(Received for publication, September 26, 1996, and in revised form, February 13, 1997)
From the Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel
The effect of gonadotropin-releasing hormone
(GnRH) upon protein kinase C (PKC) and PKC
gene expression was
investigated in the gonadotroph-derived
T3-1 cell line. Stimulation
of the cells with a stable analog
[D-Trp6]GnRH (GnRH-A) resulted in a
rapid elevation of PKC
mRNA levels (1 h), while PKC
mRNA
levels were elevated only after 24 h of incubation. The rapid
elevation of PKC
mRNA by GnRH-A was blocked by pretreatment with
a GnRH antagonist or actinomycin D. The PKC activator
12-O-tetradecanoylphorbol-13-acetate (TPA), but not the
Ca2+ ionophore ionomycin, mimicked the rapid effect of
GnRH-A upon PKC
mRNA elevation. Additionally, the rapid
stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor
GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by
Ca2+ removal. Interestingly, serum-starvation (24 h)
advanced the stimulation of PKC
mRNA levels by GnRH-A and the
effect could be detected at 1 h of incubation. The rapid effect of
GnRH-A upon PKC
mRNA levels in serum-starved cells was mimicked
by TPA, but not by ionomycin, and was abolished by down-regulation of
PKC or by Ca2+ removal. Preactivation of
T3-1 cells with
GnRH-A for 1 h followed by removal of ligand and serum resulted in
elevation of PKC
mRNA levels after 24 h of incubation.
Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKC
and PKC
. We
conclude that Ca2+ and PKC are involved in GnRH-A elevation
of PKC
and PKC
mRNA levels, with Ca2+ being
necessary but not sufficient, while PKC is both necessary and
sufficient to mediate the GnRH-A response. A serum factor masks PKC
but not PKC
mRNA elevation by GnRH-A, and its removal exposes
preactivation of PKC
mRNA by GnRH-A which can be memorized for
24 h. PKC
and PKC
gene expression evoked by GnRH-A is
autoregulated by PKC, and both isotypes might participate in the
neurohormone action.
The protein kinase C (PKC)1 family is
a family of serine/threonine protein kinase isoforms, which play key
roles in signal transduction (1-3). Conventional PKCs (,
I,
II, and
) are activated by Ca2+, diacylglycerol
(DAG), and phospholipid such as phosphatidylserine (PS) and are tightly
coupled to phosphoinositide turnover (1-3). Novel PKCs (
,
,
,
and
) are Ca2+-independent but DAG- and PS-activated
isoforms. Atypical PKCs (
and
/
) are Ca2+- and
DAG-independent but PS-activated isoforms and are also stimulated by
other lipid-derived mediators (1-4). PKCµ takes an intermediate position among the novel PKC and atypical PKC isoforms and is a
Ca2+- and DAG-independent isoform. Whereas relatively much
is known about regulation of PKC at the protein level including
cofactor requirements, translocation to the membrane, substrate
phosphorylation, and degradation (1-9), very little is known about
ligand regulation of PKC gene expression (10-12). Previous work has
implicated PKC in gonadotropin-releasing hormone (GnRH) action upon
gonadotropin secretion and gonadotropin subunits gene expression in
pituitary and
T3-1 cells (5, 6, 12-26). Recently, while examining conventional PKC regulation, we have shown that GnRH-A increases the
levels of PKC
, but not PKC
, mRNA levels in
T3-1 cells, while PKC
is not expressed in the cells (12). Since PKC
and PKC
of the novel PKC group are major subspecies in the pituitary (26), we decided to investigate the effect of GnRH-A on the mRNA
levels of both isotypes in the
T3-1 cell line. Here we demonstrate that GnRH-A directs differential autoregulation of PKC
and PKC
gene expression, which is dependent upon growth conditions and Ca2+, and reveals a memory mechanism, which might
participate in PKC
autoregulation.
