Fibroblast Growth Factor Activation of the Rat PRL Promoter is Mediated by PKC
Twila A. Jackson,
Rebecca E. Schweppe,
David M. Koterwas and
Andrew P. Bradford
Department of Obstetrics and Gynecology (T.A.J., D.M.K., A.P.B.),
Department of Biochemistry and Molecular Genetics (R.E.S., A.P.B.), and
the Colorado Cancer Center, University of Colorado Health Sciences
Center, Denver, Colorado 80262
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ABSTRACT
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Fibroblast growth factors play a critical role in cell growth,
development, and differentiation and are also implicated in the
formation and progression of tumors in a variety of tissues including
pituitary. We have previously shown that fibroblast growth factor
activation of the rat PRL promoter in GH4T2 pituitary tumor cells is
mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we
show using biochemical, molecular, and pharmacological approaches that
PKC
is a critical component of the fibroblast growth factor
signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered
the rat PRL promoter refractory to subsequent stimulation by fibroblast
growth factors, implying a role for PKC in fibroblast growth factor
signal transduction. FGFs caused specific translocation of PKC
from
cytosolic to membrane fractions, consistent with enzyme activation. In
contrast, other PKCs expressed in GH4T2 cells (
, ßI, ßII, and
) did not translocate in response to fibroblast growth factors. The
PKC
subtype-selective inhibitor, rottlerin, or expression of a
dominant negative PKC
adenoviral construct also blocked fibroblast
growth factor induction of rat PRL promoter activity, confirming a role
for the novel PKC
isoform. PKC inhibitors selective for the
conventional
and ß isoforms or dominant negative PKC
adenoviral expression constructs had no effect. Induction of the
endogenous PRL gene was also blocked by adenoviral dominant negative
PKC
expression but not by an analogous dominant negative PKC
construct. Finally, rottlerin significantly attenuated FGF-induced MAP
kinase phosphorylation. Together, these results indicate that MAP
kinase-dependent fibroblast growth factor stimulation of the rat PRL
promoter in pituitary cells is mediated by PKC
.
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INTRODUCTION
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FIBROBLAST GROWTH FACTORS (FGFs) are
members of a family of polypeptides that play a critical role in
development, embryogenesis, and angiogenesis. They are also important
regulators of cell growth, differentiation, survival, and motility.
Several FGFs have been identified as oncogenes (1, 2, 3) and
are implicated in the formation and progression of tumors in a variety
of tissues including pituitary, breast, prostate, and ovary
(3, 4, 5, 6).
FGFs mediate their biological effects via a family of at least four
distinct transmembrane tyrosine kinase receptors [FGF receptors
(FGFRs) 14] (2, 7, 8), which exhibit overlapping
recognition and redundant specificity (9). In common with
other growth factors, binding of FGFs to their receptors results in
receptor dimerization, activation of intrinsic tyrosine kinase
activity, and receptor autophosphorylation (2, 8).
Activation of FGFRs also results in increased tyrosine phosphorylation
of a number of intracellular proteins; however, very little is known
about the cellular substrates of FGFRs or the components of the FGF
signaling pathway.
Several lines of evidence implicate FGFs in pituitary tumorigenesis.
FGF-2 was originally identified in pituitary extracts and is abundant
in pituitary cells (10). It increases PRL secretion from
normal and cultured pituitary-adenoma cell lines (11, 12)
and stimulates differentiation of PRL secreting pituitary lactotrophs
(13). Furthermore, patients with multiple endocrine
neoplasia type 1 exhibit elevated plasma levels of FGF-2
(14). The oncogene FGF-4 is also expressed in human
prolactinomas (15) and activates both PRL transcription
and secretion in rat GH4T2 pituitary cells (6). GH4T2
cells that stably express FGF-4 form highly aggressive and invasive
tumors upon subcutaneous injection into rats (6). Finally,
pituitary adenomas exhibit altered FGFR subtype and isoform expression
(16). Thus, FGFs may play a critical role in the
development and pathogenesis of pituitary prolactinomas, but the
mechanism of action of these growth factors and the components of the
FGF signaling pathway in pituitary cells remain to be elucidated.
Regulation of the rat PRL (rPRL) promoter in GH4T2 rat pituitary tumor
cells provides a physiologically relevant system in which to define and
characterize mediators of FGF signaling and the role of FGFs in
tumorigenesis. GH4T2 cells are neuroendocrine cells that express the
phenotypic markers PRL and GH and maintain normal hormonal and growth
factor responses (17). Utilizing this system, we recently
demonstrated that the FGF-2 and the FGF-4 signal to the rPRL promoter
are independent of Ras and Raf-1 but act via the MAPK pathway
(18).
PKC is a family of serine/threonine kinases that has been implicated in
the regulation of numerous signaling pathways, including many that are
Ras-independent, but dependent on MAPK (19, 20, 21, 22). To date,
at least 11 PKC isoforms have been identified: the phosphatidyl serine
(PS), diacyl glycerol (DAG), and Ca+2 -dependent
conventional isoforms, which include PKC
, -ßI, -ßII, and -
;
the Ca+2 -independent novel isoforms, PKC
,
-
, -
, and -
, which require PS and DAG; and the atypical
isoforms
, µ, and
, which require only PS for activation.
Emerging data demonstrate that rather than cellular redundancy, these
isoforms have distinct functions within the cell, including
mitogenesis, apoptosis, glucose transport, gene expression, and
secretion (23, 24, 25, 26, 27, 28, 29, 30). In the present study, we have further
characterized the FGF signal transduction pathway leading to induction
of rPRL promoter activity in GH4T2 pituitary cells. We demonstrate a
critical role for PKC
in FGF-mediated activation of PRL
transcription and show that PKC
lies upstream of MAPK kinase (MEK1)
and MAPK in the FGF signaling pathway. These results represent one of
the first examples of a physiological role for a specific PKC isoform
in the regulation of pituitary lactotroph-specific gene expression.
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RESULTS
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The PKC Inhibitor, Calphostin C, Blocks FGF-2 and FGF-4 Activation
of the rPRL Promoter
FGF-2 and FGF-4 activate the rPRL promoter in GH4T2 pituitary
cancer cells (18). We have previously shown that the FGF-4
signal to the rPRL promoter is independent of Ras and Raf-1, but
requires MAPK (18). In several Ras-independent,
MAPK-dependent signaling cascades, MAPK is coupled to the receptors via
PKC. These include angiotensin II type 1 receptor signaling to the
c-fos gene, growth stimulation of endothelial cells via
vascular endothelial growth factor receptor signaling, and
lysophosphatidic acid receptor signaling (20, 21, 22). To
determine whether the MAPK-dependent FGF signal to the rPRL utilizes
PKC, we tested the ability of the broad spectrum PKC inhibitor,
calphostin C, to block FGF-2 and FGF-4 activation of the rPRL promoter.
