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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital and Department of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan (J.-C.L., F.-Y.C., C.-L.L., C.-Y.C., S.-D.L.); Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan (C.-W.C.); and Division of Allergy, Immunology, and Rheumatology, Department of Medicine, Taipei Veterans General Hospital and Department of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan (H.-Y.L.)
Received September 3, 2006; accepted October 30, 2006.
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Abstract |
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; Tarnawski et al., 2001; Yoo et al., 2002). The early-phase response occurs in the absence of cellular proliferation and is called restitution (cell migration) (Silen and Ito, 1985; Yoo et al., 2002). The late-phase response consists of cell division and cell proliferation to fill in the defect and restore mucosal architecture (Szabo et al., 2000; Tarnawski, 2005). EGF, a well studied growth factor, plays a critical role in ulcer healing and mucosal protection via mitogenic and nonmitogenic actions (Uribe and Barrett, 1997; Tarnawski and Jones, 1998).
The role of EGF and its receptor in regulating gastric mucosal cell proliferation has been well documented (Tarnawski et al., 1992; Konturek et al., 1995; Tarnawski and Jones, 1998). Binding of growth factors, such as EGF and transforming growth factor-
, to their common receptor produces the autophosphorylation of receptor tyrosine kinase on tyrosine residues, inducing cascading events that lead to activation of mitogen-activated protein kinases (MAPKs) (Lemmon and Schlessinger, 1994; Heldin, 1995). These MAPKs have a central role in the signal pathways that regulate cell proliferation, migration, and differentiation (Blenis, 1993; Vietor et al., 1993). Of the several MAPK family members, extracellular signal-regulated kinase (ERK)1 and 2 have been shown to play crucial roles in healing of ulcerated or injured mucosa (Pai et al., 1998). Once activated, MAPKs translocate into the nucleus, where they induce transcription factors, including proto-oncogenes such as c-fos, c-myc, and c-jun, and cell cycle activator cyclin D1 (Blenis, 1993; Hill and Treisman, 1995; Lavoie et al., 1996; Terada et al., 1999). EGF also up-regulates cyclooxygenase (COX)-2 expression via ERK and p38 MAPK signaling in rat gastric ulcerated mucosa and in other cell lines (Brzozowski et al., 2001; Huh et al., 2003; Sasaki et al., 2003), COX-2 expression with enhanced prostaglandin (PG) formation is essential for cell proliferation and ulcer healing (Peskar et al., 2001; Luo et al., 2003).
The pharmacological actions of glucocorticoids involve 1) inhibiting proinflammatory activities of transcription factors, in particular nuclear factor-
B; 2) altering T-helper cell type 1/T-helper cell type 2 cytokine balance; 3) inducing annexin 1 (lipocortin 1) synthesis, thereby suppressing arachidonic acid release via antagonizing phospholipase A2 activity; and 4) affecting prostaglandin synthesis via decreasing COX-2 expression (Almawi et al., 1999; Roviezzo et al., 2002; Yang and Lichtenstein, 2002). Animal studies demonstrated that dexamethasone, which is a potent corticosteroid, delayed rat gastric ulcer healing by inhibiting epithelial cell proliferation and angiogenesis at the ulcer margin and base (Carpani de Kaski et al., 1995; Luo et al., 2003, 2004). These adverse actions of glucocorticoids on gastric ulcer healing are due to their inhibiting COX-2 expression and PGE2 formation (Luo et al., 2003, 2004).
This in vitro study identified that dexamethasone significantly inhibited EGF-stimulated gastric epithelial cell proliferation in normal gastric epithelial cell line RGM-1. The study also further elucidated the possible mechanistic pathway through which dexamethasone inhibits EGF-stimulated gastric cell proliferation.
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Materials and Methods |
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Cell Culture and Cell Number Determination. Gastric epithelial cells obtained from the rat gastric mucosal epithelial cell line RGM-1 (RCB-0876; Riken Cell Bank, Tsukuba, Japan) were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium (Invitrogen) supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin, and 20% fetal bovine serum (FBS) (Invitrogen) in an incubator at 37°C and 5% carbon dioxide.
Cells were detached from the wells, using 0.25% trypsin EDTA. Trypan blue was added to the cell suspension to assess cell viability. Cell numbers were counted using Burker hemocytometer (Marienfeld GmbH, Marienfeld, Germany).
