Abstract
Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid–induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H2O2) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, PI-PLCγ1, and PI-PLCγ2, but not PI-PLCβ2 and PI-PLCδ1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCγ2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H2O2 production, and cell proliferation. In contrast, knockdown of PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, PI-PLCγ1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCγ2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCγ2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCγ2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA. Cancer Res; 70(3); 1247–55
Introduction
The incidence of esophageal adenocarcinoma (EA) has increased by more than 6-fold in the past 3 decades (1). Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for EA (2). The mechanisms of the progression from BE to EA are not known. Reactive oxygen species (ROS) may play an important role in the development of EA because levels of ROS are increased in BE (3) and EA (4, 5). ROS may cause damage to DNA, RNA, lipids, and proteins, which may result in increased mutation and altered functions of enzyme and proteins (e.g., activation of oncogene products and/or inhibition of tumor suppressor proteins; refs. 4, 6). However, the sources of ROS in these conditions have not been well defined.
Low levels of ROS, seen in nonphagocytic cells, were thought to be byproducts of aerobic metabolism. More recently, however, superoxide-generating homologues of phagocytic NADPH oxidase catalytic subunit gp91phox (NOX1, NOX3–NOX5, DUOX1, and DUOX2) and homologues of other subunits (p41phox or NOXO1 and p51phox or NOXA1) have been found in several cell types (7, 8), suggesting that ROS generated in these cells may have distinctive cellular functions related to immunity, signal transduction, and modification of the extracellular matrix. NOX5 has five isoforms: α, β, δ, γ, and NOX5-S (9, 10). NOX5 α, β, δ, and γ have EF-hand motifs at its NH2 terminus (9), whereas NOX5-S does not (11). We have shown that the NADPH oxidase isoform NOX5-S is present in EA FLO cells (12) and that levels of NOX5-S are significantly increased in Barrett's esophageal mucosa with high-grade dysplasia (13).
Bile acids may also play an important role in the progression from BE to EA (14, 15) because (a) in animal models diversion of duodenal contents into the lower esophagus leads to EA (16, 17), (b) bile acids are known to induce oxidative stress and DNA damage (18, 19), (c) bile salts may induce upregulation of cyclooxygenase-2 and c-myc expression (20, 21) and activate mitogen-activated protein (MAP) kinase and NF-κB pathways (22, 23), thereby increasing cell proliferation and decreasing cell apoptosis. However, mechanisms whereby bile acids promote the development of EA are not known.
We have shown that bile acid taurodeoxycholic acid (TDCA)–induced upregulation of NOX5-S expression and increase in cell proliferation depend on activation of TGR5 receptor (a bile acid receptor) and Gαq protein in EA cells (12). Phosphatidylinositol-specific phospholipase C (PI-PLC) has been reported to be activated by the Gαq protein family (24, 25). Gαq proteins are involved in TDCA-induced NOX5-S expression and hydrogen peroxide (H2O2) production. Whether PI-PLC plays a role in TDCA-induced NOX5 expression, H2O2 production, and cell proliferation has not been established. We now show that TDCA-induced increase in cell proliferation and upregulation of NOX5-S expression is mediated by activation of PI-PLCγ2 and extracellular signal-regulated kinase (ERK) 2 MAP kinase in EA cells.
Materials and Methods
Cell culture and TDCA treatment
Human EA cell line FLO was derived from human EA (26) and generously provided by Dr. David Beer in 2004 (University of Michigan Medical School, Ann Arbor, MI). FLO cells matched the genotype of the progenitor tumor cells from the patient's tumor tissue block. Cells were cultured in DMEM containing 10% fetal bovine serum and antibiotics at 37°C with 5% CO2 humidified atmosphere.
Human EA cell line OE33 was cultured in DMEM containing 10% fetal bovine serum and antibiotics. The cell lines were cultured at 37°C in a 5% CO2 humidified atmosphere.
For TDCA treatment, FLO cells were incubated with 10−11 mol/L TDCA for 24 h. For use of inhibitors, cells were pretreated with U73122 (10−6 mol/L), PD98059 (10−5 mol/L), or culture medium (control) for 1 h and cultured in fresh medium (pH 7.2; without phenol red) without TDCA and inhibitors (control), with TDCA (10−11 mol/L), or with TDCA and the above inhibitors for an additional 24 h. Finally, the culture medium and cells were collected for measurements.
