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Department of Molecular and Integrative Physiology, The Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: Dr. Chris Taylor, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D.C. 20007.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or -ß
(PDGF-R or PDGF-Rß). Despite this, little information is available
about which PDGF receptors are expressed in the ovary, what signaling
cascades are activated by PDGF, and the effects of PDGF on thecal cell
steroidogenesis. The present study demonstrates the expression of
immunoreactive PDGF-Rß, but not PDGF-R, in the thecal and stromal
compartments of intact porcine ovaries as well as in cultured porcine
thecal cells. Treatment of porcine thecal cells in vitro
with PDGF resulted in rapid and sustained tyrosine phosphorylation of
PDGF-Rß, activation of Src tyrosine kinase and
phosphatidylinositol-3-kinase (PI3-kinase), and serine 473
phosphorylation of Akt/protein kinase B. In addition, PDGF stimulated
an increase in GTP-Ras (activated Ras) and extracellular
signal-regulated kinase (ERK) phosphorylation. Both forms of PDGF, AB
and BB, stimulated thecal cell growth approximately 3- to 4-fold over
controls and inhibited LH-stimulated progesterone and androstenedione
secretion. Blockade of PI3-kinase activation with wortmannin had no
effect on PDGF-stimulated thecal cell growth or PDGF inhibition of
LH-stimulated steroid secretion, indicating that PI3-kinase activation
is not necessary for PDGF-stimulated thecal cell growth or inhibition
of LH-stimulated steroidogenesis. Conversely, blockade of the
MEK-ERK pathway with PD98059 completely blocked PDGF-stimulated
cell growth, indicating that activation of the MEK-ERK pathway is
required for PDGF-stimulated thecal cell growth. Additionally, the MEK
inhibitor PD98059 restored LH-stimulated steroid secretion,
demonstrating that activation of the MEK-ERK pathway can lead to
inhibition of LH-stimulated steroid secretion. The present study
demonstrates that PDGF acts on ovarian thecal cells via activation of
the PDGF ß-receptor and stimulates thecal cell growth via activation
of a Ras-mitogen-activated protein kinase-dependent,
PI3-kinase-independent pathway. The strong expression of PDGF-Rß and
the potent effects of PDGF on thecal cell growth and steroidogenesis
suggest an important role for PDGF in thecal cell recruitment and
growth during follicular development in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A thecal layer is absent from primordial follicles, but begins to assemble shortly after the onset of follicular growth (2) from mesenchymal precursor cells of the ovarian stroma (3, 4). The factors that initiate this recruitment are thought to be as yet undefined growth factor signals originating from the oocyte-granulosa cell complex. Two in vitro studies using porcine thecal cells (5) and rat thecal-interstitial cells (6) suggest the importance of platelet-derived growth factor (PDGF) as a potent mitogen for thecal cells.
PDGF has potent mitogenic, chemotactic, and antiapoptotic effects on
cells that compose connective tissue, such as fibroblasts and smooth
muscle cells, as well as other cell types, including capillary
endothelial cells and neurons (see Ref. 7 for review). PDGF exists as
one of two homodimers (AA or BB) or as a heterodimer (AB) and binds two
structurally related tyrosine kinase receptors (PDGF-R and
PDGF-Rß). Upon ligand binding, PDGF receptors dimerize and
autophosphorylate several tyrosine residues, leading to the recruitment
and activation of various downstream signaling kinases, such as
phosphatidylinositol-3-kinase (PI3-kinase) (8, 9) and Src tyrosine
kinase (10), as well as recruitment of docking proteins, such as Shc
and Grb2, leading to activation of the Ras-Raf-MEK-extracellular
signal-regulated kinase (ERK) pathway (11, 12). The end result of
activating all of these pathways is an increase in cell growth and
motility.
