1 Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098; 2 University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5; and 3 Pacific Northwest National Laboratory, Richland, Washington 99352
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ABSTRACT |
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Although ovarian surface epithelial (OSE) cells are responsible for the majority of ovarian tumors, we know relatively little about the pathway(s) that is responsible for regulating their proliferation. We found that phosphatidylinositol 3-kinase (PI3K) is activated in OSE cells in response to elevated extracellular calcium, and the PI3K inhibitors wortmannin and LY-294002 inhibited extracellular signal-regulated kinase (ERK) activation by ~75%, similar to effects of the mitogen-activated protein kinase/ERK kinase inhibitor PD-98059. However, in assays of proliferation, we found that PD-98059 inhibited proliferation by ~50%, whereas wortmannin inhibited >90% of the proliferative response to elevated calcium. Expression of a dominant negative PI3K totally inhibited ERK activation in response to calcium. These results demonstrate that ERK activation cannot account for the full proliferative effect of elevated calcium in OSE cells and suggest the presence of an ERK-independent, PI3K-dependent component in the proliferative response.
ovarian surface epithelial cells; signal transduction; extracellular signal-regulated kinase; mitogen-activated protein kinase; Akt; calcium-sensing receptor
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INTRODUCTION |
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OVARIAN CANCER is the most lethal form of female genital cancer, and it is responsible for more than half of the deaths from female genital malignancies. Malignant ovarian tumors are primarily derived from ovarian surface epithelial (OSE) cells (20), whereas tumors from other ovarian tissues are mostly benign (21). OSE cells undergo wounding at ovulation, and this may result in an increase in the formation of OSE tumors, because a positive correlation exists between the number of ovulatory cycles a woman has experienced and her risk of ovarian cancer (21, 23). Because OSE cells are the cell type responsible for ovarian tumors, an understanding of the proliferative pathways used by these cells is of obvious importance. Although OSE cells show a proliferative response to a variety of stimuli, including follicle-stimulating hormone (2), hepatocyte growth factor (13), and elevated calcium (14, 18), the relative importance of distinct signal transduction pathways in mediating the proliferation of OSE cells is not known.
In our previous work, we found that OSE cells respond to elevation of calcium levels with increased proliferation (19). Furthermore, we determined that the calcium-sensing receptor (CaR) is expressed, functional, and essential for calcium-proliferative signaling in human and rat OSE cells (14, 18). The CaR belongs to the C subfamily of seven transmembrane-spanning G protein-coupled receptors (GPCR) (5). In addition to proliferative responses, the CaR is a vital mediator of parathyroid hormone release, with the CaR-inhibiting parathyroid hormone release in the presence of high calcium levels (6). In addition to OSE cells, the CaR is also present in the kidney (22), nerve terminals (25), bone (7), and a number of other tissues.
Activation of the CaR in OSE cells is associated with increased proliferation and increased extracellular signal-regulated kinase (ERK) activity (14, 18). Additional evidence of this association is given by the observation that both the proliferative response to calcium and activation of ERK by calcium are inhibited by a dominant negative (DN) CaR construct (14, 18). The pathways involved in the proliferative response to calcium stimulation were partially elucidated by the observation that the proliferative response of OSE cells to calcium was inhibited by ~50% in the presence of the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD-98059, whereas the general tyrosine kinase inhibitor herbimycin causes a >90% inhibition (14).
In this report, we demonstrate that phosphatidylinositol 3-kinase
(PI3K) is a key mediator of the calcium response in OSE cells.
Inhibition of PI3K with chemical inhibitors or with a p110 kinase
dead DN PI3K construct prevented activation of ERK above the basal
level. Additionally, we demonstrate that the PI3K inhibitor wortmannin
and the ERK inhibitor PD-98059 both inhibit ERK to a nearly equal
extent. However, the ERK inhibitor PD-98059 only inhibits
[3H]thymidine uptake by ~50% in response to calcium
stimulation, whereas the PI3K inhibitor wortmannin inhibits
[3H]thymidine uptake by >90%. These data strongly
suggest that, although PI3K activity is essential for activation of
ERK, there are additional proliferative pathways that do not involve
ERK yet still require PI3K activity. These pathways appear to be
responsible for a significant portion of the proliferative response of
OSE cells to calcium.
