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CHEMOKINES
From the Department of Cell Biology and Neuroscience,
University of California, Riverside, CA.
Thrombin is primarily known for its role in homeostasis and
thrombosis. However, this enzyme also plays important roles in wound
healing and pathologic situations such as inflammation and tumorigenesis. Among the molecules stimulated by thrombin in these latter processes are the stress response proteins, chemokines. Chemokines are also known for their roles in inflammatory responses and
tumor development. These correlative observations strongly suggest that
chemokines may be mediators of some of thrombin's functions in these
processes. Elucidation of the molecular mechanisms of stimulation of
chemokines by thrombin may help to unravel the ways in which their
expression can be modulated. Up-regulation of the chemokine 9E3/cCAF by
thrombin occurs via its proteolytically activated receptor with
subsequent transactivation of the epidermal growth factor receptor
tyrosine kinase. This study shows that stimulation by thrombin very
rapidly activates this chemokine at the transcriptional level, that 2 Elk1 binding elements located between Thrombin is a multifunctional serine protease known
primarily for its role in homeostasis and thrombosis. However, this
enzyme also has an important role in the stress responses that occur during inflammation, wound healing, and tumor
development.1-3 Thrombin can stimulate fibroblasts,
endothelial cells, and leukocytes to initiate signal transduction
mechanisms that regulate the expression of genes important in stress
responses during these traumatic and pathologic
processes.4,5 Signaling mechanisms stimulated by thrombin
involve its 7 transmembrane receptors, trimeric G proteins, and
primarily serine/threonine (Ser/Thr) kinases.6,7 However,
tyrosine (Tyr) kinases are also important in some of the signaling
events triggered after thrombin receptor
activation.8-12
Among the molecules stimulated by thrombin after wounding and during
tumorigenesis are chemokines, small molecular weight cytokines known
primarily for their function in leukocyte trafficking and
physiology.13-15 Chemokines can be stimulated in response
to injury (be it a wound or a tumor), are immediate early
stress-response genes that initiate the immune response (eg, chemotaxis
for leukocytes), and perform functions involved in the formation of the
repair tissue and of tumor stroma (eg, angiogenesis). Understanding
regulation of thrombin-stimulated chemokine expression during wound
healing and tumor development may contribute to finding ways of
modulating these processes.
Thrombin is the most potent natural activator of the chemokine chicken
chemotactic and angiogenic factor (cCAF),12,16 which is
the product of the 9E3 gene. Henceforth we will refer to
this chemokine as 9E3/cCAF. In addition to stimulating 9E3/cCAF,
thrombin also stimulates the expression of human chemokines, such as
interleukin-8 (IL-8),17 melanoma growth stimulatory
activity (MGSA),18,19 and monocyte chemoattractant protein
1 (MCP-1)20 in a manner similar to that of 9E3/cCAF.
Furthermore, this chemokine is more homologous to IL-8 than is any
other chemokine, human or otherwise, and also is highly homologous to
MGSA/gro In vivo, 9E3/cCAF is highly expressed shortly after injury;
expression remains high during the inflammatory phase of healing and
declines to a lower level, but still elevated, during formation of the
repair tissue. In addition, it is also highly expressed in the stroma
of tumors.21,22 In these tissues, 9E3/cCAF is up-regulated
primarily in fibroblasts present in areas where interstitial collagen21,22 and tenascin23 are abundant.
Studies in the chorioallantoic membrane (CAM) show that this chemokine
is chemotactic for monocyte/macrophages and lymphocytes and is
angiogenic,5,6 functions that are important in responses
to the trauma inflicted by both wounding and tumors.