The PDGF-A polypeptide binds only the -receptor, whereas the B chain
can bind either the - or ß-form of the receptor. Thus, PDGF-AA,
-AB, or -BB can induce monomeric dimerization and activation of the
-receptor, either PDGF-AB or -BB can induce heterodimerization of
- and ß-receptors, and monomeric ß-receptor is activated
primarily by PDGF-BB, although higher concentrations of PDGF-AB may be
able to induce dimerization of monomeric ßß-receptors (7). Both May
et al. (5) and Duleba et al. (6) used PDGF-AB to
demonstrate the potent mitogenic effects of PDGF on thecal cells grown
in vitro. However, identification of which receptor is
activated, the intracellular signaling cascades that PDGF uses in
stimulating thecal cell growth, and how these impact upon LH-stimulated
steroidogenesis have not been explored. The present study was
undertaken to determine what signaling cascades are activated by PDGF
and to determine the effects of PDGF on LH-stimulated steroidogenesis
using porcine thecal cells cultured in vitro as a model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
PDGF-Rß, and phosphotyrosine (PY20) antibodies and protein AG plus
agarose were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). cAMP RIA kits were purchased from Biomedical Technologies (Staughton, MA). ECL Western blotting detection kit
was purchased from Amersham Pharmacia Biotech (Arlington
Heights, IL). [
-32P]ATP (4000 Ci/mmol) was
purchased ICN Biomedicals, Inc. (Costa Mesa, CA).
Immunocytochemistry
Ovaries from prepubertal pigs were collected from a local
slaughterhouse and transported back to the lab in a sterile container
placed on ice. Pieces (0.5–1 cm) were cut from several ovaries,
snap-frozen in liquid nitrogen, and then stored at -80 C until
processed. Sections (10 µm) were mounted on glass slides and analyzed
by immunocytochemistry for expression of PDGF receptors by standard
protocols. Briefly, sections were fixed in ice-cold acetone, rinsed
with several washes of PBS, and reacted in 0.3% hydrogen peroxide in
methanol for 5 min to quench endogenous peroxidase activity. Tissue was
then washed, blocked with 10% normal goat serum, washed in PBS, and
incubated with 1 µg/ml of the appropriate primary antibody in PBS-1%
BSA for 90 min at room temperature. Slides were washed in PBS and then
incubated with a biotinylated goat antirabbit antibody for 1 h at
room temperature. Slides were washed with PBS, incubated with
horseradish peroxidase-avidin D conjugate, washed, and reacted with
aminomethyl carbazole. The reaction was stopped by rinsing with
distilled water.
Porcine thecal cell collection and culture
Follicles in the 3- to 5-mm size range and having clear
follicular fluid were selected for isolation of thecal cells. Selected
follicles were lanced with a sterile scalpel blade, drained of
follicular fluid, and washed vigorously with DMEM/Ham’s F-12 (1:1)
using a 22-gauge needle to remove granulosa cells. Follicle linings
were then dissected using fine needle forceps and placed in DMEM/Ham’s
F-12 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1
mg/ml collagenase, and 10 µg/ml deoxyribonuclease for 30–45 min at
37 C. Follicle linings were dispersed during this incubation period by
serial passage through 18-, 20-, and 22-gauge syringe needles. After
the dispersal incubation period, cells were collected and further
purified over a Percoll gradient as previously described (13) to remove
red blood cells and granulosa cells. Thecal cells were collected,
washed, and plated in DMEM/Ham’s F-12 supplemented with antibiotics
and 5% FBS (growth medium) at a density of approximately
105 cells/cm2. Thecal cells
were allowed to plate for 24 h, after which culture wells were
rinsed vigorously to remove unattached cells, and fresh media were
added. For long term cultures (>24 h) designed to examine cell growth
and LH-stimulated steroidogenesis, cells were cultured overnight in
serum-reduced medium (1% FBS), then changed to fresh serum-reduced
medium, and treatments added as described in Results. Cells
were cultured for a further 48–72 h, after which media were collected
for steroid determination. Cells were then either collected by trypsin
digestion for cell counting or lysed in boiling 2 x treatment
buffer or radioimmunoprecipitation assay (RIPA) buffer for
subsequent SDS-PAGE. For short term cultures designed to examine
signaling cascades, cells were serum starved (serum-free media)
overnight, then media were changed to fresh serum-free media, and cells
were treated as outlined in Results. For
[3H]thymidine incorporation, cells were plated,
and treatments were initiated as outlined above. After 24 h of
treatment, [methyl-3H]thymidine
(6.7 Ci/mmol) was added (1 µCi/ml) to culture wells. Twenty-four
hours later, wells were washed extensively with ice-cold PBS, and then
cells were hydrolyzed with 0.5 N NaOH.