Materials
T3-1 cells were kindly provided by Dr. P. Mellon (University
of California San Diego, La Jolla, CA). The GnRH analog
[D-Trp6]GnRH (GnRH-A) was a gift from Dr. R. Millar (University of Cape Town Medical School, Cape Town, South
Africa). A potent GnRH antagonist [D-Glu(P)1,ClPhe(P)2,D-Trp3,6]GnRH
was kindly provided by Dr. D. Coy (Tulane University School of
Medicine, New Orleans, LA). Ionomycin was purchased from Boehringer (Mannheim, Germany). The PKC-selective inhibitor bisindolylmaleimide (GF 109203X) (27) was purchased from Calbiochem (Laufelfingen, Switzerland). Bovine serum albumin, TPA, and other chemicals were purchased from Sigma (Rehovot, Israel). Media and sera for cell culture
were from Biological Industries (Kibbutz Beth Ha'Emek, Israel).
[
-32P]dCTP was purchased from Rotem (Beersheba,
Israel). PKC
and PKC
cDNAs were kindly provided by Dr. H. Mischak (Institute of Clinical Molecular Biology, GSF, Munich, Germany)
and Dr. F. Mushinsky (NIH, Bethesda, MD) (27), and the respective
antibodies were obtained from Sigma.
Methods
Cell CultureT3-1 cells were subcultured into 60-mm
tissue culture dishes (Sterilin, Hounslow, United Kingdom). Cells were
grown in 5 ml of Dulbecco's modified Eagle's medium (DMEM) containing
5% fetal calf serum, 5% horse serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. After 3-4 days, when cells were 70-80% confluent, the cultures were washed three times with fresh DMEM, and
stimulants were added in 5 ml of DMEM at the indicated concentrations for the given length of time. For short period incubations (up to
1 h) 10 mM Hepes was added to the medium. When the
stimulation period was longer than 9 h, the medium was
supplemented with 0.1% bovine serum albumin.
At the end of the stimulation
period, total RNA was isolated from cells by extraction in guanidium
thiocyanate containing 8% 2-mercaptoethanol by the LiCl method as
described by Cathala et al. (28). For Northern blot
analysis, total RNA (15 µg) was fractionated on 1.2% denaturing
agarose gel and transferred to GeneScreen membranes (DuPont NEN).
Alternatively, RNA samples (8 µg) were slot blotted onto GeneScreen
using a slot blot manifold (Schleicher & Schüll). Following
baking and prehybridization, the membranes were hybridized overnight
with the specific cDNA probes labeled to high specific activity
using a random primer labeling kit (Boehringer). Half of each lane was
hybridized with a PKC cDNA, and the second half was hybridized with
glyceraldehyde-3-phosphate dehydrogenase cDNA as an internal
control. Thereafter, filters were washed at high stringency and were
autoradiographed at 70 °C. Steady state levels of mRNAs were
quantified with densitometric scanning of autoradiograms. The data were
corrected for variability in loading by calculation as a ratio to
glyceraldehyde-3-phosphate dehydrogenase.
Following ligand treatment, cells were washed with ice-cold Tris-buffered saline, pH 7.2, harvested with rubber policemen, and pelleted by short spin (1200 rpm for 5 min at 4 °C). Cells were resuspended in 10 mM EGTA, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 0.5 mM iodoacetic acid, and lysed by 10 strokes of a 25-gauge syringe. Following removal of nuclei (1200 rpm for 5 min at 4 °C), cytosol and membrane fractions were obtained by ultracentrifugation (100,000 × g for 2 h at 4 °C). The proteins were separated on 7.5-18% SDS-polyacrylamide gels (ratio of acrylamide to bisacrylamide, 30:0.5) and electrotransferred to nitrocellulose papers in 50 mM glycine, 50 mM Tris-HCl, pH 8.8 (100 V for 2 h at 4 °C). The papers were blocked for 60 min in 1% bovine serum albumin and 0.5% Tween 20 in Tris-buffered saline and treated overnight with the respective rabbit anti-PKC antibodies (Sigma). The signals were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG and the ECL method.