Because chemokines perform a variety of crucial functions in
vivo, it is important to determine the molecular mechanisms
involved in their activation so that their expression can be modulated. Thrombin stimulates 9E3/cCAF expression in primary normal fibroblasts independently of its mitogenic activities.12 After
activation, the G-protein-coupled receptor for thrombin transactivates
the epidermal growth factor receptor (EGFR) leading to
phosphorylation and activation of the EGFR and of Src tyrosine
kinases.12 The work presented here furthers these studies
by showing that the signaling pathways downstream from the EGFR involve
the activation of the mitogenic-activated protein (MAP) kinase cascade
despite the fact that stimulation of 9E3/cCAF by thrombin occurs
independently of mitogenesis. Furthermore, thrombin activates the Elk1
transcription factor, and these events are critical for regulation of
9E3/cCAF gene expression. These results advance our
knowledge of the mechanisms activated by the interactions of thrombin
with its receptor, which lead to stimulation of immediate early
stress-response genes.
Reagents
Cell culture
Northern blots and immunoblots These procedures were described previously in detail.12Promoter cloning and luciferase reporter construction Using polymerase chain reaction (PCR) a 1.5- kb DNA fragment from the immediate 5' upstream region of the 9E3 gene ( 1503 to +32) was amplified. Chicken genomic DNA served as
template and 2 oligonucleotides (5' primer:-GGATGAATGGCATTTCAGTG-CAC-; 3'primer: -TCGACACTAGAGAGGACAGTCTCCT-) were used for the PCR
reactions. The 1.5-kb PCR product was cloned using the TA cloning
system pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced with a Li-Cor sequencer using M13 reverse (700 nm) and forward (800 nm) primers labeled with color markers. The 1.5-kb PCR product was then subcloned into the pGL3 basic vector (Promega, Madison, WI). The 5' deletions of
this promoter were obtained using restriction enzyme digests and
re-ligations of this vector (p1503), resulting in constructs with 66, 218, 470, 683, and 1116 bp upstream from the initiation site. Other
constructs within the p683 were prepared by PCR with the following
primers: (1) p542 (542 to +32 bp), 5'-GGCAAAA-TGCAGGAATTGTTTGCAC-; 3'-TCGACACTAGAGA-GGACAGTCTCCT-); (2) p495 (495 to +28 bp),
5'-ATCAGGATGCTTTTAATACTGCACCCT-; 3'-GTGATCTCTCCTGTCAGAGGAT-); and (3)
pmElk1 (495 to +28 bp with the mutation on the Elk1 conserved binding
element, 5'-ATacGcgTGCTTTTAATACTGCACCCT-; 3'-GTGATCTCTCCTGTCAGAGGAT-). These 3 constructs were cloned into pGL3
basic vector through KpnI and SmaI sites. All PCR
products were sequenced and compared for fidelity to the published
sequence.25 To generate the mutated AP-1 binding element
(TGACTCAT, was changed into gGcCTtAT; pmAP-1), we used
the p683 construct and the QuikChange Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA). The mutation was verified by endonuclease
mapping and confirmed by sequencing. All pGL3/9E3 promoter subclones
were prepared using an Endotoxin-Free Maxiprep Kit (Qiagen,
Valencia, CA).
Transient transfection, thrombin activation, and cell lysate collection The calcium phosphate precipitation method without glycerol shock was used for the transient transfections. DNA concentrations were determined using spectrophotometry and confirmed by ethidium bromide staining of agarose gels. Primary fibroblasts were transiently transfected with a total of 6 µg DNA. Cotransfections always included 2 µg of the PCH110 vector (Pharmacia) containing lac-Z as internal transfection control and, when necessary, pcDNA 3.1 vector or calf thymus DNA was added to bring the total amount of DNA transfected to 6 µg. For all experiments, transfected primary fibroblasts were incubated in the modified 199 media for 36 hours and then stimulated with thrombin for 3 hours prior to lysis. For inhibition experiments, the fibroblasts were incubated with inhibitors for 30 minutes before thrombin stimulation. Cell extracts were prepared with reporter lysis buffer according to the protocol provided by the manufacturer (Promega) and stored in a freezer at70°C.
Luciferase and  -galactosidase assay (Promega). A minimum of triplicate
samples for each experiment was performed and the data were expressed
as mean light units of luminescence per unit -galactosidase activity.