[3H]Thymidine incorporation was determined by
scintillation counting.
Immunoprecipitation and immunoblotting
Porcine thecal cells were plated as described above. At
the end of the treatment period, cells were lysed in RIPA buffer [50
mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% SDS, 0.25%
sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1
mM PMSF, 1 mM sodium orthovanadate, 1
mM sodium fluoride, 1 µg aprotinin/ml, 1 µg
leupeptin/ml, and 1 µg pepstatin/ml] for 20 min at 4 C. Insoluble
material was cleared by centrifugation (14,000 x g for
20 min at 4 C). For immunoprecipitation, 500 µg soluble protein were
incubated with specific antibodies (1 µg) with gentle rocking at 4 C
for 1 h, after which 20 µl protein A/G plus agarose were added.
Samples were further incubated for 1 h (kinase assays) or
overnight (immunoblots). Immunoprecipitates were collected by
centrifugation and washed five times with RIPA buffer, then resuspended
in 20 µl RIPA buffer. For immunoblotting, total soluble or
immunoprecipitated proteins were mixed with an equal volume of 2
x Laemmli sample buffer. Samples were heated to 95 C for 5 min and
then subjected to SDS-PAGE. Protein was electrotransferred to
polyvinylidene difluoride membranes. Membranes were blocked with
TBST-5% milk [10 mM Tris-HCl (pH 8.0), 150
mM NaCl, 0.05% Tween-20, and 5% nonfat dry
milk] for 1 h at room temperature before incubation with an
antibody specific for the protein in question (e.g.
PDGF-Rß) in TBST-5% milk (1:1000 dilution) for 1 h. Membranes
were washed extensively with TBST, incubated with the appropriate
peroxidase-conjugated secondary antibody in TBST-5% milk for 1 h,
and then washed with TBST. Proteins were visualized by enhanced
chemiluminescence. For phosphotyrosine immunoblots, membranes were
blocked with TBST-3% BSA, incubated with antiphosphotyrosine antibody
in TBST-1% BSA, and washed with modified TBST (50
mM Tris-HCl, 200 mM NaCl,
and 0.25% Tween-20).
Src and PI3-kinase immune complex kinase assays
Cells were grown, collected, lysed, and immunoprecipitated with
the appropriate antibody as outlined above. Src activity was determined
by an in vitro immune complex kinase assay as previously
described (14).
For PI3-kinase activity determination, PI3-kinase immune complexes were
washed multiple times with RIPA, then the immunoprecipitated pellet was
resuspended in 50 µl TNE buffer [10 mM Tris (pH 7.4),
150 mM NaCl, and 5 mM EDTA] containing 20 µg
phosphatidylinositol and 25 mM MgCl2.
PI3-kinase activity was determined by 32P
incorporation into phosphatidylinositol. The reaction was started by
the addition of 1 mM cold ATP and 30 µCi
[-32P]ATP and allowed to proceed for 10 min
with constant agitation. The reaction was stopped by the addition of 20
µl 6 N HCl. CHCl3-MeOH (1:1, 160
µl) was then added to extract the lipid. The
CHCl3 phase was spotted on silicon TLC plates
treated with oxalate, and the lipids were resolved by TLC in
CHCl3-MeOH-water-ammonium hydroxide
(60:47:11.3:2). -32P incorporation into
phospholipid was visualized by autoradiography.
Ras activation assay
Porcine thecal cells were grown and treated as described above.