Flow Cytometry for DNA Synthesis Analysis. Cellular DNA replication was analyzed by flow cytometry. The two peaks of DNA contents corresponding to G1 and G2/M phase cells, respectively, and the intermediate amount of DNA corresponding to S-phase cells were counted. RGM-1 cells were seeded in a 10-cm dish at roughly 105 cells/ml and were allowed to grow in DMEM/Ham's F-12 medium containing 20% FBS for 24 h. Thereafter, cell growth was arrested in the same medium with 1% FBS for a further 24 h to synchronize cell cycles. Cells were then treated for 24 h with 1% FBS DMEM/Ham's F-12 medium containing either 10 ng/ml EGF, 10–6 M dexamethasone, or 10–6 M mifepristone, the glucocorticoid receptor antagonist. The following combinations were also applied: 10 ng/ml EGF with 10–6 or 10–8 M dexamethasone in the presence or absence of 10–6 M mifepristone; 10 ng/ml EGF with 10–8 M dexamethasone, 10–5 M NS-398; or a combination of both. After treatment, cells (
2 x 106 each dish) were trypsinized, pelleted, washed with phosphate-buffered saline, repelleted, and resuspended with lysis buffer (0.5% Triton X-100, 0.2 µg/ml Na2EDTA·2H2O, and 1% BSA) for 15 min. Cells were fixed in 80% cold methanol at –20°C overnight. The fixed cells were centrifuged, washed with phosphate-buffered saline, pretreated with RNase (5 Kunitz U/ml) at 37°C for 30 min, and then reacted with 50 µl/ml propidium iodide. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) Data were analyzed using ModFit and CellQuest software (BD Biosciences) (Huang et al., 2004).
Western Blot Analysis for ERK1/2, pERK1/2, p38 MAPK, pp38 MAPK, c-Jun NH2-Terminal Kinase 1, pJNK1, Phospholipase A2, COXs, and Cyclin D1. After synchronization as described above, cells were treated with 1% FBS DMEM/Ham's F-12 containing 10 ng/ml EGF in the presence or absence of 10–6 or 10–8 M dexamethasone or 10–6 M dexamethasone alone for different periods. In a second set of experiments, cells were pretreated with PD98059 (MEK inhibitor) (10–5 and 5 x 10–5 M) or dexamethasone (10–8 and 10–6 M) for 2 to 3 h, followed by 10 ng/ml EGF. Cells were then collected in radioimmunoprecipitation assay buffer for Western blot analysis. Following sonication and centrifugation, protein concentration was measured using a protein assay kit (Bio-Rad, Hercules, CA). Proteins were separated by SDS-polyacrylamide gels electrophoresis overlaid with a 5% acrylamide stacking gel and then transferred to Hybond C nitrocellulose membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Membranes were probed with antibodies against ERK1/2, pERK1/2, p38 MAPK, pp38 MAPK, JNK1, pJNK1, cyclin D1, cytosolic phospholipase A2 (cPLA2), COX-1, and COX-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C and incubated for 1 h with secondary antibodies conjugated with peroxidase. The membrane was developed using the enhanced chemiluminescence system (Amersham Biosciences) and was exposed to an X-ray film (Fuji Photo Film, Tokyo, Japan). Quantitation was performed using a densitometer (Scan Marker III; Microtek, Carson, NV).
Measurement of Intracellular PGE2 Level. Following treatment with 10 ng/ml EGF in the presence or absence of dexamethasone (10–8 and 10–6 M) or 10–6 M dexamethasone alone for 8 and 12 h, respectively, cells were homogenized with homogenizing buffer for 30 s (0.05 M Tris-HCl, pH 7.4, 0.1 M NaCl, 0.001 M CaCl2 ,1 mg/ml D-glucose, and 28 µM indomethacin). Then, cells were centrifuged at 12,000 rpm for 15 min at 4°C. Supernatants were assayed by using a PGE2 enzyme-linked immunosorbent assay kit (Quantikine; R&D Systems, Minneapolis, MN). Assay procedures were performed in accordance with the manufacturer's instructions. Optical densities were determined with the MRX microplate reader (Dynex Technologies, Chantilly, VA) at 405 nm. The amount of protein in the sample was determined by a protein assay kit and the medium PGE2 level was expressed as picograms per milligram of protein.
Statistical Analysis. Analytic results are expressed as means ± S.D. There were six samples in each group. Differences between the means were analyzed with the Student's t test when appropriate. Bonferroni correction was performed to adjust for the fact that multiple comparisons were done in each experiment. A gross P < 0.05 was considered statistically significant.
