2 Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555; 1 Department of Surgery, The University of Chicago Medical Center, Chicago, Illinois 60637; and 3 University of California, Davis-East Bay, Oakland, California 94602
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ABSTRACT |
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The hormone bombesin (BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) act through specific GRP receptors (GRP-R) to affect multiple cellular functions in the gastrointestinal tract; the intracellular signaling pathways leading to these effects are not clearly defined. Previously, we demonstrated that the human gastric cancer SIIA possesses GRP-R and that BBS stimulates activator protein-1 (AP-1) gene expression. The purpose of our present study was to determine the signaling pathways leading to AP-1 induction in SIIA cells. A rapid induction of c-jun and jun-B gene expression was noted after BBS treatment; this effect was blocked by specific GRP-R antagonists, indicating that BBS is acting through the GRP-R. The signaling pathways leading to increased AP-1 gene expression were delineated using phorbol 12-myristate 13-acetate (PMA), which stimulates protein kinase C (PKC)-dependent pathways, by forskolin (FSK), which stimulates protein kinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSK stimulated jun-B expression but produced only minimal increases of c-jun mRNA and AP-1 binding activity. Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKC inhibitors) inhibited the induction of c-jun and jun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor) exhibited only minimal effects. Pretreatment with tyrphostin-25, a protein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediated induction of c-jun and jun-B, but the effect was not as pronounced as with H-7. Collectively, our results demonstrate that BBS acts through its receptor to produce a rapid induction of both c-jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction of AP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimal involvement of PKA.
gastrin-releasing peptide; protein kinase C; protein tyrosine kinase; activator protein-1
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INTRODUCTION |
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BOMBESIN (BBS), a tetradecapeptide originally isolated from the skin of the frog Bombina bombina (1), and its 27-amino acid mammalian counterpart, gastrin-releasing peptide (GRP), are considered as generalized "on-switches" in the gastrointestinal tract with actions that include stimulation of peptide release, gastric and pancreatic secretion, and gut motility (8, 25, 35). GRP acts through its specific receptor, the GRP-receptor (GRP-R), which belongs to the G protein-coupled receptor superfamily (4, 39, 53), and is located throughout the gut and pancreas (8, 14, 19, 32). In addition to its effects on secretion and motility, GRP acts as a potent mitogen for certain normal and neoplastic tissues. This mitogenic effect was first reported in human small cell lung cancers that express the GRP-R and secrete GRP, thus suggesting an autocrine mechanism of action for GRP in these tumors (12). In addition, high-affinity GRP-R has been identified in gastrointestinal tract cancers such as pancreatic, gastric, and colonic adenocarcinomas (6, 8, 14, 21, 26, 43, 45, 46, 54, 55). Thus GRP acts as a potent mitogen for certain human gut and pancreatic cancers. The signal transduction pathways responsible for the multiple cellular effects of BBS remain to be clearly defined in the mammalian system.
BBS was initially thought to act as a mitogen in Swiss 3T3 fibroblasts through the activation of phospholipase C (PLC), which catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate and produces inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG; see Ref. 48). IP3 stimulates the release of Ca2+ from intracellular stores, and DAG activates protein kinase C (PKC; see Ref. 39). Both the PKC pathway and PLC-independent Ca2+-mobilizing pathway (Ca2+-calmodulin pathway) are known to stimulate cell growth (48). BBS also increases cAMP production, which is known to stimulate the growth of certain cells. Evidence to support the importance of BBS-mediated induction of cAMP is provided by the report of Benya et al. (5) showing that the inhibition of BBS activation of protein kinase A (PKA) in Swiss 3T3 cells inhibits cell proliferation. Furthermore, other reports have postulated that the mitogenic action of BBS is mediated through activation of nonreceptor protein tyrosine kinases (PTKs; see Refs. 56 and 57). The inhibitor of PTK, tyrphostin, inhibits BBS-stimulated growth of Swiss 3T3 cells (52), thus supporting this hypothesis. From these studies, it appears that GRP and BBS act through multiple, cell-specific signal transduction pathways to regulate cell proliferation (49). These signaling pathways converge upon specific transcription factors, which bind to target genes and ultimately regulate cellular growth.