At the end of the treatment period, cells were lysed in Ras lysis
buffer [25 mM HEPES (pH 7.5), 150 mM NaCl, 1%
Nonidet P-40, 100 mM MgCl2, 1
mM EDTA, 2% glycerol, 1 mM PMSF, and 1
mM sodium orthovanadate]. Cellular protein (500 µg) was
mixed with 5 µl of a Raf-1-GST-agarose conjugate fusion protein
containing the Ras-binding domain of Raf-1, which binds only activated
GTP-bound Ras. The mixture was allowed to incubate overnight at 4 C
with gentle agitation. Agarose beads were collected and washed three
times with lysis buffer. Agarose beads were then resuspended in 2
x Laemmli sample buffer, heated to 95 C for 5 min, and centrifuged at
14,000 x g. The supernatant was subjected to SDS-PAGE
(12% gel) and immunoblot analysis as described above with an antibody
specific for Ras.
Measurement of steroids and cAMP
Progesterone and androstenedione were measured in unextracted
conditioned medium by specific RIA using methods previously described
(15). Extracellular cAMP in conditioned medium was measured after
acetylation according to the protocol provided by the manufacturer of
the RIA kit (14).
Statistical analysis
Unless otherwise specified, the results are presented as
the mean ± SE of representative experiments. A
minimum of four replicates per treatment were used for each experiment,
and experiments were repeated a minimum of three times, with
qualitatively similar results. For statistical analysis, means across
treatments were subjected to ANOVA, with differences between individual
means compared by Fisher’s protected least significant differences
test (Statview 512+, SAS Inc., Cary,
NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and a 190-kDa
receptor (PDGF-Rß), coded for by different genes have been
identified. To determine which of the receptors was mediating PDGF
signaling in porcine thecal cells, several different experimental
approaches were undertaken. Immunocytochemistry with antibodies
specific for PDGF-R (Fig. 1
, a and d),
and PDGF-Rß (Fig. 1, b, e, and f) demonstrated that PDGF-R
localized only to vascular endothelial cells in the porcine ovary (Fig. 1d), whereas the ß-receptor was present throughout the thecal and
stomal compartments (Fig. 1, b and f). Granulosa cells (Fig. 1, b, e,
and f) and surface epithelial cells (Fig. 1f) were devoid of any
immunoreactive PDGF-R or PDGF-Rß.
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was undetectable (Fig. 2a). Treatment of porcine thecal cells
with either PDGF-AB or -BB (10 ng/ml) led to the rapid tyrosine
phosphorylation of an approximately 200-kDa protein (Fig. 2b),
suggesting tyrosine phosphorylation of the PDGF-Rß (190 kDa). This
was confirmed by immunoprecipitation with an antibody specific for
phosphotyrosine followed by SDS-PAGE and immunoblot analysis with an
antibody against PDGF-Rß (Fig. 2c, upper panel).
Furthermore, the reverse experiment of immunoprecipitation with an
antibody against PDGF-Rß and immunoblot analysis with the
phosphotyrosine antibody demonstrated the presence of
tyrosine-phosphorylated PDGF-Rß after PDGF treatment (Fig. 2c, lower panel). The effects of PDGF on tyrosine
phosphorylation were long lasting. Cells harvested after 48 h of
treatment with either PDGF-AB or -BB continued to display
tyrosine-phosphorylated PDGF-Rß (Fig. 2d). Concurrent treatment with
LH had no effect on PDGF-stimulated tyrosine phosphorylation of
PDGF-Rß (Fig. 2d).
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). Concurrent treatment
of cells with LH has no effect on PDGF-stimulated Src activation.
Pretreatment with herbimycin A, a reportedly Src-selective tyrosine
kinase inhibitor, completely blocked PDGF receptor tyrosine
phosphorylation, as determined by phosphotyrosine immunoblot analysis
(Fig. 3b), and all subsequent downstream signals examined (data not
shown).
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). Treatment with LH had no
effect on basal or PDGF-stimulated PI3-kinase activity.
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). This occurred
without any change in total Akt/PKB protein expression. Again treatment
with LH had no effect on basal or PDGF-stimulated Akt/PKB serine 473
phosphorylation. A 20-min pretreatment with the PI3-kinase inhibitor,
wortmannin, completely blocked PDGF-stimulated Akt/PKB serine
473-phosphorylation (Fig. 4c), demonstrating that PI3-kinase activation
is required for Akt/PKB phosphorylation.