Statistical AnalysisThe hybridization signals for PKC subtypes mRNA in each group were normalized to the hybridization signals for the housekeeping gene for glyceraldehyde-3-phosphate dehydrogenase. An arbitrary unit of 1 represents the control values. Statistical comparisons between control and treatment groups were performed using Student's t test; in the figures, a single asterisk indicates p < 0.05, a double asterisk indicates p < 0.01, and a triple asterisk indicates p < 0.001.
We first studied the cellular redistribution of PKC and PKC
following GnRH-A and TPA stimulation, since it is a criterion for PKC
activation by extracellular signals (5-8). Both GnRH-A and TPA
stimulated an increase in the molecular weight of cytosolic PKC
within 5 min (Fig. 1), consistent with the size shift
reported for PKC
phosphorylation by Src (29), and with our own
finding that GnRH-A and TPA stimulate protein-tyrosine phosphorylation in
T3-1 cells.2 PKC
in the membrane
fraction is already of the high molecular weight form and is further
elevated by GnRH-A and even more by TPA. In addition, translocation of
PKC
to the membrane fraction by GnRH-A and TPA is further validated
by the appearance of degradation products of 70 and 42 kDa (apparently
PKM; Refs. 6-8) in the membrane fraction in the ligand-treated groups
(3-4-fold stimulation by GnRH-A and TPA; Fig. 1). PKC
activation is
manifested by translocation to the membrane fraction and the appearance
of 50- and 42-kDa bands (apparently PKM) in the ligand-treated groups
(2-fold; Fig. 1). Consistent with our previous reports that
TPA-mediated down-regulation of endogenous PKC in
T3-1 cells reduced
cellular PKC activity by 90% (12, 21, 23), prolonged incubation with
TPA (100 ng/ml, 24 h) resulted in loss of most of PKC
(60 and
90% of the membrane and soluble enzyme, respectively), and all
of the detectable soluble and membrane-bound PKC
(Fig. 1).
The regulation of PKC and PKC
mRNA levels was determined by
treatment of
T3-1 cells with [D-Trp6]GnRH,
a stable GnRH analog. Addition of GnRH-A to the cells for 1 h
elevated PKC
but not PKC
mRNA levels in a dose-related
fashion, with maximal response obtained at 10 nM analog
(Fig. 2A). Stimulation of PKC
mRNA
levels by GnRH-A was rapid, with a peak at 1 h, declining thereafter to basal levels (Fig. 2B). On the other hand,
significant elevation of PKC
mRNA levels was detected only after
24 h of incubation with GnRH-A (3-fold, p < 0.001; Fig. 2B).
The effect of GnRH-A on PKC mRNA levels was investigated
further, since its rapid nature suggested that it more likely
represents a physiological response to the neurohormone. Pretreatment
of the cells with a potent GnRH antagonist (Fig.
3A) or with actinomycin D (Fig.
3B) abolished the stimulatory effect of GnRH-A upon PKC
mRNA levels, indicating a receptor-mediated effect apparently at
the transcriptional level (Fig. 3).
The potential role of PKC and Ca2+ in mediating the GnRH
response upon PKC mRNA levels was investigated since both
messengers were implicated in GnRH action upon gonadotropin release and
gonadotropin subunits gene expression (5, 6, 12-26, 30). Addition of the PKC activator TPA to
T3-1 cells for 1 h resulted in
elevation of PKC
but not PKC
mRNA levels, while the
Ca2+ ionophore, ionomycin, had no effect (Fig.
4, A and C, and data not shown).
Elevation of PKC
mRNA levels by TPA was rapid, with a peak at
1 h and a return to basal levels (Fig. 4B). The similar time responses elicited by GnRH-A and TPA suggest that PKC is involved
in GnRH-A stimulation of PKC
gene expression.