GH4T2 pituitary cells were transiently transfected with a rPRL
luciferase promoter (pA3425 PRL luciferase), pretreated with 100
nM calphostin C, and stimulated with FGF-2 or
FGF-4 as indicated. As seen in Fig. 1
, pretreatment with the general PKC inhibitor, calphostin C, reduced
basal transcription by 30%, while FGF-2 and FGF-4 stimulated the rPRL
promoter 4.5- 5 fold above basal promoter activity. Pretreatment with
100 nM calphostin C completely abolished both the
FGF-2 and FGF-4 induction of the rPRL promoter. Similar results were
obtained with the broad-spectrum PKC inhibitor, bisindolylmaleimide GF
109203, and using GH3 and GH4C1 cell lines (data not shown). These
results indicate that PKC is an important mediator of FGF-2 and FGF-4
signaling to the rPRL promoter in pituitary cells.

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Figure 1. Calphostin C Blocks FGF-2 or FGF-4 Stimulation of
the rPRL Promoter
GH4T2 pituitary cells were cotransfected with 3 µg of pA3rPRL-425luc
and 0.3 µg of pCMV ßgal and serum starved for 16 h. Cells were
pretreated with 100 nM calphostin C in the presence of
light and then treated with 2 ng/ml FGF-2 or FGF-4 or diluent, as
indicated, for 6 h before harvest. Luciferase activity was
normalized to ßgal activity, and the nonstimulated (NS) basal
activity of pA3rPRL-425luc was set at 1. Results shown are
representative of six experiments done in triplicate, and data are
expressed as fold ± SD of triplicate transfections.
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Chronic 12-O-Tetradecanoylphorbol-13 Ester (TPA)
Treatment to Down-Regulate PKC Prevents FGF-2 or FGF-4 Stimulation of
the rPRL Promoter
To confirm the role of PKC in FGF-2 and FGF-4 signaling to the
rPRL promoter, GH4T2 cells were chronically treated (16 h) with the
phorbol ester, TPA, which has been shown to deplete cells of PKC
isoforms via proteolysis (33, 34). GH4T2 pituitary cells
were transfected with the rPRL-luciferase reporter construct, serum
starved overnight, and treated with 0, 100, or 1,000 nM TPA
for 16 h. Cells were then stimulated acutely with FGF-2, FGF-4, or
TPA as indicated. As shown in Fig. 2
, chronic TPA treatment rendered the cells refractory to subsequent
stimulation by either FGF-2 or FGF-4 as well as to acute TPA
stimulation in a dose-dependent manner. The 5-fold FGF-2 and FGF-4
activation of the rPRL promoter was reduced to approximately 2-fold
after 100 nM chronic TPA treatment and completely abolished
by 1,000 nM chronic TPA treatment. It has been demonstrated
that TPA activates the rPRL promoter in GH4T2 cells (35)
and, therefore, as a control we show that the 4-fold TPA-induced
activation is also blocked by chronic TPA pretreatment. These results
provide further evidence that PKC is an essential signaling component
mediating FGF activation of the rPRL promoter in GH4T2 pituitary
cells.

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Figure 2. Chronic Phorbol Ester Treatment Renders GH4T2 Cells
Refractory to FGF-2 or FGF-4 Stimulation
GH4T2 pituitary cells were cotransfected with 3 µg of pA3rPRL-425luc
and 0.3 µg of pCMV ßgal. After transfected cells plated down ( 4
h), cells were treated with TPA at the indicated concentrations for
16 h and then stimulated with 2 ng/ml FGF-2
(squares), FGF-4 (open circles), or 100
nM TPA (solid circles) for 6 h. Cells
were harvested and assayed as described in Fig. 1 .
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GH4T2 Cells Contain Five PKC Isotypes
The PKC family of serine/threonine kinases is comprised of
numerous isoforms. To date, at least 11 isotypes have been identified
(for reviews see Refs. 36 and 37), including
the conventional
, ßI, ßII, and
isoforms, the novel
,
,
, and
isoforms, and the atypical
, µ, and
isoforms. The expression profile of PKC isoforms in GH4T2 cells has not
been characterized. To determine which isoforms of PKC are present in
GH4T2 pituitary cells, Western blot analyses of 100 µg of whole-cell
extracts were performed. As shown in Fig. 3
, five isoforms of PKC are expressed at
detectable amounts in GH4T2 cells, including the
Ca++, DAG, and PS-dependent, conventional PKC
,
-ßI, and -ßII isoforms (lanes 13), and the
Ca++- and PS-dependent novel PKC
and -
isoforms (lanes 4 and 5). PKC
, the novel PKC isoforms
and
,
and the atypical PKCs were not detected. As a control for antibody
activity for PKC
, -
, -
, -
, -µ, and -
(the PKC isoforms
that we did not detect in GH4T2 cell extracts), these isoforms were
detected by Western blotting in 100 µg of extracts of rat brain or
3T3 cells using the indicated antibodies (data not shown).
FGF-4 Induces Membrane Localization of PKC
Activation of PKC is often correlated with its movement from the
cytosol to cell membranes (reviewed in Ref. 38). To test
whether FGFs induced translocation of specific PKC isotypes, identified
in GH4T2 pituitary cells, we stimulated the cells with FGFs and then
performed subcellular fractionation followed by Western blotting of the
cytosolic and Triton X-100 soluble membrane fractions
(39). The results of a representative FGF-4 translocation
experiment are shown in Fig. 4
; similar
results were obtained using FGF-2 (data not shown). Data from multiple
experiments, analyzed by densitometry, are summarized in Table 1
. FGF-4 did not induce translocation of
the three conventional PKC isoforms (
, ßI, and ßII), whereas TPA
treatment caused substantial movement of these isoforms to particulate
fractions (Fig. 4
). A portion of both PKC
(30%) and PKC
(44%)
was present in the membrane fraction before FGF treatment. FGF induced
a dramatic redistribution of PKC
, resulting in more than 80%
membrane localization. In contrast, the relative distribution of PKC
did not change in response to FGF (Fig. 4
and Table 1
). These results
indicate that FGF treatment induces specific translocation of PKC
to
the membrane, consistent with a role for PKC
in FGF signal
transduction.

