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T3–1 Cell Line Is Mediated by Protein Kinase C, c-Src, and CDC42
Department of Biological Regulation (N.L.L., T.H., O.B., M.R.,
R.S.) The Weizmann Institute of Science Rehovot, 76100
Israel
Department of Biochemistry (M.R., D.H., N.R.,
Z.N.) George S. Wise Faculty of Life Sciences Tel Aviv
University Ramat Aviv 69978, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T3–1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as
well as tumor-promoting agent (TPA) also increased c-Src activity,
which peaked at 2 min after GnRH stimulation and was sensitive both to
PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which
serves as a Src-dominant interfering kinase, and constitutively active
forms of Src, together with JNK, confirmed the involvement of c-Src
downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant
negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1,
and MEK1 with JNK indicated that JNK activation by GnRH and TPA is
mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a
related mitogen-activated protein kinase (MAPK) pathway, did not affect
JNK activation in T3–1 cells. Taken together, our results suggest
that GnRH stimulation of JNK activity is mediated by a unique pathway
that includes sequential activation of PKC, c-Src, CDC42, and probably
also MEKK1. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GnRH, which is a hypothalamic decapeptide, serves as a key regulator of the reproductive system. It induces the synthesis and release of the pituitary gonadotropin LH and FSH. When GnRH binds to its seven-transmembrane receptor, it induces interaction of the receptor with the heterotrimeric Gq protein that leads to activation of phospholipase C and formation of inositol 1,4,5- trisphosphate and diacylglycerol, leading to elevation of Ca2+ and activation of protein kinase C (PKC). Phospholipase D, phospholipase A2, and the formation of bioactive lipoxygenase products are also activated by GnRH, although the mechanisms involved are not yet known (reviewed in Refs. 14 and 15). Recently, the stimulation of the ERK-signaling cascade by GnRH has been demonstrated by several laboratories (16, 17, 18, 19). ERK activation in response to GnRH is unique as it was shown to be fully mediated by PKC and Ca2+, which seem to act in a Ras-independent manner (19). Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein partially inhibited this pathway, which indicates the involvement of a PTK in the GnRH-induced ERK activation.
In this study, stimulation of the JNK pathway by GnRH was examined. Our
results show that GnRH-induced JNK activation was greater but occurred
more slowly than that of ERK. The signaling pathway that mediates the
GnRH stimulation of JNK appears to involve PKC, c-Src, CDC42/Rac1, and
probably also MEKK1, and to act independently of the ERK cascade. Thus,
the T3–1 cells appear to utilize a unique pathway that links a G
protein-coupled receptor to the activation of the JNK-signaling cascade
through PKC, c-Src, and CDC42.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T3–1 cells (19). To examine whether
GnRH can also activate the JNK pathway, serum-starved T3–1 cells
were stimulated with a GnRH analog (GnRH-a, 10-7
M), and JNK activity was monitored by a solid phase assay
(20). As shown in Fig. 1
, JNK activity
was significantly (25- to 55-fold) stimulated by GnRH-a, to an extent
similar to that caused by the potent stimulating agents, peroxovanadate
(VOOH, 15 min) and the PKC activator tumor-promoting agent (TPA, 30
min). This magnitude of stimulation was higher than that observed for
ERK1/2 (5- to 15-fold) in the same cells. However, the time course of
JNK activation by GnRH-a was slower than the rapid activation of the
ERKs. JNK activation was detected 5 to 10 min after the initiation of
the GnRH-a treatment, it peaked by 30 min, and it decreased over the
next 90 min (Fig. 1B). Thus, although both ERK and JNK might be key
mediators of GnRH signaling, they may regulate different cellular
processes.
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T3–1 cells. Northern blot analysis, using
c-Jun as a probe, revealed that GnRH-a induced the expression of c-Jun
mRNA in these pituitary cells (Fig. 1C
), thus confirming the findings
of Cesnjaj et al. (21). Taken together, these results
indicate that the JNK cascade does indeed play a role in GnRH signaling
in T3–1 cells.
Effects of PKC and PTK Inhibitors on JNK Activation
Activation of ERKs by GnRH-a requires PKC and to some extent also
protein tyrosine phosphorylation (16, 17, 18, 19). Therefore, the role of PKC
and PTKs in GnRH-induced JNK activation was examined. Exposure of
T3–1 cells to TPA or to VOOH (general activator of tyrosine
kinases) caused a pronounced stimulation of JNK activity (Fig. 1A),
which suggested that PKC and PTK might stimulate JNK activity in this
cell line. Whether PKC is indeed involved in GnRH signaling was
determined using PKC depletion and inhibition. Thus, T3–1 cells
were pretreated with TPA (1 µM, 16 h) which depletes
most PKC isoforms, or with the selective PKC inhibitor GF109203X. In
both cases there was a significant (80–85%) inhibition of the
GnRH-induced JNK activity (Fig. 2A).
These results suggest that, as in the case of ERK (19), activation of
JNK by GnRH-a in these cells is mainly mediated by PKC.
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T3–1 cells with the PTK inhibitor,
genistein. Genistein completely abolished the GnRH-stimulated JNK
activity for up to 1 h after stimulation (Fig. 2B) but only inhibited
ERK activation by 
30% (19), indicating the existence of a
genistein-sensitive PTK that mediates the activation of JNK by GnRH-a.
