Departments of 1 Internal Medicine and 2 Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682
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ABSTRACT |
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We previously
observed that the trophic actions of gastrin (G17) on the AR42J rat
acinar cell line are mediated by mitogen-activated protein kinase
(MAPK)-induced c-fos gene
transcription via protein kinase C (PKC)-dependent and -independent
pathways. In this study, we further investigated the signaling pathways
that target c-fos in response to G17.
G17 led to a sixfold induction in luciferase activity in cells
transfected with plasmids containing the 356+109 sequence of the
murine c-fos promoter, which includes
the Sis-inducible element (SIE), serum response element (SRE), and the
Ca2+/cAMP response element (CRE) regulatory elements.
Addition of either the selective PKC inhibitor GF-109203X or the
MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted
in an 80% reduction in luciferase activity. G17 induced the
transcriptional activity of both Elk-1 and Sap-1a, transcription
factors that bind to the E26 transformation specific (Ets) DNA sequence
of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets
sequence led to a 4-fold induction of
c-fos transcription stimulated by G17
and to a 1.3-fold induction in response to epidermal growth factor
(EGF). In contrast, mutations in the CA rich G (CArG) sequence of the
SRE prevented transcriptional activation by both G17 and EGF. G17
induction of the Ets mutant construct was unaffected by either
GF-109203X or PD-98059. Because activation of the SRE involves the
small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c-fos transcription.
Inactivation of Rho A by either the specific inhibitor C3 or by
expression of a dominant negative Rho A gene inhibited G17 induction of
both the wild-type and the Ets mutant constructs by 60%. C3 also
inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the
c-fos promoter CArG sequence via Rho
A-dependent pathways, and Rho A appears to play an important role in
the regulation of the trophic action of G17.
cellular proliferation; early response genes; protein kinases; transcriptional regulation; mitogen-activated protein kinase; extracellular signal-regulated kinases
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INTRODUCTION |
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ALTHOUGH CHARACTERIZED as a stimulant of gastric acid secretion (14), the peptide hormone gastrin also exerts growth-promoting effects on normal and malignant gastrointestinal tissues (37, 43, 45, 51). Gastrin is an important growth factor for the fetal pancreas (3, 52), and in the stomach it is a potent stimulant for the growth of the gastric mucosa (51). In addition, recent reports have indicated that gastrin induces the growth of colon carcinomas both in vivo and in vitro, thus underscoring the importance of gastrin as a growth factor for gastrointestinal neoplasms (37, 43).
The process of cellular proliferation is under the control of complex cascades of phosphorylation reactions that are triggered by the interaction of growth factors with their specific cellular receptors (24, 31, 50). Some of these receptors have intrinsic enzymatic activity (50) and are able to phosphorylate cellular target proteins directly, whereas others like those for gastrin or other gastrointestinal hormones belonging to the seven transmembrane receptor family do not. Several reports have indicated that these receptors require the assembly of multiple molecules at the ligand receptor complex to activate cytoplasmic protein kinases (7, 8, 13, 20, 35). Growth factor receptors such as those for epidermal growth factor (EGF) are prototype receptors with intrinsic tyrosine kinase activity (50). Activation of these receptors is known to lead to the induction of the extracellular signal-regulated protein kinases (ERKs) or mitogen-activated protein kinases (MAPKs). The ERKs are important elements in a signalling cascade that involves upstream protein kinases such as Raf and MAPK/ERK kinase (MEK; see Ref. 6, 11). Activation of the ERKs is known to target downstream protein kinases, such as 90-kDa S6 kinase (6, 11) and transcription factors like Elk-1 and Sap-1a, that regulate the activity of the promoter of the early response gene c-fos (27, 30, 49). The protooncogene c-fos is a member of a well-characterized family of transcription factors that are known to be expressed within minutes of cell activation (24, 32). Thus c-fos appears to be a central element in the process of transmission and integration of the extracellular signals from the cell surface to the nucleus (24, 32).