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Figure 4. The Novel PKC Isoform Is Translocated to the
Membrane Fraction in Response to FGF-2 or FGF-4
GH4T2 cells (1 x 107) were serum starved for 16
h, stimulated with 2 ng/ml FGFs for 5 min, and harvested in lysis
buffer A (see Materials and Methods). Cells were
sonicated and pelleted at 70,000 x g for 1.5
h. Supernatants obtained were stored as cytosolic fractions, and the
pellets were resuspended in lysis buffer B, sonicated, and saved as
particulate/membrane fractions. Equal volumes of cytosolic fractions
(C) and membrane fractions (M) were resolved by SDS-PAGE, transferred
to Immobilon P, and probed with the indicated antibodies. NS indicates
no stimulation with FGFs.
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The PKC
-Selective Inhibitor, Rottlerin, Blocks the FGF-2 and
FGF-4 Signal to the rPRL Promoter
To corroborate the translocation studies, we used pharmacological
isoform-selective PKC inhibitors. Gö 6976 has been shown to
selectively block the classical PKC
and -ß isoforms with an
IC50 of 2.36.2 nM
(40). As shown in Fig. 5A
, pretreatment of GH4T2 cells with Gö 6976 slightly reduced basal
activity of the rPRL promoter, but had no effect on the fold activation
by FGF-2 (2.5- to 3-fold) or FGF-4 (4- to 5-fold) at doses up to 1
µM. As a control, pretreatment of GH4T2 cells with 100
nM Gö 6976 significantly inhibited TPA stimulation of
a c-fos promoter-luciferase construct, demonstrating that
the inhibitor is functional in GH4T2 cells (data not shown).

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Figure 5. The PKC -Selective Inhibitor, Rottlerin, but Not
the Conventional PKC-Selective Inhibitor, Gö 6976, Blocks the
FGF-2 or FGF-4 Signal to the rPRL Promoter
GH4T2 pituitary cells were cotransfected with 3 µg of pA3rPRL-425luc
and 0.3 µg of pCMV ßgal. Sixteen hours after transfection, cells
were pretreated with the inhibitors at the indicated concentrations for
30 min and then stimulated with either 2 ng/ml of FGF-2 or FGF-4. Six
hours post treatment cells were harvested and assayed as described in
Fig. 1 . Results shown are representative of three experiments, and data
are expressed as fold ± SD of triplicate
transfections. Panel A shows pretreatment with Gö 6976 followed
by stimulation with FGF-2 or FGF-4, and panel B shows pretreatment with
rottlerin followed by stimulation with FGF-2 or FGF-4 as indicated.
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In contrast, the PKC
-selective inhibitor rottlerin
(IC50 3 µM) (41)
abrogated FGF induction of the rPRL promoter (Fig. 5B
). Both FGF-2 and
FGF-4 responses were completely blocked by pretreatment with 10
µM rottlerin. Although rottlerin has also been shown to
inhibit the novel PKC
, the IC50 is 100
µM, or 33-fold higher than that for PKC
. The basal
activity of the rPRL promoter was also reduced by approximately 40% in
response to rottlerin (Fig. 5B
). However, rottlerin had no effect on
the induction of rPRL promoter activity by PKA catalytic subunit
expression or forskolin treatment (data not shown). Thus, the effects
of rottlerin are not due to nonspecific cellular toxicity. These
results indicate that FGF activation of PRL gene expression is
independent of the conventional
and ß PKC isozymes. Moreover, in
combination with the specific FGF-induced translocation of PKC
, but
not PKC
, the data clearly implicate PKC
as the primary isoform
mediating the stimulatory effects of FGF-2 and FGF-4 on the rPRL
promoter.
Expression of Dominant-Negative PKC
Attenuates FGF-2 or FGF-4
Activation of the rPRL Promoter
To corroborate the pharmacological inhibitor and translocation
data, we used adenoviral vectors that express either a PKC
dominant
negative (DN) protein containing a K376-to-R mutation (DN PKC
), or
wild-type PKC
, in our transient transfection system (Carpenter, L.,
and T. Biden, unpublished observation). Using a green fluorescent
protein adenoviral expression vector, we first determined that a
multiplicity of infection of 10 yielded approximately 90% infection of
the GH4T2 cells (data not shown). Cells were infected with either
wild-type adenoviral PKC
or DN PKC
construct and then transiently
transfected with the rPRL-luciferase reporter construct. Infection with
wild-type PKC
had no effect on basal or FGF-stimulated rPRL promoter
activity (Fig. 6
). Overexpression of
wild-type PKC
did not enhance the FGF-2 or FGF-4 responses,
suggesting that endogenous PKC
is not present in limiting quantities
with respect to this signal transduction pathway. However, infection of
the cells with DN PKC
completely blocked the approximately 3-fold
FGF-2 or FGF-4 activation of the rPRL promoter. In contrast,
stimulation of rPRL promoter activity by oncogenic V12 Ras was not
affected by DN PKC
expression (Fig. 6
). Cells infected with
adenovirus encoding a DN PKC
construct (K368 to R) (2)
retained rPRL promoter FGF responsiveness (data not shown), indicating
that DN PKC
selectively blocked the FGF signaling pathway.

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Figure 6. DN PKC Blocks FGF-2 or FGF-4 Stimulation of the
rPRL Promoter
GH4T2 pituitary cells were cotransfected with 3 µg of pA3rPRL-425luc
and 0.3 µg of pCMV ßgal and then infected with a wild-type or DN
PKC adenoviral expression vector for 16 h. Cells were harvested
and assayed for luciferase activity (luciferase was normalized to
ßgal activity). Data are expressed as mean fold activation of three
independent transfection experiments performed in triplicate.
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To further test the physiological role of PKC
in FGF modulation of
PRL gene expression, we tested the ability of DN PKC
to block
transcription of the endogenous PRL gene using Northern blot analysis.
GH4T2 cells were infected with the wild-type or DN PKC
adenoviral
expression vectors described above or with DN PKC
as a control for
isozyme specificity. Cells were then stimulated with FGF-2 or FGF-4,
and total cellular RNA was probed with a radiolabeled PRL cDNA. Fig. 7A
depicts a representative Northern
blot. PRL mRNA levels from multiple experiments were normalized to
glyceraldhyde-3-phosphate dehydrogenase mRNA and the results expressed
as fold increase over basal (Fig. 7B
). DN PKC
completely blocked the
2-fold FGF stimulation of endogenous rPRL mRNA expression. In contrast,
neither wild-type PKC
nor DN PKC
affected FGF stimulation of the
endogenous rPRL gene expression. Thus, FGF induction of endogenous PRL
gene expression is also dependent upon PKC
.