To determine whether this PTK is located upstream or downstream to PKC,
genistein-treated T3–1 cells were stimulated with TPA. As shown in
Fig. 2, C and D (that quantitate the results of panels A–C),
stimulation of JNK activity by TPA was most prominent after 60 min
(30-fold), and this stimulation was completely abolished by
pretreatment with genistein. TPA bypasses the GnRH receptor-Gq complex
and activates PKC directly; these results indicate that PKC is located
upstream to the PTK(s) in the pathway that mediates the activation of
JNK by GnRH. Sequential activation of PKC and PTKs that leads to JNK
activation in G-protein coupled receptors signaling seems to be unique
to GnRH-stimulated T3–1 cells, as will be discussed below.
c-Src Is Involved in the GnRH-JNK Signaling Pathway
The involvement of a putative PTK that was implicated by the use
of genistein initiated an effort to identify the PTK that is involved
in JNK activation by GnRH. c-Src or other member(s) of the Src family
of PTKs are genistein-sensitive protein kinases that have been
previously reported to participate in G-protein signaling (22, 23, 24). To
examine whether a c-Src family member is the PTK that mediates the
effect of GnRH on JNK, we first asked whether these kinases are
expressed in the T3–1 cells. Western blot analysis revealed that
the T3–1 cells contain a substantial amount of c-Src, a small
amount of Fyn, and, as expected, no Lyn (Fig. 3A). Activation of c-Src by GnRH-a was
probed by immunoprecipitating c-Src from GnRH-a-stimulated cells using
specific antibodies and measuring its activity using enolase as a
substrate (25). GnRH-a caused an 3.2-fold increase in c-Src
activity, which peaked at 5 min after stimulation and was maintained
for an additional 15 min (Fig. 3, B and C). Although the fold
activation seems to be small, it is comparable to the activation
reported in other cellular systems (22) and might be due to a high
basal activity of this PTK. c-Src was activated also by TPA (2.6-fold;
Fig. 3, B and C), and the activation of c-Src by GnRH was sensitive
both to PKC and to PTK inhibitors, thus indicating that the activation
of c-Src in response to GnRH-a is downstream of PKC.
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T3–1 cells. GnRH-stimulated JNK activity was markedly inhibited by
the overexpression of Csk, whereas the basal activity of JNK was not
affected (Fig. 4A), indicating that most,
if not all, the GnRH signal toward JNK is indeed mediated by c-Src. To
further verify this point we also cotransfected the constitutively
active form of c-Src, Y527F-Src, together with HA-JNK2 into the same
cells. Y527F-Src by itself activated JNK up to 4-fold above basal
activity, and this activity was not enhanced by the addition of GnRH-a
to the cells (Fig. 4, B and C), further indicating that most of the
GnRH signal is funneled through c-Src. Moreover, the activation of JNK
by Y527F-Src was sensitive to genistein (Fig. 4, B and C), indicating
that c-Src by itself is a target for genistein and therefore might be
the genistein- sensitive PTK in the GnRH-JNK pathway. As expected,
GF109203X did not influence the Y527F-Src activation of JNK (up to 60
min treatment with the inhibitor). Therefore, GnRH signal toward JNK
appears to be mediated mainly via the genistein-sensitive c-Src (or a
family member), operating downstream of PKC.
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T3–1 cells together with HA-JNK2. After cell
treatment with the desired stimuli and inhibitors, lysis of the cells
and immunoprecipitation with anti-HA antibodies, JNK2 activity was
measured using GST-Jun as a substrate. All three inactive forms of the
sGP had an inhibitory effect on the GnRH-stimulated JNK2 activity (Fig. 5, A and B). T17N-CDC42
inhibited GnRH-stimulated JNK activity by as much as 62 ± 5%,
the T17N-Rac1 by 36 ± 4%, and the S17N-H-Ras by only 17 ±
4%.
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T3–1 cells together with HA-JNK2, and the
stimulation of JNK activity was determined as above. The constitutively
active CDC42 stimulated JNK activity greatly [8.8 ± 1.3 fold
(n = 3), which is 65% of GnRH stimulated JNK activity under these
conditions]. The constitutively active form of Rac1 had a moderate
effect (3.8 ± 0.5 fold), and the constitutively active H-Ras had
only a marginal effect (1.9 ± 0.7 fold; Fig. 5C). The stimulation
of HA-JNK2 by Q61L-CDC42 or by Q61L-Rac1 was not affected by the PKC
inhibitor GF109203X nor by the PTK inhibitor genistein (Fig. 5C). In
addition, TPA stimulation of JNK activity was significantly inhibited
by the T17N-CDC42 (Fig. 5D), which supports CDC42 and also, to a lesser
extent, Rac1 acting downstream of PKC and c-Src. The effects of the PKC
and PTK inhibitors on cells cotransfected with the constitutively
active form of H-Ras could not be determined because of the low
stimulation with this construct. The above results indicate that most of the genistein-sensitive, GnRH-induced JNK activation is mediated by CDC42. Although in many cases CDC42 and Rac1 seem to use similar guanine nucleotide exchange factors, the effect of Rac1 on the GnRH-JNK pathway seems to be limited. Similar phenomenon, where CDC42 transmits downstream signals without the involvement of Rac1, was recently reported for serum-induced proliferation (28). Ha-Ras does not seem to be significantly involved in GnRH-induced signaling that contributes to the JNK cascade.