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Signal Transduction after EGF Treatment. After cells were treated with 10 ng/ml EGF for 0 (control) to 120 min, it was found that 10 ng/ml EGF markedly increased the expression of pERK1/pERK2 at 10 min, but the expression of total ERK1/ERK2 at 10 to 120 min remained the same (Fig. 2A). When the RGM-1 cells were treated with 10 ng/ml EGF for a longer time (1–18 h), it was observed that the expression of COX-2 increased significantly at 3 h, and the response lasted up to hour 18. In addition, cyclin D1 expression also increased significantly at 8 and 12 h (Fig. 2B). In contrast, the expression of COX-1 and cPLA2 was similar to that of the control (data not shown). The expression of another two activators of COX-2, namely, p38 MAPK/pp38 MAPK and JNK1/pJNK1 did not show any change following EGF treatment for 10 to 120 min, respectively (Fig. 2A).
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Signal Transduction after EGF Cotreated with Dexamethasone. Incubation of RGM-1 cells with 10 ng/ml EGF and dexamethasone combined (10–8 and 10–6 M) for 10 min did not alter expression of ERK1/ERK2, pERK1/pERK2, p38 MAPK/pp38 MAPK, or JNK1/pJNK1 compared with the EGF-treated group (Fig. 3). However, pretreatment of RGM-1 cells with dexamethasone (10–8 and 10–6 M) for 3 h significantly reduced EGF-stimulated expression of pERK1/pERK2 (Fig. 4), and it did not influence expression of ERK1/ERK2, p38 MAPK/pp38 MAPK, or JNK1/pJNK1 compared with those in the EGF-treated group at 10 min (data not shown). In contrast, incubation of RGM-1 cells with 10 ng/ml EGF and dexamethasone combined (10–8 and 10–6 M) for 3 and 6 h significantly decreased EGF-stimulated COX-2 expression (Fig. 5, A and B) and not COX-1 and cPLA2 expression compared with the 10-ng/ml EGF group. Incubation of RGM-1 cells with 10 ng/ml EGF and 10–8 and 10–6 M dexamethasone for 8 and 12 h decreased EGF-stimulated cyclin D1 expression compared with that in the 10-ng/ml EGF group (Fig. 6, A and B).
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Signal Transduction after EGF Cotreated with PD98059. Incubation of RGM-1 cells with 10 ng/ml EGF and PD98059 (MEK inhibitor at 10–5 and 5 x 10–5 M) combined for 10 min did not affect expression of ERK1/ERK2 and pERK1/pERK2 compared with those in the EGF-treated group (Fig. 7). Pretreatment of RGM-1 cells with 5 x 10–5 M PD98059 for 2 h significantly reduced EGF-stimulated expression of pERK1/pERK2 compared with that in the EGF-alone group at 10 min (Fig. 7). However, PD98059 (10–5 and 5 x 10–5 M) significantly decreased EGF-stimulated COX-2 expression at 6 h (Fig. 5B). These experimental results indicate that EGF-induced expression of phosphorylated ERK1/ERK2, COX-2 and cyclin D1 and not that of phosphorylated p38 MAPK and JNK1.
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The PGE2 Concentration following EGF Cotreated with Dexamethasone. Treatment of RGM-1 cells with 10 ng/ml EGF for 8 and 12 h significantly increased the intracellular PGE2 concentration (Fig. 8, A and B). Again, dexamethasone (10–8 and 10–6 M) significantly reduced EGFinduced PGE2 levels compared with the EGF-treated group.
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; Tarnawski, 2005). Furthermore, EGF up-regulated gastric epithelial COX-2 expression, which is important for ulcer healing, in in vivo and in vitro studies (Brzozowski et al., 2001; Sasaki et al., 2003). This study indicated that EGF activated ERK1/ERK2, then increased COX-2 expression and PGE2 synthesis, finally promoting RGM-1 cell proliferation; dexamethasone, a glucocorticoid frequently used clinically, suppressed EGF-induced pERK1/pERK2, COX-2 expression, and then decreased PGE2 synthesis and cell proliferation in the RGM-1 cells. Current experimental findings are coincident with those obtained in our previous in vivo study, which demonstrated that dexamethasone at 0.1 and 0.2 mg/kg/day delayed ulcer repair via limiting epithelial cell proliferation at the ulcer margin by suppressing COX-2 expression and PGE2 formation in rat stomachs (Luo et al., 2003). Studies by Filaretova demonstrated that glucocorticoid had a gastric protective role during stress and administration of nonsteroidal anti-inflammatory drugs in rats (Filaretova et al., 1998, 2002). In our opinion, our results and their observations on the functional role of glucocorticoid on ulcer healing are not contradictory since endogenous glucocorticoids may have a physiologically protective role (Filaretova et al., 1998, 2002), whereas its administration in pharmacological doses would in turn delay ulcer repair and wound healing (Carpani de Kaski et al., 1995; Luo et al., 2003; Lee et al., 2005).