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Figure 7. DN PKC Inhibits FGF Induction of Endogenous rPRL
mRNA
A, GH4T2 cells were infected with the indicated adenoviral PKC
construct and subsequently treated ± FGF-2 for 8 h. RNA was
isolated and probed for rPRL message by Northern blotting as described
in Materials and Methods. B, Northern blots were quantitated
using a Phosphorimager (Molecular Dynamics, Inc.,
Sunnyvale, CA). PRL message was normalized to glyceraldhyde-3-phosphate
dehydrogenase mRNA and FGF responses expressed as fold increase over
control levels. Data are means ± SD of
three to five experiments. *, P < 0.05; **,
P < 0.005 by the paired t test.
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MAPK Is Downstream of PKC in the FGF-2 and FGF-4 Signal
Transduction Pathway to the rPRL Promoter
We have previously shown, using DN or inhibitory expression
plasmids, that the FGF-2 or -4 signal to the rPRL promoter is
independent of Ras/Raf-1, but mediated via MAPK (18). To
further investigate the role of MAPK in FGF regulation of the rPRL
promoter and in GH4T2 pituitary cells, we used the MEK1 inhibitor, PD
98059, which inhibits activation of MAPK (44). As shown in
Fig. 8A
, treatment with PD 98059 inhibits
FGF-2 or FGF-4 induction of rPRL promoter activity in a dose-dependent
manner. These results confirm the critical role of MAPK activation in
FGF-2 and FGF-4 signaling to the rPRL promoter.

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Figure 8. FGF Stimulation of the rPRL Promoter Is Mediated by
PKC -Dependent Activation of MAPK
A, GH4T2 pituitary cells were cotransfected with 3 µg of
pA3rPRL-425luc and 0.3 µg of pCMV ßgal. Sixteen hours after
transfection, cells were pretreated with PD98059 at the indicated
concentrations for 30 min and then stimulated with either 2 ng/ml of
FGF-2 or FGF-4. Six hours post treatment cells were harvested and
assayed as described in Fig. 1 . Results shown are representative of
three experiments, and data are expressed as fold ±
SD of triplicate transfections. B, GH4T2 cells were treated
for 16 h with 100 nM TPA (cTPA) and then stimulated
acutely with TPA (100 nM) (acTPA), FGF-2 or FGF-4 (10
ng/ml). Ten minutes after stimulation, cells were harvested and protein
concentration was determined. Fifty micrograms of total protein were
analyzed by Western blotting using either a phospho-specific MAPK
primary antibody or a pan MAPK antibody. Purified, nonphosphorylated
MAPK (MK) and phosphorylated MAPK (pMK) were included as controls for
antibody specificity. The pan MAPK panel is included as an equivalent
loading control. C, Cells were pretreated with the general PKC
inhibitor, calphostin C, the conventional PKC inhibitor, Gö6976,
MEK1 inhibitor, PD98059, or the PKC -selective inhibitor rottlerin
for 30 min and then stimulated with 10 ng/ml FGF-4. Ten minutes after
stimulation, cells were harvested and protein concentration was
determined. Fifty micrograms of total protein were analyzed by Western
blotting using either a phospho-specific MAPK primary antibody or a pan
MAPK antibody.
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Our previous work documented FGF-induced activation of MAPK in GH4T2
cells (18). Here we examine the role of PKCs in FGF
activation of MAPK using an antibody specific for the phosphorylated,
active form of MAPK (Fig. 8
, B and C). As shown in Fig. 8B
, down-regulation of PKC isoforms by chronic TPA treatment dramatically
inhibits both FGF-2 and FGF-4 stimulation of MAPK phosphorylation and
activation. As a control, chronic TPA treatment also inhibited acute
TPA induction of MAPK phosphorylation. These results indicate that FGF
activation of MAPK is dependent on PKC.
To address the role of specific PKC isoforms in FGF-mediated MAPK
activation, we used a panel of selective PKC inhibitors (Fig. 8C
). The
general PKC inhibitor calphostin C (lanes 4 and 5) and the
PKC
-selective inhibitor, rottlerin (lanes 4 and 8) substantially
reduced FGF-induced MAPK phosphorylation. However, Gö 6976, which
selectively inhibits the classical PKCs (
, ßI, and ßII), had
no effect on the ability of FGFs to activate MAPK (lanes 4 and 6). The
MEK1 inhibitor, PD 98059, which inhibits FGF activation of the rPRL
promoter (Fig. 8A
), also inhibited FGF-induced MAPK
phosphorylation/activation (Fig. 8C
, lanes 4 and 7). Taken together,
these results (Fig. 8
, AC) indicate that FGF activation of MAPK and
rPRL promoter activity are primarily mediated via PKC
and further
suggest that PKC
is upstream of MAPK in the FGF rPRL signal
transduction pathway.
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DISCUSSION
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FGFs play a critical role in pituitary development and lactotroph
physiology and have been implicated in the formation and progression of
pituitary tumors. FGF-2 is highly expressed in normal pituitary, where
it plays a role in PRL secretion (11, 12) and has recently
been shown to initiate differentiation of GHFT lactotroph
precursor cells (45). FGF-2 also induces pituitary
transforming gene (pttg) expression, which coincides with early
lactotrophic hyperplasia, angiogenesis, and prolactinoma development
(46). In humans, many malignant pituitary tumors including
prolactinomas express the FGF-4 (hst) oncogene, which results in
aggressive prolactinoma formation in SCID mice (47).
Despite this evidence that FGFs are important in normal and
pathological pituitary functions, FGF signal transduction pathways have
not been extensively characterized.