The large family of activator protein-1 (AP-1) transcription factors, constituted by members of the jun (c-Jun, JunB, JunD) and fos gene families, binds to the phorbol ester-responsive enhancer (TGAGTCA) located in certain genes as either homo- or heterodimers (9, 11, 49). Both jun and fos were originally recognized as viral oncogenes, and their cellular counterparts are the "immediate early genes" that are induced by a wide variety of mitogens, including BBS (16, 37); the immediate early genes also mediate proliferation in certain cell types (2, 29). Previously, we have shown that the human gastric adenocarcinoma SIIA, which was established in our laboratory (3), possesses a native GRP-R that binds BBS with high affinity (6); treatment of SIIA cells with BBS results in increased intracellular Ca2+ mobilization and induction of AP-1 gene and protein expression and DNA binding activity (31).
The signaling mechanisms responsible for BBS-mediated AP-1 activation in SIIA cells are not known. Therefore, the purpose of our present study was to determine whether AP-1 activation occurs as a result of PKC, PKA, and/or PTK stimulation in SIIA cells. Delineation of these mechanisms will provide a better understanding of BBS-mediated AP-1 gene activation in gastric cancers and may have potential implications in the adjuvant treatment of some of these neoplasms.
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MATERIALS AND METHODS |
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Materials.
Restriction, ligation, and other DNA-modifying enzymes were purchased
from Promega (Madison, WI) or Stratagene (La Jolla, CA). Nylon
membranes (Zetaprobe) were from Bio-Rad Laboratories (Hercules, CA),
and oligo(dT)-cellulose (type III) was purchased from Collaborative
Biomedical Products (Bedford, MA). Nucleotides and poly(dI · dC)
were purchased from Pharmacia LKB Biotechnology (Piscataway, NJ), and
radioactive compounds were obtained from NEN (Wilmington, DE). The
GRP-R antagonists BIM-26226 and
[D-Tyr6]Bn-(6---13) methyl ester (ME) were
from Biomeasure (Milford, MA) and Dr. Robert Jensen (National Institute
of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, MD), respectively. Protein kinase inhibitors (H-7,
H-8, and H-89), and the tyrosine kinase inhibitor (tyrphostin-25 and
its control tyrphostin-1) were obtained from Biomol (Plymouth Meeting,
PA). The phorbol ester phorbol 12-myristate 13-acetate (PMA), the cAMP agonist forskolin (FSK), and actinomycin D were obtained from Sigma
(St. Louis, MO). The AP-1 cDNA probes and -actin were obtained from
American Type Culture Collection (Rockville, MD) and were labeled using
a random primer kit (Random Prime-it II) from Stratagene. The
phosphorylated c-Jun antibody (KM-1) and the antibody to c-Fos (4-10G) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was from Amersham (Arlington Heights, IL). The oligonucleotide containing consensus AP-1 binding sites was from Promega. Tissue culture media and reagents were obtained from GIBCO BRL (Grand Island,
NY). All other reagents were of molecular biology grade and were
obtained from either Sigma or Amresco (Solon, OH).
Tissue culture. The human gastric cancer cell line SIIA was established and characterized in our laboratory (3). SIIA cells (passages 11-28) were maintained in F-10 media with 10% FCS in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
RNA extraction and Northern blot analysis.
Cells were harvested, and RNA was extracted by the method of Schwab et
al. (51). Polyadenylated [Poly(A)+] RNA was
selected from all samples using oligo(dT)-cellulose, electrophoresed in
1.2% agarose-formaldehyde gels, transferred to nylon membranes, and
hybridized with the cDNA probes. Hybridization and washing conditions
were described previously (18, 31). Blots
were stripped and reprobed with the constitutively expressed -actin
gene to ensure intact RNA samples and equality of loading.
Protein extraction and Western immunoblot analysis. Nuclear and cytoplasmic protein was extracted from SIIA cells as described by Schreiber et al. (50); concentrations were measured using the method of Bradford (7). Protein lysates were resolved by 10% SDS-PAGE and electroblotted to nitrocellulose membranes as described previously (18, 31). Membranes were blocked overnight in 5% milk with 0.05% Tween 20 in Tris-buffered saline solution and were incubated for 3 h with either an antibody that recognizes phosphorylated c-Jun (1:500) or an antibody to c-Fos (1:1,000). Filters were washed and then incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody (1:500 dilution) for 1 h. The blots were developed using ECL detection.
Preparation of nuclear extracts and electrophoretic mobility
shift assays.
Crude nuclear extracts were prepared from SIIA cells according to the
method described by Schreiber et al. (50). The extracts were quick-frozen, stored in aliquots at 80°C, and used within 2 mo
of extraction.
