1 Division of Gastroenterology, 3 Department of Medicine and Molecular Biology and Pharmacology, Washington University, St. Louis, Missouri 63110; and 2 Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, Virginia 22904
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ABSTRACT |
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The intestinal cell line I407 responds to
basic fibroblast growth factor (bFGF) by upregulating cyclooxygenase-2
(COX-2) mRNA and protein expression and increasing PGE2
production. bFGF treatment of I407 cells results in phosphorylation of
p38, and the p38 inhibitor SB-203580 abrogates bFGF-induced
PGE2 synthesis. Wild-type p38 (p38
WT) and
dominant-negative p38
(p38
DN) stable transfectant clones of I407
cells were used to examine the role of the p38 MAP kinase pathway in
the events controlling PGE2 synthesis after treatment with
bFGF. Treatment of p38
WT clones with bFGF resulted in increased
COX-2 protein levels and PGE2 synthesis similar to those
seen in bFGF-treated control-transfected cells. In contrast, the
p38
DN clones failed to upregulate COX-2 protein or increase PGE2 synthesis when treated with bFGF. Exogenous
arachidonate did not restore PGE2 synthesis by p38
DN
cells. bFGF treatment increased COX-2 mRNA stability, and the p38
inhibitor SB-203580 attenuated COX-2 mRNA stability in bFGF-treated
I407 cells. These data demonstrate a crucial role for p38
in growth
factor-induced PGE2 synthesis by intestinal cells.
Furthermore, they indicate that p38 activity is required at a step
distal to arachidonate release, most likely COX-2 upregulation, because
exogenous arachidonate did not restore PGE2 synthesis.
intestinal epithelial cells; intestinal injury and repair; arachidonic acid metabolism; mRNA stability.
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INTRODUCTION |
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THE FIBROBLAST GROWTH FACTOR (FGF) family affects the proliferation, differentiation, and migration of intestinal epithelial cells in vitro (3, 5, 22, 45). In addition, members of this family are known to be associated with intestinal pathology, including inflammatory bowel disease and cancer (3, 5, 10, 22, 44, 45). The effect of FGFs on biological events involved in repair as well as the broad range of cell types that respond to these growth factors make them prime candidates as mediators of the intestinal response to injury (3, 4, 22, 27, 45). Indeed, one member of the FGF family, basic FGF (bFGF), has been shown to increase intestinal stem cell survival in the mouse after radiation injury, as measured by the number of regenerative crypts (18). The signaling events initiated by bFGF and how these downstream targets ultimately converge to affect stem cell survival remains unclear.
Prostaglandins are known modulators of the intestinal response to
radiation injury. bFGF induces prostaglandin synthesis in a variety of
cell types, often in the context of angiogenesis or tissue
remodeling/healing (23, 26, 30). When a stable analog of
PGE2, dimethyl PGE2, is given to mice before
radiation, the number of surviving crypts is increased
(15). Although dimethyl PGE2 given alone after
radiation does not change crypt survival, it does reverse the ability
of indomethacin to suppress the number of crypts surviving radiation
injury (6). Similarly, irradiated cyclooxygenase
(COX)-1/
mice show a reduced number of
surviving crypts and an increased number of apoptotic cells
compared with their wild-type littermates (19). Together,
these data raise the possibility that bFGF modulates radiation injury
through the induction of prostaglandins.
Much of the work regarding FGF signal transduction has centered on the
proliferative effects of these growth factors and has emphasized the
ERK signal-transduction module. Less is known regarding the ability of
FGFs to interact with other signal-transduction modules such as p38
MAPK, Src, protein kinase C, phospholipase C, and
phosphatidylinositol 3' kinase. The relative contributions of
activation of these different signaling pathways to the biological effects of bFGF are unknown. At least two FGF-induced effects, proliferation and migration, are activated through different
signaling pathways (Src and p38, respectively) (4,
27). Most studies regarding the ability of bFGF to activate p38
have concentrated on endothelial cells and the role of p38 in migration
and angiogenesis. Whether bFGF stimulation of intestinal epithelial
cells results in activation of p38 and the downstream consequences of
this activation has not been explored previously.
