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T3–1 Cell Line: Differential Roles of Calcium and Protein Kinase C1
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University (N.R., S.S., D.H., Z.N.), Ramat Aviv 69978; and the Department of Membrane Research and Biophysics, The Weizmann Institute of Science (L.N.-L., R.S.), Rehovot 76100, Israel
Address all correspondence and requests for reprints to: Zvi Naor, Ph.D., Department of Biochemistry, Tel Aviv University, Tel-Aviv 69978, Israel. E-mail: NAORZVI{at}CCSG.TAU.AC.IL
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Abstract |
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T3–1 cell
line. GnRH-a as well as the protein kinase C (PKC) activator
12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a
sustained response of MAPK activity, whereas epidermal growth factor
(EGF) stimulated a transient response. MAPK kinase (MEK) is also
activated by GnRH-a, but in a transient manner. GnRH-a and TPA
apparently activated mainly the MAPK isoform ERK1, as revealed by
Mono-Q fast protein liquid chromatography followed by Western blotting
as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine
phosphorylation of several proteins, and this effect as well as the
stimulation of MAPK activity were inhibited by the PKC inhibitor GF
109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies
nearly abolished the effect of GnRH-a and TPA on MAPK activity.
Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein
inhibited protein tyrosine phosphorylation and reduced
GnRH-a-stimulated MAPK activity by 50%, suggesting the participation
of genistein-sensitive and insensitive pathways in GnRH-a action.
Although Ca2+ ionophores have only a marginal stimulatory
effect, the removal of Ca2+ markedly reduced MAPK
activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA
stimulation of protein tyrosine phosphorylation. Interestingly, the
removal of Ca2+ also partly inhibited the activation of
MAPK by EGF and vanadate/H2O2. Thus, a
calcium-dependent component(s) downstream of PKC and PTK might also
participate in MAPK activation. Elevation of cAMP by forskolin exerted
partial inhibition on EGF, but not on TPA or GnRH-a action, suggesting
that MEK activators other than Raf-1 might be involved in GnRH action.
We conclude that Ca2+, PTK, and PKC participate in the
activation of MAPK by GnRH-a, with Ca2+ being necessary
downstream to PKC and PTK. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Signal transduction elicited by hormones and growth factors has been found to involve sequential activation of several cytosolic protein kinases collectively known as the mitogen-activated protein kinase (MAPK) cascade (4, 5, 6, 7, 8, 9, 10). The MAPK gene family converges signals from G protein-coupled receptors (GPCRs) or growth factor receptor tyrosine kinases (RTKs), leading to cellular responses such as differentiation and proliferation. The mechanism of MAPK activation seems to differ among various stimuli. RTKs signal via an adaptor molecule, GRB2, and a guanine nucleotide exchange factor, mSOS, to activate the small GTP-binding protein (G protein), Ras, followed by activation of the MAPK cascade: Raf-1, MAPK kinase (MAPKK; MEK), and MAPK. On the other hand, GPCRs are thought to act via diverse mechanisms, including PKC-dependent and independent pathways, to activate the above cascade (6, 7, 8, 9, 10).
The signals transmitted through the MAPK cascade lead to activation of
a set of regulatory molecules that eventually initiates cellular
responses such as growth and differentiation (4, 5, 6, 7, 8, 9, 10). Recently, it has
been shown that GnRH is capable of activating MAPK in pituitary organ
culture (11) and the T3–1 gonadotroph cell line (12, 13) via PKC,
and that MAPK is involved in regulation of gene expression of the
gonadotropin -subunit (12, 13). We decided to follow the above
reports and study further the mechanism of MAPK (ERK1 and ERK2)
activation by GnRH. We found that GnRHR signaling results in
stimulation of mainly the ERK1 isoform and involves a
Ca2+-dependent pathway that does not include
Gi and Go and seems to require MEK
activators other than Raf1 as well as a protein tyrosine kinase (PTK)
upstream to the MAPK cascade. Furthermore, we show that
Ca2+ is required, but not sufficient, and its site of
action is downstream to PKC and PTK signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP was purchased from Rotem Industries
(Beersheba, Israel). Genistein, phenylmethylsulfonylfluoride, EGTA,
12-O-tetradecanoyl phorbol-13-acetate (TPA), EGF
(recombinant), forskolin, and other chemicals were purchased from Sigma
Chemical Co. (St. Louis, MO). Recombinant ERK2 used in the MAPKK assay
was prepared as previously described (15). Anti-P-tyrosine antibodies
were obtained from Zymed (San Francisco, CA). Anti-MAPK serum was
generously donated by Dr. Y. Granot, Ben-Gurion University (Beer-Sheva,
Israel). The selective PKC inhibitor bisindolylmaleimide (GF 109203X)
(16) was purchased from Calbiochem (Laufelfingen, Switzerland).