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RESULTS |
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BBS-mediated AP-1 activation is blocked by specific GRP-R antagonists and actinomycin D. Previously, we have demonstrated that administration of BBS increases the expression of the AP-1 genes c-jun and jun-B, their corresponding proteins, and AP-1 binding activity in the human gastric cancer cell line SIIA (31). The increases noted in mRNA expression occur early, suggesting that BBS is acting through its receptor to activate AP-1 gene transcription in SIIA cells.
To confirm these assumptions, we first determined the effect of pretreatment with two different GRP-R antagonists (15, 38), BIM-26226 and ME, on BBS-mediated induction of c-jun, jun-B, c-fos, and jun-D mRNAs. Treatment with BBS (20 nM) produced a rapid induction of both c-jun and jun-B mRNA levels by 30 min (3- and 2.8-fold induction, respectively) with maximal levels achieved by 1 h after treatment (5- and 3-fold induction compared with control levels, respectively; Fig. 1A). Pretreatment for 2 h with either BIM-26226 (100 nM) or ME (10 nM) and harvesting the RNA 1 h after the addition of BBS demonstrated that both agents effectively blocked BBS-mediated induction of c-jun and jun-B (Fig. 1A, lanes 7 and 9) compared with cells treated with BBS and the vehicle (i.e., 0.05% DMSO) used to dissolve the BIM-26226 compound (Fig. 1A, lane 5). These antagonists had no effect on the levels of c-jun and jun-B when given alone (lanes 6 and 8). Expression of c-fos, which is constitutively elevated in SIIA, was only minimally increased at 30 min and 1 h after BBS addition (~1.5-fold compared with control). Both BIM-26226 and ME blocked this minimal induction of c-fos mRNA. In addition, we elevated the effect of BBS on jun-D expression and found, similar to c-fos, high basal levels of jun-D in untreated SIIA cells, with only a minimal induction noted with addition of BBS (data not shown). The blot was stripped and reprobed with
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Inhibition of BBS-mediated AP-1 activation by actinomycin D
suggests regulation at the transcriptional level.
The rapid induction (within 30 min) of c-jun and
jun-B suggested that BBS regulates AP-1 activation at the
transcriptional level. To better ascertain whether regulation was due
to an increase in transcription, actinomycin D (5 µg/ml), which
inhibits RNA synthesis (40), was added to SIIA cells at
the time of BBS treatment, and cells were harvested 1 h later
(Fig. 2). Actinomycin D effectively blocked BBS-mediated AP-1 induction (lane 5) compared with
BBS treatment alone (lane 2) or BBS combined with the
vehicle (i.e., 0.01% methanol) used to dilute the actinomycin D
(lane 3). Also, addition of actinomycin D alone had no
effect on c-jun or jun-B expression (lane
4) compared with control (untreated) cells (lane 1).
These findings, in combination with the rapid induction of these AP-1
genes, provide evidence to suggest that BBS regulates the transcription
of these genes in SIIA cells.
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Influence of PKC and PKA activation on AP-1 induction in SIIA
cells.
Depending on the cell type and the particular stimulus, activation of
various AP-1 factors may occur through stimulation of the PKC and/or
the PKA signaling pathways (29, 30). To begin to elucidate the role of these protein kinases in BBS-mediated AP-1
induction, SIIA cells were treated with BBS (10 nM), PMA (100 nM), or
FSK (50 µM; Fig. 3A). The
phorbol ester PMA activates both conventional and novel PKC isozymes
(42), whereas the diterpene FSK activates the
PKA-dependent pathways directly through adenylyl cyclase/cAMP
(34). For the remainder of our studies, we have focused on
c-jun and jun-B, since BBS had only minimal
effects on c-fos and jun-D mRNA levels in SIIA
cells.
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PKC inhibitors block BBS-mediated AP-1 activation.
To further delineate the role of PKC and PKA pathways in BBS-mediated
induction of AP-1 expression, various protein kinase inhibitors were
added before the addition of BBS. SIIA cells were pretreated for 2 h with the protein kinase inhibitors H-7, H-8, or H-89 (25 µM each),
and then BBS (10 nM) was added for an additional 1 h (Fig.