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MATERIALS AND METHODS |
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Cell culture. These studies were conducted on the immortalized OSE (IOSE)-120 and IOSE-80 cell lines, derived from primary cultures of normal ovarian surface epithelial cells by transfection with SV40 large T antigen to expand the life span of these cells in culture. Although these IOSE cells can no longer be considered normal, they are not immortal (senescing after ~50 cell doublings), and they are anchorage-dependent for growth and nontumorigenic in nude mice (1, 17). IOSE cells were routinely cultured in D/F-12+ medium [phenol red-free 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 (calcium concentration 0.8 mM)], supplemented with hydrocortisone, selenium, insulin, transferrin, triiodothyronine, and 10% calf serum at 37°C in 5% CO2 and 95% air. Cells passaged between 8 and 11 times since isolation as primary cultures were used for experiments, and cells were never split more than 1:5.
Immunoprecipitation and immunoblotting.
IOSE-120 or IOSE-80 cells were harvested at the indicated times after
calcium addition by lysis in M-triton glycerol buffer [1%
Triton X-100, 10% glycerol, 20 mM HEPES, pH 8.0, 2 mM
Na3VO4, 150 mM NaCl, 1 mM NaF, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1% aprotinin, and leupeptin
1%], and lysates were cleared by centrifugation at 6,000 g. Protein concentration was determined by using the Bio-Rad
protein assay, and equal amounts of protein were incubated with primary
antibody for 4 h, followed by incubation with protein A/G-agarose
(Santa Cruz Biotechnology) for
1 h. The immune complex was collected
by centrifugation at 2,000 g for 5 min. The pellet was
washed extensively with M-TG buffer and boiled for 3 min in 1× Laemmli buffer.
PI3K assay.
PI3K was immunoprecipitated from IOSE-120 cells that were made
quiescent by overnight culture in serum-free low-calcium medium (0.3 mM
Ca, -
, and -
(06-195, Upstate
Biotechnology). The pellet was washed in 0.1 mM orthovanadate, 137 mM
NaCl, 1 mM CaCl2, and 1 mM MgCl2 in 20 mM Tris,
pH 7.4. To assay lipid kinase activity, the PI3K bound to protein
A/G-agarose beads was resuspended in 0.05 ml of Tris-NaCl-EDTA,
phosphatidylinositol was added to a final concentration of 0.27 mg/ml,
and the reaction was initiated by the addition of 30 µCi of
[
-32P]ATP. After 10 min at 37°C, the reaction was
quenched with 0.02 ml of 6 N HCl. The radiolabeled lipid phosphates
were extracted by the addition of 0.16 ml of chloroform:methanol (1:1),
and, after recovery of the organic phase, the lipid phosphates were resolved by TLC using chloroform:methanol:water:ammonium hydroxide (60:47:11.3:2) as the developing solvent. The products were visualized by autoradiography and identified by comigration with unlabeled PI3P
standards that were visualized by iodine staining, and the radioactive
bands were quantified by PhosphorImager or by excision and liquid
scintillation spectrometry.
ERK activity assays.
In vitro kinase assays were conducted on immunoprecipitates as
previously described (24). IOSE-80 cells at 80%
confluence were transfected with hemagglutinin (HA)-tagged ERK in 10-cm
plates or with empty vector as described in
Transfections. The cells were cultured in serum-free
Ham's F-12 (0.3 mM Ca-32P]ATP) and incubated at 30°C for 30 min. Glutathione-S-transferase (GST)-Elk1 (3 µg per
reaction) was added as a substrate for phosphorylation. Phosphorylated
proteins were resolved by SDS-PAGE in 12% acrylamide gels. Proteins
were electrophoretically transferred to PVDF membranes (Millipore), and
radioactivity was visualized and quantified with a Molecular Dynamics
PhosphorImager and IP LabGel software. The membranes were then stripped
and immunoblotted with anti-ERK1/2 antibody (Santa Cruz Biotechnology)
to verify equal loading of protein.
Raf assays.
For Raf assays, IOSE-80 cells were incubated with DMSO, PD-98059, or
wortmannin for 1 h. Unstimulated control cells and cells treated
with 2.0 mM calcium for 15 min were lysed in ice-cold 1% Nonidet P-40
buffer containing 10 mM Tris, pH 7.4, 5 mM EDTA, 50 mM NaCl, and 1 mM
PMSF. Immune complex kinase assays were performed as described
(26) by using MEK-1 as a substrate and
[-32P]ATP as a phosphate donor with equal protein
amounts per assay. The reaction products of all kinase assays were
resolved by 10% SDS-polyacrylamide gel and analyzed with a
PhosphorImager (Molecular Dynamics).