We have previously identified FGF response elements in the rPRL
promoter and shown that FGF induction of the rPRL promoter in GH4T2
cells is mediated via MAPK. However, in contrast to other systems, FGF
activation of the rPRL transcription is independent of Ras and Raf-1
(18). In this report we show that PKC is required for FGF
induction of the rPRL promoter and activation of MAPK. Furthermore, we
identify the specific PKC isoform, PKC
, as the primary mediator of
the FGF signal. General inhibitors of PKC or down-regulation of PKC by
chronic TPA treatment blocks FGF induction of the rPRL promoter (Figs. 1
and 2
). Use of the isotype-selective PKC inhibitors, Gö 6976
and rottlerin, which target the conventional PKCs (
, ß,
) and
, respectively, indicate that FGF activation of the rPRL promoter is
dependent on the novel PKC
isoform (Fig. 5
). Consistent with this
hypothesis, FGFs translocate PKC
, but not the conventional
, ß,
and
or the novel
isoforms from soluble to particulate
fractions, implying selective activation of PKC
in response to FGF
treatment (Fig. 4
and Table 1
).
To confirm the role of PKC
, we used adenoviral constructs encoding a
kinase dead PKC
mutant (DN PKC
), which functions as a specific DN
inhibitor. Expression of DN PKC
abrogated activation of the rPRL
promoter by FGF-2 and -4 (Fig. 6
). Viral mediated expression of
wild-type PKC
did not significantly potentiate the FGF response,
implying that PKC
is not limiting in GH4T2 cells. Induction of
exogenous rPRL promoter activity by oncogenic Ras was not affected by
DN PKC
expression (Fig. 6
), indicating a selective inhibition of
the FGF signaling pathway and excluding nonspecific viral toxicity. The
use of adenoviral vectors also allowed us to investigate the role of
PKC
in FGF induction of the endogenous rPRL gene. Northern blot
analysis of rPRL mRNA shows that DN PKC
completely blocked FGF
induction of PRL transcription, whereas neither wild-type PKC
nor
DN PKC
had an effect (Fig. 7
). Thus, using a variety of
experimental approaches, our results demonstrate a critical role for
PKC
in transducing the FGF-inductive signal to the rPRL
promoter.
We have previously shown that FGF-2 and FGF-4 activate the rPRL
promoter via MAPK but do not utilize Ras or Raf-1 as upstream
activators of MAPK in the GH4T2 pituitary cell line. Treatment of GH4T2
cells with the MEK1 inhibitor PD 98059 completely inhibited FGF
stimulation of rPRL promoter in a dose-dependent manner (Fig. 8A
).
These results suggest that the FGF signal is mediated via MEK1
activation of MAPK. PKC-dependent activation of MAPK has been
documented in two other pituitary cell signaling systemsTRH in GH3
lactotrophs and GnRH in
T31 cells (42, 43, 48). In
other cell types, PKC has been implicated in several signal
transduction pathways that are Ras-independent yet coupled to MAPK.
These include angiotensin II type 1 receptor signaling to the
c-fos gene, growth stimulation of endothelial cells via
vascular endothelial growth factor receptor signaling, and
lysophosphatidic acid receptor signaling (20, 21, 22). FGFs
stimulate MAPK phosphorylation and activation in GH4T2 cells (Fig. 8
, B
and C, and Ref. 18). Down-regulation of PKCs blocked FGF
activation of MAPK, placing PKC upstream of MAPK in the FGF signaling
pathway (Fig. 8B
). The PKC inhibitor, calphostin C or the
PKC
-selective antagonist, rottlerin, also abrogated FGF induction of
MAPK (Fig. 8C
). However, the conventional PKC (
, ß, and
)
inhibitor Gö 6976 had no effect on FGF induction of MAPK. Thus,
we have shown that FGF activation of the rPRL promoter is dependent
upon both PKC
and MAPK. Moreover, since inhibitors of PKC
block
FGF activation of MAPK, our results suggest that PKC
is upstream of
MAPK in the FGF signal transduction pathway impacting on the rPRL
promoter.
The PKC family has long been implicated in the control of cellular
functions and modulation of signal transduction in the hypothalamic
pituitary axis, regulation of hormonal synthesis and secretion, and
cell type ontogeny (49, 50, 51). Recent evidence suggests that
the multiple PKC isoforms are not redundant but exert specific effects
to up- or down-regulate cell growth, gene expression, cell
differentiation, and apoptosis (21, 23, 52, 53). For
example, PKC plays a key role in the GnRH stimulation of LH and FSH
synthesis and secretion from pituitary gonadotropes (54).
GnRH treatment of gonadotroph-derived
T31 cells results in
differential up-regulation of PKC
and -
mRNA levels with
concomitant translocation of both PKC isoforms to the membrane
(52). Multiple PKC isoforms are also involved in distinct
aspects of the control of PRL synthesis and secretion. PKC
has been
implicated in TRH signaling and PRL secretion in pituitary cells
(50, 55), whereas the effects of TRH on actin cytoskeletal
reorganization are independent of PKC
(27). TRH
treatment of pituitary GH3B6 cells resulted in translocation of PKC
to regions of cell-cell contact (56). PKC
is also
involved in mediating the antiproliferative effects of dopamine
(57), while increased PRL release from rat pituitary
lactotrophs induced by dopamine withdrawal is associated with selective
translocation and activation of PKCs
and ß (51).
Inhibition of PRL gene expression by TGFß2 in GH3 cells is thought to
be mediated by decreases in the activity of a select subset of PKC
isozymes (58). Finally, in this report, we show that
PKC
is a critical component of the FGF signaling pathway leading to
induction of PRL gene expression.
Thus, differential activation of functionally distinct PKC isoforms,
such as PKC
, in response to specific signal transduction pathways,
e.g. FGF, provides a mechanism to coordinate and integrate
both inductive and inhibitory stimuli regulating pituitary hormone
synthesis and secretion and gene expression.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transfections
GH4T2 rat pituitary tumor cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 12.5%
horse serum and 2.5% FCS (referred to as full serum) (Life Technologies, Inc.) and 50 µg/ml penicillin and streptomycin
(Life Technologies, Inc.). Cells were maintained at 37 C
in 5% CO2.
Transient transfections were carried out by electroporation, as
described previously (31, 32). Briefly, media were changed
412 h before each transfection, and cells were harvested at 5070%
confluency and electroporated in full serum as described
(32). After electroporation, 200 µl cells (35 x
106) were plated in 3 ml DMEM without serum for a
final concentration of 0.94% serum to achieve low levels of endogenous
growth factors. Cells were incubated for 16- 24 h and treated with
FGF-2 or FGF-4 (R & D Systems, Minneapolis, MN), or
diluent (0.1% BSA in PBS) at a final concentration of 2 ng/ml.
FGF responses were assayed 6 h after treatment. Electroporations
were performed in triplicate for each condition within a single
experiment, and experiments were repeated using different plasmid
preparations of each construct at least three times. Luciferase and
ß-galactosidase assays were performed as previously described
(31, 32).