4A). Both H-7 and H-8 are
primarily PKC inhibitors (28), whereas H-89 is a selective
PKA inhibitor (10). BBS treatment resulted in increased
expression of both c-jun and jun-B at 1 h
(lane 2); pretreatment with the vehicle (i.e., 0.01%
ethanol) used to dissolve H-7, H-8, and H-89 did not affect
BBS-mediated c-jun and jun-B induction
(lane 3). Pretreatment of SIIA cells with either H-7 or H-8
(lanes 4 and 5, respectively) inhibited the
BBS-mediated induction of both c-jun and jun-B,
with the H-7 compound more effective. In contrast, pretreatment with
H-89 had little effect on the AP-1 induction by BBS (lane
6).
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Attenuation of BBS-mediated AP-1 induction by tyrphostin-25.
In addition to activation of PKC, BBS can activate nonreceptor PTKs in
certain cells (8, 41, 56);
tyrphostin, an inhibitor of these kinases, can block BBS-mediated
growth of Swiss 3T3 cells (52). Therefore, we next
determined whether BBS could affect AP-1 induction in SIIA cells
through a PTK pathway (Fig. 6). As demonstrated previously, BBS treatment stimulated c-jun and
jun-B mRNA levels at 1 h (lane 2);
pretreatment with 1 µM of the PTK inhibitor tyrphostin-25
(22, 23) attenuated the induction of both
c-jun and jun-B (lane 5). In contrast,
pretreatment with either the vehicle (i.e., 0.05% DMSO) or
tyrphostin-1 (a negative control for tyrphostin-25) did not affect AP-1
induction after BBS treatment (lanes 3 and 4,
respectively). Taken together, our results demonstrate involvement of
both the PKC- and the PTK-dependent pathways in the induction of the
AP-1 genes c-jun and jun-B in response to BBS.
Conversely, PKA-dependent pathways do not appear to play a significant
role in AP-1 induction in SIIA cells.
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DISCUSSION |
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Previously, we identified the high-affinity GRP-R in the human gastric cancer SIIA (6). BBS stimulates Ca2+ mobilization and AP-1 gene induction in SIIA cells (31). In our present study, we have confirmed the induction of various AP-1 genes after addition of BBS, noting that this induction was both rapid (within 30 min) and transient (ending in 4 h). Specifically, we found that the induction of c-jun and jun-B was more pronounced than either c-fos or jun-D, which are both expressed at high levels in untreated SIIA cells. Pretreatment with the specific GRP-R antagonists BIM-26226 and ME effectively blocked the BBS-mediated AP-1 gene induction, further confirming that BBS is acting directly through its high-affinity receptor to stimulate AP-1 gene expression in SIIA cells. Furthermore, we demonstrate that the BBS-mediated induction of c-jun and jun-B in SIIA cells likely occurs as a result of increased gene transcription.
Depending on the cell type and the particular mitogen, the induction of the AP-1 genes can occur by a variety of signaling mechanisms, including both PKC- and PKA-dependent pathways (29, 30). The activation of c-jun is thought to be primarily through a PKC-dependent pathway, since treatment of various cells with phorbol ester rapidly induces transcription of the c-jun gene (27). In contrast, jun-B can be activated through both PKC- and PKA-dependent pathways (13, 30). We assessed whether these pathways contribute to the BBS-mediated induction of AP-1 gene expression and DNA binding activity in SIIA cells. Treatment of SIIA cells with the phorbol ester PMA resulted in stimulation of c-jun and jun-B gene expression and AP-1 DNA binding, which were both comparable to the increases noted with BBS. In contrast, treatment with FSK increased jun-B gene expression but resulted in no significant changes in c-jun expression or DNA binding. To further determine the role of these signaling pathways in AP-1 gene induction, SIIA cells were pretreated with the protein kinase inhibitors H-7, H-8 (which primarily inhibit PKC-dependent pathways; see Ref. 28), or H-89 (which is a predominant inhibitor of PKA; see Ref. 10). Our results demonstrate that addition of either H-7 or H-8 inhibited the induction of c-jun and jun-B. On the other hand, H-89 had little effect on the BBS-mediated induction. These findings demonstrate the involvement of PKC-dependent signaling pathways in BBS-mediated AP-1 induction of SIIA cells, whereas PKA does not appear to play a significant role in this response.
