From the Department of Biology, University of California, Riverside, California 92521
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ABSTRACT |
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The 9E3/CEF4 gene codes for a chemokine that is
highly homologous to human interleukin-8 and melanoma
growth-stimulating activity/gro. These chemokines belong
to a family of molecular mediators that are importantly involved in
inflammation, wound healing, tumor development, and viral entry into
cells. On the chorioallantoic membrane the 9E3 protein is chemotactic
for monocyte/macrophages and lymphocytes and is angiogenic. In cultured
chicken embryo fibroblasts, which have many of the properties of wound
fibroblasts, the gene is stimulated by a variety of agents including
oncogenes, growth factors, phorbol esters, and thrombin. The strong
stimulation of 9E3 by thrombin in culture correlates well with the
observation that in young chicks this gene is stimulated to very high
levels in fibroblasts upon wounding and remains high throughout wound repair. Activation of 9E3 by thrombin: (i) occurs very rapidly, one
minute exposure to thrombin is sufficient to initiate the signals
necessary for gene activation; (ii) is independent of mitogenesis;
(iii) operates through the proteolytically activated receptor for
thrombin; (iv) is mediated by tyrosine kinases, including c-src and the epidermal growth factor (EGF) receptor,
rather than Ser/Thr kinases such as protein kinase C and protein kinase
A. Inhibition of either c-src or the EGF receptor tyrosine
kinase inhibits the stimulation of 9E3 by thrombin. We show here for the first time that activation of the EGF receptor through a
cell-surface receptor that does not have tyrosine kinase activity can
lead to expression of an immediate early response gene which encodes for a secreted protein, a chemokine. This rapidly activated tyrosine kinase pathway may be a general stress response by which in
vivo a localized cell population reacts to emergency situations
such as viral infection, wounding, or tumor growth.
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INTRODUCTION |
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The avian gene 9E3/CEF4 encodes a secreted protein that is a
member of the chemokine superfamily (1-4). Of all the members of this
superfamily, the 9E3 protein shows the highest homology to
interleukin-8 and also is highly homologous to
MGSA1/gro. This
gene is constitutively overexpressed in chicken embryo fibroblasts
(CEFs) transformed by Rous sarcoma virus, but its expression in normal
quiescent cells is tightly regulated (5, 6); serum stimulation of
normal CEFs results in transient expression of 9E3 as a consequence of
entry into the cell cycle (7). In addition to serum, a wide variety of
oncogenes, growth factors, and inflammatory agents also stimulate 9E3
expression in normal quiescent CEFs (5, 6, 8, 9). In vivo,
the 9E3 gene is expressed at very low levels in connective tissue,
tendon, and bone, but it is highly expressed in the endothelial cells of young blood vessels (10-12). Furthermore, this gene is rapidly overexpressed upon wounding, remains very highly elevated during the
inflammatory phase of healing, and 36 h after wounding declines to
a plateau of elevated expression that remains constant throughout granulation tissue formation (10). The 9E3 protein is chemotactic for
monocyte/macrophages and lymphocytes (12, 13); the high levels of
expression shortly after wounding could be responsible for this
function. In addition, this protein is angiogenic in vivo
(12), which might occur in response to the steady expression of the 9E3
gene during granulation tissue formation. The observations in
vivo and in vitro combined with the biochemical
properties of the molecule (14) indicate that this gene could play an
important role in the inflammatory response and healing of wounds.
We have found that of the growth factors, inflammatory agents, and other molecules released upon wounding, thrombin is the most potent natural activator of 9E3 expression (14, 15). Thrombin is a serine protease that is generated at sites of vascular injury and is known to regulate hemostasis and thrombosis via its serine protease activity by inducing platelet aggregation and clot formation. However, many other actions of thrombin are independent of its thrombogenic activity and suggest that this protease is also important directly or indirectly during the inflammatory response and in the proliferative phase of wound healing (16-19). In these situations, thrombin is known to be chemotactic for monocytes and mitogenic for lymphocytes, fibroblasts, and smooth muscle cells (20-23). Furthermore, thrombin stimulates endothelial cells to express the neutrophil adhesion protein GMP-140 (24-26) and to produce platelet-derived growth factor that stimulates smooth muscle cells to grow (27, 28).