Electrophoretic mobility shift assays For electrophoretic mobility shift assays (EMSAs) fibroblasts transfected with the pCMV3.1 (Invitrogen) or pCMV-Elk1 expression vector26 were treated with thrombin for 1 hour before extraction. The methods to prepare the nuclear extracts have been described previously.27 Protein concentration was determined with the D-C protein assay kit (BioRad). Briefly, EMSA buffer (50 mmol/L HEPES pH 7.9, 10 mmol/L MgCl2, 0.2 mol/L KCl, 0.5 mmol/L EGTA, 5 mmol/L DTT, 20% Ficoll) containing 10 µg extracted nuclear protein and 2 ng -32P-adenosine
triphosphate-labeled probe containing the conserved Elk1 binding
element located between 493 and 483 bp
(5'-TTTGCAAAATGCAGGAATTGTTTTCACAGT-3') was used. As
a control, mutated oligonucleotide
(5'-TTTGCAAAATaCgatAATTGTTTTCACAGT-3') was labeled and used
for EMSA. Unlabeled probe (50 ng) was used for the competition assay
and 2 to 3 µg anti-SAP1a (Santa Cruz), anti-Elk1 (New England
Biolab), or antiphospho-Elk1 (Ser383) antibody (New England Biolab) was
added for the supershift assay. For peptide competition experiments,
GST-Elk1310-428 vector was used to produce the protein as
previously described.27 GST-P-Elk1307-428 (Ser 383 phosphorylated C-terminus of Elk1) was purchased from New England
Biolab. It was concentrated and sodium dodecyl sulfate (SDS) washed
away with 2.5% Triton X-100 before use. All samples were assayed in
5% nondenaturing polyacrylamide gel electrophoresis (PAGE)
(acrymide:bis as 60:1).
Transcription activation of the 9E3/cCAF gene To determine the gene activation pattern of 9E3/cCAF after thrombin stimulation, we treated primary fibroblasts with thrombin for varying periods of time and collected samples for RNA analysis. Seven minutes after exposure to thrombin, a 4.0-kb RNA band corresponding to the full-length gene (Kaiser and Hughes, published in GeneBank; see legend for Figure 1) was visible by Northern blot analysis (Figure 1A, lane 5). This RNA species increased to a maximum at 3 hours and declined slowly thereafter, returning to basal levels by 18 hours (Figure 1A, lanes 6-10). Shortly after the appearance of this band, we also observed a marked increase in the level of the processed (1.2 kb) 9E3/cCAF messenger RNA (mRNA; Figure 1A, lane 6). Figure 1B shows that when cells were treated with thrombin in the presence of the transcription inhibitor actinomycin D, the 4.0-kb band was not observed. Cells treated with cytochalasin D, an inhibitor that does not affect transcription but rather inhibits actin cystoskeleton functions, did not eliminate the 4.0-kb band, indicating that the effect of actinomycin D was not due to general disruption of cellular functions. These results show that stimulation of the 9E3/cCAF gene by thrombin is very rapid and suggest that this activation occurs first at the transcriptional, then at the posttranscriptional level. To further verify that activation of this gene by thrombin occurs at the transcriptional level, we cloned the 9E3/cCAF promoter (1503 bp to +32 bp25) into a reporter
construct containing the firefly luciferase gene. When this construct
(p1503) was transfected into primary fibroblasts, expression of
luciferase was greatly stimulated by thrombin (4-5-fold; Figure 1C). To
determine whether this promoter contains the recently published
thrombin response region that consists of a repeat of a CCACCC element
in an ABBA configuration,28 we used the Transcription Element Search Software (TESS assay).29 Although
no such thrombin response region was found in p1503, a number of
potentially functional transcription factor binding elements known to
be important in the regulation of chemokine gene expression and in gene
activation by thrombin were identified (Figure
2).30-34
Analysis of the 9E3/cCAF promoter responses to activation by thrombin To further analyze the contribution of the various consensus transcription factor binding elements to the activation of 9E3/cCAF expression, we produced 5' deletions in p1503, linked them to the luciferase gene, and analyzed the constructs by transient tranfections (Figure 2A). The683-bp to +32-bp promoter construct (p683) contains