MAPK signal transduction pathways are important in regulating cell proliferation, migration, and differentiation (Blenis, 1993; Vietor et al., 1993). Previous studies have showed that COX-2 expression is regulated by MAPK subtypes, such as ERK1/ERK2, p38 MAPK, and JNK, depending on the types of extracellular stimuli and cells (Sheng et al., 1998; Matsuura et al., 1999; Lasa et al., 2000). Our results clearly showed that EGF activated ERK1/ERK2 in RGM-1 cells within 10 to 20 min (Fig. 2), but it did not affect expression of the phosphorylated forms of p38 MAPK and JNK1 from 10 to 120 min. This reaction was followed by increased COX-2 expression at 3 h after EGF treatment (Fig. 5). Moreover, dexamethasone suppressed EGF-induced COX-2 expression by reducing ERK1/ERK2 phosphorylation and not reducing phosphorylated forms of p38 MAPK and JNK1. However, it is interesting that an effective dose of PD98059 (5 x 10–5 M) reduced ERK activation by EGF (Fig. 7), yet it did not completely block EGF-induced COX-2 expression (Fig. 5B). This suggests that EGF-induced COX-2 activation may involve mechanisms other than ERK pathway.
Our results demonstrated that dexamethasone alone had no inhibitory effect on RGM-1 cells growth (Fig. 1), but the EGF-induced ERK response was blocked only when RGM-1 cell were pretreated for 3 h (Fig. 4) and not by simultaneous treatment with dexamethasone and EGF (Fig. 3). Similar to our results, a study by Xu et al. (2005) showed that gene 33, an adaptor protein, suppressed EGF receptor autophosphorylation and subsequent activation of downstream signaling in Rat 2 cells. In that study, dexamethasone induced gene 33 expression and 2-h pretreatment with dexamethasone similarly limited EGF-induced EGF receptor phosphorylation. These findings suggest that dexamethasone may have indirectly interfered with EGF binding to its receptor or EGF activation of ERK1/ERK2.
The fact that the glucocorticoid-receptor antagonist mifepristone significantly and completely reversed the action of dexamethasone on EGF-stimulated cell proliferation suggested that the inhibitory action of dexamethasone was glucocorticoid receptor-mediated. The current study also revealed that combination of dexamethasone and COX-2-selective inhibitor did not have an additive effect in the inhibition of EGF-stimulated cell proliferation compared with the individual drug-treated group (Fig. 9). The findings suggested that the main inhibitory effect of dexamethasone on EGF-stimulated cell proliferation was likely through blocking COX-2 activity.
Cyclin D1 is linked to the cell cycle progression by shortening the G1 phase and predisposition of cells to enter the S phase (Jiang et al., 1993; Resnitzky et al., 1994); Cyclin D1 accumulation via ERK activation is needed to pass the G1 restriction point and enter the S phase (Talarmin et al., 1999; Terada et al., 1999). Results from this study clearly indicated that EGF induced ERK1/ERK2 activation within minutes and then activated COX-2 function 3 to 6 h later, leading to increased cyclin D1 expression at 8 and 12 h, and finally it promoted DNA synthesis in RGM-1 cells at 24 h.
In conclusion, EGF enhanced gastric ulcer healing by stimulating epithelial cell proliferation, whereas glucocorticoid delayed ulcer healing partially via inhibiting epithelial cell proliferation. The study demonstrated that dexamethasone significantly suppressed EGF-stimulated gastric epithelial cell proliferation partially by inhibiting EGF-induced activation of ERK1/ERK2, followed by inhibition of COX-2, cyclin D1 expression, and DNA synthesis in a rat gastric epithelial cell line.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; COX, cyclooxygenase; PG, prostaglandin; NS-398, N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide; MEK, mitogen-activated protein kinase kinase; PD98059, 2'-amino-3'-methoxyflavone; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; pERK, phosphorylated extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; pJNK, phosphorylated c-Jun NH2-terminal kinase; cPLA2, cytosolic phospholipase A2; Dexa, dexamethasone; Mif, mifepristone.
Address correspondence to: Dr. Full-Young Chang, Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, 201 Shih-Pai Rd., Section 2, Taipei, Taiwan 11217. E-mail: changfy{at}vghtpe.gov.tw
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