In addition to the stimulation of PKC-dependent pathways by BBS, other studies indicate that the GRP-R is coupled to nonreceptor PTK activation in certain cell types. For example, BBS stimulates expression of the immediate early genes c-myc and c-fos through a PTK-mediated pathway in Swiss 3T3 cells (35). In addition, a recent report by Nishino et al. (41) demonstrated that BBS acts through three different signaling pathways, including PTK, in rat pancreatic acini to affect secretion. Therefore, we next determined whether PTK pathways contribute to the AP-1 gene induction noted with BBS treatment in SIIA cells. Addition of tyrphostin-25, a potent general PTK inhibitor (22, 23, 41), to SIIA cells before BBS treatment markedly attenuated c-jun and jun-B induction, whereas treatment with tyrphostin-1, a negative control for tyrphostin-25, had no effect on the BBS-mediated response. These findings provide compelling evidence that both PKC- and PTK-dependent pathways are involved in the BBS-mediated induction of c-jun and jun-B in the SIIA cell line. Consistent with our findings using BBS, others have shown that stimulation of both PKC- and PTK-dependent pathways is required for optimal AP-1 induction in both myogenic cells and the Jurkat T cell line in response to reactive oxygen intermediates (36, 44). In fact, depending on the extracellular stimulus, PTK activation may be the predominant mechanism leading to AP-1 induction, as noted by the stimulation of c-jun expression with arachidonic acid treatment in stromal cells (47).
Although not as common in the United States, gastric cancer is estimated to be one of the more common cancers worldwide, with an increased incidence in certain endemic areas such as Japan, Eastern Europe, and South America (33). Therapeutic options are limited, since radiation and chemotherapy are, for the most part, ineffective in increasing survival in patients with metastatic disease; the cellular mechanisms regulating gastric cancer growth remain unknown. Previously, we have identified expression of GRP-R in two gastric cancer cell lines (SIIA and MKN45) and in three of five human gastric cancer xenografts (31). Consistent with our findings, Preston et al. (43) demonstrated that 13 of 23 gastric cancers expressed high-affinity GRP-R. The presence of GRP-R in gastric cancers appears to play a functional role in the growth of these tumors, as noted by Qin et al. (45) who demonstrated that the GRP antagonist RC-3095 blocks the growth of the human gastric cancer Hs 746T both in vitro and when placed as xenografts in nude mice. Recently, Ferris et al. (20) reported that the constitutive activation of GRP-R in nonmalignant colonic epithelial cells results in cellular proliferation, suggesting a role for GRP-R as a potential oncogene in certain cancers; therefore, it is interesting to speculate whether the expression of GRP-R in SIIA cells contributes to the high basal expression of the c-fos and jun-D genes noted in our study. We suspect that the induction of AP-1 proteins may play a role in the mitogenic effect of GRP in certain gut neoplasms, since the activation of these transcription factors is important for the mitogen-induced growth of other tumors (2, 29). Our future studies may address which specific PKC isozymes and nonreceptor PTKs mediate agonist-stimulated AP-1 activation in SIIA cells. Furthermore, we will assess the effects of blocking specific AP-1 genes or second messenger pathways (e.g., PKC or PTK; see Ref. 24) on the growth regulation of GRP-R-expressing gastric cancers.
In conclusion, we have confirmed the rapid induction of AP-1 gene expression and DNA binding in the SIIA gastric cancer cell line in response to BBS. This induction of AP-1 genes was selective, with a more pronounced effect noted on the expression levels of c-jun and jun-B; both c-fos and jun-D were constitutively expressed in SIIA cells and were only minimally affected by BBS. The induction of AP-1 is mediated through the high-affinity GRP-R and appears to be due to an increase in gene transcription. Finally, using various protein kinase inhibitors, we have established that the BBS-induced increase in c-jun and jun-B expression involves both PKC- and PTK-dependent signaling pathways, with the PKA pathway appearing not to play a significant role. Delineation of the intracellular signaling pathways may provide the cellular basis for improved adjuvant agents to target specific cellular pathways in gastric cancers.
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ACKNOWLEDGEMENTS |
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We thank Eileen Figueroa and Karen Martin for manuscript preparation and Jell Hsieh and Xiaofu Wang for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants PO1 DK-35608, RO1 DK-48345, RO1 DK-48498, and T32 DK-07639 and the James E. Thompson Molecular Biology Laboratory for Surgical Research.
Address for reprint requests and other correspondence: B. M. Evers, Dept. of Surgery, The Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-0533. (E-mail: mevers{at}utmb.edu).
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.
Received 13 August 1998; accepted in final form 10 February 2000.
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