Most of our understanding of signal transduction pathways turned on by thrombin comes from work with platelets (29, 30), Chinese hamster lung fibroblasts (31, 32), and endothelial cells (33). In these systems, upon binding to its seven transmembrane domain receptor, thrombin cleaves part of the N terminus of the receptor, exposing a new peptide that then binds to one of the external loops of the receptor (34). This proteolytically activated receptor for thrombin causes activation of Gi or Gq,11 that turn on mitogenic and nonmitogenic events (18). Gi inhibits adenylyl cyclase and cAMP and reduces the levels of PKA (35), whereas Gq,11 activates phosphatidylinositol turnover, PKC, Na+/H+ antiport, and induces c-fos and c-myc (19, 36-38). In platelets, the Gq,11 can also activate phospholipase A2 with the subsequent generation of arachidonate-derived metabolites (29). These lipids, known as eicosanoids, include prostaglandins, thromboxanes, and leukotrienes, which are often involved in the inflammatory process. More recently, a new proteinase-activated receptor 2 was identified that has 30% homology to proteolytically activated receptor for thrombin but for which the signaling events are not yet worked out (39).
The signaling events activated by thrombin, which are described above, are mediated by Ser/Thr kinases stimulated shortly after receptor activation. However, more recently tyrosine phosphorylation has also been implicated in the signaling events triggered upon thrombin receptor activation. It has been shown that tyrosine phosphorylation in platelets occurs independently of the activation of PKC (40) and that cells that do not undergo mitogenesis in response to thrombin stimulation require tyrosine kinase signals initiated by EGF and platelet-derived growth factor (41) to trigger cell division. In growth-responsive Chinese hamster fibroblasts, thrombin activates the c-src and fyn tyrosine kinases, which provide a link between the thrombin receptor and the downstream events leading to activation of ras and the mitogenic kinase cascade (42).
It is now abundantly clear that thrombin is a multifunctional molecule.
The fact that thrombin and chemokines are important in the inflammatory
response and granulation tissue formation, and the demonstration that
thrombin stimulates the expression of chemokines such as
MGSA/gro (43, 44), 9E3 (14, 15, 45), and interleukin-8
(46) may indicate that these chemokines could be the mediators of some
of thrombin's effects on healing. Identification and understanding of
the signal transduction pathways generated by thrombin leading to
activation of chemokines can give insight into ways of manipulating the
function of this latter class of molecules. Because the 9E3 gene is
stimulated to high levels shortly after wounding and because thrombin
is the most potent natural activator of this gene, we are investigating
the signal transduction mechanisms by which thrombin activates 9E3 expression. To perform these studies we use primary cell cultures of
CEFs. This culture system allows us to make inferences to the situation
in vivo because the properties of the fibroblasts in wounded
tissue and in granulation tissue resemble those of embryonic fibroblasts (47). Our results show that the Ser/Thr kinases PKC and PKA
are not directly involved in the stimulation of this gene by thrombin,
but instead we find that tyrosine phosphorylation is a key event that
occurs when CEFs are activated by thrombin to produce 9E3. We show here
that the EGF receptor and c-src are two of the tyrosine
kinases importantly involved in this process.
