Human esophageal epithelial cells play a key role in esophageal inflammation in response to acidic pH during gastroesophageal reflux disease (GERD), increasing secretion of IL-6 and IL-8. The mechanisms underlying IL-6 and IL-8 expression and secretion in esophageal epithelial cells after acid stimulation are not well characterized. We investigated the role of PKC, MAPK, and NF-κB signaling pathways and transcriptional regulation of IL-6 and IL-8 expression in HET-1A cells exposed to acid. Exposure of HET-1A cells to pH 4.5 induced NF-κB activity and enhanced IL-6 and IL-8 secretion and mRNA and protein expression. Acid stimulation of HET-1A cells also resulted in activation of MAPKs and PKC (α and ε). Curcumin, as well as inhibitors of NF-κB (SN-50), PKC (chelerythrine), and p44/42 MAPK (PD-098059) abolished the acid-induced expression of IL-6 and IL-8. The JNK inhibitor SP-600125 blocked expression/secretion of IL-6 but only partially attenuated IL-8 expression. The p38 MAPK inhibitor SB-203580 did not inhibit IL-6 expression but exerted a stronger inhibitory effect on IL-8 expression. Together, these data demonstrate that 1) acid is a potent inducer of IL-6 and IL-8 production in HET-1A cells; 2) MAPK and PKC signaling play a key regulatory role in acid-mediated IL-6 and IL-8 expression via NF-κB activation; and 3) the anti-inflammatory plant compound curcumin inhibits esophageal activation in response to acid. Thus IL-6 and IL-8 expression by acid may contribute to the pathobiology of mucosal injury in GERD, and inhibition of the NF-κB/proinflammatory cytokine pathways may emerge as important therapeutic targets for treatment of esophageal inflammation.
- gastroesophageal reflux disease
gastroesophageal reflux disease (GERD) is a commonly encountered condition in Western societies, occurring in 7–10% of individuals in the United States. Pathogenesis of GERD is linked to the prolonged contact of acidic gastric contents with the epithelial luminal surface of the esophagus. The magnitude of mucosal injury in GERD is highly variable. Mild disease may demonstrate no visible mucosal damage, while more severe manifestations may present with erosions, ulcerations, stricture formation, and Barrett's metaplastic transformation, which is associated with the development of adenocarcinoma (24). The molecular and cellular mechanisms underlying this differential response to acidic injury in the esophagus remain largely undefined. Much of the investigation into the mechanisms of mucosal defense against esophageal acid exposure has focused on damage to tight junctions and loss of epithelial integrity in response to acid contact (21, 22). One of the major limitations preventing further characterization of the molecular and cellular basis of acid-induced esophageal inflammation is the relative inaccessibility of in vitro esophageal epithelial cell populations for study.
Interleukin (IL)-6 is a proinflammatory cytokine that plays a central role in host defense against infections and tissue injury (19, 30). Studies in peritonitis models suggest that IL-6 signaling is of crucial importance in the transition from acute to chronic phases of inflammatory processes (17). Involvement of IL-6 in various diseases including carcinogenesis has been established, and recent investigation supports the role of the IL-6 signaling pathways in the development of GERD (29).
It has been shown that classic cytokines are important mediators of human esophageal inflammation, and exposure to gastric juice stimulates esophageal epithelial cells to produce IL-6, which alters esophageal contractility (29). However, the intracellular signaling mechanisms underlying acid-induced activation of the esophageal epithelium in GERD remain incompletely defined. IL-8 is a potent chemokine implicated in neutrophil recruitment during inflammatory reactions. Increased levels of IL-8 have been demonstrated in GERD, and esophageal epithelium is believed to be the major source of production of this cytokine (18).