PDGF activates Ras-ERK1/2 kinase pathway
Stimulation of many cell types with growth factors results in
activation of the ERK1/ERK2 MAP kinase pathway via activation of Ras
and Raf. PDGF in several cell types has been shown to activate such a
pathway; thus, it was of interest to determine whether PDGF treatment
resulted in the activation of Ras and phosphorylation of ERK1/2.
Treatment of porcine thecal cells with PDGF led to an increase
GTP-bound (activated) Ras compared with that in controls (Fig. 5a). The increase in activation of Ras
was accompanied by the phosphorylation of both ERK1 and ERK2 at 10 min,
but not at 48 h (Fig. 5b). Wortmannin, the PI3-kinase inhibitor,
had no effect on PDGF-stimulated ERK phosphorylation. In contrast, the
MEK inhibitor PD98059 completely blocked ERK phosphorylation (Fig. 5c).
None of the treatments had any effect on total ERK expression, as
determined by the total ERK immunoblots.
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). PDGF-BB appeared to be
more potent (ED50, 
2.6 ng/ml) than PDGF-AB
(ED50, 5.1 ng/ml). Maximal stimulating
concentrations of PDGF increased cell number after 72 h 3- to
4-fold compared with controls, which showed little increase in cell
number from the initial seeding number (i.e. 100,000 cells).
LH, either alone or in combination with PDGF, had no effect on thecal
cell growth (Fig. 6). Both herbimycin A, a Src-selective tyrosine
kinase inhibitor, and the MEK inhibitor PD98059 completely inhibited
PDGF-stimulated cell growth (Fig. 7). In
contrast, wortmannin, a PI3-kinase inhibitor had no effect on
PDGF-stimulated thecal cell growth. In a second series of experiments,
[3H]thymidine incorporation was increased in
PDGF-BB-treated cells (1142 ± 103.3 cpm) compared with controls
(558.5 ± 43.9 cpm). As with the cell counts, herbimycin and
PD98059 blocked the effect of PDGF on
[3H]thymidine uptake (308.5 ± 21.6 and
425.3 ± 19.7 cpm, respectively), whereas wortmannin had little
effect (941 ± 61.6 cpm).
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). Addition of either PDGF-AB or -BB
inhibited both LH-stimulated progesterone and androstenedione
accumulation in a dose-dependent manner. As with cell growth, PDGF-BB
showed a higher potency than PDGF-AB. Wortmannin had no effect on the
PDGF inhibition of LH-stimulated cAMP, progesterone, or androstenedione
secretion (Table 1), indicating that
PI3-kinase is not involved in the inhibition of steroidogenesis.
Conversely, herbimycin A, the Src tyrosine kinase inhibitor, completely
restored, and in fact greatly enhanced, LH-stimulated cAMP and steroid
secretion. The MEK inhibitor, PD98059 also restored, but did not
enhance, LH-stimulated progesterone secretion (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression was limited to ovarian vascular endothelial cells, whereas
PDGF-Rß was expressed throughout the thecal and stromal compartments.
Neither receptor appears to be expressed in either granulosa cells or
ovarian surface epithelial cells. A previous study examining expression
of PDGF receptors in human ovarian cancers also reported PDGF-Rß in
the ovarian stroma from normal ovaries (16). Both PDGF-AB and PDGF-BB
have similar in vitro effects on porcine thecal cells,
although PDGF-BB appears to be more potent. A previous study reported
that PDGF-AB could stimulate porcine thecal cell growth, albeit with
relatively low potency. The present study suggests that the likely
reason for the relatively low potency of PDGF-AB is the exclusive
expression of PDGF-Rß and the lack of expression of the -form of
the receptor. The PDGF-A polypeptide does not bind the ß-receptor
with high affinity; however, previous reports have demonstrated that
higher concentrations of PDGF-AB can induce ßß-receptor
homodimerization and receptor activation (17, 18). The present study
confirms these observations by the demonstration that PDGF-AB
stimulates the phosphorylation of the PDGF-Rß. The present study demonstrates that PDGF inhibits LH-stimulated steroid secretion. A previous report suggested that PDGF stimulated the growth of steroidogenically inactive cells based upon histochemical staining for 3ß-hydroxy- steroid dehydrogenase (6). The present findings suggest that PDGF may maintain thecal cells in a steroidogenically inactive state, as addition of LH did not stimulate steroid secretion in the presence of PDGF.