GnRH Stimulation of JNK Is Mediated by MEKK1 But Not by MEK1
Several reports have recently suggested that the stress-activated
JNK cascade consists of JNK, JNKK (SEK1, MKK7), MEKK1 (6), and either
PAK1 (29) or MLK (30). To determine whether the genistein-sensitive
GnRH signal toward JNK is mediated via this cascade, T3–1 cells
were cotransfected with HA tagged-JNK2 and the truncated,
constitutively active form of MEKK1 (31) and processed as described for
the sGPs above. The constitutively active MEKK1 elicited a strong
activation (25 fold) of JNK2, which was somewhat lower than the
activation caused by GnRH-a (data not shown) but could not be further
activated by treatment of the transfected cells with GnRH-a (Fig. 6). The lack of synergistic activation
with MEKK1 and GnRH indicates (although does not prove) that MEKK1
might be involved in the GnRH-JNK signaling pathway. Indeed, the MEKK1
effect was not blocked by GF109203X or genistein when added for 15
(Fig. 6), 60, or 120 min (data not shown), thus indicating that PKC and
the genistein-sensitive PTK(s) are probably situated upstream of MEKK1,
(although they may lie on a distinct pathway in case MEKK1 is not the
activatory component as suggested above). On the other hand,
coexpression of HA-JNK2 with the constitutively active form of MEK1
[
N-EE-MEK (32, 33)] which is involved in the distinct ERK
signaling cascade, did not affect JNK activity under conditions in
which MEKK1 caused its strong activations (data not shown). This later
observation is in agreement with our previous findings on the effects
of N-EE-MEK in COS7 cells (32). Thus, in T3–1 and COS7 cells,
but unlike in U937 cells (34), the JNK signaling cascade appears to be
completely distinct from the MEK-ERK cascade.
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T3–1 cells. A divergence of the
GnRH-stimulated ERK and JNK pathways at the level of PKC stimulation of
PTK activity was suggested by the differential sensitivity of the two
MAPKs to genistein (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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)
and is similar to time courses observed for the induction of JNK by
other heterotrimeric G protein-coupled receptors (35) and tyrosine
kinase receptors (36). However, this slower time course does not occur
in all cells. For example, in nerve growth factor-treated PC12 cells,
the short-term JNK activation is rapid (10–30 min) and transient (37).
Thus, like the ERK pathway (38), the duration of JNK activity may
determine the outcome and specificity of the signal. The delayed
response of JNK observed here (Fig. 1) may indicate that JNK is
involved in a later stage of transcription regulation and suggests that
the signaling machinery that is involved in the activation of JNK is
different from that leading to ERK activation. This upstream machinery
was elucidated in this study, and our results suggest that GnRH binding
to its receptors triggers the activation of PKC, which occurs most
probably via the heterotrimeric Gq protein and PLC. The signal is then
transmitted to a Src-family PTK and further to the sGPs CDC42 and
possibly Rac1. The sGPs activate, in turn, a sGP-activated kinase whose
identity is still controversial and which might be PAK1 (29), MLK (30),
or another unknown protein kinase. Finally, the JNK cascade, including
JNKKK (probably MEKK1), JNKK, and JNK (reviewed in Ref.6), is
activated, causing the stimulation of several transcription factors
including, most likely, c-Jun (6) and the expression of c-Jun protein
(Fig. 7).
It is well established by now that PKC mediates many of the downstream
effects of GnRH, including the activation of the ERK cascade (14). In
T3–1 cells, JNK activation, like that of ERK (19), is activated by
TPA and markedly inhibited (80–85%) by the PKC-specific inhibitor
GF109203X or by TPA depletion of PKC (Fig. 2). This degree of
activation would imply that most of the GnRH signal toward JNK is
mediated by PKC, although the small amount of residual activity upon
PKC inhibition could suggest an additional signaling machinery. This
involvement of PKC in JNK activation by GnRH seems to be unique to our
system. In other cell lines TPA does not stimulate JNK activity (35, 39, 40) or may even cause a decrease in the basal activity of JNK (32).
A clue regarding the identity of the putative components of GnRH
signaling was first achieved by the effect of PTK inhibitors on JNK
activity. Thus, the PTK inhibitor, genistein, completely blocked
GnRH-stimulated JNK activation (Fig. 2) whereas it inhibited only
30% of ERK activation by GnRH (19). Moreover, TPA-induced JNK
activation was also blocked by genistein (Fig. 2), indicating that the
PTK is located downstream to PKC. Since TPA bypasses the GnRH
receptor-Gq complex and activates PKC directly, our results indicate
that the PKC-PTK step is located in the pathway leading to GnRH
activation of JNK. This is reminiscent of the involvement of PTKs in
PKC signaling toward ERK in other systems (19, 41, 42). The difference
in the extent of inhibition by genistein suggests that two (or more)
distinct PTKs may be involved in the activation of ERK and JNK by GnRH.
Therefore, the activation of PTKs by PKC might be the point of
divergence between the JNK and ERK cascades in T3–1 cells (Fig. 7).
Recently, Src family protein kinases have been implicated in some G
protein-coupled receptor signaling pathways. Lyn is involved in the
activation of MEK in DT40 cells (23), and c-Src is involved in
Angiotensin II signaling in COS cells (43). Our results showing that
c-Src is involved in the stimulation of JNK activity (Figs. 3 and 4) in
a GF109203X-independent, but genistein-dependent, manner implies that
c-Src (or a member of its family) is the PTK that mediates
PKC-dependent signaling toward JNK. Since JNK activation by GnRH-a was
completely blocked by a Src-dominant interfering kinase (Csk), and JNK
could not be further activated by GnRH in cells transfected with the
constitutively active form of c-Src, this PTK seems to act as a central
component that funnels all the PKC-mediated GnRH signals toward JNK.
