g, 4 C, 10 min).
Pelleted cells were resuspended in Western lysis buffer (50
mM Tris-Cl, pH 7.4, 1 mM EDTA, 20 µg/ml
phenylmethylsulfonylfluoride, 0.5 µg/ml leupeptin, 0.7 µg/ml
Pepstatin A, 10 nM Microcystin LR), lysed by sonication,
and processed for anti-CREB immunoblotting as described previously (7)
using a rabbit anti-rat CREB antibody directed against the first 205
amino acids of the amino-terminal region of CREB (Upstate Biotechnology, Inc., Lake Placid, NY).
Immunoprecipitation and in Vitro PKA
Phosphorylation
Ad CREB M1-infected and control granulosa cells were harvested
in ice-cold RIPA buffer [150 mM NaCl, 10 mM
Tris-Cl (pH 7.5), 0.1% (wt/vol) SDS, 1% (vol/vol) NP-40]
supplemented with protease and phosphatase inhibitors (20 µg/ml
phenylmethylsulfonylfluoride, 0.5 µg/ml leupeptin, 10 nM
Microcystin LR, 200 µM sodium vanadate).
Immunoprecipitation of CREB was performed using the antibody directed
against the amino-terminal region of CREB as described above, and
in vitro phosphorylation of immunoprecipitates by PKA were
performed as described previously (43).
ß-Galactosidase Assays
Identification of ß-galactosidase-expressing cells in
granulosa cell cultures was assessed by histochemistry using X-gal as
substrate (42). Granulosa cell monolayers were fixed in 2%
paraformaldehyde for 60 min, washed three times in PBS, and incubated
in Xgal solution containing 2 mM
K3Fe(CN)6, 2 mM
K4Fe(CN)6, 1 mM MgCl2,
and 1 mg/ml Xgal. Quantification of ß-galactosidase activity was
performed by fluorometric analysis (44). Granulosa cells were harvested
by scraping into ice-cold PBS, and centrifuged (16,000 x
g, 4 C, 10 min) to pellet the cells. The PBS was removed,
and the cell pellets were frozen on dry ice and then stored at -80 C
until use. Cell pellets were thawed on ice with the addition of 100
µl ß-galactosidase assay buffer (150 mM Tris-Cl, pH
7.5, 10 mM MgCl2, 25 mM NaCl, 10
mM 2-mercaptoethanol). Identical volumes of cell extract
were added to a reaction mixture containing 150 mM Tris-Cl,
pH 7.5, 10 mM MgCl2, 25 mM NaCl, 10
mM 2-mercaptoethanol, 150 nM
4-methylumbelliferyl ß-galactoside, and 1 mg/ml BSA and incubated at
30 C for 30 min, after which reactions were stopped by the addition of
0.5 ml of 0.25 M glycine (pH 10.65). Three hundred
microliters of each assay were transferred to a 96-well dish, and
fluorescence was measured using an excitation wavelength of 340 nm and
emission wavelength of 460 nm.
Steroid Production
Estradiol and progesterone concentrations of culture medium were
determined by RIAs as described previously (45).
Cell Viability Assay
Viability of granulosa cells was assessed using the CellTiter
assay (Promega Corp., Madison WI) in which a tetrazolium
salt is bioreduced into a formazan by metabolically active cells.
Assays were performed according to the manufacturers direction and
were terminated 4 h after the addition of substrate, at which time
the production of the formazan, measured by recording the absorbance at
490 nm, was linear as a function of time.
Statistics
Results were assessed for statistical significance by ANOVA
followed by comparison of group means with Duncans multiple range
analyses (46).
 |
ACKNOWLEDGMENTS
|
---|
We thank Joseph Alcorn, Ph.D. (Department of Pediatrics,
University of Texas Medical School, Houston, TX) for providing us with
the Ad ß-gal and valuable suggestions for the preparation of
adenoviruses; Andrea Gambotto, M.D. (Department of Molecular Genetics
and Biochemistry, University of Pittsburgh School of Medicine) for the
Ad EGFP; Marc Montminy, M.D., Ph.D., (Joselin Diabetes Center, Boston,
MA) for providing us with the CREB M1 cDNA; The National Hormone and
Pituitary Program (NIDDK, NIH) for the hFSH; and Ms. Lynda Little-Ihrig
for performing RIAs.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Anthony J. Zeleznik, Ph.D., Magee-Womens Research Institute, 204 Craft Ave., Pittsburgh, Pennsylvania 15213.
This work was supported by a Johnson & Johnson Focused
Giving Award and NIH Grant HD-16842 (A.J.Z.).
Received for publication February 2, 1999.
Revision received May 3, 1999.
Accepted for publication May 13, 1999.
 |
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