Small interfering RNA and plasmid transfection
Twenty-four hours before transfection at 70% to 80% confluence, cells were trypsinized and diluted 1:5 with fresh medium without antibiotics (1–3 × 105/mL) and transferred to 12-well plates (1 mL/well). Transfection of small interfering RNAs (siRNA) was carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. Per well, 75 pmol of siRNA duplex of PI-PLCγ1, PI-PLCγ2, PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, ERK1, ERK2, or control siRNA formulated into liposomes were applied; the final volume was 1.2 mL/well. Forty-eight hours after transfection, cells were treated with or without TDCA in culture medium (pH 7.2; without phenol red) for 24 h, and then the culture medium and cells were collected for measurements. Transfection efficiencies were determined by fluorescence microscopy after transfection of BLOCK-iT fluorescent oligonucleotide (Invitrogen) and were ∼70% at 48 h.
Reverse transcription-PCR
Total RNA was extracted by Trizol reagent (Invitrogen) for the cultured cells and purified by the total RNA purification system (Invitrogen). According to the protocols of the manufacturers, 1.5 μg of total RNAs from cultured cells were reversely transcribed by using a SuperScript First-Strand Synthesis System for reverse transcription-PCR (Invitrogen).
Quantitative real-time PCR
Quantitative real-time PCR was carried out on a Stratagene Mx4000 multiplex quantitative PCR system. The primers used were as follows: NOX5-S, 5′-AAGACTCCATCACGGGGCTGCA-3′ (sense) 5′-CCTTCAGCACCTTGGCCAGA-3′ (antisense); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CATGACCACAGTCCATGCCATCAC-3′ (sense) and 5′-AGGTCCACCACCCTGTTGCTGTA-3′ (antisense). All reactions were performed in triplicate in a 25 μL total volume containing a 1× concentration of Brilliant SYBR Green QPCR Master Mix (Stratagene), and the concentration of each sense and antisense primer was 100 nmol/L, 1 μL cDNA, and 30 nmol/L reference dyes. Reactions were carried out in a Stratagene Mx4000 multiplex quantitative PCR system for 1 cycle at 94°C for 5 min; 40 cycles at 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s; 1 cycle at 94°C for 1 min; and 1 cycle at 55°C for 30 s. Fluorescence values of SYBR Green I dye, representing the amount of product amplified at that point in the reaction, were recorded in real time at both the annealing step and the extension step of each cycle. The Ct, defined as the point at which the fluorescence signal was statistically significant above background, was calculated for each amplicon in each experimental sample using Stratagene Mx4000 software. This value was then used to determine the relative amount of amplification in each sample by interpolating from the standard curve. The transcript level of each specific gene was normalized to GAPDH amplification.
Western blot analysis
Cells were lysed in Triton X-100 lysis buffer. The suspension was centrifuged at 15,000 × g for 5 min, and the protein concentration in the supernatant was determined. Western blot was done as described previously (27). Briefly, after these supernatants were subjected to SDS-PAGE, the separated proteins were electrophoretically transferred to a nitrocellulose membrane at 100 V for 1 h. The nitrocellulose membranes were blocked in 5% nonfat dry milk and then incubated with appropriate primary antibodies followed by 60-min incubation in horseradish peroxidase–conjugated secondary antibody (Amersham Biosciences). Detection was achieved with an enhanced chemiluminescence agent (Amersham Biosciences).
Primary antibodies used were as follows: PI-PLCγ1 and PI-PLCγ2 antibody (1:1,000), PI-PLCβ1 antibody (1:1,000), PI-PLCβ3 and PI-PLCβ4 antibody (1:1,000), phospho–MAP kinase antibody (1:1,000), ERK2 antibody (1:1,000), and GAPDH antibody (1:2,000). NOX5 antibody was prepared against a mixture of unique NOX5 peptides (NH2-YESFKASDPLGRGSKRC-COOH and NH2-YRHQKRKHTCPS-COOH) and used at a dilution of 1:1,000.
[3H]Thymidine incorporation
Twenty-four hours after pretreatment with U73122 or PD98059, or transfection with siRNAs of PI-PLCγ1, PI-PLCγ2, PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, ERK1, ERK2, or control, cells were treated with or without TDCA for 24 h and then incubated with [methyl-3H]thymidine (0.05 μCi/mL) for 4 h. After washing thrice with PBS to remove unincorporated radioactivity, cells were collected and homogenized with a lysis buffer containing (pH 7.4) 50 mmol/L HEPES, 50 mmol/L NaCl, 1% Triton X-100, 1% NP40, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 1 mmol/L DTT. [Methyl-3H]thymidine uptake was measured in a scintillation counter. The level of protein in the homogenates was also determined, and the level of [methyl-3H]thymidine incorporation was normalized to protein content.