The mechanisms responsible for the activation of the ERKs and c-fos in response to ligand binding to seven transmembrane receptors have been much less characterized. Gastrin is known to stimulate inositol phospholipid turnover, to stimulate protein kinase C (PKC) activation, and to induce intracellular calcium release (10, 26, 36). Gastrin also induces tyrosine kinase activity, and it stimulates both the ERKs and the early response genes c-fos and c-jun (10, 26, 40, 46-48). In a recent study, we have reported that gastrin stimulates the growth of the rat pancreatic adenocarcinoma cell line AR42J through induction of the ERKs and of c-fos via PKC-dependent and -independent pathways (48).
Many growth factors can target c-fos through a specific portion of its promoter known as the serum response element (SRE; see Ref. 49). Transcriptional activation of the SRE is under the control of numerous nuclear proteins that are known to assemble on the gene forming specific DNA-protein complexes (49). Recent studies have indicated that the SRE comprises DNA regulatory sequences that appear to receive input from different signal transduction pathways. Of these elements, the CA rich G (CArG) box and the E26 transformation specific (Ets) motif have been the focus of extensive investigations. The CArG box binds the serum response factor (SRF) while the Ets motif (CAGGAT) binds ternary complex factors (TCFs) such as Elk-1 and Sap-1a (Ets proteins), which serve as SRF accessory proteins (49). Elk-1 and Sap-1a are phosphorylated and activated by signaling pathways that involve both PKC and different members of the MAPK family of protein kinases (27, 30, 49, 53), whereas transcriptional activation of SRF appears to be regulated, at least in part, by serum stimulation through a yet poorly characterized signal transduction pathway that requires the activation of the small GTP-binding proteins Ras and Rho (25, 49). Recent studies have indicated that ligands for seven transmembrane receptors might signal to the CArG box of the c-fos SRE via MAPK/PKC-independent pathways (15, 25, 49).
In this study, we sought to dissect the signal transduction pathways that target c-fos in response to gastrin stimulation and to investigate the role of the small GTP-binding protein Rho A in gastrin signaling to the c-fos SRE. Furthermore, we examined whether Rho A would be involved in the mediation of the growth-promoting effect of gastrin.
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MATERIALS AND METHODS |
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Plasmids. 5xGal-Luc (22) and Rho A
(N19) (33) were gifts from M. Karin (San Diego, CA), Gal4-ElkC (30) was
a gift from R. Treisman (London, UK), Gal4-Sap-1a (27) was a gift from
T. Hunter (San Diego, CA), Rho A (V14) (38) was a gift from A. Hall
(London, UK), pET3a/C3 (34) was a gift from S. Narumiya (Kyoto, Japan),
and pCMV-Gal was a gift from M. Uhler (Ann Arbor, MI). The plasmid
356wt-fos-CAT and the point
mutants pm12- and pm18-fos-CAT were
gifts from M. Gilman (Cambridge, MA; see Refs. 16, 17). The
356Wt-fos-LUC,
pm12-fos-LUC, and
pm18-fos-LUC were constructed by
excising the Hind
III/Xba I fragments of
356wt-fos-CAT, pm12-fos-CAT, and
pm18-fos-CAT containing the genomic
DNA sequences
356+109 of the murine
c-fos promoter and by subcloning them
into the Hind
III/Xba I sites of the pBK-RSV
(Promega, Madison, WI) multiple cloning site. The fragments were then
excised as Sac I/Xba I fragments and directionally
subcloned into the Sac
I/Nhe I sites of pGL3LUC (Promega)
(42). The resulting constructs were verified by restriction mapping and sequencing.
Cell culture, transient transfection, and luciferase
assays. For our experiments, we used the rat exocrine
pancreatic cell line AR42J, which is known to express receptors for
both gastrin (G17) and EGF (5). The cells (obtained from American Type
Culture Collection, Rockville, MD) were grown at 37°C in 35-mm
dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 5%
CO2-95%
O2. Subconfluent AR42J cells were
transfected with 2 µg of the luciferase reporter plasmid and, where
indicated, with 2 µg of the expression vectors. Transfections were
carried out using lipofectamine (GIBCO-BRL, Grand Island, NY) as
previously described (48). The day after transfection, the media were
removed, and the cells were fed with serum-free media (DMEM) for 24 h
and then incubated for 5 h with or without human gastrin
heptadecapeptide (G17; 10 nM; Bachem, Torrance, CA), EGF (10 nM),
12-O-tetradecanoylphorbol 13-acetate
(TPA; 100 nM; Sigma, St. Louis, MO), or serum (10%). In some
experiments, bisindolylmaleimide I (GF-109203X; 3.5 µM; Calbiochem,
La Jolla, CA) and PD-98059 (50 µM; New England Biolabs, Beverly, MA;
see Ref. 1) were added 30 min before the addition of gastrin. In additional studies, the cells were also preincubated for 24 h in 40 µg/ml exoenzyme C3 from Clostridium
botulinum before the addition of gastrin. GF-109203X,
TPA, and PD-98059 were dissolved in dimethyl sulfoxide (Sigma). All
other test substances were dissolved in water. Control experiments were
performed by incubating the cells in either incubation buffer or
vehicle without the test substances. At the end of the incubation
period, the cells were washed and lysed, and luciferase assays were
performed as described previously (48). Luciferase activity was
expressed as relative light units and was normalized for
-galactosidase activity.