The mechanism by which PKC induces c-Src activation in the GnRH-JNK
system is not clear. PKC has already been shown to directly activate
c-Src by phosphorylation of its serines 12 and 48 (44), which are
required for the enhanced response to ß-adrenergic agonists in cells
overexpressing c-Src. The importance of the c-Src phosphorylation on
serine 12 was supported also by Liebenhoff et al. (45), who
suggested that this phosphorylation by PKC induces cytoskeletal
association necessary for c-Src activation. Furthermore, G
protein-coupled receptors may mediate their signal through a
cascade of PTK that involves activation of c-Src by Pyk2 (22) or c-Src
and FAK (46). Therefore, in the GnRH-JNK pathway studied here, PKC may
activate an upstream PTK, which in turn activates c-Src. However, since
some reports have implied that PKC may be located downstream of c-Src
in the signaling pathway leading to proliferation (47, 48), the
relationship between PKC and c-Src is not yet fully understood, and
some other signaling components may be involved in the activation of
c-Src by PKC.
The activation of JNK involves in most cases the sGPs, CDC42 and Rac1,
that might operate downstream to Ras (26, 27). Both dominant-negative
and constitutively active forms of these G proteins were used to
determine whether they participate in the genistein-sensitive
GnRH-stimulated pathway leading to JNK activation. Here we show that
CDC42 and, to some extent, also Rac1 are involved in this activation,
whereas the involvement of Ras is unlikely (Fig. 5). The activation of
Rac1 and CDC42 by stress-activated signals was previously shown to be
Ras-dependent (27). Therefore, the activation of JNK by GnRH in
T3–1 cells seems to differ and involves a Ras-independent
activation of Rac1 and CDC42 by c-Src. Although the mechanism of
activation is not yet known, it might involve a distinct set of adaptor
molecules and nucleotide exchange factors.
In summary, we demonstrated here the activation of the JNK cascade by a unique signaling pathway that involves PKC, c-Src family PTK, CDC42/Rac1, and probably MEKK1, which leads to JNK activation and most likely to the c-Jun induction in response to GnRH. Similar to the ERK cascade, the activation of JNK by GnRH is PKC dependent, and the point of divergence between the two cascades seems to be the activation of PTK downstream of PKC. Furthermore, the JNK pathway in these cells seems to be different from most other signaling pathways that link G protein-coupled receptors to the activation of JNK in that it includes the activation of CDC42/Rac1 by PKC and c-Src in a Ras-independent mechanism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Stimulants, Inhibitors, Antibodies, and Miscellaneous
Reagents
[D-Trp6]-GnRH, a stable GnRH analog,
genistein (PTK inhibitor), enolase, and protein A-Sepharose were
obtained from Sigma Chemical Co. (St. Louis, Mo). GF109203X (a PKC
inhibitor) and TPA were purchased from Calbiochem (La Jolla, CA).
GST-Jun (1–97) was prepared as previously described (20). Mouse
monoclonal antihemagglutinin (HA)-antibodies were produced by the
Antibody Unit of The Weizmann Institute of Science. Rabbit polyclonal
anti-c-Src antibodies, N-16 and SRC 2, and anti-Fyn and anti-Lyn
antibodies and their control peptides were purchased from Santa Cruz
(San Diego, CA). Mouse monoclonal anti-active MAPK antibodies were from
Sigma (Rehovot, Israel).
Plasmids
The CDC42 constructs (Q61L and T17N-CDC42) were a gift from Dr. G.
Bokoch (The Scripps Research Institute, La Jolla CA). The Csk construct
was a gift from Dr. S. Courtneidge (Sugen, Inc. Redwood City, CA).
MEKK1, GST-c-Jun JNK2, and Rac constructs were provided by Dr. Y.
Ben-Neriah (Hebrew University, Jerusalem, Israel). N-EE-MEK was
cloned in pCDNA1 (Invitrogen, San Diego, CA) as previously described
(32).
Solid Phase Assay for JNK Activity
Pituitary T3–1 cells were grown to 80% confluency serum
starved for 16 h, and the examined stimulants were added for
various time intervals. The cells were then washed (twice with PBS and
once with buffer H), scraped into 250 µl of buffer H and sonicated
(50 W, 2 x7 sec), all at 4 C. After centrifugation (20,000 x
g, 15 min, 4 C), aliquots of the resulting supernatant were
assayed by the Coomassie protein assay (Pierce, Rockford, IL) for
protein. JNK activity was detected according to Hibi et al.
(20). Briefly, aliquots (100–150 µg protein) of the cell extracts
were incubated (2 h, 4 C) with GST-Jun to allow the JNKs to bind to the
substrate. After extensive washing, the JNK activity was measured by
phosphorylation of the GST-Jun, which was mediated by the bound kinase
in the presence of 20 mM MgCl2, 20
µM [
-32P]-ATP (300 cpm/pmol) for 20 min
at 30 C. The reactions were terminated by the addition of sample
buffer, and the samples were subjected to SDS-PAGE analysis. The gels
were dried, autoradiographed on Kodak X-100 films, and the
phosphorylation of GST-Jun was quantitated by densitometry (Bio-Rad 690
densitometer, Bio-Rad, Richmond, CA).