Amplex Red H2O2 fluorescent assay
Levels of H2O2 in culture medium were determined by using the Amplex Red H2O2 assay kit (Molecular Probes, Inc.). This assay uses the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) to detect H2O2. In the presence of peroxidase, the Amplex Red reagent reacts with H2O2 in a 1:1 stoichiometry to produce the red fluorescent oxidation product resorufin. Fluorescence is then measured with a fluorescence microplate reader using excitation at 550 nm and emission detection at 590 nm.
Protein measurement
The amount of protein was determined by colorimetric analysis (Bio-Rad) according to the method of Bradford (28).
Materials
[3H]Thymidine was purchased from Perkin-Elmer Life Sciences, and human phospho–MAP kinase antibody was from Cell Signaling, Inc. PI-PLCγ1, PI-PLCγ2, PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, ERK2 antibody, GAPDH antibody, PI-PLCγ1 siRNA, PI-PLCγ2 siRNA, PI-PLCβ1 siRNA, PI-PLCβ3 siRNA, PI-PLCβ4 siRNA, and ERK1 siRNA were from Santa Cruz Biotechnology, Inc.; ERK2 siRNA was from Ambion, Inc. EA cell line OE33, Triton X-100, NP40, PMSF, dl-DTT, HEPES sodium, and other reagents were purchased from Sigma.
Statistical analysis
Data are expressed as mean ± SE. Statistical differences between two groups were determined by Student's t test. Differences between multiple groups were tested using ANOVA and checked for significance using Fisher's protected least significant difference test. All experiments were repeated for at least thrice.
Results
PI-PLC involved in TDCA-induced NOX5-S expression in FLO cells
In this study, we found that low dose of TDCA (10−11 mol/L, 24 hours) caused 4-fold increase in NOX5-S expression, 1.6-fold increase in H2O2 production, and 2-fold increase in cell proliferation in FLO cells when compared with control (Fig. 1A–C). These changes were statistically significant. We also found that the PI-PLC inhibitor U73122 (29) decreased TDCA-induced NOX5-S protein expression from 395.1 ± 87.1% to 135.1 ± 11.3% control (P < 0.02; Fig. 1A), H2O2 production from 155.1 ± 3.5% to 63.2 ± 1.9% control (P < 0.001; Fig. 1B), and thymidine incorporation from 202.4 ± 38.0% to 127.7 ± 13.2% control (P < 0.02; Fig. 1C) in FLO cells. These data suggest that PI-PLC may be involved in TDCA-induced NOX5-S expression and H2O2 production in FLO cells.
Seven major PI-PLC isoforms have been identified thus far (30). To further determine which PI-PLC isoforms mediate TDCA-induced NOX5-S expression, we used Western blot analysis to examine which PI-PLC isoforms are present in FLO cells. Isoforms of PI-PLC (β1, β3, β4, γ1, and γ2, but not β2 and δ1) were detectable in FLO cells (Fig. 1D). To examine whether these isoforms of PI-PLC regulate TDCA-induced NOX5-S in FLO cells, we used PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, PI-PLCγ1, and PI-PLCγ2 siRNA to knock down expression of these proteins. PI-PLCγ2 siRNA significantly decreased its protein expression (Fig. 2A) 48 hours after transfection. Knockdown of PI-PLCγ2 protein expression with PLCγ2 siRNA decreased TDCA-induced NOX5-S expression from 339.7 ± 50.2% to 136.7 ± 32.4% control (P < 0.001; Fig. 2B), H2O2 production from 131.5 ± 2.6% to 106.7 ± 9.7% control (P < 0.001; Fig. 2C), and thymidine incorporation from 162.5 ± 19.4% to 103.6 ± 8.8% control (P < 0.001; Fig. 2D) in FLO cells. Knockdown of PI-PLCγ2 protein also remarkably decreased TDCA-induced cell proliferation in OE33 EA cells (Fig. 2D). However, knockdown of PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, or PI-PLCγ1 protein expression did not affect TDCA-induced NOX5-S expression (Supplementary Figs. S1 and S2). The data suggest that TDCA-induced NOX5-S expression may depend on activation of PI-PLCγ2 protein, but not PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, or PI-PLCγ1 protein.