Adenoviral infections were performed as follows: adenoviruses
were added to cells in one-third volume of normal media with full serum
at a multiplicity of infection of 1020. Plates were shaken every 10
min for 1 h after which media containing full serum were added to
regular volume. Cells were incubated for 24 h, harvested, and used
for electroporation as described above. Adenoviral constructs were a
generous gift of Drs. Lee Carpenter and Trevor Biden (Garvan Institute
of Medical Research, St. Vincents Hospital, Sydney, Australia).
Pharmacological Reagents
Calphostin C, Gö6976, rottlerin, and TPA were obtained
from Calbiochem (San Diego, CA), and PD98059 was obtained
from New England Biolabs, Inc. (Beverly, MA). All
pharmacological reagents were prepared and stored according to the
manufacturers specifications and used at the concentrations indicated
in the specific experiments. Calphostin C pretreatment was 30 min to
1 h in the presence of light and Gö6976, PD98059, and
rottlerin pretreatments were 30 min to 1 h at 37 C. For PKC
down-regulation studies, cells were treated with TPA for 16 h
(chronic TPA treatment).
Plasmid Constructs
The promoter construct pA3 -425 rPRLluc and pCMV ßgal
(cytomegalovirus ßgalactosidase) (CLONTECH Laboratories, Inc., Palo Alto, CA) have been described previously
(18).
Membrane Localization Assay
GH4T2 cells were serum starved (0% serum) for 24 h and
subsequently stimulated with FGFs or TPA for the indicated times. Cells
were washed once with ice-cold PBS and harvested in lysis buffer A (20
mM Tris, pH 7.5, 2 mM EDTA, 2 mM
EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.1%
ß-mercaptoethanol and 1x Complete Protease Inhibitor Cocktail
(Roche Molecular Biochemicals, Indianapolis, IL). Cells
were sonicated for 10 sec, output 4 on constant duty cycle, using a
Branson Sonicator (Branson Ultrasonics Corp., Danburg, CT) and pelleted
at 70,000 x g for 1.5 h. The supernatant was
collected as the cytosolic fraction. The pellet was resuspended in
lysis buffer B (lysis buffer A + 1% Triton X-100) by 10 sec sonication
and repelleted for 15 min at 13,000 x g in a
microfuge. The supernatant was collected as the Triton-soluble
particulate fraction.
Western Blot Analysis
GH4T2 cells were serum starved overnight and treated with 2
ng/ml FGF-2 or FGF-4 or the equivalent volume of diluent for the
indicated times. Cells (107) were washed in cold
PBS and harvested in 500 µl RIPA buffer [PBS, 1% NP40, 0.5% sodium
deoxycholate, 0.1% SDS, Complete Protease Inhibitor Cocktail
(Roche Molecular Biochemicals)]. Equal amounts of protein
(50100 µg), as determined by the Pierce Mini BCA protein assay
(Pierce Chemical Co., Rockford, IL), were resolved by
electrophoresis on 10% polyacrylamide-SDS gels and transferred to an
Immobilon-P membrane (Millipore Corp., Bedford, MA)
according to the manufacturers protocol. Membranes were blocked
1 h at room temperature or overnight at 4 C in blocking buffer
[5% nonfat dry milk in TBS + 0.1% Tween 20]. They were then
incubated with primary antibodies directed against various
isoform-specific epitopes of PKC (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) according to the manufacturers protocol
(1:500 in blocking buffer, 1 h at room temperature). For Western
blot analyses using MAPK phospho-specific or pan primary antibodies
(New England Biolabs, Inc.) the primary antibody
incubations were done for 20 h at 4 C. As a control for phospho-
and nonphospho-MAPK antibody specificity, 20 ng of bacterially
expressed phospho- or nonphospho-MAPK 2 were included (New England Biolabs). Membranes were incubated with a horseradish
peroxidase-conjugated, goat antirabbit secondary antibody
(Life Technologies, Inc.) diluted 1:5,000 in blocking
buffer for 1 h at room temperature or overnight at 4 C. Protein
was detected using the Super Signal chemiluminescence assay
(Pierce Chemical Co.) according to the manufacturers
protocol. Where indicated, membranes were stripped of antibody for 30
min at 50 C according to the Super Signal protocol and reprobed
as described above.
Northern Blot Analysis
GH4T2 cells were serum starved for 24 h and stimulated with
10 ng/ml FGF-2 or FGF-4 for 8 h. Cells were then resuspended in
RNA-STAT60 (Tel-Test, Inc., Friendswood, TX), and RNA was
prepared as per manufacturers protocol. Ten micrograms of RNA were
electrophoresed on a 1.4% agarose/1.75% formaldehyde
3-(N-morpholino)propanesulfonic acid gel and
transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). RNA was cross-linked to the
filter using 50 mM NaOH for 5 min and 2x SSC
(300 mM sodium chloride, 30
mM sodium citrate, pH 7.0) for 5 min. Blots were
prehybridized 1 h at 42 C in Ultrahyb (Ambion, Inc.,
Austin, TX) and then probed overnight at 42 C with a labeled
full-length rPRL probe (5 x 106 cpm/blot)
in Ultrahyb hybridization solution. Blots were washed three times with
0.2x SSC, 0.5% SDS at 65 C to remove background signal followed by
autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Carpenter and Biden for PKC adenoviral constructs
and Dr. J. J. Tentler and J. D. Graham for assistance with
Northern blots. We also thank Drs. D. F. Gordon, A.
Gutierrez-Hartmann, M. E. Reyland, J. J. Tentler, and W.
M. Wood for critical reading and discussion of this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Twila A. Jackson, Department of Obstetrics/Gynecology, Colorado Cancer Center, University of Colorado Health Science Center, 4200 East Ninth Avenue, Box B-198, Denver, Colorado 80262. E-mail: Twila.Jackson{at}UCHSC.edu
This work was supported by NIH Grant DK-53496 (to A.P.B.). T.A.J. is
supported by National Research Service Award DK-10031.
Abbreviations: CMV ßgal, Cytomegalovirus
ß-galactosidase; DAG, diacyl glycerol; DN, dominant negative; FGF,
fibroblast growth factor; FGFR, FGF receptor; PS, phosphatidyl serine;
rPRL, rat PRL; TPA,
12-O-tetradecanoylphorbol-13-ester.
Received for publication October 25, 2000.