Investigation of novel anti-inflammatory compounds has recently focused increased attention on the natural product curcumin. Curcumin, the major yellow coloring pigment found in the household spice turmeric (Curcuma longa Linn, Zingiberaceae), has been used for centuries in food preparation as well as in Ayurvedic traditional medicine to treat inflammatory disorders (32). Curcumin has low toxicity, and it has already been demonstrated to benefit gastrointestinal inflammation in both animal models and human inflammatory bowel disease in a randomized crossover clinical trial (12). Also, curcumin has been shown to have antineoplastic potential, inhibiting the development of chemically induced tumors of the oral cavity, skin, forestomach, duodenum, and colon in rodents (7, 14–16). Curcumin has been shown to decrease secretion of proinflammatory cytokines, including IL-6 and IL-8, in various cells through inhibition of intracellular signaling cascades (4, 20, 28). The effect of curcumin on esophageal inflammation associated with GERD has not been defined.
The aims of this study were to 1) characterize the role of the MAPK and PKC signaling pathways and NF-κB activation in the transcriptional regulation of IL-6 and IL-8 expression and secretion in human esophageal epithelial cells (HET-1A) after acidic pH stimulation and 2) define the effect of curcumin on acid-mediated activation. Here we report that acid is a potent stimulator of IL-6 and IL-8 production in HET-1A cells, which involve activation of the MAPK and PKC signaling pathways as well as NF-κB and that curcumin is a potent inhibitor of both IL-6 and IL-8 expression after acid stimulation.
MATERIALS AND METHODS
Bronchial Epithelial Cell Medium (BEGM BulleKit; catalog no. CC-3170) was obtained from Clonetics (Walkersville, MD). The alternative growth medium LHC-9 (catalog no. P181-500) was from Biosource (Camarillo, CA). The additive BEGM SingleQuots Kit (catalog no. CC-4175) was obtained from Cambrex Bio Science (Walkersville, MD). MAPK antibodies (phosphorylated and nonphosphorylated) were obtained from Cell Signaling (New England BioLabs, Beverly, MA). Pan-PKC antibody was from Upstate Biotechnology. Anti-PKCα, anti-PKCδ, and anti-PKCε were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKCζ, anti-PKCβ, and anti-PKCλ were from Transduction Laboratories (San Diego, CA). SB-203580, PD-098059, SP-600125, and chelerythrine were obtained from Calbiochem (San Diego, CA), and SN-50 was from BioMol (Plymouth Meeting, PA). Unless otherwise indicated, all other chemicals used in this study were purchased from Sigma (St. Louis, MO). IL-6 and IL-8 enzyme-linked immunosorbent assay (ELISA) kits were from R&D Systems (Minneapolis, MN).
Human esophageal epithelial (HET-1A) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and were grown in complete growth medium [Bronchial Epithelial Cell Medium (BEGM BulleKit)]. Immortalized and transformed human esophageal epithelial cells (EPC1-hTERT and EPC2-hTERT) with functional p53 gene (13) were the generous gifts of Dr. Hiroshi Nakagawa, Gastroenterology Division, University of Pennsylvania (Philadelphia, PA). Cells were grown in monolayer culture according to a standard protocol. Primary human esophageal epithelial cells (HEEC) were obtained from ScienCell Research Laboratories (Carlsbad, CA) and were grown according to the manufacturer's protocol.
Activation and pharmacological modulation of HET-1A cells.
HET-1A cells were incubated in acidified growth medium (pH 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5) for 1, 5, 10, 30, 45, and 60 min. Because we were primarily interested in the effect of prolonged acid exposure, HET-1A cells were incubated in acidified growth medium (pH 4.5) for 60 min (unless otherwise stated). pH 4.5 was chosen because over a period of 60 min there was <2% cell death as tested by Trypan blue exclusion and monolayers remained intact as examined microscopically. Cells were then rinsed twice with basal medium and incubated in neutral pH growth medium for the indicated time periods (3, 6, 12, 24, and 48 h). The contribution of signal transduction pathways to HET-1A cell activation and gene expression was defined with specific MAPK inhibitors including 5 μM SB-203580 (p38 MAPK), 10 μM PD-098059 (p44/42 MAPK), 10 μM SP-600125 (JNK), 1 μM chelerythrine (PKC), 18 μM SN-50 (NF-κB), and curcumin (0.1, 1, 10, and 20 μM). HET-1A cells were pretreated with the inhibitors for 30 min before acidic pH activation as indicated above.