Transfection of T3–1 Cells
Subconfluent T3–1 cells were cotransfected with 5 µg
each of HA-JNK2-SR and one of the following plasmids
(MEKK-pCMV5, Q61L-Rac1-pCDNA3, T17N-Rac1-pCDNA3,
Q61L-CDC42-pCDNA3, T17N-CDC42-pCDNA3, Q61L-H-Ras-pCMV5,
S17N-H-Ras-pCMV5, Y527F-Src-RK5, and Csk-pRK5) using the calcium
phosphate technique (49). The total amount of plasmid was adjusted to
10 µg with vector DNA in the control experiments. The transfection
efficiency was 10–30%, as determined by transfection with a plasmid
that contained ß-Gal and appropriate staining. Two days after
transfection, the cells were serum starved for 16 h and incubated
for the desired time intervals with GnRH-a in the presence or absence
of various inhibitors. The cells were then lysed with 250 µl of HEPES
lysis buffer at 4 C, vortexed, and kept on ice for 5 min. After
centrifugation (20,000 x g, 20 min), the supernatant
was assayed for protein content as above.
Immunoprecipitation with Anti-HA Antibodies
Lysates from transfected cells (400–500 µg protein in HEPES
lysis buffer) were incubated (1 h, 4 C) with anti HA-antibodies (3 µg
Ab/reaction). The immunocomplexes were precipitated with Protein
A-Sepharose, and the resulting precipitates were washed twice with PBS
containing 1% Nonidet P-40 and 2 mM sodium vanadate, once
with 100 mM Tris (pH 7.5) containing 0.5 M LiCl
and once with JNK buffer. The immunoprecipitates were then resuspended
in 30 µl of JNK buffer, and the JNK activity was measured (30 min, 30
C with constant mixing) using GST-c-Jun as a substrate. Phosphorylation
of GST-c-Jun was monitored by autoradiography.
Immunoprecipitation with Anti c-Src Antibodies
Cell lysates (400–500 µg protein in HEPES lysis buffer) were
incubated (1 h at 4 C) with anti-c-Src-antibodies (5 µg/reaction) and
then precipitated with Protein A-Sepharose. The immunocomplexes were
washed twice with PBS containing 1% Nonidet P-40 and 2 mM
sodium vanadate, once with 100 mM Tris (pH 7.5) containing
0.5 M LiCl, and once with PTK buffer. The washed
immunoprecipitates were resuspended in PTK buffer and the c-Src
activity was determined by using acid-denatured enolase (3
mM) as substrate (final volume of 40 µl) in the presence
of 20 µM [32P]-ATP (8,000 cpm/pmol). The
enzymatic reactions were terminated by the addition of sample buffer.
The samples were then subjected to SDS-PAGE, autoradiography, and
densitometry analysis.
RNA Extraction and Northern Blot Analysis
Total RNA was isolated from cells by extraction in guanidium
thiocyanate containing 8% ß-mercaptoethanol by the LiCl method. For
Northern blot analysis, total RNA (15 µg) was fractionated on 1.2%
denaturing agarose gel and transferred to GeneScreen membrane (Dupont,
NEN, Boston, MA). After baking and prehybridization, the membranes were
hybridized (16 h) with the specifc cDNA probes labeled to high specific
activity using a random primer labeling kit (Boehringer Mannheim,
Indianapolis, IN). The filters were washed at high stringency and
autoradiographed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the Israeli Ministry of Health, the Forschheimer Fund, and Keren Naftali (to R.S.) and a postdoctoral fellowship award from the Israel Cancer Research Fund (to N.L.L). R.S. is an incumbent of the Samuel and Isabela Friedman Career Development Chair.