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MATERIALS AND METHODS |
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Reagents--
Concentrations of activators and inhibitors of
signal transduction cited here were selected for efficacy from a wider
range of concentrations initially tested. A23187 (1 µM),
arachidonic acid (100 µM), and bovine thrombin (9 units/ml) were obtained from Sigma. Calphostin C (200 nM),
cholera toxin (100 ng/ml), genistein (1-25 µM), H-7 (200 nM), HA 1004-HCl (20 µM), H89 (100 nM), herbimycin A (1 µM), ionomycin (1 ng/ml), lavendustin A analogue RG 14355 (100-200 nM),
pertussis toxin (100 ng/ml), staurosporine (200 nM), and
tyrphostin AG1478 (500 nM) came from Calbiochem. 1-Oleoyl-2-acetyl-glycerol (300 µM) was obtained from
Serdary Research Laboratories, London, Ontario, and human recombinant TGF (10 ng/ml) and EGF (10 and 100 ng/ml) were obtained from Intergen. 100% Me2SO was used as a solvent when
appropriate. Anti-phosphotyrosine PY20 was purchased from Transduction
Labs and 4G10 from Upstate Biotechnology Inc. ECL reagents and
antibodies conjugated to horseradish peroxidase were obtained from
Amersham. TRDP (200 µM) is a thrombin receptor-derived
peptide with the following sequence:
H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-OH (Bachem).
Cell Culture-- Primary cultures of CEFs were prepared from 10-day-old chicken embryos as described previously (48). On the fourth day, secondary cultures were prepared by trypsinizing and plating the primary cells in 199 medium containing 0.3% tryptose phosphate broth and 2% donor calf serum at a density of 1.2 × 106/60-mm plate. Transformed CEFs (tCEF) were obtained by infecting these cells with Rous sarcoma virus XD-2 (an engineered form of Schmidt-Ruppin strain A) as described previously (49). To study the effects of thrombin on 9E3 expression and the effects of known activators and inhibitors of signal transduction, we used quiescent confluent CEF (qcCEF) cultures and incubated them in serum-free 199 medium containing the specific treatment at various concentrations and for varying times. After the initial experiments, the inhibitors and activators were used at the doses shown above and incubated for 1 h; thrombin was added for another hour and then the cells were washed and incubated in serum-free medium for up to 18 h. At the end of the incubation period the supernatant was collected, protease inhibitors were added, and cell debris was removed by centrifugation. Because qcCEFs are primary cells rather than a cell line, there are small variations in the basal levels of 9E3 expression from batch to batch of cells. Therefore, for each experiment and/or treatment of a different batch of cells we used internal positive (thrombin-treated cells) and negative (untreated cells) controls.
Western Blotting-- Volumes of the cell culture supernatant corresponding to equal amounts of protein in the cells extracts were loaded on 20% polyacrylamide-glycerol gels and electrophoresed at 16 mA for about 4 h. The concentration of the protein was determined using the Bio-Rad DC-protein kit. Transfer was performed using a semidry transfer apparatus (Millipore). After transfer, to check for evenness of protein loading, the upper part of the gel was cut and stained with silver staining for a protein that is ~35 kDa. This protein is present in the supernatant of qcCEFs, and its concentration does not vary with treatment of the cells. The efficiency and evenness of transfer of the 9E3 protein was monitored by silver staining the gel after the transfer. The 9E3 protein was detected using polyclonal antibodies raised in rabbit (14) and enhanced chemiluminescence (ECL) reagents (Amersham).
Northern Blot Analysis-- CEFs were homogenized and total RNA was prepared using TRIzolTM reagent (Life Technologies, Inc.). RNA samples (20 µg each) were denatured in formamide-formaldehyde buffer containing ethidium bromide and separated on formaldehyde-agarose gel. After electrophoresis, the RNA was transferred to MagnaGraph nylon membrane (MSI Inc.) that were photographed to visualize the quality of the rRNA and confirm equal loading and even transfer. The RNA was UV cross-linked to the membrane for 2 min and baked at 60 °C for 2 h. Prehybridization was performed for 6-9 h, and hybridization was carried out for 36 h following the procedures described in Ref. 10. Microdensitometry analysis was performed by laser densitometric scanning in a LKB microdensitometer.