The COX-2 gene is commonly upregulated by two general classes of
mediators: growth factors and inflammatory cytokines. Depending on the
stimulus and cell target, the NF-B signaling pathway and/or any one
of the MAPK modules may be involved in activating COX-2 expression
(40). The COX-2 promoter contains a number of potential regulatory elements; one of these elements, the ATF/CRE site, binds
transcription factor complexes whose formation can be modulated by
activation of p38 (40). Although small molecule inhibitors of p38 have been used to demonstrate a requirement for p38 in upregulated COX-2 expression (8, 11, 12, 14, 21, 37, 41),
the contribution of p38-dependent transcriptional activation to the
observed increase in COX-2 expression has been explored less
extensively. Posttranscriptional regulation also plays a significant
role in induced COX-2 expression (7, 9, 16). Run-on
experiments indicate that increased transcription accounts for only a
small component of the observed increase in COX-2 mRNA in some cell
types treated with inflammatory stimuli. For stimuli such as IL-1
,
LPS, and TNF-
, message stabilization appears to be the primary
mechanism in COX-2 induction (2, 20). Not surprisingly,
the stress-activated MAPK, p38, appears to be required for this
enhanced stability (8, 11, 12, 21, 29, 35). Message
stabilization also contributes to the induced levels of COX-2 observed
in "growth-stimulated" cells such as colonic carcinomas (37), transforming growth factor (TGF)-
-stimulated
(39), or ceramide and bile acid-treated ras-transformed
rat intestinal cells (47), IFN-
-stimulated
keratinocytes (31), and nucleotide-stimulated (46) or angiotensin II (33)-treated smooth
muscle cells. In most but not all of these instances, p38 also appears
to play a role in COX-2 mRNA stability (31, 33, 47). The
ability of bFGF to upregulate COX-2 through a p38-dependent mechanism has not previously been evaluated.
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MATERIALS AND METHODS |
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Cell culture and transfected cell lines.
The human intestinal cell line I407 (American Type Culture Collection;
Manassas, VA) was maintained in basal medium Eagle with Earl's
salts (GIBCO-BRL; Grand Island, NY) supplemented with 6 mM glutamine,
10% heat-inactivated fetal calf serum, and antibiotics (penicillin 50 U/ml and streptomycin 50 µg/ml). Control-transfected, p38
wild-type (p38
WT), and p38
dominant-negative (p38
DN) stable transfectant cell lines were maintained in the above media supplemented with G-418 as the antibiotic, but were cultured for 1 wk in the absence
of G-418 before use in experiments. Briefly, 50-80% confluent cultures of I407 cells seeded in six-well tissue culture plates were
transfected with FuGENE 6 reagent (Roche; Indianapolis, IN) according
to the manufacturer's guidelines at a ratio of 3:2 FuGENE/DNA and 2 µg DNA/well. pCMV-p38
wild-type and pCMV-p38
dominant-negative (Thr180 and Tyr182 replaced with Ala and Phe,
respectively) expression plasmids used for transfection were obtained
from Dr. R. Davis, Howard Hughes Medical Institute. pcDNA 3.1 (Invitrogen; Carlsbad, CA) was used as the empty vector for control
transfections. After overnight exposure to the DNA/FuGENE complex, the
media was changed, and two days later, cultures were placed into media
containing 1.2 mg/ml G-418 for selection of stable transfectant clones.
COX mRNA and protein levels. The ability of bFGF to regulate COX-2 was determined by stimulating subconfluent I407 cells with bFGF (Scios-Nova; Mountainview, CA) at 10 ng/ml plus heparin at 10 µg/ml. Subconfluent HT-29 or Caco-2 cells were serum starved (0.1% fetal calf serum) overnight and then stimulated with bFGF (as for I407 cells) in media containing 0.1% fetal calf serum with or without the p38 inhibitor SB-203580 (10 µM). At the indicated times, cells were lysed with Laemmli buffer for subsequent detection of COX-2 protein by Western analysis or lysed with a guanidine thiocyanate-based solution (Ambion Direct Protect kit) for subsequent ribonuclease protection assays.