-Galactosidase activity was measured by
the luminescent light derived from 10 µl of each sample incubated in
100 µl of Lumi-Gal 530 (Lumigen, Southfield, MI) and was used to
correct the luciferase assay data for transfection efficiency.
ADP-ribosylation of Rho in AR42J cells. Recombinant C3 was expressed in Escherichia coli using a plasmid encoding C. botulinum exoenzyme C3 (pET3a/C3) and was purified as previously described (34). To verify the effectiveness of ADP-ribosylation of Rho by C. botulinum exoenzyme C3, the AR42J cells were either left untreated or were incubated with increasing concentrations of the toxin (10-200 µg/ml) for 24-48 h. The cells were then extensively washed with PBS and were lysed by three cycles of freezing and thawing in 100 µl of hypotonic lysis buffer [20 mM Tris (pH 8), 3 mM MgCl2, 0.4 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochlorine (AEBSF; ICN Biomedicals, Aurora, OH), 5 µg/ml aprotinin, 2 µg/ml trypsin inhibitor, and 20 µM leupeptin]. Cell debris was separated by centrifugation at 14,000 rpm at 4°C for 20 min, and the supernatant was subjected to ADP-ribosylation assay. Cell lysates containing equal amounts of protein were incubated with 4 µg/ml recombinant C. botulinum exoenzyme C3 and 1 µCi [32P]NAD (Amersham Life Science, Arlington Heights, IL) in 20 µl of buffer containing 10 µM NAD, 50 mM triethanolamine hydrochloride (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mM AEBSF, 10 mM thymidine, and 100 µM GTP at 37°C for 60 min. Protein concentrations were measured by the Bradford method (2). Samples were resolved by SDS-PAGE on 15% gels, and the ADP-ribosylated Rho was visualized by autoradiography.
Proliferation studies. These studies were conducted according to previously described techniques (41, 48). Briefly, the AR42J cells were grown in 35-mm dishes in DMEM supplemented with 10% FBS in 5% CO2 at 37°C. Subconfluent AR42J cells were cultured for 24 h in serum-free DMEM containing 0.2 mM unlabeled thymidine. After being washed with serum-free medium, the cells were treated with gastrin (1 nM) for 18 h. In some experiments, 40 µg/ml of exoenzyme C3 from C. botulinum was added 24 h before the addition of gastrin. Control experiments were performed by incubating the cells in either incubation buffer or vehicle without the test substances. DNA synthesis was estimated by measurement of [3H]thymidine incorporation in the trichloroacetic acid-precipitable material according to the method of Seva et al. (41). [3H]thymidine (0.1 µCi/ml, 10 Ci/mmol) was added during the last hour of the treatment period. Cells were washed with serum-free media to remove unincorporated [3H]thymidine, and the DNA was precipitated with 5% trichloroacetic acid at 4°C for 15 min. Precipitates were washed two times with 95% ethanol, dissolved in 1 ml of 0.1 N NaOH, and analyzed in a liquid scintillation counter (Beckman Instruments, Palo Alto, CA). In some experiments, the growth-promoting effect of gastrin was quantitated by cell counting using a Coulter counter (Coulter Electronics, Hialeah, FL). Measurement of cell proliferation with either method yielded identical results (data not shown).
Data analysis. Data are presented as means ± SE, wherein n is equal to the number of separate transfections performed with the AR42J cells. Statistical analysis was performed using Student's t-test. P values <0.05 were considered to be significant.