Enzyme-linked immunosorbent assay.
HET-1A cells were exposed to acidic pH as described above with or without pharmacological inhibitors. Supernatants from cultured HET-1A cells were harvested at different time points, and IL-6 and IL-8 secretion from control and acidic pH-stimulated cells was assessed with commercially available ELISA kits (R&D Systems) according to the manufacturer's protocol. Unconditioned growth medium was used as a negative control. All conditions were assessed in triplicate.
Semiquantitative and real-time RT-PCR.
IL-6 gene expression was assessed in control and acidic pH-stimulated cultures of HET-1A cells with or without pharmacological inhibitors. Total RNA was extracted with RNAzol B (Teltest, Friendswood, TX) and quantitated by optical density as described previously (27). Primer sequences for IL-6 were 5′-TCA ATG AGG AGA CTT GCC TG-3′ [forward (F)] and 5′-GAT GAG TTG TCA TGT CCT GC-3′ [reverse (R)] (35 cycles). Primer sequences for β-actin were 5′-CCA GAG CAA GAG AGG CAT CC-3′ (F) and 5′-CTG TGG TGG TGA AGC TGT AG-3′ (R) (25 cycles).
For real-time PCR, HET-1A cells were exposed to pH 4.5 for 10 min and then incubated in neutral medium for the indicated time. RNA was isolated from confluent 60-mm plates at 3 h and 6 h from HET-1A cells with Qiagen's RNA Cell Protect reagent followed by Qiagen's RNeasy Plus Mini Kit according to the manufacturer's instructions. cDNA was then synthesized from 1 μg of RNA with Bio-Rad's iScript cDNA synthesis kit according to the manufacturer's instructions. Real-time PCR was performed with Bio-Rad's Sybr Green Master Mix, with 250 nM primer concentration and 1 μl of cDNA per 25-μl reaction. Each sample was run in triplicate. The cycling parameters were as follows: 1 cycle at 95°C for 3 min, 50 cycles at 95°C for 30 s and 53.2°C for 30 s, and finally a melt cycle to confirm the absence of nonspecific products. Primer sequences for IL-6 and IL-8 were designed with Beacon Designer software. IL-6 primer sequences were ACA GCC ACT CAC CTC TTC (F) and AAG TCT CCT CAT TGA ATC CAG (R). IL-8 primer sequences were CTC TCT TGG CAG CCT TCC (F) and CTC AAT CAC TCT CAG TTC TTT G (R). For GAPDH the primer sequences were TGC ACC ACC AAC TGC TTA GC (F) and GGC ATG GAC TGT GGT CAT GAG (R). Normalized gene expression was analyzed with Bio-Rad's iQ5 software.
SDS-PAGE and Western blotting.
Gel electrophoresis and Western blot analysis were performed as described previously (26, 27). In brief, HET-1A cells were assessed unstimulated or after acid activation (as above) with or without pharmacological inhibitors and were analyzed by SDS-PAGE and Western blotting with specific antibodies (phosphorylated and nonphosphorylated).
Analysis of PKC subcellular distribution.
The subcellular distribution of PKC was analyzed by separation of particulate and cytosolic fractions by ultracentrifugation (26).
NF-κB gel electrophoretic mobility shift assay.