This notion was further supported by inhibition and depletion of PKC.
Addition of the selective PKC inhibitor GF 109203X (27, 31) to the
cells resulted in a dose-related inhibition of the GnRH-A stimulated
PKC mRNA levels with half-maximal inhibition (IC50)
observed at 0.8 µM of the drug, in good agreement with IC50 values of PKC inhibition in cellular systems such as
Swiss 3T3 fibroblasts (31) (Fig. 5A). The
drug alone (1 µM) reduced the basal level by about 50%,
suggesting that PKC is also involved in the maintenance of basal PKC
gene expression. We also used down-regulation of endogenous PKC by
prolonged incubation with TPA. Pretreatment of the cells with TPA (100 ng/ml, 24 h) reduced cellular PKC activity by 90% as measured by
enzymatic activity assay and Western blot analysis (Fig. 1 and Refs.
12, 21, and 23). The stimulatory effect of GnRH-A and TPA upon PKC
mRNA levels was abolished in the down-regulated cells (Fig.
5B). In addition, we observed no additivity between GnRH-A
and TPA upon PKC
mRNA levels (Fig. 6), lending
further support to the role of PKC in mediating the GnRH-A effect on
PKC
gene expression. The Ca2+ ionophore, ionomycin, had
no effect on basal PKC
mRNA levels or on the stimulatory
response elicited by GnRH-A or TPA (Fig. 6). On the other hand,
transfer of
T3-1 cells to Ca2+ free medium, in the
presence or absence of EGTA, abolished stimulation of PKC
mRNA
levels by GnRH-A (Fig. 7). It therefore seems that Ca2+ is necessary but not sufficient for mediation of the
GnRH-A response.
Since GnRH stimulated PKC mRNA levels only after 24 h of
incubation in medium without serum (Fig. 2B), it was
possible that growth conditions are involved in PKC
gene expression.
We therefore examined the role of serum in PKC
and PKC
gene
expression. Transfer of the cells to medium with low serum (0.5%) for
24 h had no effect on GnRH-A-stimulation of PKC
mRNA levels
(Fig. 8). On the other hand, serum starvation advanced
the stimulation of PKC
mRNA levels by GnRH-A to 1 h of
incubation that could not be observed in serum-grown cells (Fig. 8).
Time course of PKC
mRNA levels in serum-starved cells revealed a
rapid effect of GnRH-A at 1 h of incubation with no effect at
24 h, as seen in non-starved cells (Fig.
9A). Similarly, serum starvation exposed a
rapid response (peak at 30 min) of TPA on PKC
mRNA levels (Fig.
9B), suggesting a role for PKC in mediating PKC
gene
expression. Indeed, down-regulation of endogenous PKC by prolonged
incubation with TPA abolished GnRH-A and TPA stimulation of PKC
mRNA levels in serum-starved cells (Fig. 10). Transfer of the cells to Ca2+ free medium, in the presence
or absence of EGTA, abolished the rapid stimulation of PKC
in
serum-starved cells (Fig. 11).
As shown above, when cells are transferred to serum-free medium and
exposed to GnRH-A, elevation of PKC mRNA levels is observed at
24 h, but not at 1 h of incubation (Fig. 12,
columns 1-3). On the other hand, when cells are first
serum- starved (24 h) and later exposed to GnRH-A, elevation of PKC
mRNA levels is observed after 1 but not 24 h of incubation
(Fig. 12, columns 4-6). We therefore exposed normal cells
to GnRH-A for 1 h, washed the cells several times to remove serum
and GnRH-A, and further incubated the cells for 24 h. As seen in
Fig. 12 (columns 7 and 8), PKC
mRNA levels were elevated at 24 h by pretreatment (1 h) with GnRH-A. Thus, the
late effect of GnRH-A on PKC
mRNA levels (Fig. 12, column 3) is due to generation of a rapid signal (1 h, column
5), which is "memorized" during the long starvation period
(t1/2
12 h) required for manifestation
of the early signal by means of removal of the inhibitory effect of the
serum.