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RESULTS |
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We have recently reported that the specific MEK inhibitor PD-98059
potently inhibits gastrin-stimulated SRE transcriptional activity in
AR42J cells transfected with plasmids containing the c-fos SRE upstream of the thymidine
kinase gene minimal promoter and the luciferase reporter gene (SRE-Luc;
see Refs. 48, 54). In this study, we observed that PD-98059 had similar
inhibitory effects on both gastrin- and EGF-stimulated
356wt-fos-luciferase activity,
indicating that activation of MEK and MAPK is essential for efficient
transcription of c-fos in response to
both gastrin and EGF (Fig.
1A).
Gastrin is also known to induce the
c-fos SRE, at least in part, through
PKC-dependent signaling pathways (48). Therefore, we examined the
effect of the selective PKC inhibitor GF-109203X on
356wt-fos-luciferase activity
stimulated by either gastrin or EGF. In a previously published study,
we reported that this compound completely inhibited both TPA-stimulated
MAPK induction and SRE-luciferase activity (48), demonstrating that
GF-109203X is an effective inhibitor of PKC in the AR42J cells. As
depicted in Fig. 1B, GF-109203X
significantly inhibited gastrin-stimulated
356wt-fos-luciferase activity,
whereas it failed to exert any effect on the induction observed in the
presence of EGF. Thus gastrin specifically targets the
c-fos promoter via pathways that appear to require activation of PKC.
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Activation of both PKC and MAPK is known to induce
c-fos gene expression by
phosphorylation of transcription factors like Elk-1 and Sap-1a, which
bind to the Ets DNA sequence of the
c-fos SRE (30, 49). To examine the
effect of gastrin on Elk-1 and Sap-1a transcriptional activation, we
used a system involving cotransfection of the cells with chimeric
GAL4-ElkC and Gal4-Sap-1a expression vectors and the 5XGAL-luciferase
reporter plasmid, which contains five copies of the Gal4 DNA binding
domain linked to the luciferase reporter gene. In this system,
induction of PKC and MAPK leads to enhancement of both GAL4-ElkC and
Gal4-Sap-1a transcriptional activity, resulting in stimulation of
luciferase gene expression. We first examined the role of PKC in
gastrin and EGF signaling to either Elk-1 or Sap-1a. As depicted in
Fig. 2A,
GF-109203X completely inhibited gastrin-stimulated Elk-1
transcriptional activity. In contrast, no inhibition was detected when
the cells were stimulated with EGF. Similar results were observed when
the cells were transfected with a vector expressing Sap-1a (Fig.
2B). These data confirm the notion
that gastrin but not EGF specifically targets the
c-fos promoter via PKC-dependent
pathways.
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We then investigated the role of MEK and MAPK in the transcriptional
activation of both Elk-1 and Sap-1a in response to gastrin and EGF. As
shown in Fig.
3A,
PD-98059 completely inhibited both gastrin- and EGF-stimulated Elk-1
transcriptional activity. Similar results were observed when the cells
were transfected with a vector expressing Sap-1a (Fig.
3B). Taken together, these data
indicate that activation of MEK appears to be necessary for both
gastrin and EGF induction of Elk-1 and Sap-1a transcriptional activity.
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Because the
356wt-fos-luciferase construct
comprises multiple regulatory elements that are known to receive inputs
from different signal transduction pathways, we examined the effect of
point mutations in either the Ets
(pm18-fos-LUC construct) or the CArG (pm12-fos-LUC construct) DNA sequences
of the SRE on luciferase activity stimulated by either gastrin or EGF.
Both gastrin and EGF induced
356wt-fos-luciferase activity
(Fig.
4A).
Point mutations in the Ets DNA sequence of the SRE, recognized by p62
TCFs such as Sap-1a and Elk-1, led to a 4-fold induction of
c-fos transcription stimulated by G17
and to a 1.3-fold induction in response to EGF (Fig.
4B). These data suggest that gastrin
can activate c-fos transcription, at
least in part, through a TCF-independent mechanism. In contrast,
mutations in the CArG sequence of the SRE, which binds the SRF,
prevented transcriptional activation by both gastrin and EGF (Fig.