Phosphotyrosine Immunoblots-- Cell extracts were prepared by lysing the cells in boiling 2 × Laemmeli buffer and shearing by passing through a 26-gauge needle 10 times, followed by boiling in a water bath for 5 min. The samples were electrophoresed on an 8% SDS-polyacrylamide gel and transferred to the nitrocellulose membrane (Schleicher & Schuell) using a wet transfer system (Bio-Rad) and Towbin's buffer for 13 h at 30 V or 5 h at 60 V. The blots were blocked for 2 h at 37 °C in Tris-buffered saline containing 0.1% Tween 20, 2% bovine serum albumin, and 0.02% thimerasol (TBST-BSA) and then incubated with the anti-phosphotyrosine antibodies (PY20 at 1:2500 and 4G10 at 1:1000 dilutions) in TBST containing 2% bovine serum albumin for 2 h at 37 °C. The excess antibodies were washed 3 times for 5 min each, plus a longer 20-min wash with TBST. A second blocking step was done for 1 h with TBST containing 5% nonfat milk. This was followed by incubation with goat anti-mouse antibodies conjugated to horseradish peroxidase (1:5000) for 1 h at room temperature. The washings were performed as described for the immunoblots. The antibody detection was done using ECL reagents (Amersham).
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RESULTS |
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Nonmitogenic Receptor-dependent Activation of 9E3 by Thrombin-- Addition of serum to CEFs in culture stimulates 9E3 expression as cells enter the cell cycle (5, 6, 8, 9). Because thrombin is mitogenic for fibroblasts, we expected that stimulation of the 9E3 protein by thrombin occurs via the same mitogenic pathway. To test this possibility, we plated CEFs in serum-free medium, added thrombin at 10 units/ml, and fed them with serum-free medium plus 10 units/ml of thrombin every 24 h for a period of 4 days. Parallel cultures were treated with medium containing 5% donor calf serum and 1% chicken serum. At the end of this period the numbers of cells were counted and the 9E3 protein present in the supernatant for the final 24 h was analyzed by immunoblot with an antibody specific for the 9E3 protein (14). The results showed that although the total number of cells was approximately the same for both treatments (Fig. 1A), the amount of protein secreted into the supernatant was at least 15-fold higher in the cells treated with thrombin (Fig. 1B). For the same period of time, the amount of 9E3 protein produced by the CEFs treated with thrombin was similar to that produced by Rous sarcoma virus transformed CEFs (tCEFs), which express the 9E3 gene constitutively (Fig. 1B). Our observations at the protein level were confirmed by analysis of the mRNA under the same conditions (Fig. 1C); the level of the protein in cells with the various treatments correlates well with levels of mRNA under the same conditions. To determine if the stimulation of 9E3 by thrombin is via the proteolytically activated receptor for thrombin receptor, we treated qcCEFs, which do not express 9E3, with the human thrombin receptor derived peptide (TRDP), which can activate proteolytically activated receptor for thrombin much like thrombin (50). We found that this peptide could stimulate qcCEFs to express 9E3 to the same level as those stimulated by thrombin itself (Fig. 1D).
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Additive Effect of v-src and Thrombin on 9E3 Expression-- Our observation that activation of 9E3 by thrombin is independent of mitogenesis, coupled with a previous determination that activation of 9E3 by v-src is via a mitogenic pathway (51) raised the possibility that thrombin and the oncogene might activate 9E3 via different signal transduction pathways. If so, when added together, they should stimulate an additive effect on the expression of 9E3. To test this possibility, we treated tCEFs with thrombin and analyzed them for both protein (Fig. 3A) and mRNA (Fig. 3B) and observed that thrombin and v-src have additive effects. These results suggest that thrombin activates 9E3 by a pathway independent of that turned on by v-src (which involves activation of PKC by the v-src oncogene) (51) but do not preclude the possibility that it could be a superactivation of the same pathway involving PKC.