Binding of PDGF to its receptor induces receptor dimerization and trans-autophosphorylation of several tyrosine residues in the cytoplasmic tail (7). These tyrosine-phosphorylated residues then act as docking sites for SH2-containing kinases, such as Src (10) and PI3-kinase (8, 9), as well as adaptor molecules, such as Grb7 (19) and Shc (20). Binding of Shc and or Grb to the PDGF receptor ultimately leads to activation of the Ras-Raf-MEK-ERK pathway. The present study demonstrates that PDGF treatment of porcine thecal cells leads to rapid tyrosine phosphorylation of the ß-receptor, stimulation of Src and PI3-kinase activity, serine 473 phosphorylation of Akt/PKB (suggesting Akt activation), activation of Ras, and phosphorylation of ERK1 and 2. The end result of this stimulation is an increase in cell growth and inhibition of LH-stimulated steroid secretion.
A possible source for PDGF may be granulosa cells. Granulosa cells harvested from immature equine CG/hCG-treated rats express PDGF-B polypeptide with a time-dependent decrease in PDGF-B expression in response to hCG (our unpublished observations). In addition, PDGF has been detected in human follicular fluid (21). These observations set the stage for a paracrine granulosa cell-thecal cell interaction, such that PDGF from granulosa cells of the developing follicle may stimulate thecal cell recruitment and growth. As the follicle matures and acquires granulosa cell-LH receptors, pituitary LH then inhibits granulosa cell-derived PDGF expression, lifting the PDGF-induced inhibition of thecal cell steroidogenesis. Of course, much work remains to confirm this speculation.
As has been documented in other cell types (see Ref. 22 for review), activation of PI3-kinase in porcine thecal cells leads to the phosphorylation and activation of the serine-threonine kinase Akt/PKB. Wortmannin, a PI3-kinase inhibitor, completely blocked PDGF-stimulated phosphorylation of Akt/PKB, demonstrating that activation of PI3-kinase is required for PDGF-stimulated Akt/PKB phosphorylation in porcine thecal cells. The effect of activation of the PI3-kinase-Akt/PKB pathway in porcine thecal cells is as yet unclear. Wortmannin had no effect on PDGF-stimulated thecal cell growth, nor did it restore LH-stimulated steroid secretion, demonstrating that activation of PI3-kinase and Akt/PKB is not necessary for PDGF-stimulated cell growth or PDGF inhibition of LH-stimulated steroid secretion. Activation of Akt/PKB is associated with protection from apoptotic stimuli via phosphorylation of several proapoptotic molecules, such as BAD (23, 24) and procaspase 9 (25), blocking their activation. In addition, Akt has very recently been demonstrated to phosphorylate some members of the forkhead family of transcription factors, leading to their sequestration in the cytoplasm, thus blocking their ability to activate forkhead-responsive genes, which include insulin-like growth factor-binding proteins and Fas ligand (26). Thus, it seems likely that activation of PI3-kinase is important in protecting thecal cells from apoptotic stimuli.
In stark contrast, it would appear that activation of the MEK-ERK pathway via Ras is necessary for PDGF-stimulated thecal cell growth. As expected, the MEK inhibitor PD98059 blocked PDGF-stimulated ERK phosphorylation. PD98059 also completely inhibited PDGF-stimulated thecal cell growth, demonstrating that activation of the MEK-ERK pathway is necessary for PDGF-stimulated porcine thecal cell growth. Activation of the MEK-ERK pathway also appears to be capable of inhibiting LH-stimulated steroid secretion. Blocking activation of this pathway with PD98059 restored LH-stimulated progesterone and androstenedione secretion. An interesting observation was that ERK phosphorylation was not sustained. This was in contrast to PDGF-Rß and Akt phosphorylation, which was sustained for 48 h of treatment. This perhaps suggests an interesting difference between growth-stimulating signals, which can be transient, and survival signals, which may need to be continuously activated to protect cells from apoptotic stimuli.