Received for publication December 15, 1997. Revision received February 4, 1998. Accepted for publication February 19, 1998.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 F. Tatin, C. Varon, E. Genot, and V. Moreau A signalling cascade involving PKC, Src and Cdc42 regulates podosome assembly in cultured endothelial cells in response to phorbol ester J. Cell Sci., February 15, 2006; 119(4): 769 - 781. [Abstract] [Full Text] [PDF]
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M. Enomoto, M. Utsumi, and M. K. Park Gonadotropin-Releasing Hormone Induces Actin Cytoskeleton Remodeling and Affects Cell Migration in a Cell-Type-Specific Manner in TSU-Pr1 and DU145 Cells Endocrinology, January 1, 2006; 147(1): 530 - 542. [Abstract] [Full Text] [PDF]
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H. N. Jabbour, K. J. Sales, S. C. Boddy, R. A. Anderson, and A. R. W. Williams A Positive Feedback Loop that Regulates Cyclooxygenase-2 Expression and Prostaglandin F2{alpha} Synthesis via the F-Series-Prostanoid Receptor and Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway Endocrinology, November 1, 2005; 146(11): 4657 - 4664. [Abstract] [Full Text] [PDF]
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K. J. Sales, T. List, S. C. Boddy, A. R.W. Williams, R. A. Anderson, Z. Naor, and H. N. Jabbour A Novel Angiogenic Role for Prostaglandin F2{alpha}-FP Receptor Interaction in Human Endometrial Adenocarcinomas Cancer Res., September 1, 2005; 65(17): 7707 - 7716. [Abstract] [Full Text] [PDF]
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M. Lu, L. Zhang, R. S. Maul, M. R. Sartippour, A. Norris, J. Whitelegge, J. Y. Rao, and M. N. Brooks The Novel Gene EG-1 Stimulates Cellular Proliferation Cancer Res., July 15, 2005; 65(14): 6159 - 6166. [Abstract] [Full Text] [PDF]
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 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
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 K.-Y. Kim, K.-C. Choi, S.-H. Park, N. Auersperg, and P. C. K. Leung Extracellular Signal-Regulated Protein Kinase, But Not c-Jun N-Terminal Kinase, Is Activated by Type II Gonadotropin-Releasing Hormone Involved in the Inhibition of Ovarian Cancer Cell Proliferation J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1670 - 1677. [Abstract] [Full Text] [PDF]
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 A. Rose, P. Froment, V. Perrot, M. J. Quon, D. LeRoith, and J. Dupont The Luteinizing Hormone-releasing Hormone Inhibits the Anti-apoptotic Activity of Insulin-like Growth Factor-1 in Pituitary {alpha}T3 Cells by Protein Kinase C{alpha}-mediated Negative Regulation of Akt J. Biol. Chem., December 10, 2004; 279(50): 52500 - 52516. [Abstract] [Full Text] [PDF]
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 L L Burger, D J Haisenleder, A C Dalkin, and J C Marshall Regulation of gonadotropin subunit gene transcription J. Mol. Endocrinol., December 1, 2004; 33(3): 559 - 584. [Abstract] [Full Text] [PDF]
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S. Kraus, G. Levy, T. Hanoch, Z. Naor, and R. Seger Gonadotropin-Releasing Hormone Induces Apoptosis of Prostate Cancer Cells: Role of c-Jun NH2-Terminal Kinase, Protein Kinase B, and Extracellular Signal-Regulated Kinase Pathways Cancer Res., August 15, 2004; 64(16): 5736 - 5744. [Abstract] [Full Text] [PDF]
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J. S. Jorgensen, C. C. Quirk, and J. H. Nilson Multiple and Overlapping Combinatorial Codes Orchestrate Hormonal Responsiveness and Dictate Cell-Specific Expression of the Genes Encoding Luteinizing Hormone Endocr. Rev., August 1, 2004; 25(4): 521 - 542. [Abstract] [Full Text] [PDF]
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K.-Y. Kim, K.-C. Choi, S.-H. Park, C.-S. Chou, N. Auersperg, and P. C. K. Leung Type II Gonadotropin-Releasing Hormone Stimulates p38 Mitogen-Activated Protein Kinase and Apoptosis in Ovarian Cancer Cells J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 3020 - 3026. [Abstract] [Full Text] [PDF]
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D. Bonfil, D. Chuderland, S. Kraus, D. Shahbazian, I. Friedberg, R. Seger, and Z. Naor Extracellular Signal-Regulated Kinase, Jun N-Terminal Kinase, p38, and c-Src Are Involved in Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone Follicle-Stimulating Hormone {beta}-Subunit Promoter Endocrinology, May 1, 2004; 145(5): 2228 - 2244. [Abstract] [Full Text] [PDF]
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 A. Ulloa-Aguirre, J. A. Janovick, A. Leanos-Miranda, and P. M. Conn Misrouted cell surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism Hum. Reprod. Update, March 1, 2004; 10(2): 177 - 192. [Abstract] [Full Text] [PDF]
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L. Davidson, A. J. Pawson, R. P. Millar, and S. Maudsley Cytoskeletal Reorganization Dependence of Signaling by the Gonadotropin-releasing Hormone Receptor J. Biol. Chem., January 16, 2004; 279(3): 1980 - 1993. [Abstract] [Full Text] [PDF]
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T. A. Jackson, D. M. Koterwas, M. A. Morgan, and A. P. Bradford Fibroblast Growth Factors Regulate Prolactin Transcription via an Atypical Rac-Dependent Signaling Pathway Mol. Endocrinol., October 1, 2003; 17(10): 1921 - 1930. [Abstract] [Full Text] [PDF]