For Western analysis, lysates were separated by SDS-PAGE, blotted to Immobilon-P membrane (Millipore; Bedford, MA), and COX-2 protein detected using an anti-COX-2 antibody from Santa Cruz Biotechnology (Santa Cruz, CA) according to the protocol suggested by the supplier. Immunoreactive protein was detected by enhanced chemiluminescence (ECL) reagent (Amersham, Piscataway, NJ) and corrected to actin determined by reprobing the same blot (antibody from Santa Cruz). COX-1 protein expression was determined in the same manner using a COX-1 antibody from Santa Cruz. COX-2 mRNA levels were evaluated using the Direct Protect ribonuclease protection kit from Ambion (Austin, TX). The 400-nucleotide antisense human COX-2 32P-labeled probe was generated using SalI linearized plasmid (gift of Dr. J. Masferrer, Pharmacia) and T7 polymerase (Maxiscript kit, Ambion). The 32P-labeled cyclophilin probe was generated using a template from Ambion. After gel purification of the probes, they were combined with the cell lysate, and the ribonuclease protection assay was performed as directed in the kit instructions. Protected fragments were separated by electrophoresis, and radioactivity was detected using autoradiography on Kodak Biomax MR film or using the PhosphorImager SI (Molecular Dynamics; Sunnyvale, CA). The COX-2 signal was corrected to cyclophilin. COX-1 mRNA levels were determined in a similar manner. For COX-1 RPAs, a 240-nucleotide antisense COX-1 32P-labeled probe was generated from an EcoRV-linearized plasmid from Dr. J. Masferrer (Pharmacia) using T7 polymerase.COX-2 mRNA stability. For experiments determining the stability of COX-2 mRNA, COX-2 mRNA levels were evaluated using real-time RT-PCR. Cells were stimulated for 1 h with bFGF followed by the addition of dichlorobenzimidazole riboside (DRB; 100 µM) or DRB and the p38 inhibitor SB-203580 (10 µM) for the indicated times. RNA was isolated using TriZOL (Invitrogen) and reverse transcribed using Superscript II RT (Invitrogen) with random hexamers according to the manufacturer's instructions. Real-time PCR was performed in an iCycler (Bio-Rad; Hercules, CA) using SYBR Green PCR master mix (Applied Biosystems). COX-2 primers (0.2 µM each) were forward: 5'-ATC CTG AAT GGG GTG ATG AG-3'; reverse: 5'-GCC ACT CAA GTG TTG CAC AT-3'. GAPDH primers (0.3 µM each) were forward: 5'-GAA GGT GAA GGT CGC AGT C-3'; reverse: 5'-GAA GGT GAT GGG ATT TC-3'. Reaction conditions were as follows: 10 min at 95°C, then 40 cycles of 95°C (15 s) and 60°C (60 s), with data acquisition during the 1-min 60°C step. Melt analysis was used to confirm PCR products.
Transcriptional activation of the COX-2 promoter by bFGF.
I407 cells were transfected with a human COX-2 promoter/luciferase
reporter plasmid containing the region 1432/+59 of the human COX-2
promoter in the pGL3 reporter plasmid (generously supplied by Dr. T. Tanabe, National Cardiovascular Research Institute, Japan). Control
vectors for promoter activity were pGL3-basic (negative control;
Promega) and pGL3-control (positive control; Promega).
pSV-
-galactosidase vector (Promega) was cotransfected as a
transfection control. Subconfluent cells in 12-well plates were
transfected using FuGENE 6 as suggested by the manufacturer at a ratio
of 3:1 FuGENE 6 to DNA. Each well of cells received a total of 0.3 ug
DNA (0.2 ug of the promoter reporter plasmid and 0.1 ug of the
transfection control plasmid). Twenty-four hours later, the transfected
cells were placed into fresh media with or without bFGF as described
above. For experiments using the p38 MAPK inhibitor, SB-203580 (10 µM) was added 1 h before the media change and was included
during the treatment period. At the indicated times after stimulation,
cells were lysed and luciferase and
-galactosidase activity was
assayed using kits from Promega.