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Stimulation of 9E3 by Thrombin Is Enhanced by Inhibition of Ser/Thr
Kinases--
Thrombin is known to stimulate several signal
transduction pathways resulting in activation of a variety of genes and
leading to many different biological responses. The pathway stimulated by thrombin that activates PKC involves activation of Gq,11
which activates phospholipase C that, in turn, cleaves
phosphatidylinositol phosphate into IP3 and
diacylglycerol. The latter activates PKC by interacting directly with this kinase, whereas IP3
interacts with its receptor, present in the endoplasmic reticulum
resulting in intracellular Ca2+ release. To determine if
PKC is involved in the stimulation of 9E3 by thrombin, we treated
qcCEFs with OAG (oleoyl-2-acetyl-glycerol), an analogue of
diacylglycerol that activates PKC directly (52), and found that OAG did
not stimulate the production of the 9E3 protein (Fig. 4A).
To further these studies, we treated qcCEFs with calphostin C (Fig.
4B), a specific inhibitor of PKC (53) and found that this
treatment did not inhibit stimulation of 9E3 by thrombin but instead it
potentiated thrombin stimulation. Furthermore, when we treated qcCEFs
with the broad spectrum Ser/Thr kinase inhibitors H7 and staurosporine
(53, 54), we found that both inhibitors potentiated the stimulation of
9E3 by thrombin and that staurosporine by itself stimulated 9E3
expression (Fig. 4B). In addition, we also found that an
increase in cytosolic Ca2+ by treatment with the ionophores
ionomycin and A23187 (Fig. 4C) had little effect on the
levels of the 9E3 protein and did not enhance that stimulated by
thrombin. These results suggest that the signaling pathways activated
by Gq,11, and in particular the activation of PKC, do not
play a major role in stimulation of 9E3 by thrombin.
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Tyrosine Kinases Are Involved in the Activation of 9E3 Expression by Thrombin-- Our findings that Ser/Thr kinases are not significantly involved in 9E3 stimulation by thrombin, led us to investigate the role of tyrosine kinases in this process (40-42, 60). We found that genistein, a general inhibitor of tyrosine kinases (61), abolished the expression of 9E3 induced by thrombin both at the protein (Fig. 6A) and mRNA levels (Fig. 6B). To investigate the nature of the proteins phosphorylated on tyrosines, we performed Western blot analysis of extracts prepared from cells treated with thrombin, using anti-phosphotyrosine antibodies. A time course study showed that proteins of molecular masses 220, 170, 60, 44, and 42 kDa were phosphorylated on tyrosines in a biphasic manner upon thrombin stimulation of qcCEFs with the first peak at 5-7 min and the second at 3-6 h after activation (Fig. 7). By reprobing the same blot or identical blots with specific antibodies, we found that the 170-kDa band was labeled by an antibody to the EGF receptor, the 60-kDa band was labeled by an antibody to the c-src protein and the 44- and 42-kDa bands were labeled by antibodies to ERK1 and ERK2, respectively. In this study we focus on the potential involvement of the c-src and the EGF receptor tyrosine kinases.
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DISCUSSION |
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Previous work has shown that serum factors stimulate 9E3 expression (5, 6) and that expression under these conditions is initiated during the G0/G1 transition of the cell cycle and declines in S-phase (8). The results presented here show that for the same number of cells, stimulation of 9E3 by thrombin is 15-fold higher than that stimulated by serum. Furthermore, in all of the other experiments performed here, we used qcCEFs that are contact inhibited; under these conditions only a small number of cells will undergo cell division during 18 h of exposure to serum or thrombin. The reduced amounts of 9E3 in the supernatant of qcCEFs treated with serum is reflective of the small number of cells entering G1 (8), whereas the increased amounts of 9E3 after thrombin treatment must reflect expression by many of the preexisting quiescent cells. Therefore, thrombin stimulation of the 9E3 gene occurs by a pathway that does not involve mitogenesis.