several elements that can potentially be involved in the stimulation of
9E3/cCAF by thrombin. In addition to the TATA box, it contains the
consensus binding sequences for the transcription activators
Elk1,35 AP-1,36 PDRII B (the chicken equivalent of nuclear factor B37-39); and the CAAT box
for C/EBP. This construct also contains the binding element for the
chemokine gene repressor Oct-1,32 which is found a short
distance upstream from the AP-1 binding site. The 1116-bp to +32-bp
construct (p1116) contains in addition to these elements 3 Ets binding
sites and another PDRIIB element. The full-length p1503 promoter
contains an additional 2 AP-1, 1 Oct-1, and 2 Ets binding sites. These 3 constructs and an additional one containing only the
9E3/cCAF gene TATA box (p66) were transiently transfected
into primary fibroblasts and the cells were assayed for luciferase
activity in the presence or absence of thrombin stimulation (Figure
2A). Because primary cells were used, variations in transfection
efficiencies between batches of cells can occur. Therefore, for each
experiment, internal controls were always included. Furthermore, the
levels of activation are not of the magnitude of those generally seen when molecules are overexpressed or cell lines are used. Although all
constructs yielded some basal level of activation, treatment with
thrombin resulted in significantly higher levels of activation. The
response of the full-length construct, p1503, to thrombin resulted in a
3-fold stimulation over the control (Figure 2A,I), whereas the promoter
truncated at 1116 (p1116) yielded a 6-fold stimulation by thrombin
(Figure 2A,II), and the one truncated at 683 (p683) exhibited
activity comparable to that of p1503. These results indicate that the
region encompassing 683 to +32 bp contains most, if not all, of the
elements necessary for basal transcription as well as responsiveness to
thrombin in intact cells. They also suggest that there might be
negative regulatory elements between 1503 and 1116 bp.
Because most of the activation induced by thrombin was achieved with
the p683 construct, we concentrated on determining which element(s) in
this region of the promoter are the thrombin response elements. For
these studies, further 5' deletions of p683 followed by transient
transfections into primary fibroblasts were performed. The results show
that eliminating the sequence from Elk1 is important for the activation of the 9E3/cCAF gene by thrombin To further address the possibility that the Elk1 consensus binding sites are critical for the transcription activation of the 9E3/cCAF gene, we deleted the Elk1 site starting at534
bp, mutated the Elk1 site starting at 493 bp to obtain a construct containing 495 bp to +32 bp (pmElk1), and performed transient transfection studies similar to those described above. In contrast to
the normal p495 (that contains the unaltered putative Elk1 binding
site), the p495 with the mutated Elk1 yielded a much lower level of
transcription and was not responsive to thrombin (Figure 3).
To determine whether a transcription factor in primary normal
fibroblasts binds to the sites present in the 9E3/cCAF promoter, we
performed EMSAs using a radiolabeled probe containing the sequence of
the putative Elk1 binding element (
Activation of the Elk1 transcription factor involves the
phosphorylation of this factor on Ser383 and Ser389.40,41
To determine if thrombin stimulates the phosphorylation of this
transcription factor, we performed the supershift assays with an
antibody specific for the Ser383 phosphorylated form of Elk1
(anti-Ser383 phosphor-Elk1). This antibody caused a supershift to occur
with extracts from cells stimulated with thrombin (lanes 10 and 14),
but not with extracts from cells that were not stimulated (lane 6).