Buffers
Detergent lysis buffer consisted of 50 mM Tris-HCl
(pH 6.8), 2 mM EGTA, 1 mM sodium orthovanadate,
20 mM NaCl, 10 mM ß-glycerophosphate, 28
mM ß-mercaptoethanol, 1 mM
phenylmethylsulfonylfluoride, 20% glycerol, 50 nM each of
okadaic acid and calyculin A, and 0.5% Nonidet P-40.
Buffer A consisted of 50 mM ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium orthovanadate.
Buffer H consisted of 50 mM ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, 1 mM benzamidine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 2 mg/ml pepstatin A.
Cell culture
T3–1 cells were grown in DMEM supplemented with 5% FCS, 5%
horse serum, penicillin (100 U/ml), and streptomycin (0.1 mg/ml). At
70–80% confluence, the cells were serum-starved for 18 h in
DMEM, and 0.25% each of FCS and horse serum and stimulants were added
in DMEM at the indicated concentration for the given length of
time.
Fractionation and assay of MAPK and MAPKK
For determination of MAPK activity in cell extracts, the cells
were rinsed twice with ice-cold PBS and lysed for 10 min in detergent
lysis buffer. Samples of 5 µl (
2 µg protein) were subjected to
MAPK assay at 22 C for 20 min. The reaction mixture (25 µl) contained
50 mM HEPES (pH 7.8), 50 µM
[-32P]ATP (1–2 cpm/fmol), 2 mM EGTA, 28
mM ß-mercaptoethanol, 2 mM MnCl2,
10 mM MgCl2, 0.8 mM sodium
orthovanadate, and EGF receptor peptide (1 mg/ml). Reactions were
quenched by spotting 10 µl of the reaction mixture on
phosphocellulose paper squares (P81, Whatman, Clifton, NJ), which were
washed in 150 mM phosphoric acid. Phosphate incorporation
was measured by the Cerenkov method.
An in-gel kinase assay was performed as previously described (17), using myelin basic protein (MBP; 0.5 mg/ml gel) as a substrate.
For determination of fractionated MAPK and MAPKK activities (15), cells were harvested in buffer H and disrupted by two 7-sec sonication (50 watts) on ice, followed by centrifugation at 20,000 x g for 15 min at 4 C.
For DE-52 cellulose fractionation, the cytosolic extracts (0.5 ml) were applied to minicolumns (0.35 ml). The flow-through and wash in 0.02 M NaCl in buffer A were collected and measured as previously described (18) for MAPKK activity in a double couple assay in the presence or absence of recombinant ERK2. The eluate of 0.22 M NaCl in buffer A (0.75 ml) contained more than 85% MAPK activity, measured toward MBP as recently described (15).
For fast protein liquid chromatography fractionation, the cytosolic extracts (6 mg protein from 2 x 108 cells) were loaded on a 1 ml Mono-Q column and fractionated using a 0–0.4 M linear NaCl gradient in buffer A (1 ml/min; 1-ml fractions) as described by Ahn et al. (19). Even fractions were assayed for MAPKK and MAPK activities as described below.
Immunoblotting
Proteins in the cell extracts were separated on 7.5–18%
PAGE-SDS gels (ratio of acrylamide to bisacrylamide, 30:0.5) and
electrotransferred to nitrocellulose papers at 4 C and 100 V in 50
mM glycine and 50 mM Tris-HCl, pH 8.8. For
detection of phosphotyrosine-containing proteins, the papers were
blocked for 60 min in 1% BSA and 0.5% Tween-20 in TBS (20
mM Tris, pH 8.3, and 150 mM NaCl) and treated
overnight with 0.1 µg/ml affinity-purified rabbit anti-P-tyrosine
antibodies (Zymed). After intensive washing, the signals were
visualized using horseradish peroxidase-conjugated goat antirabbit IgG
and the enhanced chemiluminescence method.
Experiments, in duplicate or triplicate, were repeated two or three times, and the results shown are from a representative experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells with GnRH-a or TPA resulted in a rapid
activation of MAPK (within 1 min), as revealed by phosphorylation of
EGF receptor peptide or MBP (Fig. 1
). Maximal activity
(4- to 5-fold over basal levels) was noticed around 10 min of
incubation and persisted for the entire period examined here (60 min).