It has been reported previously that when cells are infected with viruses that carry the oncogenes v-fps or v-src, they express the 9E3 gene constitutively; these oncogenes activate PKC, which in turn is responsible for triggering mitogenic events that lead to 9E3 expression (51, 67, 68). Our observation that stimulation of 9E3 by thrombin occurs through a nonmitogenic pathway, coupled with the additive effect with v-src stimulation, suggests that v-src and thrombin operate primarily through different independent pathways. Indeed, thrombin activation of 9E3 does not involve PKC and is potentiated by a variety of inhibitors of Ser/Thr kinases, indicating that inhibition of these kinases in general potentiates the stimulation of 9E3 by thrombin. In support of this proposal are the findings of Ishii et al. (69) who showed that the receptor for thrombin is turned off by Ser/Thr kinases that phosphorylate these amino acids present in the C terminus of the receptor. Thus, inhibition of these kinases might allow the thrombin receptor to act for longer periods and produce greater effects.
We show here that tyrosine kinases are importantly involved in stimulation of the 9E3 gene by thrombin. Five specific proteins (four that co-migrate with the c-src, the EGF receptor, and the ERK 1/ERK 2 kinases) are phosphorylated on tyrosines within 5-7 min of stimulation by thrombin, and this stimulation is synchronous (Fig. 7). This observation, coupled with the rapid activation of the gene (15), suggests direct involvement of these proteins in 9E3 stimulation. Their possible roles in 9E3 activation are the subject of ongoing research in our laboratory.
Our observation that herbimycin A, which preferentially inhibits kinases of the c-src family (62, 70), inhibits 9E3 expression stimulated by thrombin, suggests involvement of c-src in this activation. In support of involvement of c-src is the observation that when chinese hamster fibroblasts are treated with thrombin, the c-src tyrosine kinase is activated (42). Also, staurosporine, a broad spectrum Ser/Thr kinase inhibitor, not only potentiated the action of thrombin but by itself stimulated low levels of 9E3 expression. It has recently been found that staurosporine induces spreading of a human colon cancer cell line (70, 71) and that this effect is inhibited by herbimycin A. One of the proteins phosphorylated on tyrosines upon staurosporine treatment was the c-src tyrosine kinase (70, 71). Therefore, it is possible that the stimulation of 9E3 by staurosporine alone is due to the activation of this kinase by this inhibitor, further supporting the involvement of c-src in the expression of 9E3. It was surprising that c-src is involved in this tyrosine-kinase-dependent pathway of 9E3 activation by thrombin, given that activation of this gene by v-src occurs via a PKC-dependent pathway (51). However, we note that it has been shown (72) that v-src can turn on both PKC-dependent and PKC-independent pathways leading to gene expression, showing that src can participate in multiple signaling pathways.
We have used inhibitors of the tyrosine kinase activity of the EGF
receptor to address the involvement of this receptor in the stimulation
of 9E3 expression by EGF, TGF, and thrombin. The inhibitors we used
have varying specificity for the EGF receptor tyrosine kinase.
Genistein inhibits tyrosine kinase activity by competing with these
enzymes for the ATP binding site and therefore is a broad spectrum
inhibitor. Genistein eliminated thrombin stimulation of 9E3 (Fig. 6).