Role of ERK MAP kinases in TDCA-induced NOX5-S expression
Activation of PI-PLC produces inositol triphosphate and diacylglycerol. Diacylglycerol has been shown to activate the protein kinase C–MAP kinase pathway (31). Therefore, we examined whether ERK MAP kinases mediate TDCA-induced NOX5-S expression. The MAP kinase kinase inhibitor PD98059 (32) decreased TDCA-induced NOX5-S mRNA expression from 131.4 ± 3.9% to 59.8 ± 7.6% control (P < 0.001; Fig. 3A), H2O2 production from 155.1 ± 3.5% to 71.1 ± 5.2% control (P < 0.001; Fig. 3B), and thymidine incorporation from 202.9 ± 33.7% to 135.6 ± 16.4% control (P < 0.01; Fig. 3C), indicating that TDCA-induced increase in NOX5-S expression, H2O2 production, and cell proliferation may depend on activation of ERK MAP kinases.
We used ERK1 and ERK2 siRNA to knock down ERK1 and ERK2 expression, respectively. ERK1 and ERK2 siRNA significantly decreased its corresponding protein expression (Figs. 4A and 5A) 48 hours after transfection. Knockdown of ERK2 protein expression with ERK2 siRNA decreased TDCA-induced NOX5-S expression from 228.7 ± 70% to 89.4 ± 11.7% control (P < 0.05; Fig. 4B), H2O2 production from 116.3 ± 8.5% to 73.6 ± 1.7% control (P < 0.05; Fig. 4C), and thymidine incorporation from 173.8 ± 7.4% to 103.7 ± 2.9% control (Fig. 4D) in FLO cells. Knockdown of ERK2 protein expression with ERK2 siRNA also significantly decreased thymidine incorporation in response to TDCA treatment in OE33 cells. Conversely, knockdown of ERK1 protein expression had no statistically significant effect on NOX5-S expression induced by TDCA stimulation (Fig. 5B). The data suggest that TDCA-induced NOX5-S expression, H2O2 production, and cell proliferation may be mediated by activation of ERK2 MAP kinases, but not ERK1.
To further confirm the role of ERK2 MAP kinases, we examined ERK2 phosphorylation in FLO cells. We found that TDCA significantly increased ERK2 phosphorylation by 4.5-fold (Fig. 6A), indicating that ERK2 may be activated by TDCA in FLO cells. In addition, TDCA-induced ERK2 phosphorylation was significantly reduced by U73122 (Fig. 6A), suggesting that activation of ERK2 may depend on activation of PI-PLC. Knockdown of PI-PLCγ2 expression significantly decreased TDCA-induced ERK2 phosphorylation from 571 ± 167.9% to 179.3 ± 67.9% control (P < 0.05; Fig. 6B). The data suggest that TDCA-induced NOX5-S expression may depend on sequential activation of PI-PLCγ2 and ERK2 MAP kinase.
Discussion
Bile acids have been shown to contribute to the development of EA in a rat model of BE (17). Therefore, we examined whether bile acids upregulate NOX5-S expression and increase ROS production, thereby increasing cell proliferation and contributing to the development of EA. Bile acids, a group of structurally diverse molecules that are primarily synthesized in the liver, are the major components of bile. Besides their well-established roles in dietary lipid absorption and cholesterol homeostasis, it has recently emerged that bile acids are also signaling molecules in cell metabolism and signal transduction (33, 34). TDCA has been reported to be one of the major bile acids in the refluxate of patients with BE (35) and is more toxic than primary bile acids; thus, we used TDCA in our studies. Although reflux episodes are usually in terms of minutes or hours, particularly in long-segment BE and in the supine position, percent total time pH <4 ranges from 10.0 to 46.0 in BE patients (35); that is, esophageal mucosa is exposed to the refluxate for 2.4 to 11.0 hours per day. It is possible that low concentration of bile acids is present in the mucus layer that covers the surface of metaplastic cells for a much longer time after reflux episodes. In addition, it has been reported that short-term bile acid treatment does not alter cell proliferation in BAR-T cells (36). Therefore, 24-hour treatment with TDCA was used in our studies. The concentrations of bile acids in micromolar range are commonly found in the refluxate of BE patients (35), but metaplastic cells may be exposed to a much lower concentrations of bile acids due to the mucus layer that covers the surface of these cells. This notion is implicated by an in vivo study showing that the interstitial pH is conserved in normal rat esophagus when luminal pH is reduced to 1.0 (37). In addition, we have previously shown that 10−11 mol/L TDCA, but not higher doses, increases cell proliferation (12). Therefore, 10−11 mol/L TDCA was used in the present study. We found that TDCA increased NOX5-S expression, H2O2 production, and cell proliferation.