These results show that Elk1 binds the To confirm that the Elk1 transcription factor is phosphorylated on Ser383 in response to thrombin, the nuclear extracts used in Figure 4A were examined by immunoblot analysis with the anti-Ser383 phosphor-Elk1 antibody. The results show that thrombin stimulation resulted in a significant increase of Elk1 phosphorylation on Ser383 (Figure 4B). Similar expression levels and even loading of the Elk1 protein were demonstrated by reprobing the blot with the antibody to the C-terminus of Elk1 (Figure 4C). Because this antibody detects all of the Elk1 protein in the cells (phosphorylated and unphosphorylated), 2 bands were observed; the lower band represents the unphosphorylated form of the Elk1 protein, whereas the higher band corresponds to phosphorylated Elk1. To further verify the specificity of the shift complex, we show an EMSA using the mutated Elk-1 binding element (Figure 4D). The shift complex formed with the wild-type oligonucleotide (lane 2) but did not form with the mutated oligonucleotide (lane 3). Moreover, the complex was competed out by excess (50-fold) unlabeled wild-type (lane 4) but not mutated oligonucleotide (lane 5). These findings indicate that this complex is specific for the conserved Elk1 binding element. To verify the specificity of the supershift complex, we used a control antibody and the C-terminal peptides of Elk-1 (phosphorylated and unphosphorylated) as competitors for the transcription factor binding to its antibodies. The supershift complex obtained with the anti-Elk1 antibody (lane 6) did not form with the control antibody (lane 7) and was competed out when the antiElk1 antibody was preincubated with unphosphorylated peptide competitor (lane 8; the latter was obtained by expressing a GST-Elk1310-428 fusion protein that does not contain the DNA binding domain but contains the unphosphorylated Elk1 C-terminus). Furthermore, in control cells, the supershift complex also did not form when the anti-P-Elk1 antibody was used (lane 9) but, when cells were stimulated with thrombin, the supershift occurred with both Elk1 antibodies (compare lanes 10, 11, and 13). Unlabeled GST-Elk1310-428 was able to compete out the supershift obtained with anti-Elk1 (lane 12) but not the supershift obtained with anti-P-Elk1 (lane 13). The latter was competed out (lane 14) by a GST-P-Elk1307-428 C-terminus peptide phosphorylated on Ser383. Figure 4E shows that the GST-Elk1310-428 is recognized by the anti-Elk1 antibody but not by anti-P-Elk1, whereas the phosphorylated GST-P-Elk1307-428 protein was recognized by both antibodies. These results show that the supershift is specific for Elk1. To determine whether the thrombin-induced phosphorylation of Elk1
enhances the activation of this factor, we used a heterologous expression system with a Gal4-Elk1 fusion protein and a Gal4 luciferase reporter (Stratagene). Cells were cotransfected with the pFR-Luc construct, which contains 6 Gal4 DNA binding elements in tandem upstream of the luciferase gene, and with the pFA-Gal4dbd-Elk1AD construct, which is a cytomegalovirus-driven expression vector for a fusion protein containing the Gal4 DNA binding domain (Gal4dbd) and the Elk1 activation domain (Elk1AD). The Gal4dbd part of this fusion protein binds to the Gal4 response elements in pFR-Luc and the
Elk1AD portion of the fusion protein activates the luciferase gene
after phosphorylation. As a negative control we used an expression vector for the Gal4 DNA binding domain alone (pFC-Gal4dbd). It was
observed that, when pFC-Gal4dbd was cotransfected with the reporter
construct (pFR-Luc) followed by treatment with thrombin, the activation
of the luciferase gene was not significantly different from that of the
control (Figure 5Ai-ii); the same was
observed when pFA-Gal4dbd-Elk1AD was cotransfected with the reporter
construct in the absence of thrombin treatment (Figure 5Aiii). However, when thrombin was used to stimulate the cells cotransfected with the
pFA-Gal4dbd-Elk1AD and the reporter construct, we observed a 16-fold
increase in activation (Figure 5Aiv). These results show that the
signals generated after stimulation of cells by thrombin activate the
Elk1 transcription factor.