p38 Activation. To determine the time course of p38 activation, equivalent amounts of protein from bFGF-stimulated I407 cells were separated by SDS-PAGE, blotted to Immobilon-P, and probed with an antibody specific for the dually phosphorylated form of p38 (Cell Signaling Technology; Beverly, MA) using the protocol suggested by the supplier.
PGE2 enzyme immunoassay. PGE2 levels were determined by analyzing the media from cells stimulated with bFGF using a kit from Cayman (Ann Arbor, MI) as described above. For time course experiments, media were collected every 12 h and replaced with fresh media. Inhibitors were added at the time of stimulation and at the time of media replacement. In experiments testing the effect of exogenous arachidonate on PGE2 synthesis, cells were stimulated for either 2 or 24 h with bFGF; the media was then replaced with fresh media containing vehicle or 10 µM arachidonate and collected after 15 min.
FGF receptor expression.
Whole cell lysates were prepared from I407 cells grown to various cell
densities in 25-ml flasks. The monolayers were washed with PBS, 1 ml
lysate buffer [N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES); 20 mM], pH 7.4, 2 mM DTT, 10% glycerol, 25 µg/ml each antipain, aprotinin, leupeptin, and chymostatin, 50 µM
phenanthrolene, 10 ug/ml pepstatin A, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 200 µM sodium orthovanadate, 0.1 mM PMSF, and 1%
Triton X-100 was pipetted onto each. The flasks were incubated for 30 min at 4°C, and lysates were collected into 1.5-ml tubes and
cleared by spinning 15 min at 10,000 g at 4°C. The
supernatants were transferred to a clean tube, then aliquotted and
frozen at 20°C. Protein concentration was determined by the
Bradford protein assay (Bio-Rad).
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RESULTS |
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In our study of the regulation of COX-2 expression by bFGF, we
first examined the expression of FGFRs in I407 cells by Western blot
analysis (Fig. 1). Each of the FGFR
antibodies used was directed toward an epitope on the COOH terminus of
the receptor. FR-3 was easily detectable by Western blotting in both
preconfluent (50%) and confluent cultures of I407 cells. Low levels of
FR-1, -2, and -4 could also be detected on prolonged exposures of
Western blots from both preconfluent and confluent cultures. There was no change in expression of any of the FGFRs with the degree of confluence of the cell cultures. RT-PCR indicated the presence of FR-1,
-3, and -4 in I407 cells (Fig. 1E)
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I407 cells stimulated with bFGF began synthesizing PGE2
within 12 h (Fig. 2).
PGE2 production remained elevated for 36 h and was
inhibited by both indomethacin, a nonselective COX inhibitor, and by
NS-398, a selective COX-2 inhibitor (Fig. 2). Concomitant with
increased PGE2 synthesis, bFGF-stimulated cells also
rapidly upregulated COX-2 mRNA (Fig.
3A) and COX-2 protein (Fig.
3B). Neither COX-1 mRNA, nor protein levels were increased
with bFGF treatment (data not shown). These data indicate that bFGF
upregulates PGE2 synthesis in I407 cells through a
COX-2-dependent mechanism.
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Treatment of I407 cells with bFGF induced phosphorylation of p38 MAPK
within 5 min; p38 remained phosphorylated for 3 h. This time
course is consistent with a role for p38 activation in bFGF-stimulated PGE2 synthesis (Fig. 4). We,
therefore, examined the role of p38 activation in the events
controlling PGE2 production by stimulated cells. The p38
inhibitor SB-203580 completely abrogated PGE2 synthesis in
bFGF-treated cells (Fig. 5). The role of
p38 in the events leading to PGE2 production by
bFGF-stimulated cells was established using I407 cells stably
transfected with either p38WT, p38
DN, or empty vector (control
transfected). Transfection with p38
WT did not increase basal
PGE2 synthesis nor did it further enhance the ability of
I407 cells to synthesize PGE2 after bFGF stimulation (Fig.