The stimulatory effect of GnRH-a and TPA on MAPK activity declined to
near-basal levels only after 2–4 h of incubation (not shown). On the
other hand, EGF stimulated a transient response of MAPK activation that
peaked 5 min after treatment and declined to basal levels within 20 min
(Fig. 1, inset). The similarity of the GnRH-a and TPA time
course was consistent with PKC being involved in GnRH-a stimulation of
MAPK, as recently shown by Sundaresan et al. (13), whereas
EGF might operate via a different mechanism (4, 5, 6, 7, 8, 9, 10).
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). On the other hand, the
Ca2+ ionophores, ionomycin and A23187 (not shown), had
marginal or no stimulatory effect on MAPK activity (Fig. 2) even when
combined with TPA (data not shown).
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).
No MAPKK activity was observed in nonstimulated cells (not shown).
However, unlike that of MAPK, activation of MAPKK by GnRH-a was of
short duration, peaked at 5 min of stimulation, and declined to
near-basal levels at 10 min, suggesting different regulatory
termination mechanisms at the two levels of the MAPK signaling
cascade.
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and data not shown). As MAPKK is highly specific toward its MAPK
substrates (18), its activation generally results in MAPK stimulation.
Indeed, as shown in Fig. 4B, two peaks of MBP phosphorylating
activities are markedly activated by GnRH-a. Their elution in peak
fractions 36 and 46 corresponds to the elution of ERK2 and ERK1 as
previously described (15) and verified here by Western immunoblotting
using anti-MAPK serum (Fig. 4B, inset, lanes 2 and 3), which
recognizes both MAPK isoforms to the same extent (data not shown). An
in-gel kinase renaturation assay of crude T3–1 cell lysate also
revealed the enhanced activation of ERK1 by GnRH-a (not shown). Similar
to GnRH-a, TPA caused preferential activation of ERK1 (not shown). As
the ratio of ERK2 to ERK1 in T3–1 cells is about 3:1 (Fig. 4B, inset, lane 1), the data suggest that in pituitary
gonadotrophs, GnRH-a as well as TPA stimulate mainly the ERK1 isoform
of MAPK. This is in contrast to most other cells and ligands that seem
to activate mainly the ERK2 isoform (9). Thus, ERK1 might be involved
in regulation of differentiated cellular functions of GnRH.
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T3–1 cells with PTX (100 ng/ml; 18 h) had no
significant effect on stimulation of MAPK activity by GnRH-a, TPA, and
EGF (Fig. 5). Thus, the effects of GnRH-a, TPA, and EGF
on MAPK activity in T3–1 cells do not involve PTX-sensitive G
proteins such as Gi or Go. On the
other hand, PTX inhibited (by 47%) serum (10% FCS) stimulation of
MAPK activity (Fig. 5).
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T3–1 cells with GnRH-a resulted in the elevation
of tyrosine phosphorylation of several proteins of 42, 46, 85, 95, 100,
125, and 170 kDa (Fig. 6 and data not shown). The
magnitude of the response ranged from 2- to 4-fold over basal levels,
and the ED50 ranged from 1 nM, similar to that
observed for MAPK activation (Fig. 2
). A similar protein
phosphorylation pattern was noticed with TPA (not shown).
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), suggesting the participation of PTK-dependent and
independent pathways in MAPK activation by GnRH-a. Genistein also
inhibited the stimulation of MAPK by EGF by 90% (not shown).
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).
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), protein tyrosine phosphorylation
stimulated by GnRH-a and TPA was also inhibited by GF 109203X, with an
IC50 ranging from 0.4–0.8 µM, with the
exception of pp125 phosphorylation by GnRH-a (IC50, 30
µM; Fig. 8B and data not shown). The data strongly
suggest that PKC mediates the activation of MAPK and PTKs by GnRH-a.
The drug alone (up to 30 µM) had no effect on MAPK
activity. In addition, the drug showed no inhibition of MAPK activity
in vitro, of the PTK activity of EGF receptor (from A431
cells), or of the total PTK activity of T3–1 cell lysate (not
shown).
We also examined the activation of MAPK in PKC down-regulated cells.
Prolonged pretreatment of the cells with TPA (100 ng/ml; 16 h)
reduced endogenous PKC activity by about 90% (26, 27). GnRH-a and TPA
stimulation of MAPK activity was abolished in the PKC down-regulated
cells (Fig. 9). Further examination of GnRH-a
stimulation of MAPK in control and PKC down-regulated cells at
different time points revealed loss of responsiveness at all time
points examined (Fig. 9, inset). The data rules out a
possible PKC-independent transient response elicited by GnRH-a.