Whereas much has been learned concerning the regulation of PKC and
its subspecies at the protein level (1-9), very little is known about
ligand regulation of PKC subtypes gene expression (10-12).
Differentiation regulators of the human promyelocytic leukemia cell
line (HL-60) such as 1,25-dihydroxyvitamin D3, retinoic
acid, and dimethyl sulfoxide, were shown to increase the expression of
PKC
and PKC
mRNA levels (10, 11). Furthermore, transcriptional activation of PKC
and PKC
expression was reported to result in increased PKC enzymatic activity (10, 11, 32). Here we
demonstrate that GnRH-A, which does not promote growth or
differentiation, is capable of activating differential nPKC isoforms
gene expression. To the best of our knowledge, this is the first
demonstration of a natural ligand stimulation of PKC
and PKC
mRNA levels.
Activation of PKC mRNA levels, but not that of PKC
, is
dependent upon growth conditions, suggesting the presence of a serum factor that is involved in regulation of PKC
gene expression, possibly via a serum response element.
The differential activation of PKC and PKC
mRNA levels by
GnRH-A suggests that the isoforms might specialize in different functions. PKC
is the major subspecies in the 6-day-old rat
pituitary and is markedly reduced in the 3-month-old pituitary (26).
The opposite is observed for pituitary PKC
, which increases with age
(26). Therefore, PKC
and PKC
might play different roles during
pituitary development. It was also shown that while PKC
is involved
in exocytosis, PKC
participates in feed-back inhibition of
phospholipase C activity in rat basophilic RBL-2H3 cells (33). In a
recent study, GnRH was shown to translocate PKC
and PKC
in
T3-1 cells while PKC
was not detected (30). We report here that
both the PKC
and PKC
isoforms are expressed in the
T3-1 cells
at the mRNA and protein levels and that they are translocated to
the membranes and activated in response to GnRH-A, as also validated by
the apparent formation of the PKM species (6-8). The differences
between the reports are most likely due to the use of different PKC
type-specific antibodies.
While overexpression of PKC resulted in inhibition of growth rate in
NIH 3T3 cells, overexpression of PKC
increased growth rate, and the
transformed cells (NIH 3T3 or Rat 6 cells) formed tumors in nude mice
(27, 34). Since GnRH affect differentiated responses, it is possible
that PKC
and PKC
are involved in separate functions such as
gonadotropin release and gonadotropin subunit gene expression during
the hormone action. Elevation of mRNA of a given PKC isoform by
ligands in general and by GnRH-A in particular might be a step in the
life cycle of PKCs during hormone action to replenish the enzymes after
translocation and degradation as shown in Fig. 1.
The rapid effect (peak at 60 min) of GnRH-A upon PKC and PKC
(in
serum-starved cells) mRNA levels might be physiologically relevant,
since GnRH is released from the hypothalamus in a pulsatile manner at
intervals of 1-2 h according to the species and its half-life is about
2-4 min (35-37). Thus, prolonged responses such as those observed in
Fig. 2 (24 h) are more difficult to interpret, since it is not clear
whether multiple pulses of GnRH are capable of eliciting a similar
response. Hence, we investigated in more detail the rapid effects of
GnRH, which prompted us to identify the second messengers involved in
the neurohormone action. Indeed, the PKC activator TPA mimicked the
GnRH-A rapid responses and stimulated PKC
mRNA levels in
serum-grown cells and PKC
mRNA levels in serum-starved cells
with a similar time course. Additionally, the stimulatory effect of
GnRH-A on PKC
and PKC
mRNA levels was abolished in
PKC-down-regulated cells or by the use of the selective PKC inhibitor
GF 109203x (27, 31) (present results and data not shown). We therefore
suggest that GnRH-A stimulation of PKC
and PKC
gene expression is
autoregulated by PKC.