Lavendustin A RG14355 is a cell-permeable selective inhibitor for the
EGF receptor tyrosine kinase (IC50 = 11 nM) and
for pp60src (IC50 = 500 nM) (62). At
the concentrations used in our study (100 nM) it is
selective for the EGF receptor tyrosine kinase. We found that
lavendustin A RG14355 virtually inhibited the stimulation of 9E3 by
thrombin (Fig. 8A). We also used tyrphostin AG1478, a more
potent and selective inhibitor for this receptor tyrosine kinase
(62-64). The evidence that tyrphostin AG1478 is highly specific for
the EGF receptor tyrosine kinase is now extensive. Fry et al. (63), in a paper directed specifically to selectivity of inhibition for the EGF receptor, showed that a quinazoline identical to
tyrphostin AG1478 except for substitution of bromine for chlorine on
the free benzene ring of the molecule is a highly selective inhibitor
for the EGF receptor at concentrations from a few nanomolar to a few
micromolar. Furthermore, in a comprehensive review of tyrosine kinase
inhibition, Levitzki and Gazit (62) showed that tyrphostin AG1478
inhibits the EGF receptor tyrosine kinase in vitro with an
IC50 = 3 nM, whereas the corresponding
IC50 values for inhibition of other tyrosine kinases by
this molecule were all greater than 50 µM (HER2-neu
IC50 > 100 µM; platelet-derived growth
factor IC50 > 100 µM). In the present study
in vivo, concentrations of 500 nM tyrphostin
AG1478 completely obliterated stimulation of 9E3 by thrombin, hrTGF
,
and hrEGF. Finally, Daub et al. (64) also showed specificity
of tyrphostin AG1478 when studying the stimulation of the
fos gene by thrombin via transactivation of the EGF receptor
tyrosine kinase in Rat-1 fibroblasts. They confirmed their results by
use of a dominant negative mutant of the receptor that is capable of
inhibiting events downstream from the receptor by forming signaling
defective heterodimers with the wild-type receptor. Both tyrphostin
AG1478 and this mutation eliminated the EGF receptor phosphorylation
stimulated by thrombin and abolished activation of the fos
gene. Experiments using a dominant negative mutant of the EGF receptor,
similar to those of Daub et al. (64), are not possible in
our system because we are using primary cell cultures, and there are no
chicken cell lines to perform such studies. However, use of such a
mutant by these authors confirmed the specificity of tyrphostin AG1478,
enabling us to infer that the stimulation of 9E3 by thrombin involves
analogous transactivation of the EGF receptor as an important step in
the 9E3 gene activation cascade triggered by thrombin.
Transactivation of a growth factor receptor by another cell-surface
receptor is only a recently recognized phenomenon (64, 73). These two
previous studies also involve transactivation of the EGF receptor; the
former via the thrombin receptor (discussed in the previous paragraph)
and the latter via the TNF receptor. In both cases, demonstration of
transactivation involved detection of fos gene expression.
fos is an immediate early response gene that is activated by
phosphorylation of the Elk1 transcription factor by mitogen-activated
protein kinases (74). Here we have shown activation of another
immediate early response gene, 9E3 (5), which codes for a secreted
protein, a chemokine. The promoter of this gene also contains an Elk1
binding site (75) and mitogen-activated protein kinase is also involved
in 9E3 gene expression stimulated by
thrombin.2 Further work will be required to
determine if transactivation of the EGF receptor could represent a
general mechanism for turning on immediate early response genes by
receptors that do not have an intrinsic tyrosine kinase activity.
The greater stimulation of 9E3 by TGF than by EGF in this study is
explained by differences in affinity for the chicken EGF receptor.
Although hrEGF and hrTGF
bind with equal affinity to the receptor in
humans, hrTGF
binds with 100-fold greater affinity than does hrEGF
to the chicken receptor (66), hence EGF requires much higher doses to
cause the same effect. Our observation that EGF and TGF
are less
efficient than thrombin in stimulating 9E3 expression is consistent
with the fact that these growth factors are mitogenic for CEFs (76) and
stimulate 9E3 via entry into the cell cycle (8). We previously showed
using in situ hybridization that only some cells in qcCEF
cultures can be stimulated to enter cell division and that in
experiments involving growth factors, only the dividing cells express
9E3 (8). On the other hand, thrombin stimulation does not require cell
division, hence it will stimulate 9E3 expression in a large fraction of
cells resulting in greater integrated expression. An additional factor
that could contribute to this difference between these growth factors
and thrombin is that growth factors interact with their receptors and
down-regulate them, whereas the activation of the EGF receptor by
thrombin is intracellular and therefore may stay on for longer periods
of time.