6A). However, I407 cells
expressing p38
DN were unable to increase PGE2 synthesis
in response to bFGF (Fig. 6A).
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Western blot analysis of cell lysates revealed that in contrast
to nontransfected, control-transfected, and p38WT-transfected cells,
cells transfected with p38
DN did not upregulate COX-2 protein
expression following bFGF treatment (Fig. 6B). Thus
induction of COX-2 by bFGF appears to require the
-isoform of p38.
The failure of p38DN cells to produce PGE2 and to
upregulate COX-2 synthesis in response to bFGF suggests that
bFGF-stimulated PGE2 synthesis requires COX-2 induction.
However, it is possible that the observed levels of COX-2 are
sufficient and that the defect in bFGF-induced PGE2
synthesis is at the level of arachidonate release. cPLA2
can be phosphorylated by p38, and phosphorylation is a critical event
in cPLA2 activation (40). To investigate the
possibility that the defect in bFGF-induced PGE2 synthesis in p38
DN cells was due to diminished PLA2 activation,
we examined the effect of exogenous arachidonate on
PGE2 synthesis in bFGF-stimulated cells. In resting
control-transfected, nontransfected, p38
DN, and p38
WT
cells, PGE2 synthesis was ~8 pg/ml (data not shown). All
cell types showed an approximate fourfold increase in PGE2 production (~33 pg/ml) when supplied with exogenous arachidonate (data not shown). Two time points following bFGF stimulation were examined, an early (2 h) time point at which time COX-2 protein levels
are just beginning to increase (and therefore might be limiting) and a
late (24 h) time point at which COX-2 protein levels have plateaued
(and arachidonate release may be limiting). Exogenous arachidonate did
not further increase PGE2 synthesis in any of the cells
stimulated for 2 h with bFGF, indicating that at this time, the
amount of PGE2 produced is limited by COX-2 protein (Fig.
7). However, exogenous arachidonate
resulted in increased PGE2 synthesis by nontransfected,
control-transfected, and p38
WT-transfected cells stimulated for
24 h with bFGF (Fig. 7). These data indicate that late after bFGF
stimulation, the induced level of COX-2 enzyme activity is sufficient
to metabolize all the available endogenous substrate; thus arachidonate
release is the limiting factor in PGE2 synthesis 24 h
after bFGF stimulation. At neither time point was exogenous
arachidonate able to overcome the defect in PGE2 synthesis
in p38
DN cells. Thus, in I407 cells, bFGF stimulates
PGE2 synthesis through a p38
-dependent mechanism that
requires upregulation of COX-2 protein.
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COX-2 protein levels may be regulated by both transcriptional and
posttranscriptional mechanisms. We investigated the effect of bFGF on
COX-2 transcription using a COX-2 promoter/luciferase reporter
construct containing the region 1432/+59 of the human COX-2 promoter.
I407 cells transfected with the luciferase vector without a promoter
(pGL3-basic) averaged 3.8 × 104 relative light units
(RLU) at the end of transfection, and cells transfected with
the luciferase vector under the control of the SV40 promoter
(pGL3-control) averaged 4.6 × 107 RLU (data not
shown). We found that the COX-2 promoter is active in unstimulated I407
cells (3.1 × 106 RLU) and that promoter activity is
not further increased by bFGF treatment (Fig.
8A). Treatment of transfected
cells with SB-203580 (1 h before and during stimulation) did not alter
COX-2 promoter activity (Fig. 8B). We confirmed our ability
to detect changes in activity of the transfected hCOX-2
promoter/luciferase plasmid using various stimuli. As shown in Fig.