The mechanisms of bile acid–induced increase in cell proliferation are poorly understood. We have found that TDCA-induced increase in NOX5-S expression and cell proliferation is mediated by activation of the TGR5 receptor and Gαq protein in FLO EA cells (12). It has been reported that Gαq protein family may activate PI-PLC (24, 25). Because Gαq proteins are involved in TDCA-induced NOX5-S expression and H2O2 production, we examined the role of PI-PLC in bile acid–induced NOX5-S expression in FLO cells. Based on comparison of the sequences and structural studies, PI-PLC has three kinds of eukaryotic isozymes: PI-PLCβ, PI-PLCγ, and PI-PLCδ (30, 38). We found that TDCA-induced increase in NOX5-S expression and H2O2 production may depend on activation of PI-PLCγ2 protein because (a) TDCA-induced increase in NOX5-S expression and H2O2 production was significantly reduced by the PI-PLC inhibitor U73122; (b) knockdown of PI-PLCγ2 protein significantly reduced NOX5-S expression, H2O2 production, and cell proliferation in response to TDCA treatment in FLO and/or OE33 cells; (c) knockdown of PI-PLCβ1, PI-PLCβ3, PI-PLCβ4, and PI-PLCγ1 protein had no significant effect on TDCA-induced NOX5-S expression (Supplementary Data). The mechanisms whereby Gαq proteins activate PI-PLCγ2 are not clear. It is possible that Gαq first activates tyrosine kinase (39, 40) and then activates PI-PLCγ isoforms (41–43).
PI-PLC has been shown to regulate ERK2 phosphorylation in osteoblasts and macrophages (44, 45). ERK MAP kinases are key kinases in cell proliferation and cell cycle regulation (46, 47). In addition, we have shown that activation of ERK MAP kinases contributed to platelet-activating factor–induced NOX5-S expression (48). Therefore, we examined whether ERK MAP kinase plays a role in TDCA-induced NOX5-S expression and H2O2 production. We found that TDCA-induced increase in NOX5-S expression, H2O2 production, and cell proliferation was significantly reduced by the MAP kinase inhibitor PD98059, suggesting that ERK MAP kinase is involved in TDCA-induced NOX5-S expression and H2O2 production. ERK MAP kinase has two isoforms: ERK1 and ERK2. These two isoforms can be activated respectively or together in different cell biological process (49, 50). To examine which isoform(s) of ERK MAP kinases participates in TDCA-induced NOX5-S expression and H2O2 production, ERK1 and ERK2 protein expression were downregulated with their siRNAs, respectively. Knockdown of ERK2 protein significantly reduced TDCA-induced NOX5-S expression, H2O2 production, and cell proliferation. Conversely, knockdown of ERK1 protein did not have significant effect. The data suggest that TDCA-induced NOX5-S expression may depend on activation of ERK2 MAP kinase, but not ERK1. To further confirm the role of ERK2, we examined ERK2 phosphorylation after TDCA simulation. We found that TDCA significantly increased ERK2 phosphorylation, which was significantly reduced by U73122 and by knockdown of PI-PLCγ2 protein, suggesting that TDCA-induced activation of ERK2 MAP kinase may depend on activation of PI-PLCγ2. The data suggest that TDCA-induced NOX5-S expression may depend on sequential activation of PI-PLC and ERK2 MAP kinase.
In conclusion, TDCA may induce increase in NOX5-S expression, H2O2 production, and cell proliferation in EA cells. This increase may depend on sequential activation of PI-PLCγ2 and ERK2 MAP kinase. It is possible that bile acid reflux present in patients with BE may increase ROS production and cell proliferation via activation of PI-PLCγ2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby causing DNA damage and gene mutation, which contribute to the development of EA. Our data may provide potential targets to prevent and/or treat Barrett's EA.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Grant Support: National Institute of Diabetes and Digestive and Kidney Diseases grant R01 DK080703.
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