At first thought, it might seem surprising that the EGF receptor can
function as the entry point to a signal-transduction pathway leading to
gene expression through a mitogenic pathway (e.g. for EGF
and TGF) and also serve as a major early step in a nonmitogenic
pathway (e.g. for thrombin). However, it is already known
that after activation by EGF and consequent dimerization, the EGF
receptor autophosphorylates on several tyrosine residues that serve
as docking sites for a variety of molecular mediators for different
signal-transduction pathways that emanate from this receptor (77). The
details of the role played by this receptor in the stimulation of 9E3
by thrombin presumably are different from that for either EGF or TGF
because thrombin activates the EGF receptor intracellularly. Therefore,
our results suggest the possibility of still greater complexity of
behavior of the EGF receptor than previously recognized.
Our results do not yet clarify whether c-src activation
precedes or succeeds EGF receptor activation. However, upon thrombin interaction with its receptor, it has been shown that c-src
is rapidly activated by both pertussis toxin-sensitive and -insensitive G-proteins (78). In addition, in Rat-1 fibroblasts, v-src
phosphorylates Gq,11 on tyrosines and increases its
activity (79, 80). This is also true for several other
G
-proteins (81). Therefore, it appears that upon
thrombin activation of its receptor, there is mutual interaction
between a G
protein and c-src (82, 83).
Furthermore, the association of c-src with the plasma
membrane (84) and its known ability to bind to the EGF receptor (85) suggests that it may be involved in the transactivation event. These
and other aspects of the signaling pathways stimulated by thrombin and
leading to 9E3 expression are currently being pursued in our
laboratory.
In summary, thrombin stimulation of the 9E3 chemokine gene in qcCEFs
occurs via a nonmitogenic pathway and is accompanied by synchronous,
biphasic, phosphorylation of several proteins including
c-src and the EGF receptor. Similar stimulation of 9E3 by
EGF and TGF verify that the EGF receptor is functional in these
cells, and a series of inhibitors that are highly selective for
c-src and the EGF receptor tyrosine kinases eliminate 9E3 stimulation by thrombin. This study is now the third to show
stimulation of an immediate early response gene via transactivation of
the EGF receptor after initial binding of a ligand to a receptor that does not have an intrinsic tyrosine kinase activity, suggesting that
this could be a general mechanism for activation of immediate early
response genes by the latter class of receptors.
On a broader scale, the stimulation of the 9E3 gene via entry into the cell cycle may be akin to that occurring in normal tissues where expression is very low and probably represents the result of a small number of cells that are undergoing cell division (8, 10). In contrast, the high levels of 9E3 expression triggered by thrombin could represent a response from all cells to an emergency situation such as wounding, tumorigenesis or viral entry into cells. The signal transduction pathway by which this is accomplished, involving transactivation of the EGF receptor and other tyrosine kinases, offers the opportunity for amplification of the signals and a prolonged response, much like the activation of adenylcyclase by the epinephrine receptor. This type of activation could provide an advantage when a rapid and strong tissue response is needed.
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ACKNOWLEDGEMENTS |
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We thank H. W. Green, D. Strauss, and R. Zidovetzki for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by NIGMS, National Institutes of Health Grant GM48436 (to M. M.-G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 909-787-2585;
Fax: 909-787-4509; E-mail: mmgreen{at}ucrac1.ucr.edu.
1
The abbreviations used are: MGSA, melanoma
growth-stimulating activity; CEF, chicken embryo fibroblast; EGF,
epidermal growth factor; IP3, inositol trisphosphate; OAG,
1-oleoyl-2-acetyl-glycerol; PKA, protein kinase A; PKC, protein kinase
C; qcCEF, quiescent confluent CEF; tCEF, transformed CEF; TGF,
transforming growth factor
; TRDP, thrombin receptor-derived
peptide.
2 S. Vaingankar, H. M. Green, M. Martins-Green, manuscript in preparation.
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