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). Pretreatment of the
cells with forskolin (50 µM; 15 min) stimulated basal
MAPK activity (50%; P < 0.05). The EGF response
(5-min treatment) was partially inhibited (31%; P <
0.02), but no significant effect on GnRH-a and TPA stimulation of MAPK
activity was exerted by forskolin at 15 min.
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). On the other hand, transfer of the cells to
Ca2+-free medium containing EGTA (250 µM)
markedly reduced the stimulatory effect of both GnRH-a and TPA on MAPK
activity (Fig. 11), but not on protein tyrosine
phosphorylation (Fuchs, T., M. Liskovitch, and Z. Naor, in
preparation). Thus, Ca2+ is necessary, but not sufficient,
for mediating the effect of GnRH-a on MAPK activity. Surprisingly,
removal of Ca2+ resulted in partial inhibition (50%) of
EGF or vanadate/H2O2 stimulation of MAPK
activity, but had no effect on PTK activity stimulated by
vanadate/H2O2 (Fig. 11 and data not shown). The
data suggest the participation of a Ca2+-dependent
component distinct from PKC in the activation mechanism of MAPK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transient activation of MAPK is thought to be associated with cellular
growth and proliferation, whereas prolonged activation is involved in
differentiation (31, 32). GnRH-a, which triggers differentiated
cellular functions such as gonadotropin secretion and synthesis, is
also found capable of inducing a sustained MAPK signal in the
gonadotroph T3–1 cell line. Thus, prolonged activation of MAPK
might be associated with alternative cellular functions. Furthermore,
GnRH-a is shown here to apparently activate mainly the ERK1 isoform,
whereas other ligands were reported to activate mainly ERK2 (4, 5, 6, 7, 8, 9, 10).
The significance of the differential activation of ERK1 vs.
ERK2 is under investigation. Interestingly, unlike the prolonged MAPK
activation, stimulation of MAPKK (MEK) by GnRH-a was transient,
suggesting that distinct termination signals and protein phosphatases
operate on these two members of the signaling cascade during GnRH-a
action.
Different mechanisms were proposed for MAPK activation by ligands
operating via GPCRs. Thrombin and lysophosphatidic acid stimulate MAPK
via Gi in a PTX-sensitive, PKC-independent
pathway (20, 21), whereas endothelin-1 acts in a PTX-sensitive and
PKC-dependent mechanism (22). The agents acting via muscarinic
receptors m1 and m2 stimulate MAPK apparently via the ß-subunits
of G proteins (Gi and Gq) in a
Ras-dependent, PKC-independent process (33). Other
Gq-linked receptors, such as that for bombesin, are thought
to mediate activation of MAPK almost entirely via PKC (34), or in the
case of TRH, activation is mediated partly by PKC and also by a
Ras-dependent pathway (35). The results presented here for GnRH-a
represent another aspect of signaling, emphasizing the differential
role of PKC and Ca2+ in MAPK activation. In contrast to a
previous report (23), our results indicate that
Gi and Go are not involved in GnRH-a
activation of MAPK by demonstrating the lack of inhibition of the
GnRH-a response by pertussis toxin. The reason for the discrepancy is
not known. Thus, GnRH-a stimulation of MAPK activity is most likely
mediated by Gq via activation of phospholipase C (36, 37).
Activation of MAPK by RTKs, GPCRs, and phorbol esters was inhibited by
elevation of cAMP (17, 28, 29; see Ref. 30 for review). However, in the
PC12 cell line, forskolin seems to slightly elevate basal MAPK activity
without affecting its maximal stimulated activity (17). The reason for
this discrepancy is probably due to the pattern of MEK activator
expression in PC12 cells (17). Indeed, the stimulation of basal MAPK
activity by forskolin and the lack of inhibition of the GnRH-a and TPA
responses observed here resemble the effect of forskolin in PC12 cells.
However, the partial inhibition of EGF-stimulated activity suggests
that more than one signaling mechanism leads to MAPK activation in
T3–1 cells. Thus, EGF stimulates MAPK activity by both
cAMP-sensitive and insensitive pathways, whereas GnRH-a and TPA act by
a cAMP-insensitive mechanism. One possible explanation for these
distinct pathways is that GnRH-a and TPA signals might be transmitted
by MEK activators that are distinct from the cAMP-sensitive Raf-1 (30, 38), whereas EGF signals use both Raf-1 and the other MEK
activators.