Herbimycin A, a Src-selective inhibitor, had profound effects on both
PDGF-stimulated cell growth and LH-stimulated steroid secretion.
Herbimycin completely inhibited PDGF-stimulated cell growth. This was
associated with a potentiation of LH-stimulated steroid secretion, as
has been previously reported for rat thecal interstitial cells (14, 27). The effects of herbimycin on the potentiation of LH-stimulated
steroid secretion can largely be attributed to inhibition of Src
tyrosine kinase activity. MA10 Leydig cells expressing a dominant
negative Src kinase are more responsive to LH stimulation than control
cells (28). Conversely, MA10 cells expressing a temperature-sensitive
viral Src are completely unresponsive to LH stimulation at the
kinase-permissive temperature (28). Src modulates LH-stimulated steroid
secretion at least partially via regulation of phosphodiesterase
activity (27, 28), although it is not yet known whether Src directly
interacts with phosphodiesterase. Src may also be acting at other
levels to modulate LH-stimulated steroid secretion, including
uncoupling the LH receptor from Gs and LH
receptor internalization.
Herbimycin also has a profound effect on PDGF-stimulated cell growth. It is not clear whether this is via inhibition of Src or perhaps through blockade of PDGF-Rß activation. Pretreatment of porcine thecal cells with herbimycin diminishes PDGF-induced receptor tyrosine phosphorylation, thus inhibiting all downstream signals. However, Src is also thought to phosphorylate secondary tyrosine sites within the cytoplasmic tale of PDGF-Rß (29); therefore, the decrease in PDGF-Rß phosphorylation could be due to inhibition of Src activity. Whatever the mechanism, the results demonstrate a potential requirement for PDGF signal activation for normal thecal cell growth and survival.
In summary, the present study demonstrates that porcine thecal cells express only the ß-form of the PDGF receptor. Treatment of thecal cells in vitro with PDGF stimulates tyrosine phosphorylation of PDGF-Rß and the activation of several different signaling networks, including PI3-kinase-Akt/PKB and Ras-Raf-MEK-ERK pathways, leading to an increase in thecal cell growth and inhibition of LH-stimulated steroidogenesis.
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Footnotes |
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Received October 25, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor in vascular smooth muscle cells. J Biol Chem 268:17045–17050-subunits for high affinity, but not
low affinity, binding and signal transduction. J Biol Chem 268:4473–4480- and ß-receptors. J Biol Chem 271:30942–30949This article has been cited by other articles:
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D. A. Dewi, D. R. E. Abayasekara, and C. P. D. Wheeler-Jones Requirement for ERK1/2 Activation in the Regulation of Progesterone Production in Human Granulosa-Lutein Cells Is Stimulus Specific Endocrinology, March 1, 2002; 143(3): 877 - 888. [Abstract] [Full Text] [PDF]
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A.L. Johnson, J.T. Bridgham, and J.A. Swenson Activation of the Akt/Protein Kinase B Signaling Pathway Is Associated with Granulosa Cell Survival Biol Reprod, May 1, 2001; 64(5): 1566 - 1574. [Abstract] [Full Text]
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R. B. Slee, S. G. Hillier, P. Largue, C. R. Harlow, G. Miele, and M. Clinton Differentiation-Dependent Expression of Connective Tissue Growth Factor and Lysyl Oxidase Messenger Ribonucleic Acids in Rat Granulosa Cells Endocrinology, March 1, 2001; 142(3): 1082 - 1089. [Abstract] [Full Text] [PDF]
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S. M. Quirk, R. M. Harman, and R. G. Cowan Regulation of Fas Antigen (Fas, CD95)-Mediated Apoptosis of Bovine Granulosa Cells by Serum and Growth Factors Biol Reprod, November 1, 2000; 63(5): 1278 - 1284. [Abstract] [Full Text]
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