Antibodies. ERK, phospho-ERK, Akt, and phospho-Akt antibodies were purchased from New England Biolabs. Anti-Raf and anti-p85, the regulatory subunit of PI3K, were purchased from Upstate Biotechnology.
Incorporation of tritiated thymidine. IOSE-120 or IOSE-80 cells in 12-well plates were grown to 70-80% confluence as described in Cell culture. Cells were then changed into serum-free low-calcium medium (0.3 mM calcium) for 24 h before being changed into medium containing the indicated concentration of calcium chloride. Tritiated thymidine (1 µCi/ml) was added 16 h later, and cells were harvested after a 4-h period of [3H]thymidine incorporation. [3H]thymidine incorporation was determined by precipitation of the DNA in 10% trichloroacetic acid, solubilization in 0.2 M sodium hydroxide, and liquid scintillation counting.
Transfections.
IOSE-80 cells were transfected by using Transfast transfection reagent
from Promega according to the manufacturer's protocol. Briefly,
IOSE-80 cells were grown to ~80% confluence in 100-mm dishes and
placed in serum-free DMEM containing 15 µg of total DNA and 45 µl
of the Transfast reagent, resulting in a 1:1 charge ratio of DNA to
Transfast reagent. Cells were transfected with the construct containing
the gene indicated, and control cells were transfected with vector
lacking insert. The cells were incubated with the transfection mixture
for 1 h, and then the transfection mix was removed and 10 ml of
DMEM/F-12+ with 10% calf serum were added. After overnight growth, the
medium was replaced with serum-free Ham's F-12 (0.3 mM
Ca
Vectors and constructs. The preparation of the constitutively active (CA) and the DN PI3K has been described previously (3, 15). The expression vector for HA-ERK was a gift from D. Cohen (9). Vectors used as a transfection control were the parental vectors lacking inserted DNA.
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RESULTS |
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PI3K plays an important role in the proliferative response of OSE
cells to elevated Ca activation of the p110
catalytic subunit of PI3K
(12). If PI3K is involved in signal transduction
downstream of the CaR, we would expect to see increased phosphorylation
of Akt, a major PI3K target, in response to activation of the CaR. As
shown in Fig. 1, IOSE cells responded to
2 mM Ca
0.05; Fig. 2).
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Activation of PI3K by extracellular calcium.
The efficacy of wortmannin and LY-294002 as inhibitors of Akt and ERK
phosphorylation in response to 2 mM Ca
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Effects of DN and CA PI3K constructs on ERK activation.
Although pharmacological inhibitors such as wortmannin are very useful
tools in understanding signaling pathways, there is always doubt as to
the specificity of the effects of an inhibitor on other pathways. For
this reason, we chose to transiently transfect IOSE cells with a DN
mutant of PI3K that consists of the p110 subunit of PI3K mutated to
lack kinase ability (DN PI3K). The expression of the DN PI3K construct
has previously been demonstrated to be an effective inhibitor of PI3K
activity (3). Transient transfection of IOSE-80 cells with
the DN PI3K inhibited ERK activation in low-calcium medium to 5% of
control basal levels (Fig. 7). When IOSE
cells were shifted from 0.05 to 2.0 mM Ca containing the inter-SH2 domain of
the p85
subunit that confers constitutive kinase activity in a
variety of systems (15), had relatively little effect on
ERK activity levels in IOSE cells (Fig. 7). Expression of CA PI3K
failed to significantly increase either basal or calcium-stimulated ERK
kinase activity compared with vector-transfected IOSE cells. Although
there was a trend toward increased ERK activity in response to 2 mM
Ca
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Relative sensitivity of OSE proliferation to MEK vs. PI3K
inhibitors.
To determine the relative contribution of MEK-dependent and
PI3K-dependent pathways to the proliferative response induced by 2 mM
Ca
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DISCUSSION |
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In this report, we present evidence that PI3K plays an important role in signal transduction from the CaR to calcium-sensitive proliferative pathways including but not necessarily limited to ERK. This study was performed using human OSE cell lines to expand our knowledge of the regulation of a proliferative response in the cell type that forms the most aggressive and lethal ovarian tumors (20, 21). Work in this report and others (14) indicates that normal OSE cells modulate their rate of proliferation in response to the concentration of extracellular calcium. Therefore, understanding the proliferative pathways activated by the CaR in normal OSE cells is a prerequisite to identifying possible disruptions of these pathways in ovarian adenocarcinomas.