While we show here positive regulation of PKC mRNA levels by
GnRH-A and TPA, others have recently reported that PKC
mRNA is
down-regulated by TPA (4 h) in the mouse B lymphoma cell line A20 (38).
The difference between the results might be due to the presence of a
soluble destabilizing factor, which specifically accelerates
degradation of PKC
mRNA in A20 cells (38).
Removal of Ca2+ abolished the effect of GnRH-A upon PKC
and PKC
mRNA levels, but Ca2+ ionophore had no
stimulatory effect. We therefore conclude that Ca2+ is
necessary but not sufficient, while PKC is both necessary and
sufficient to mediate the GnRH response. Furthermore, since removal of
extracellular Ca2+ per se is not sufficient to
block Ca2+ mobilization in pituitary cells (39), the data
suggest that GnRH-A-induced PKC
and PKC
gene expression is mainly
mediated by Ca2+ influx, apparently via L-type
voltage-sensitive channels (39). The data also suggest that a
Ca2+-dependent PKC isoform might be involved in
GnRH-A action. Since PKC
and PKC
are differentially regulated by
GnRH-A, it is unlikely that one regulates the expression of the other
during the neurohormone action. Furthermore, it is unlikely that PKC
is involved in the process since its membrane-bound form (the active
form) was reduced only by 60% by down-regulation, whereas the effect
of GnRH-A was abolished. Further studies are required to identify the
PKC isoforms and the site of Ca2+ action in the GnRH-A
response.
In addition to mediation by Ca2+ and PKC, elevation of
PKC but not PKC
mRNA levels by GnRH-A and TPA required
removal of a serum factor. Therefore, although PKC
and PKC
gene
expression share some common mechanisms, which are mediated by
Ca2+ and PKC, they differ in sensitivity to the serum
factor. Changes in the concentrations of the serum factor under
physiological conditions might therefore enable preferential activation
by GnRH of the two isotypes. Moreover, it was possible to first
stimulate the cells with GnRH-A, and remove the hormone and serum for
24 h, at the end of which elevation of PKC
mRNA levels by
GnRH-A was detected. The observation is in contrast to previous
observations, in which GnRH-stimulated LH release in perifused
pituitary cells was terminated immediately after removal of the
neurohormone (Ref. 40 and data not shown). Since PKC is the main
mediator of GnRH actions (5, 6, 12-26, 30), it is likely that the
half-life of the phosphoproteins involved in exocytosis is very short
(t1/2
8 min; Ref. 40), while those mediating
the neurohormone effect on PKC
gene expression is relatively long
(t1/2
12 h). The presence of the serum
factor does not block the formation of downstream effectors involved in
PKC
gene expression, but only masks the effectors activity. Since
growth conditions also affected TPA stimulation of PKC
gene
expression, it seems that the site of action of the serum factor is
downstream to PKC activation. Since both Ca2+ and PKC
participate in GnRH-stimulated PKC
and PKC
gene expression, it is
likely that transcriptional regulation of both isotypes by GnRH-A
involves similar transcription factors but different coactivators and
composite response elements (41). Future analysis of the gene structure
of both isotypes will reveal the different response elements involved
in ligand regulation of PKC
and PKC
gene expression.
The present report demonstrates differential activation of PKC and
PKC
mRNA levels by GnRH-A that is dependent upon growth conditions and Ca2+ influx, and is autoregulated by PKC.
Since both isotypes are shown here to be translocated by GnRH from the
cytosol to the membrane (an index of PKC activation; Refs. 1-3 and
5-8), PKC
and PKC
are therefore likely candidates to participate
in GnRH action, the first key hormone of the reproductive cycle.
We thank Drs. P. Mellon for the T3-1 cell
line, H. Mischak and F. Mushinsky for PKC subtype cDNAs. We also
thank Sharon Shacham for help during the studies and Angela Cohen and
Erica Vallenci for editorial assistance.