Accepted for publication May 16, 2001.
 |
REFERENCES
|
---|
-
Friesel R, Maciag T 1995 Molecular mechanisms of
angiogenesis: fibroblast growth factor signal transduction. FASEB J 9:919925[Abstract/Free Full Text]
-
Johnson D, Williams L 1993 Structural and functional
diversity in the FGF receptor multigene family. Adv Cancer Res 60:
141
-
Mason I 1994 The ins and outs of fibroblast growth factors.
Cell 78: 547552
-
Birnbaum D, DeLapeyriere O, Adnane J, et al. 1991 Role of
FGFs and FGF receptors in human carcinogenesis. Ann NY Acad Sci 638:409411[Medline]
-
Leung H, Dickson C, Robson C, Neal D 1996 Overexpression of
growth factor-8 in human prostate cancer. Oncogene 11:18331835
-
Shimon I, Huttner A, Said J, Spirina O, Melmed S 1996 Heparin-binding secretory transforming gene (hst) facilitates rat
lactotrope cell tumorigenesis and induces prolactin gene expression.
J Clin Invest 97:187195[Abstract/Free Full Text]
-
Gabbay R, Sutherland C, Gnudi L, et al. 1996 Insulin
regulation of phosphoenolpyruvate carboxykinase gene expression does
not require activation of the ras/mitogen-activated protein kinase
signaling pathway. J Biol Chem 271: 18901897
-
Schlessinger J, Lax I, Lemmon M 1995 Regulation of growth
factor activation by proteoglycans: what is the role of the low
affinity receptors. Cell 83: 357360
-
Burgess W, Maciag T 1989 The heparin binding (fibroblast)
growth factor family of proteins. Annu Rev Biochem 58:575[CrossRef][Medline]
-
Gospodarowicz D 1975 Purification of a fibroblast growth
factor from bovine pituitary. J Biol Chem 250:25152520[Abstract]
-
Atkin S, Landolt A, Jeffreys R, Diver M, Radcliffe J, White M 1993 Basic fibroblast growth factor stimulates prolactin secretion from
human anterior pituitary adenomas without affecting adenoma cell
proliferation. J Clin Endocrinol Metab 77:831837[Abstract]
-
Baird A 1993 Editorial: Fibroblast growth factors: whats in
a name? Endocrinology 132:487488[Medline]
-
Porter T, Wiles C, Stephan Frawley L 1994 Stimulation of
lactotrope differentiation in vitro by fibroblast growth
factor. Endocrinology 134:164168[Abstract]
-
Zimering M, Brandi M, deGrange D, et al. 1990 Circulating
fibroblast growth factor-like substance in familial endocrine neoplasia
type 1. J Clin Endocrinol Metab 70:149154[Abstract]
-
Gonsky R, Herman V, Melmed S, Fagin J 1991 Transforming DNA
sequences present in human prolactin-secreting tumors. Mol Endocrinol 5:16871695[Abstract]
-
Asghar Abbass S, Asa S, Ezzat S 1997 Altered expression of
fibroblast growth factor receptors in human pituitary adenomas. J
Clin Endocrinol Metab 82:11601166[Abstract/Free Full Text]
-
Gourdji D, Laverriere J-N 1994 The rat prolactin gene: a
target for tissue-specific and hormone-dependent transcription factors.
Mol Cell Endocrinol 100: 133142
-
Schweppe R, Frazer-Abel A, Gutierrez-Hartmann A, Bradford A 1997 Functional components of fibroblast growth factor signal
transduction in pituitary cells. J Biol Chem 272:3085230859[Abstract/Free Full Text]
-
Alblas J, van Etten I, Moolenaar WH 1996 Truncated,
desensitization-defective neurokinin receptors mediate sustained MAP
kinase activation, cell growth and transformation by a Ras-independent
mechanism. EMBO J 15:335160[Abstract]
-
Arai H, Escobedo JA 1996 Angiotensin II type 1 receptor
signals through Raf-1 by a protein kinase C-dependent, Ras-independent
mechanism. Mol Pharmacol 50:522528[Abstract]
-
Takahashi T, Ueno H, Shibuya M 1999 VEGF activates protein
kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for
DNA synthesis in primary endothelial cells. Oncogene 18:22212230[CrossRef][Medline]
-
Takeda H, Matozaki T, Takada T, et al. 1999 PI 3-kinase
and protein kinase C-
mediate RAS-independent activation of MAP
kinase by a Gi protein-coupled receptor. EMBO J 18:386395[Abstract/Free Full Text]
-
Acs P, Wang QJ, Bogi K, et al. 1997 Both the catalytic and
regulatory domains of protein kinase C chimeras modulate the
proliferative properties of NIH 3T3 cells. J Biol Chem 272:2879328799[Abstract/Free Full Text]
-
Billiard J, Koh DS, Babcock DF, Hille B 1997 Protein kinase C
as a signal for exocytosis. Proc Natl Acad Sci USA 94:1219212197[Abstract/Free Full Text]
-
Gschwendt M 1999 Protein kinase C
. Eur J Biochem 259:555564[Abstract/Free Full Text]
-
Hata A, Akita Y, Suzuki K, Ohno S 1993 Functional divergence
of protein kinase C (PKC) family members. PKC
differs from PKC
and ß II and nPKC
in its competence to
mediate-12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive
transcriptional activation through a TPA-response element. J Biol
Chem 268:91229129[Abstract/Free Full Text]
-
Kiley SC, Parker PJ, Fabbro D, Jaken S 1992 Hormone- and
phorbol ester-activated protein kinase C isozymes mediate a
reorganization of the actin cytoskeleton associated with prolactin
secretion in GH4C1 cells. Mol Endocrinol 6:120131[Abstract]
-
Lozano J, Berra E, Municio MM, et al. 1994 Protein kinase C
isoform is critical for
B-dependent promoter activation by
sphingomyelinase. J Biol Chem 269:1920019202[Abstract/Free Full Text]
-
Ozawa K, Szallasi Z, Kazanietz MG, et al. 1993 Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C
mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells.