9, activity of the hCOX-2 promoter was
increased when serum-starved cells were incubated with media containing
10% fetal calf serum. Similarly, serum-starved cells stimulated with
20 ng/ml IL-1
showed a detectable increase in promoter activity
(Fig. 9). C2-ceramide (10 µM) did not stimulate promoter
activity (data not shown).
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We next assessed whether the observed p38-dependent net increase in
COX-2 mRNA following bFGF stimulation was due to message stabilization.
For these experiments, I407 cells were treated for 1 h with or
without bFGF and then incubated with DRB with and without the p38
inhibitor SB-203580. In untreated I407 cells, COX-2 mRNA rapidly
decayed, with a half-life (t1/2) of ~15 min and a 90% loss within the first 30 min after DRB addition (Fig. 10). In cells treated with bFGF, COX-2
mRNA stability was increased to a t1/2 of 60 min
(Fig. 10). Inhibition of p38 with SB-203580 reduced the
t1/2 of the COX-2 message from 60 min observed
in bFGF-stimulated cells to 30 min (Fig. 10). Thus the major mechanism responsible for increased COX-2 mRNA levels in bFGF-stimulated I407
cells is message stabilization, and p38 plays an important role in this
stabilization.
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bFGF-stimulated p38 MAPK-dependent COX-2 expression appears to be a
general phenomenon in gastrointestinal epithelial cell lines, because
bFGF also upregulated COX-2 expression in the colonic epithelial cell
lines HT-29 and Caco-2 (Fig. 11). Both
cell lines are reported to express receptors for bFGF (24,
32). bFGF increased COX-2 protein expression in both
serum-starved HT-29 (Fig. 11A) and serum-starved Caco-2
(Fig. 11B) approximately twofold. These cell lines express
high levels of COX-2 protein under normal growth conditions (10% fetal
calf serum); therefore, the cells had to be serum-starved overnight to
detect bFGF-induced upregulation. The p38 MAPK inhibitor SB-203580
attenuated bFGF-induced COX-2 protein expression by both cell lines
(Fig. 11).
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DISCUSSION |
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In this study, we have demonstrated that bFGF stimulates
PGE2 synthesis in a human intestinal epithelial cell line
via a p38-dependent increase in COX-2 mRNA stability. In the absence
of COX-2 upregulation, bFGF was unable to stimulate PGE2
synthesis in a p38
DN cell line even in the presence of exogenous
arachidonate. Although bFGF has been demonstrated to affect epithelial
stem cell survival in vivo after radiation injury (18),
this is the first demonstration of a direct effect of bFGF on
intestinal epithelial cells. Both HT-29 and Caco-2 cells have been
reported to express receptors for bFGF (24, 32). Similar
to I407 cells, we found that both of these cell lines increased COX-2
expression in response to bFGF and that the p38 MAPK inhibitor
SB-203580 abrogated bFGF-induced COX-2 expression. These findings
support the suggestion that the effects of bFGF on intestinal stem cell
survival after radiation are direct effects on epithelial stem cells
rather than being mediated by other cell types (34, 38).
COX-2 mRNA peaked within 1 h after bFGF treatment of I407 cells, with a subsequent decline to approximately threefold over basal at 24 h. COX-2 protein expression lagged slightly, with a maximal increase observed 3 h after bFGF. However, after the initial increase, COX-2 protein levels remained essentially stable up to 24 h. The relatively high levels of COX-2 protein at 24 h compared with the level of COX-2 mRNA may reflect the differences in COX-2 protein stability vs. COX-2 mRNA stability. Zhang et al. (47) observed a similar pattern in which COX-2 mRNA was near baseline 24 h after chenodeoxycholate treatment of rat intestinal epithelial cells, whereas COX-2 protein was still elevated. In an extensive comparison of COX-2 expression in colon carcinoma cell lines, Shao et al. (37) observed a good correlation between COX-2 mRNA and protein for some cell lines (e.g., HCA-7, Moser), whereas others appeared to have relatively low levels of message while expressing easily detectable protein (LS-174) or vice versa (HT-29). They also observed significant cell line-to-cell line variation in COX-2 protein stability (37).