4C). In agreement with previously
published observations (16, 21), we observed that serum (10%) induced
the
356wt-fos-luciferase construct fivefold, whereas it stimulated the
pm12-fos-LUC construct only twofold
(data not shown). These data underscore the importance of the CArG
sequence of the SRE for transcription of
c-fos in the AR42J cells.
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We then performed studies to examine whether either PKC or MAPK would
be involved in gastrin induction of the
pm18-fos-LUC construct. As depicted in
Fig. 5, gastrin induced
pm18-fos luciferase activity, and this
effect was unaffected by preincubation of the AR42J cells with either
the PKC inhibitor GF-109203X or the MEK inhibitor PD-98059. Similarly,
TPA did not have any effect on pm18-fos luciferase activity,
indicating that gastrin targets the CArG sequence of the SRE via MAPK-
and PKC-independent pathways.
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Because the small GTP-binding protein Rho A plays an important role in
the regulation of c-fos transcription
(15, 25), we undertook studies to examine whether Rho A would be
involved in gastrin signaling to the
c-fos SRE. For these studies, we used recombinant exoenzyme C3 from C. Botulinum, a toxin known to ADP-ribosylate and
inactivate Rho A. We first performed ADP-ribosylation assays to monitor
the dose of toxin required to ADP-ribosylate Rho A in vivo. As shown in
Fig. 6, the AR42J cells were either left untreated (lanes 1 and
6) or were incubated with increasing
concentrations of C3 (lanes
2-5). After 48 h, the cells were lysed, and equal amounts of protein were subjected to
an in vitro ADP-ribosylation assay using C3 (4 µg/ml) and
[32P]NAD. As indicated
in Fig. 6, C3 was able to ADP-ribosylate Rho A in vitro only when the
cells were left untreated (lane 1),
whereas no effect was detected in lysates from cells incubated in vivo with C3 (10-200 µg/ml). These data indicate that a dose of C3 as
low as 10 µg/ml could almost completely ADP-ribosylate Rho A in vivo.
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We then tested the effect of C3 (40 µg/ml) on gastrin induction of
both 356wt-fos-LUC and
pm18-fos-LUC. Preincubation of the
cells with C3 inhibited gastrin induction of both
356wt-fos-LUC (Fig.
7A) and
pm18-fos-LUC (Fig.
7B). We then confirmed that Rho A is
able to activate c-fos transcription
in the AR42J cells. For these experiments, we cotransfected the cells
with the pm18-fos-LUC construct
together with a vector expressing a constitutively active Rho A gene.
As shown in Fig.
8A,
constitutively active Rho A induced pm18-fos luciferase activity
threefold. Identical results were observed in the presence of the
356wt-fos-LUC construct (data not shown). To support further the involvement of Rho A in gastrin signaling to the CArG sequence of the SRE, we cotransfected the AR42J
cells with the pm18-fos-LUC construct
together with a vector expressing a dominant negative Rho A gene. As
depicted in Fig. 8B, dominant negative
Rho A inhibited gastrin induction of
pm18-fos luciferase activity by
>50%, suggesting that gastrin requires activation of Rho A to
stimulate c-fos transcription.
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We finally examined if Rho A would be involved in gastrin induction of
AR42J cell proliferation. For these experiments, the cells were
incubated in the presence of gastrin, alone or in combination with C3.
Gastrin induced
[3H]thymidine
incorporation in AR42J cells, and this effect was inhibited in the
presence of C3, demonstrating that activation of Rho A is required for
the growth-promoting effect of gastrin (Fig.
9).
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DISCUSSION |
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The protooncogene c-fos is a member of a well-characterized family of transcription factors that are known to be expressed within minutes of cell activation (32, 24). The c-Fos protein is known to interact with the product of another member of this family of genes, c-Jun, to form a heterodimeric transcription factor complex [activator protein-1 (AP-1)] that binds specifically to the consensus sequence TGAG(C)TCA (4, 9, 18, 29). Both c-fos and c-jun appear to take part in several programs of cellular activation that include growth and differentiation (44, 23). The transcription of c-fos is regulated by numerous hormones, growth factors, and neurotransmitters via the activation of different signal transduction pathways. Activation of the c-fos promoter by many growth factors requires a cis-acting element, the SRE (16, 17, 49). The c-fos SRE contains at least two DNA sequence motifs that bind different transcription factors: a CArG motif that binds the SRF and an Ets motif (CAGGAT) that binds TCFs such as Elk-1 and Sap-1 (Ets proteins), which serve as SRF accessory proteins (49). These transcription factors are known to be phosphorylated and activated by signaling pathways that involve both PKC and different members of the MAPK family of protein kinases (28, 30, 49, 53).