Reconstitution of secretory responses with Ca2+ and purified isozymes
in washed permeabilized cells. J Biol Chem 268:17491756[Abstract/Free Full Text]
-
Taylor MJ, Clark CL 1988 Stimulatory effect of phorbol diester
on relaxin release by porcine luteal cells in culture. Biol Reprod 39:743750[Abstract]
-
Conrad KE, Oberwetter JM, Vallaincourt R, Johnson GL,
Gutierrez-Hartmann A 1994 Identification of the functional components
of the Ras signaling pathway regulating pituitary cell-specific gene
expression. Mol Cell Biol 14:15531565[Abstract]
-
Bradford AP, Conrad KE, Wasylyk C, Wasylyk B,
Gutierrez-Hartmann A 1995 Functional interaction of c-Ets-1 and
GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene
expression: mapping of the essential c-Ets-1 domain. Mol Cell Biol 15:28492857[Abstract]
-
Hug H, Sarre TF 1993 Protein kinase C isoenzymes: divergence
in signal transduction? Biochem J 291:329343[Medline]
-
Kishimoto A, Mikawa K, Hashimoto K, et al. 1989 Limited
proteolysis of protein kinase C subspecies by calcium-dependent neutral
protease (calpain). J Biol Chem 264:40884092[Abstract/Free Full Text]
-
Oberwetter JM, Conrad KE, Gutierrez-Hartmann A 1993 The Ras
and protein kinase C signaling pathways are functionally antagonistic
in GH4 neuroendocrine cells. Mol Endocrinol 7:915923[Abstract]
-
Csukai M, Mochly-Rosen D 1999 Pharmacologic modulation of
protein kinase C isozymes: the role of RACKs and subcellular
localisation. Pharmacol Res 39:253259[CrossRef][Medline]
-
Liu WS, Heckman CA 1998 The sevenfold way of PKC regulation.
Cell Signal 10:529542[CrossRef][Medline]
-
Shirai Y, Sakai N, Saito N 1998 Subspecies-specific targeting
mechanism of protein kinase C. Jpn J Pharmacol 78:411417[CrossRef][Medline]
-
Chen N, Ma W, Huang C, Dong Z 1999 Translocation of protein
kinase C
and protein kinase C
to membrane is required for
ultraviolet B-induced activation of mitogen-activated protein kinases
and apoptosis. J Biol Chem 274: 1538915394
-
Martiny-Baron G, Kazanietz MG, Mischak H, et al. C 1993 Selective inhibition of protein kinase C isozymes by the
indolocarbazole Go 6976. J Biol Chem 268: 91949197
-
Gschwendt M, Muller HJ, Kielbassa K, et al. 1994 Rottlerin, a
novel protein kinase inhibitor. Biochem Biophys Res Commun 199:9398[CrossRef][Medline]
-
Ohmichi M, Sawada T, Kanda Y, et al. 1994 Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3
cells by divergent pathways. J Biol Chem 269:37833788[Abstract/Free Full Text]
-
Reiss N, Llevi L, Shacham S, Harris D, Seger R, Naor Z 1997 Mechanism of mitogen-activated protein kinase activation by
gonadotropin-releasing hormone in the pituitary of
T31 cell line:
differential roles of calcium and protein kinase C. Endocrinology 138:16731682[Abstract/Free Full Text]
-
Pang L, Sawada T, Decker SJ, Saltiel AR 1995 Inhibition of MAP
kinase kinase blocks the differentiation of PC-12 cells induced by
nerve growth factor. J Biol Chem 270:1358513588[Abstract/Free Full Text]
-
Lopez-Fernandez J, Palacios D, Castillo A, Tolon R, Aranda A,
Karin M 2000 Differentiation of lactotrope precursor GHFT cells in
response to FGF-2. J Biol Chem 275:2165321660[Abstract/Free Full Text]
-
Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S 1999 Early involvement of estrogen-induced pituitary tumor transforming gene
and fibroblast growth factor expression in prolactinoma
pathogenesis. Nat Med 5:13171321[CrossRef][Medline]
-
Shimon I, Hinton DR, Weiss MH, Melmed S 1998 Prolactinomas
express human heparin-binding secretory transforming gene (hst) protein
product: marker of tumour invasiveness. Clin Endocrinol (Oxf) 48:2329[CrossRef][Medline]
-
Wang YH, Maurer RA 1999 A role for the mitogen-activated
protein kinase in mediating the ability of thyrotropin-releasing
hormone to stimulate the prolactin promoter. Mol Endocrinol 13:10941104[Abstract/Free Full Text]
-
Garcia-Navarro S, Marantz Y, Eyal R, et al. 1994 Developmental
expression of PKC subspecieis in rat brain-pituitary axis. Mol Cell
Endocrinol 103:133138[CrossRef][Medline]
-
Akita Y, Ohno S, Yajima Y, et al. 1994 Overproduction of a
Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases
the secretion of prolactin from thyrotropin-releasing
hormone-stimulated rat pituitary GH4C1 cells. J Biol Chem 269:46534660[Abstract/Free Full Text]
-
Mau S 1997 Effects of withdrawal of dopamine on translocation
of PKC isozymes and prolactin secretion in rat lactotroph enriched
pituitary cells. J Mol Endocrinol 18:181191[Abstract]
-
Harris D, Reiss N, Naor Z 1997 Differential activation of
PKC
and
gene expression by GnRH in
T31 cells. J Biol
Chem 272:1353413540[Abstract/Free Full Text]
-
Reyland ME, Anderson SM, Matassa AA, Barzen KA, Quissell DO 1999 Protein kinase C
is essential for etoposide-induced
apoptosis in salivary gland acinar cells. J Biol Chem 274:1911519123[Abstract/Free Full Text]
-
Naor Z, Harris D, Shacham S 1998 Mechanism of GnRH receptor
signaling: combinatorial cross-talk of Ca and PKC. Front
Neuroendocrinol 19:119[CrossRef][Medline]
-
Kiley S, Parker P, Fabbro D, Jaken S 1991 Differential
regulation of protein kinase C isoforms by thyrotropin releasing
hormone in GH4C1 cells. J Biol Chem 266:2376123768[Abstract/Free Full Text]
-
Vallentin A, Prevostel C, Fauquier T, Bonnefont X, Joubert D 2000 Membrane targeting and cytoplasmic sequestration in the
spatiotemporal localization of human protein kinase C
. J Biol
Chem 275:60146021[Abstract/Free Full Text]
-
Senogles S 1994 The D2 dopamine receptor mediates inhibition
of growth in GH4ZR7 cells: involvement of protein kinase C epsilon.
Endocrinology 134:783789[Abstract]
-
Chuang C, Tan S, Tai L, Hsin J, Wang F 1998 Evidence for
the involvement of PKC in the inhibition of prolactin gene expression
by TGFß 2. Mol Pharmacol 53:10541061[Abstract/Free Full Text]