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S. Kraus, O. Benard, Z. Naor, and R. Seger c-Src Is Activated by the Epidermal Growth Factor Receptor in a Pathway That Mediates JNK and ERK Activation by Gonadotropin-releasing Hormone in COS7 Cells J. Biol. Chem., August 29, 2003; 278(35): 32618 - 32630. [Abstract] [Full Text] [PDF]
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D. J. Haisenleder, L. L. Burger, K. W. Aylor, A. C. Dalkin, and J. C. Marshall Gonadotropin-Releasing Hormone Stimulation of Gonadotropin Subunit Transcription: Evidence for the Involvement of Calcium/Calmodulin-Dependent Kinase II (Ca/CAMK II) Activation in Rat Pituitaries Endocrinology, July 1, 2003; 144(7): 2768 - 2774. [Abstract] [Full Text] [PDF]
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D. J. Haisenleder, H. A. Ferris, and M. A. Shupnik The Calcium Component of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Subunit Gene Transcription Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase Type II Endocrinology, June 1, 2003; 144(6): 2409 - 2416. [Abstract] [Full Text] [PDF]
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 L. C. Sheldahl, D. C. Slusarski, P. Pandur, J. R. Miller, M. Kuhl, and R. T. Moon Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos J. Cell Biol., May 26, 2003; 161(4): 769 - 777. [Abstract] [Full Text] [PDF]
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B. S. Ellsworth, B. R. White, A. T. Burns, B. D. Cherrington, A. M. Otis, and C. M. Clay c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in {alpha}T3-1 Cells Endocrinology, March 1, 2003; 144(3): 839 - 849. [Abstract] [Full Text] [PDF]
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D. Harris, D. Chuderland, D. Bonfil, S. Kraus, R. Seger, and Z. Naor Extracellular Signal-Regulated Kinase and c-Src, But Not Jun N-Terminal Kinase, Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone {alpha}-Subunit Promoter Endocrinology, February 1, 2003; 144(2): 612 - 622. [Abstract] [Full Text] [PDF]
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B. H. Shah, J.-W. Soh, and K. J. Catt Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation J. Biol. Chem., January 24, 2003; 278(5): 2866 - 2875. [Abstract] [Full Text] [PDF]
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 P. Robin, I. Boulven, C. Desmyter, S. Harbon, and D. Leiber ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells Am J Physiol Cell Physiol, July 1, 2002; 283(1): C251 - C260. [Abstract] [Full Text] [PDF]
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S. C. Parnell, B. S. Magenheimer, R. L. Maser, C. A. Zien, A.-M. Frischauf, and J. P. Calvet Polycystin-1 Activation of c-Jun N-terminal Kinase and AP-1 Is Mediated by Heterotrimeric G Proteins J. Biol. Chem., May 24, 2002; 277(22): 19566 - 19572. [Abstract] [Full Text] [PDF]
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V. V. Vasilyev, F. Pernasetti, S. B. Rosenberg, M. J. Barsoum, D. A. Austin, N. J. G. Webster, and P. L. Mellon Transcriptional Activation of the Ovine Follicle-Stimulating Hormone-{beta} Gene by Gonadotropin- Releasing Hormone Involves Multiple Signal Transduction Pathways Endocrinology, May 1, 2002; 143(5): 1651 - 1659. [Abstract] [Full Text] [PDF]
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F. Liu, D. A. Austin, P. L. Mellon, J. M. Olefsky, and N. J. G. Webster GnRH Activates ERK1/2 Leading to the Induction of c-fos and LH{beta} Protein Expression in L{beta}T2 Cells Mol. Endocrinol., March 1, 2002; 16(3): 419 - 434. [Abstract] [Full Text] [PDF]
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D. Harris, D. Bonfil, D. CHuderland, S. Kraus, R. Seger, and Z. Naor Activation of MAPK Cascades by GnRH: ERK and Jun N-Terminal Kinase Are Involved in Basal and GnRH-Stimulated Activity of the Glycoprotein Hormone LH{beta}-Subunit Promoter Endocrinology, March 1, 2002; 143(3): 1018 - 1025. [Abstract] [Full Text] [PDF]
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 D.J. Haisenleder, L.J. Workman, L.L. Burger, K.W. Aylor, A.C. Dalkin, and J.C. Marshall Gonadotropin Subunit Transcriptional Responses to Calcium Signals in the Rat: Evidence for Regulation by Pulse Frequency Biol Reprod, December 1, 2001; 65(6): 1789 - 1793. [Abstract] [Full Text] [PDF]
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T. Zhang, M. W. Wolfe, and M. S. Roberson An Early Growth Response Protein (Egr) 1 cis-Element Is Required for Gonadotropin-releasing Hormone-induced Mitogen-activated Protein Kinase Phosphatase 2 Gene Expression J. Biol. Chem., November 30, 2001; 276(49): 45604 - 45613. [Abstract] [Full Text] [PDF]
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G. Maya-Nunez and P. Michael Conn Cyclic Adenosine 3',5'-Monophosphate (cAMP) and cAMP Responsive Element-Binding Protein Are Involved in the Transcriptional Regulation of Gonadotropin-Releasing Hormone (GnRH) Receptor by GnRH and Mitogen-Activated Protein Kinase Signal Transduction Pathway in GGH3 Cells Biol Reprod, August 1, 2001; 65(2): 561 - 567. [Abstract] [Full Text] [PDF]
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M. S. Johnson, E. M. Lutz, C. J. MacKenzie, W. B. Wolbers, D. N. Robertson, P. J. Holland, and R. Mitchell Gonadotropin-Releasing Hormone Receptor Activation of Extracellular Signal-Regulated Kinase and Tyrosine Kinases in Transfected GH3 Cells and in {alpha}T3-1 Cells Endocrinology, September 1, 2000; 141(9): 3087 - 3097. [Abstract] [Full Text] [PDF]