To extend the studies with the heterologous expression system to the full-length Elk1 protein, we determined whether the overexpression of Elk1 in the presence of thrombin stimulation would cause a higher level of activation than with the endogenous factor alone. Thrombin induced about a 3-fold increase in activation by the endogenous Elk1 with p683 alone (Figure 5Bii; see also Figure 2A-B, but note the difference in the units of the Y-axis), whereas overexpression of Elk1 in the absence of thrombin caused an approximate 2-fold increase above the p683 basal level (Figure 5Biii) (this could be due to the trace amounts of phosphorylated Elk1 seen when Elk1 is overexpressed in the absence of thrombin, Figure 4B). However, simultaneous Elk1 overexpression and thrombin treatment of cells transfected with p683 produced a strong synergistic effect (about 28-fold over control; Figure 5Biv). To correlate these activation levels with those of the cCAF protein, cells were treated in a similar manner and the supernatants were analyzed by immunoblotting using an antibody to cCAF. We found that the levels of endogenous cCAF protein parallel the levels of the luciferase activity driven by the cCAF promoter (Figure 5C). MEK1/ERK2 involvement in the stimulation of 9E3/cCAF by thrombin We have previously shown that ERK2 is differentially phosphorylated on tyrosines after primary fibroblasts are stimulated by thrombin.12 These findings raise the possibility that ERK2 is involved in the stimulation of 9E3/cCAF by thrombin. Western blot analysis (Figure 6A) using an antibody against activated ERK2 (phosphorylation on Thr202 and Tyr204) showed that this kinase was highly phosphorylated and activated after stimulation by thrombin. This effect was inhibited by PD98059, the highly selective inhibitor of MEK1, which is the kinase that is directly upstream from ERK2 in the MAP kinase cascade and phosphorylates and directly activates ERK2.42 The inhibitor to MAP kinase p38, SB203580, used under the same conditions, showed minimal effects on thrombin-induced ERK2 phosphorylation. Similarly, thrombin stimulation of these cells caused an increase in production of cCAF, whereas treatment by thrombin in the presence of PD98059 resulted in inhibition of cCAF production (Figure 6B). The specificity of the PD98059 effects were further confirmed by transient transfection analysis. Activation of the p683 promoter by thrombin was down-regulated by PD98059 in a dose-dependent manner (Figure 6C). Similarly, EMSA experiments (Figure 7A) performed with the nuclear extracts from cells treated with thrombin in the presence of PD98059 showed that this inhibitor decreases the binding of Elk1 to the probe (compare lanes 2 and 6). Furthermore, this complex (lane 6) was supershifted by anti-Elk1 (lane 8) but not by anti-Ser383 phosphor-Elk1 (lane 9) indicating that MEK1/ERK2 is involved in the phosphorylation of Elk1 by thrombin in primary fibroblasts. To confirm that the decreased Elk1 binding with PD98059 treatment is not due to the down-regulation of nuclear Elk1 protein levels, but through the Ser383 phosphorylation, Western blot analysis was performed with the same nuclear extracts used in the EMSA. The results indicate that a similar amount of Elk1 is overexpressed and present in the nuclei even with the inhibitors. PD98059 dramatically decreases Elk1 Ser383 phosphorylation, whereas the p38 MAP kinase inhibitor SB203580 had only a very small effect (Figure 7B). To show that MEK1/ERK2 acts via Elk1 in our cells, the Gal4-Elk1 fusion construct described above (pFA-Gal4dbd-Elk1AD) was cotransfected with a MEK1 expression vector (pFC-MEK1) and the reporter construct (pFR-Luc). Even in the absence of thrombin stimulation, the reporter gene was activated by MEK1 overexpression (Figure 7Civ), indicating that overexpression of this kinase activated its substrate ERK2 followed by activation of the Elk1 transcription factor. Furthermore, addition of PD98059 eliminated the activation of the reporter gene by MEK1/ERK2 (Figure 7Cv). Taken together, these results support the conclusion that the Elk1 transcription factor is activated by thrombin through the MEK1/ERK2 kinase cascade.