Elevation of Ca2+ by ionomycin had marginal effect on MAPK
activity in T3–1 cells. The results differ from those reported in
other cell systems, where elevated Ca2+ activated MAPK (39, 40). Furthermore, raising the levels of extracellular Ca2+
inhibited EGF-induced ERK2 activity in human primary keratinocytes
(41). On the contrary, in our system, removal of Ca2+
markedly reduced the GnRH-a and EGF responses. Thus, Ca2+
is necessary, but not sufficient, for mediating GnRH-a elevation of
MAPK activity. On the other hand, TPA elevates MAPK activity, whereas
PKC inhibition or its depletion resulted in inhibition of the GnRH-a
response. Thus, PKC is necessary and sufficient for mediating the
GnRH-a effect. As activation of phosphoinositide turnover by GnRH is
Ca2+ independent (36), it is possible that the
Ca2+-requiring step involves the activation of a
Ca2+-dependent PKC subtype, consistent with the report that
PKC activates Raf-1 by direct phosphorylation (42). However, the
inhibitory effect of Ca2+ removal on the action of GnRH-a
as well as that of EGF or vanadate/H2O2 on MAPK
activity, but not on protein tyrosine phosphorylation or PTK activity,
suggests that another Ca2+-binding protein might
participate in the activation process downstream from PKC and PTK.
One of the targets of MAPK is the cytosolic phospholipase A2, which is phosphorylated and activated by MAPK (43). As the release of arachidonic acid and formation of its lipoxygenase products play a role in GnRH-induced gonadotropin release and gonadotropin subunit gene expression (44, 45, 46, 47, 48, 49), it is likely that activation of MAPK by GnRH is required to enable stimulation of the arachidonate cascade. In addition, some potential MAPK substrates, such as c-fos, Elk1, Ets2, and TAL-1 (6, 7, 8, 9, 10), might be required for GnRH actions as regulators of nuclear transcriptional activity.
Consistent with the reported effect of a general PKC activator such as
TPA on protein tyrosine phosphorylation (50, 51), GnRH-a, stimulated
the tyrosine phosphorylation of proteins of 42, 46, 85, 95, 100, 125,
and 170 kDa, which might represent p42/p44 MAPKs, focal adhesion kinase
(p125FAK) (21), and others. Interestingly, the phosphotyrosyl proteins
induced by GnRH-a were inhibited by the selective PKC inhibitor GF
109203X, with an IC50 of about 1 µM, with the
exception of pp125, which required higher doses of the drug
(IC50, 30 µM). As inhibition of GnRH-a- and
TPA-induced MAPK activation by GF 109203X was observed at an
IC50 of 1.8 µM, some of the
tyrosine-phosphorylated proteins, but not pp125, might be linked to
MAPK in a manner yet to be determined. This is further supported by the
inhibitory effect of the PTK inhibitor genistein on GnRH-a stimulation
of MAPK activity. Furthermore, as the GnRH effect on protein tyrosine
phosphorylation and MAPK activation is fully dependent on PKC, whereas
genistein only partly inhibits (50%) the activation of MAPK by GnRH,
it seems likely that at least two PKC subspecies that specialize in
PTK-dependent and -independent signaling participate in the action of
GnRH on MAPK. Nevertheless, PKC-sensitive mechanisms are involved in
GnRH-a-induced protein tyrosine phosphorylation, which apparently are
manifested upstream of MAPK activation. Based on the data presented
here, we propose a model for GnRH-induced MAPK activation (Fig. 12). The present study opens a new vista in elucidating
the mechanism of action of the neurohormone GnRH, which is the first
key hormone of reproduction, and sheds further light on the pathways
involved in RTKs vs. GPCRs leading to activation of
MAPK.
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Acknowledgments |
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T3–1 cells, and Ms. T. Fuchs
and Drs. U. Zor, Y. Granot, and M. Liscovitch for their interest and
help during the studies. We also thank Mrs. Angela Cohen for editorial
assistance. |
Footnotes |
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2 Incumbent of the Samuel and Isabela Friedman Career Development
Chair.
Received October 3, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter.
Mol Cell Biol 15:3531–3539[Abstract]
and -
in NIH 3T3 cells
induces opposite effects on growth, morphology, anchorage dependence,
and tumorigenecity. J Biol Chem 268:6090–6096-subunit mRNA levels in the T3–1 cell line: role of
Ca2+ and protein kinase C. Biochem J 309:325–329
-subunit mRNA levels in pituitary T3–1 cell line: role in
gonadotropin releasing hormone. Biochemistry 33:12795–12799[CrossRef][Medline]
T3–1 cell line: role in
gonadotropin-releasing hormone action. Biochem J 316:667–670
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