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J. M. Mulvaney and M. S. Roberson Divergent Signaling Pathways Requiring Discrete Calcium Signals Mediate Concurrent Activation of Two Mitogen-activated Protein Kinases by Gonadotropin-releasing Hormone J. Biol. Chem., May 5, 2000; 275(19): 14182 - 14189. [Abstract] [Full Text] [PDF]
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A. Kimura, M. Ohmichi, K. Tasaka, Y. Kanda, H. Ikegami, J. Hayakawa, K. Hisamoto, K.-i. Morishige, S. Hinuma, H. Kurachi, et al. Prolactin-releasing Peptide Activation of the Prolactin Promoter Is Differentially Mediated by Extracellular Signal-regulated Protein Kinase and c-Jun N-terminal Protein Kinase J. Biol. Chem., February 4, 2000; 275(5): 3667 - 3674. [Abstract] [Full Text] [PDF]
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A. Chen, B. H. Davis, M. Bissonnette, B. Scaglione-Sewell, and T. A. Brasitus 1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation J. Biol. Chem., December 10, 1999; 274(50): 35505 - 35513. [Abstract] [Full Text] [PDF]
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J. M. Mulvaney, T. Zhang, C. Fewtrell, and M. S. Roberson Calcium Influx through L-type Channels Is Required for Selective Activation of Extracellular Signal-regulated Kinase by Gonadotropin-releasing Hormone J. Biol. Chem., October 15, 1999; 274(42): 29796 - 29804. [Abstract] [Full Text] [PDF]
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A. Kimura, M. Ohmichi, H. Kurachi, H. Ikegami, J. Hayakawa, K. Tasaka, Y. Kanda, Y. Nishio, H. Jikihara, N. Matsuura, et al. Role of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Cascade in Gonadotropin-releasing Hormone-induced Growth Inhibition of a Human Ovarian Cancer Cell Line Cancer Res., October 1, 1999; 59(20): 5133 - 5142. [Abstract] [Full Text] [PDF]
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 P. Ping, J. Zhang, Y.-T. Zheng, R. C. X. Li, B. Dawn, X.-L. Tang, H. Takano, Z. Balafanova, and R. Bolli Demonstration of Selective Protein Kinase C–Dependent Activation of Src and Lck Tyrosine Kinases During Ischemic Preconditioning in Conscious Rabbits Circ. Res., September 17, 1999; 85(6): 542 - 550. [Abstract] [Full Text] [PDF]
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R. A. Abbud, R. K. Ameduri, J. S. Rao, T. M. Nett, and J. H. Nilson Chronic Hypersecretion of Luteinizing Hormone in Transgenic Mice Selectively Alters Responsiveness of the {alpha}-Subunit Gene to Gonadotropin-Releasing Hormone and Estrogens Mol. Endocrinol., September 1, 1999; 13(9): 1449 - 1459. [Abstract] [Full Text]
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G. B. Call and M. W. Wolfe Gonadotropin-Releasing Hormone Activates the Equine Luteinizing Hormone {beta} Promoter Through a Protein Kinase C/Mitogen-Activated Protein Kinase Pathway Biol Reprod, September 1, 1999; 61(3): 715 - 723. [Abstract] [Full Text]
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M. W. Wolfe and G. B. Call Early Growth Response Protein 1 Binds to the Luteinizing Hormone-{beta} Promoter and Mediates Gonadotropin-Releasing Hormone-Stimulated Gene Expression Mol. Endocrinol., May 1, 1999; 13(5): 752 - 763. [Abstract] [Full Text]
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B. R. White, D. L. Duval, J. M. Mulvaney, M. S. Roberson, and C. M. Clay Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Partially Mediated by Protein Kinase C Activation of an Activator Protein-1 Element Mol. Endocrinol., April 1, 1999; 13(4): 566 - 577. [Abstract] [Full Text]
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 D. I. Johnson Cdc42: An Essential Rho-Type GTPase Controlling Eukaryotic Cell Polarity Microbiol. Mol. Biol. Rev., March 1, 1999; 63(1): 54 - 105. [Abstract] [Full Text] [PDF]
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M. S. Roberson, T. Zhang, H. L. Li, and J. M. Mulvaney Activation of the p38 Mitogen-Activated Protein Kinase Pathway by Gonadotropin-Releasing Hormone Endocrinology, March 1, 1999; 140(3): 1310 - 1318. [Abstract] [Full Text]
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M. A. Silverman, O. Benard, H. Jaaro, A. Rattner, Y. Citri, and R. Seger CPG16, a Novel Protein Serine/Threonine Kinase Downstream of cAMP-dependent Protein Kinase J. Biol. Chem., January 29, 1999; 274(5): 2631 - 2636. [Abstract] [Full Text] [PDF]
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O. Benard, Z. Naor, and R. Seger Role of Dynamin, Src, and Ras in the Protein Kinase C-mediated Activation of ERK by Gonadotropin-releasing Hormone J. Biol. Chem., February 9, 2001; 276(7): 4554 - 4563. [Abstract] [Full Text] [PDF]
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S. Murasawa, H. Matsubara, Y. Mori, H. Masaki, Y. Tsutsumi, Y. Shibasaki, I. Kitabayashi, Y. Tanaka, S. Fujiyama, Y. Koyama, et al. Angiotensin II Initiates Tyrosine Kinase Pyk2-dependent Signalings Leading to Activation of Rac1-mediated c-Jun NH2-terminal Kinase J. Biol. Chem., August 25, 2000; 275(35): 26856 - 26863. [Abstract] [Full Text] [PDF]
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T. Yokoi, M. Ohmichi, K. Tasaka, A. Kimura, Y. Kanda, J. Hayakawa, M. Tahara, K. Hisamoto, H. Kurachi, and Y. Murata Activation of the Luteinizing Hormone beta Promoter by Gonadotropin-releasing Hormone Requires c-Jun NH2-terminal Protein Kinase J. Biol. Chem., July 7, 2000; 275(28): 21639 - 21647. [Abstract] [Full Text] [PDF]
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, GnRH-a+ TPA depletion; 
,
GnRH-a+GF109203X; x, GnRH-a+genistein; 
, TPA; 
, TPA +
genistein. All results are from a representative experiment that was
reproduced three times.