AP-1 is not critical for the activation of 9E3/cCAF in normal fibroblasts It has been shown that phorbol-12-myristate-13-acetate (PMA) activates the IL-8 chemokine gene through the AP-1 complex43 in human Jurkat T cells, and that thrombin can stimulate mitogenesis of CCL39 hamster fibroblasts through rapid and persistent activation of AP-1 via the MAP kinase cascade.44,45 Furthermore, it has also been shown that AP-1 is involved in the activation of 9E3/cCAF in Rous sarcoma virus-transformed fibroblasts.38,46 Although the results presented above (Figures 2B and 3A) show that in primary normal fibroblasts the promoter construct containing the AP-1 binding element but lacking the Elk1 sites (p470) does not respond to thrombin stimulation, it is possible that in this particular case AP-1 cooperates with Elk1. To address this possibility, we first overexpressed c-Fos and c-Jun simultaneously in primary cells and used a reporter construct containing 7 AP-1 consensus binding elements linked to the luciferase gene (pFR-AP1; Stratagene) to determine whether thrombin can activate the AP-1 complex. Expression of c-Fos and c-Jun simultaneously should result in the formation of the Fos/Jun complex that constitutes the active AP-1 transcription factor.36 We found that pFR-AP1 yielded a very low level of basal activity (Figure 8Ai), whereas treatment with thrombin resulted in a 3-4 fold increase in activation (Figure 8Aii). Similar results were observed when c-Fos and c-Jun were overexpressed together in the absence of thrombin treatment (Figure 8Aiii), but when both thrombin treatment and overexpression of the AP-1 complex occurred, the transcription activity increased significantly (Figure 8A, compare iii and iv). MEK kinase was used as positive control to verify that the pFR-AP-1 was working correctly (Figure 8Av). Thus, the AP-1 transcription factor complex made from the expression vectors is functional in primary fibroblasts and can be activated by thrombin.
To determine whether the AP-1 element in the context of the 9E3/cCAF promoter plays a role in transcription activation stimulated by thrombin, the cells were cotransfected with the p683 reporter construct (which contains the AP-1 element and both Elk1 binding sites) and the expression vectors for c-Fos and c-Jun in the presence or absence of thrombin treatment. Expression of c-Fos and c-Jun individually or in combination did not stimulate transcription activation above that of the control (Figure 8Biii-v), and overexpression of the complex in cells treated with thrombin resulted in a level of activation that was not significantly different from that in cells treated with thrombin alone (Figure 8B, compare ii and vi). Furthermore, transfections performed with the promoter containing the mutated Elk1 elements (pmElk1) showed that treatment with thrombin (Figure 8Bviii) or coexpression of c-Fos and c-Jun in cells treated with thrombin, did not result in transcription activation of the reporter system (Figure 8Bxii). These results show that in primary normal fibroblasts, overexpression of a functionally competent c-Fos/c-Jun complex does not activate the 9E3/cCAF promoter. To confirm these results, we performed site-directed mutagenesis on the AP-1 binding site in the p683 construct and found that neither the basal level nor the thrombin-stimulated level of transcription was significantly altered (Figure 9C). Thus, we conclude that AP-1 is not a significant factor in the thrombin activation of the 9E3/cCAF gene in primary fibroblasts.
Thrombin, an enzyme released on wounding, is known to play key
roles in wound healing and in the development of tumor stroma and to
stimulate the expression of chemokines to high levels in cells
importantly involved in these processes. Understanding the mechanism of
activation of chemokines by agents such as thrombin could lead to ways
of modulating expression of these small cytokines in conditions of
injury and tumorigenesis where thrombin plays important functional
roles. Here we show that in primary normal fibroblasts, cells that are
critical in wound healing and in the development of tumor stroma: (1)
thrombin very rapidly activates the expression of the 9E3/cCAF
chemokine at the transcriptional level; (2) the 2 Elk1 binding elements