In previous studies, we indicated that gastrin induces the transcriptional activity of a plasmid expressing the luciferase reporter gene under the control of two copies of the c-fos SRE (54), thus demonstrating that the SRE is a gastrin response element (48). Furthermore, we showed that this effect was mediated at least in part by activation of both PKC and MAPK (48). In this report, we have further characterized the signaling pathways that target c-fos in response to gastrin. We first investigated the role of TCFs in gastrin signaling to the SRE. In these studies, we noted that gastrin induced the transcriptional activity of both Sap-1a and Elk-1 via MAPK- and PKC-dependent mechanisms, since this effect was blocked by GF-109203X and PD-98059, agents known to inhibit PKC and MEK, respectively. In addition, activation of PKC appeared to be a signaling pathway employed specifically by gastrin and not by EGF, since induction of c-fos transcription by EGF was insensitive to PKC inhibition. Taken together, these observations confirm the notion that activation of TCFs in response to gastrin plays an important role in induction of c-fos transcription. Interestingly, gastrin exerted a stronger stimulatory effect on activation of Sap-1a than Elk-1. Recent reports (27) have suggested that, while MAPK preferentially phosphorylates Sap-1a, Elk-1 appears to be more efficiently activated by JNK. Because gastrin induces MAPK more effectively than JNK in the AR42J cells (47), the relatively modest stimulatory effect observed on Elk-1 activation might reflect the low level of JNK induction achieved by gastrin.
We then investigated whether additional signaling pathways would
mediate gastrin stimulation of c-fos
transcription. For these experiments, we took advantage of a series of
luciferase constructs comprising the genomic DNA sequences
356+109 of the murine c-fos promoter. These constructs contain the SRE and other DNA
regulatory elements such as the Sis-inducible element (SIE),
Ca2+/cAMP response element (CRE), and the AP-1-like
element, which are known to bind several transcription factors (16, 17,
28). In the presence of point mutations in the Ets DNA sequence of the SRE, gastrin was still able to induce
c-fos transcription. In contrast,
these mutations compromised the response to either EGF or TPA,
suggesting that gastrin can sustain
c-fos transcription through
TCF-independent mechanisms. This observation was further supported by
the finding that G17 induction of the
pm18-fos-LUC construct was unaffected
by pretreatment of the cells with either PD-98059 or GF-109203X.
Mutations in the CArG sequence of the SRE
(pm12-fos-LUC construct), which binds
the SRF, have been shown to lead to a marked decrease in
c-fos transcription in response to
serum, phorbol 12-myristate 13-acetate, and endothelin-1 in several
cell types (16, 21). In agreement with these observations, we observed
that the pm12-fos-LUC construct was
not stimulated by either gastrin or EGF, whereas it exhibited a modest
twofold induction in response to serum (10%; data not shown),
underscoring the importance of the CArG sequence for transcription of
c-fos in the AR42J cells. Thus gastrin
can target the SRE via both TCF-dependent and -independent mechanisms.
In addition, these data suggest that maximal activation of the SRE by
gastrin requires an intact Ets binding site. In fact, the
transcriptional activity of
pm18-fos-LUC was less than that
observed in the presence of
356wt-fos-LUC, and gastrin
induction
of
356wt-fos-LUC was
potently inhibited by both GF-109203X and PD-98059. Taken together,
these observations indicate that, in the presence of an intact SRE,
phosphorylation of the TCFs by PKC and MAPK is crucial for efficient
transcription of the c-fos promoter.
Because the construct bearing mutations in the SRF binding site
(pm12-fos-LUC) was transcriptionally
inactive in response to both gastrin and EGF, it is unlikely that the
CRE, SIE, or the AP-1-like elements play an important role in gastrin induction of the c-fos promoter,
although we can not exclude that these elements might cooperate with
the Ets or the SRF binding sites in directing transcription of
c-fos in response to gastrin. Additional studies using constructs bearing mutations in these elements
will be necessary to further elucidate this issue.