The biological responses of bFGF are mediated through specific cell
surface receptors that possess tyrosine kinase activity (3,
27). Binding of bFGF to its receptor activates a number of
signaling pathways including phospholipase C, ERK-1, ERK-2, Src, and
p38, which ultimately converge to elicit a particular biological effect
(4, 27). Here, we demonstrate that the induction of COX-2
expression by bFGF in I407 cells is mediated through the -isoform of
p38. Although signaling through p38 has been associated with bFGF
binding, this is the first association of bFGF with a specific p38
isoform. The induction of COX-2 expression by bFGF suggests the
presence of one or more FGFRs on these cells. Western analysis
indicated that I407 cells express FR-1, -2, -3, and -4, although FR-3
appeared to be most prominent. RT-PCR indicated the expression of
FR-1, -3, and -4. This is consistent with previous reports of FR-3
expression in other intestinal epithelial cell lines including HT-29
and Caco-2 (24, 32).
Transcriptional regulation of COX-2 is mediated by a variety of
cytokines and growth factors including transforming growth factor-
(TGF-
) (40). bFGF has been demonstrated to induce COX-2
expression in gastric epithelial cells (36), Syrian
hamster embryo cells (1), osteoblasts (26),
endothelial cells derived from bone (23), and aortic
smooth muscle cells (25). In these studies, the only
attempt to define the intracellular signaling involved in bFGF
induction of COX-2 expression was the demonstration that PD-98059, an
ERK-pathway inhibitor, inhibited bFGF-induced COX-2 expression in
aortic smooth muscle cells (25). Here, we demonstrate that
in intestinal epithelial cells, COX-2 induction is mediated through
p38
, because I407 cells transfected with p38
DN failed to
upregulate PGE2 or COX-2 expression following bFGF
treatment. However, I407 cells transfected with p38
WT did not
exhibit elevated basal levels of COX-2 or PGE2, nor did
they exhibit any further increase in COX-2 expression or
PGE2 synthesis following bFGF compared with nontransfected
or control-transfected cells. These data are similar to the findings of
Guan et al. (13), with rat primary mesangial cells stably
transfected with similar constructs. However, transient transfection of
a mammary epithelial cell line (41), human synovial
fibroblasts (11), immortalized human articular
chondrocytes (43), and the liver parenchymal-like cell
line RL34 (28) with p38
WT did increase basal COX-2
promoter activity or protein expression. This difference in
response may simply reflect experimental differences (i.e., different
cell types, transient vs. stable transfection) or may reflect
differences in the basal activity of upstream modulators of p38 activity.
PGE2 synthesis is dependent not only on the expression of
COX, but also on the availability of arachidonic acid, the substrate for COX. bFGF is known to affect both COX-2 expression and arachidonate availability. In pancreatic acini, bFGF stimulates arachidonate release
through sequential activation of tyrosine kinase, phospholipase C,
protein kinase C, and diacylglycerol lipase (17).
Unstimulated I407 cells produce ~8 pg/ml PGE2. When
supplied with exogenous arachidonate PGE2, synthesis
increases approximately fourfold for nontransfected,
control-transfected, p38WT, and p38
DN cells, most likely
reflecting metabolism by COX-1. Within 2 h after bFGF stimulation,
control-transfected and nontransfected I407 cells exhibited an
approximate 10-fold increase in the ability to synthesize PGE2, which was not further increased by exogenous
arachidonately. These data suggest that bFGF-stimulated
PGE2 synthesis occurs via increased metabolism by COX-2 and
that endogenous levels of arachidonate are sufficient to saturate the
cyclooxygenase present. After 24 h, exogenous arachidonate does
increase PGE2 synthesis by nontransfected,
control-transfected, and p38
WT cells, indicating that, at this time,
the ability to metabolize arachidonate is greater than the amount of
endogenous substrate. In p38
DN I407 cells, bFGF does not stimulate
PGE2 production, even in the presence of exogenous
arachidonate. This demonstrates that the p38
-mediated increase
in PGE2 synthesis induced by bFGF requires COX-2 synthesis and is not mediated solely via arachidonate availability.