in the promoter of this chemokine gene starting at Previous work using the region of the 9E3/cCAF promoter between The results presented here showing that Elk1 but not AP-1 is crucial for the stimulation of 9E3/cCAF gene activation by thrombin are supported by previous findings. For example, the kinetics of gene activation stimulated by thrombin and v-src are different. As shown in Figure 1A, thrombin stimulation of 9E3/cCAF mRNA occurred very rapidly after thrombin addition to the cells, reached a maximum within 3 hours, and declined to near background levels by 18 hours. Similar patterns of activation by thrombin have also been shown for MCP-1 in vascular smooth muscle cells47 and for IL-8 in human umbilical vein endothelial cells and in fibroblasts stimulated by serum, which contains thrombin.48,49 On the other hand, stimulation by v-src is first seen at 3 hours after switching transformed cells to the permissive temperature, requires 8 to 12 hours to reach its peak, and remains high for 24 hours.38,46 The differences in the 2 patterns of stimulation imply that different mechanisms are involved in the activation processes stimulated by thrombin and by the src oncogene. Indeed, stimulation of 9E3/cCAF by thrombin and v-src are additive.12 Our results also show that Elk1 can stimulate 9E3/cCAF expression in absence of the serum response element (SRE) and serum response factor (SRF). Elk1 was originally characterized as a component of a ternary complex, which contains an Elk1 transcription factor and 2 SRFs. Elk1 contains various functional domains: an N-terminal DNA binding domain, an SRF-interacting region, a C-terminal transactivation domain that contains phosphorylation sites that regulate transcription,41 and a region for binding of ERK2, which then phosphorylates and activates Elk1.26 In the ternary complex, Elk1 can bind to the SRE with the cooperation of the 2 SRFs and activate gene expression.50-53 However, the capability of Elk1 to activate transcription is not dependent on its interaction with the SRF.54 Rather, activation of transcription by Elk1 depends on phosphorylation of the (S/T)P motif in the transcription domain,40,50 which is carried out by the ERK/JNK on binding of the pertinent kinase. Therefore, it has been proposed that Elk1-dependent gene activation can occur independently of SRE/SRF if it can bind to the promoter efficiently.50 This proposition is supported by the finding that the phosphorylation of Elk1 by ERK2 increases its ability to bind to DNA without interacting with SRF54,55 and by the fact that Elk1 binding is facilitated in transfection experiments with a reporter gene containing multiple high-affinity Elk1 binding sites40,56 or phosphorylation in its C-terminus.55,57 Even though the 9E3/cCAF promoter does not contain an SRE, Elk1 binds to the Elk1 elements and activates transcription, suggesting that thrombin-induced Elk1 activation does not require cooperation with SRF. Furthermore, the 9E3/cCAF promoter contains several Elk1-Ets elements in tandem, which may be instrumental in promoting efficient binding of Elk1 to its element. Thus, the work presented here strongly supports the previous proposition that Elk1 can function independently of SRF/SRE.50,58 Our results taken together suggest that the Elk1 transcription factor may normally be bound to its response element on the promoter of the 9E3/cCAF gene and that signal transduction pathways triggered by thrombin lead to the phosphorylation and activation of this transcription factor. In support of our findings are the observations showing that Elk1 can bind its DNA response element even when it is not activated.52,56,58 However, only when the cells are stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) was Elk1 able to activate transcription.58,59 Furthermore, very recently it has been shown that MAP kinases can stabilize Elk1 binding and activation and that this is mediated by phosphorylation of the C-terminus of the protein.57 In conclusion, our studies show that thrombin can stimulate stress responses leading to chemokine gene expression through the classical MAP kinase cascade (MEK1/ERK2) normally associated with mitogenesis, but that it does so by activating the Elk1 transcription factor rather than SAP1a or AP-1. These observations lend support to the previous proposition that Elk1-driven gene activation occurs in the absence of SRE/SRF cooperation. The consistent presence of Elk1 binding elements in the promoter regions of many immediate early response genes (eg, c-Fos60; Zif268, MKP-161; egr-162; pip9263) and the rapid kinetics of stress-induced transduction signals suggest that this pathway may play an important role in the responses of normal cells in vivo to other stress-inducing agents.
We thank W. Wong for technical assistance and ACG Design for assistance with computer graphics used to prepare the figures. We are indebted to Drs R. Tjian (University of California, Berkeley, CA) for the expression vector for c-Fos and c-Jun, R. Davis (University of Michigan Medical Center) for the expression vector of Elk1, A. Sharrocks (University of Manchester, United Kingdom) for the vector of GST-Elk1310-428, and D. Strauss and S. Gill (University of California, Riverside) for use of equipment in their laboratories.
Submitted January 5, 2000; accepted July 25, 2000.
Supported in part by National Institutes of Health grant GM48436 (M.M-G.) by National Institute of General Medical Sciences.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Manuela Martins-Green, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521; e-mail: mmgreen{at}ucrac1.ucr.edu.
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Abstract
Introduction
and 