Recent studies have indicated that ligands for seven transmembrane receptors such as endothelin-1 (21, 42), acetylcholine (15), or lysophosphatidic acid (25) signal to the CArG box of the c-fos SRE via MAPK/PKC-independent cascades. These novel signaling pathways appear to involve the small GTP-binding proteins Ras and Rho and Src protein-tyrosine kinases (21, 42). The downstream kinases, which receive input from Ras, Rho, and Src, as well as the specific transcription factors that are phosphorylated by this signaling cascade are still unknown. Recent reports have suggested that novel protein kinases known as p21-activated kinases or protein kinase N could be targets for activated Rho proteins and that these kinases could in turn phosphorylate a "recognition factor" bound to the SRF (49).
The small GTP-binding proteins Ras and Rho are important molecular switches in the cellular activation process (12, 19, 38, 39). Ras, in particular, is involved in several signaling cascades, leading to the activation of protein kinases such as MAPK and JNK (28, 33). Rho proteins include Rac, Cdc 42, and Rho A (12). These small GTP-binding proteins have been implicated both in the activation of gene transcription and in the regulation of the cytoskeleton (12, 38, 39). Recent reports have shown that Rho proteins can activate c-fos transcription through TCF-independent pathways (15, 25). In this study, we have examined the role of Rho A in gastrin signaling to the c-fos SRE. For these experiments, we employed two different inhibitors of Rho function: a dominant negative Rho A gene and exoenzyme C3 from C. botulinum. Our observations indicate that Rho A is a crucial element in gastrin signaling to the c-fos promoter, since gastrin induction of c-fos transcription was significantly inhibited by both dominant negative Rho A and exoenzyme C3. Interestingly, this inhibition was not complete, thus suggesting the possible involvement of Rho A-independent pathways. Additional studies will be necessary to define if, in addition to Rho A, Cdc 42 or Rac-1 might also be involved in gastrin signaling to c-fos. The complexity of the system is underscored by the observation that, in some cell types such as in fibroblasts, Ras seems to be upstream of and required for the activation of Rho A (38, 39). Furthermore, Ras appears to be necessary for endothelin-1 signaling to the CArG box of the SRE (21). The role of Ras in gastrin signaling is currently poorly understood. A recent report (26) has suggested that Ras is not involved in gastrin signaling, although gastrin has been shown to induce the assembly of several adapter molecules involved in the process of Ras activation such as Shc and Grb2 (10, 26). Additional studies will be necessary to elucidate the role of Ras in gastrin signaling and its relationship with Rho A and to further characterize the cascade of biochemical reactions activated by Rho A in the AR42J cells.
The physiological importance of our observations was underscored by the finding that inhibition of Rho A blocked gastrin stimulation of cell proliferation. Thus Rho A appears to be a crucial element in the complex series of events that regulate the growth factor action of gastrin.
In conclusion, our data suggest that gastrin, binding to specific cholecystokinin B receptors on the AR42J cells, induces a complex cascade of reactions that target multiple signaling cascades. Activation of the c-fos SRE, in particular, seems to occur through at least two distinct pathways. One, requiring activation of both MAPK and PKC, leads to the transcriptional activation of the TCFs Elk-1 and Sap-1a, and the other, which is independent of TCF activation, involves the small GTP-binding protein Rho A, and it targets the CArG box of the SRE. Finally, activation of Rho A seems to play an important role in the regulation of the trophic action of gastrin.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jessica Schwartz for helpful advice.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1DK-33500 and RO1DK-34306 and by funds from the University of Michigan Gastrointestinal Peptide Research Center (NIDDK Grant P30DK-34933). A. Todisco is a recipient of an American Gastroenterological Association Industry Research Scholar Award, a Clinical Investigator Award from the National Institutes of Health (NIDDK Grant K08DK-02336), and a grant from the Charles E. Culpeper Foundation Health Program.
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. §1734 solely to indicate this fact.
Address for reprint requests: A. Todisco, 6520 MSRB I, Ann Arbor, MI 48109-0682.
Received 5 May 1998; accepted in final form 19 October 1998.
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