COX-2 mRNA levels are regulated both by the rate of transcription and
by mRNA stability (16, 40). Which mechanism is dominant is
a function of the stimulus; IL-1, for example, increases COX-2 mRNA
levels primarily by enhancing mRNA stability (11, 20, 35).
p38 Activation can affect mRNA levels either by increasing transcription or by stabilizing mRNA. In human mammary epithelial cells, taxol induces COX-2 through a p38-dependent mechanism; in this
system, p38 increases COX-2 mRNA by enhancing transcription rather than
by stabilizing mRNA (42). Our study demonstrates that bFGF
increases COX-2 mRNA levels predominantly by increasing COX-2 mRNA
stability. In resting I407 cells, ~90% of the COX-2 message decays
within 30 min. Similarly, Sheng et al. (39) found that
COX-2 mRNA is short lived in rat intestinal epithelial cells (t1/2 = ~13 min). When ras expression in
these cells was induced or when these cells were treated with TGF-,
COX-2 mRNA message was stabilized (t1/2 = ~30 min). When cells were induced to express ras and concomitantly
treated with TGF-
, COX-2 mRNA t1/2 increased to ~1 h. In two colonic carcinoma cell lines with high basal COX-2 expression, LS-174 and HCA-7, COX-2 mRNA t1/2
was ~80 and >120 min, respectively (37). The
t1/2 of COX-2 mRNA in bFGF-stimulated cells is
~60 min and is reduced to ~30 min in the presence of the p38
inhibitor SB-203580. In HeLa cells treated with IL-1, COX-2 mRNA,
t1/2 is ~60 min and is reduced to ~30 min in
the presence of SB-203580 (35). Using a COX-2
promoter/luciferase construct, we found that in resting I407
cells, the COX-2 promoter is quite active and that no further increase
in transcription from this promoter following bFGF treatment could be
detected. However, stimuli, such as IL-1
and serum, previously
demonstrated to increase COX-2 promoter activity in other cell lines
(20, 40), did increase COX-2 promoter activity in
serum-starved I407 cells. In I407 cells, IL-1
moderately increased
COX-2 transcriptional activity similar to the effect of IL-1
on
COX-2 transcription in human macrophages (20). The
increase in COX-2 message stability appears to be mediated through a
p38
-dependent mechanism, because in the absence of p38
activation, bFGF fails to increase COX-2 expression and mRNA stability
in bFGF-stimulated cells is attenuated by an inhibitor of p38.
bFGF mediates a variety of biological functions in injury repair,
including stimulation of epithelial migration (3, 4, 22,
27) and enhanced stem cell survival after radiation
(18). Here, we demonstrate that bFGF induces COX-2 in an
intestinal epithelial cell line through a p38-dependent mechanism.
The ability of bFGF to induce COX-2 expression in a p38-dependent
manner was not confined to I407 cells. Both the HT-29 and Caco-2
gastrointestinal cell lines showed bFGF-induced increases in COX-2
protein expression were abrogated by the p38 inhibitor SB-203580. These
data raise the possibility that the effects of bFGF observed in vivo,
in the gut, may be due to a direct effect on the epithelial cells and
that other biological effects of bFGF on the epithelium may also be
mediated through p38
activation.
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ACKNOWLEDGEMENTS |
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We thank J. M. Buzan for expert technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-33165 and R01-DK-55753 (to W. F. Stenson), RO1-DK-50924 (to S. M. Cohn), and DK-52574 (to the Washington Univ. Digestive Disease Center).
Address for reprint requests and other correspondence: T. G. Tessner, Gastroenterology, Campus Box 8124, Washington Univ. School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: stensnlb{at}im.wustl.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. Section 1734 solely to indicate this fact.
First published October 9, 2002;10.1152/ajpgi.00226.2002
Received 11 June 2002; accepted in final form 2 October 2002.
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