The effective inhibition of ERK kinase activity by chemical inhibitors of PI3K has been observed in murine interleukin (IL)-3-BaF/3 cells in which LY-294002 inhibited IL-3 activation of MEK, ERK1, and ERK2 (10). LY-294002 has also been shown to inhibit insulin-like growth factor-I-induced activation of both MEK and ERK in MCF-7 cells (27) as well as staurosporine-induced phosphorylation of ERK1/ERK2 in peritoneal macrophages (28). In contrast, wortmannin had no effect on ERK activation in response to the ligand-stimulated chemokine receptor CXCR3 in hepatic stellate cells (4). LY-294002 had no effect on ERK activation in erythropoietin-stimulated erythroid cells (8), and LY-294002 and wortmannin had no effect on activation of ERK in response to epidermal growth factor in glioblastoma cells (16). These mixed observations led us to examine whether PI3K may be activated in response to stimulation of the CaR. We saw a substantial increase in phosphatidylinositol 3-phosphate levels 5 min after shifting calcium from 0.05 to 2.0 mM, indicating that activation of the CaR is coupled to activation of PI3K.
The selective ERK inhibitor PD-98059 has previously been shown to
inhibit phosphorylation of ERK in rat OSE cells and inhibit proliferation (14). We have expanded on this observation
in dissecting the respective roles of ERK and PI3K in the proliferative response to elevated calcium concentration. Our observations
demonstrate that PD-98059 does not affect signaling through PI3K, i.e.,
it does not prevent phosphorylation of Akt (Fig. 4) and does not interfere with Raf activation (Fig. 6). This clearly shows that PI3K is
active upstream of MEK. In contrast, the PI3K inhibitor wortmannin
prevents the stimulation of Akt, Raf, and ERK. Furthermore, we
strengthened the evidence for inhibition of ERK by wortmannin and
PD-98059 by measuring ERK kinase activity as well as phosphorylation and obtaining similar results in terms of the level of ERK
phosphorylation. However, when we examined the effects of MEK
inhibitors and PI3K inhibitors on [3H]thymidine uptake in
response to elevated calcium, the PI3K inhibitors completely inhibited
any increase in thymidine incorporation in response to 2 mM calcium,
whereas the MEK inhibitors appeared to act predominantly on basal (low
calcium) levels of thymidine incorporation, with a 50-70%
increase in thymidine incorporation still evident upon switching to 2 mM calcium. The observation of a residual Ca
Although wortmannin is known to inhibit PI3K without interfering
with upstream events, pharmacological inhibitors always raise concerns
about their specificity. To address this concern, we transiently
transfected IOSE-80 with either a DN or a CA PI3K. The DN PI3K totally
inhibited ERK activation in response to elevated calcium (Fig. 7). This
result convincingly demonstrates that PI3K is required for activation
of ERK through the CaR in OSE cells. However, when the same cells were
transfected with the CA PI3K, we saw no significant change in the
levels of ERK activation. The CA PI 3-kinase construct did not activate
ERK above basal levels at low calcium levels, and it did not
significantly increase ERK activation at high calcium levels. Together,
these results demonstrate that, although PI3K is necessary for ERK
activation in response to elevated calcium signaling through the CaR,
increased p110 activity alone may not be sufficient for ERK
activation. One possible explanation for this discrepancy is the
possibility that ERK activation may be dependent on activation of
p110
, such that the DN PI3K interferes with signaling from any
catalytic PI3K subunit, whereas the CA PI3K can only mimic the effects
of p110
activation.
In summary, we have demonstrated that PI3K is involved in signal transduction from the CaR to ERK. Our data suggest that there may be other calcium-sensitive proliferative pathways that are independent of ERK but require the activation of PI3K. Further investigation is needed to fully understand the pathway by which the CaR signals through PI3K to ERK. There is also a need to define the pathways involved in the proliferative response to calcium mediated through PI3K but independent of ERK.
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ACKNOWLEDGEMENTS |
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We thank Erika Hammarlund and Gabrielle Whitney for expert technical assistance.
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FOOTNOTES |
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This work was supported by grants from National Cancer Institute Grants CA-74272 and CA-78722 to K. D. Rodland. T. R. Bilderback was supported by a National Research Service Award.
Address for reprint requests and other correspondence: K. D. Rodland, Pacific Northwest National Laboratory, 790 Sixth St., PO Box 999, Richland WA 99352 (E-mail: Karin.Rodland{at}pnl.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 20, 2002;10.1152/ajpcell.00437.2001
Received 10 September 2001; accepted in final form 15 March 2002.
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