Nuclear protein extracts from HET-1A cells were prepared with the NE-PER kit from Pierce Biotechnology (Rockford, IL) according to the manufacturer's protocol. For the DNA binding assay, an end-labeled biotinylated double-stranded NF-κB oligonucleotide (5′-GCC CGG GGA GGA TTC CTG GGC CCC-3′) was used. Binding reactions were carried out with the nonradioactive LightShift Chemiluminescence electrophoretic mobility shift assay (EMSA) Kit (Pierce Biotechnology) according to the manufacturer's protocol. Reaction products were separated through a 6% DNA retardation gel (Invitrogen, Carlsbad, CA) and transferred to Biodyne B membrane (Pierce Biotechnology). Membranes were exposed to X-ray films and were developed with the Chemiluminescent Nucleic Acid Detection Module (Pierce Biotechnology).
Immunofluorescence staining of p65 NF-κB subunit.
HET-1A monolayers were grown on coverslips to 80% confluence, and immunofluorescence staining was performed as described previously (27).
Mucosal biopsy was obtained from the esophagus (proximal and distal) of patients with a history of reflux esophagitis (n = 2). The use of human tissue was approved by the Institutional Review Board of the Medical College of Wisconsin.
Mucosal biopsy was placed on a cell culture plate containing 1 ml of RPMI 1640 medium with penicillin and streptomycin (BioWhittaker, Rockland, ME). The culture was performed in 5% CO2 at 37°C overnight, and then the culture medium was removed and kept at −80°C until being assayed for cytokine secretion by ELISA.
Cytokine/chemokine mRNA expression in HET-1A cells.
Because we were primarily interested in the effect of prolonged acid exposure, HET-1A cells were incubated in acidified growth medium (pH 4.5) for 60 min. pH 4.5 was chosen because over a period of 60 min there was <2% cell death as tested by Trypan blue exclusion (not shown) and HET-1A monolayers remained intact as examined microscopically (Fig. 1A).
With real-time PCR and respective primers, levels of mRNA for IL-6 and IL-8 were examined in control and acidic pH-activated HET-1A cells. The levels of mRNA between control and activated HET-1A cells were compared after normalization to β-actin, which functioned as an internal control. Figure 1B shows that the level of both IL-6 and IL-8 mRNA expression was increased after acidic stimulation of HET-1A cells compared with unstimulated control cells. The induction of both IL-6 and IL-8 mRNA by pH 4.5 was time dependent and maximized by 6 h. These data demonstrate that HET-1A cells respond to the stress of acid by increasing expression of IL-6 and IL-8.
Cytokine/chemokine protein expression in HET-1A cells.
Next we determined cytokine protein expression in HET-1A cells exposed to acid. Using antibodies to human IL-6 and IL-8, we demonstrated that exposure of HET-1A cells to pH 4.5 for 60 min followed by incubation in neutral medium resulted in an increase of IL-6 protein level by 6 h that was maximized by 12 h and declined to basal level by 24 h. The level of IL-8 protein was increased by 3 h and was maximized by 6 h (Fig. 1C).
IL-6 and IL-8 secretion in HET-1A cells.
To investigate IL-6 and IL-8 secretion in HET-1A cells, ELISAs of cell culture supernatants were performed. Exposure of HET-1A cells to acidic pH 4.5 followed by incubation in neutral medium resulted in both IL-6 and IL-8 secretion as assessed for multiple time points (6, 12, 24, 48 h) in culture supernatants (Fig. 1D). After exposure to acid, both IL-6 and IL-8 secretion were maximized by 12 h as assessed after incubation in neutral medium, then declined by 48 h compared with control, unstimulated cells. We also assessed these acid-stimulated HET-1A cells for secretion of the eosinophil chemoattractant chemokine eotaxin. No eotaxin was detectable in either acid-exposed or unstimulated control HET-1A cell supernatants (data not shown).
MAPK signaling in HET-1A cells after acidic pH exposure.
It has been shown that acid exposure significantly enhances MAPK phosphorylation in esophageal epithelium (31). To determine whether the duration of acid exposure may also lead to activation of these signaling pathways in HET-1A cells, we exposed the cells to acidified growth medium (pH 4.5) for multiple time points (0, 5, 15, 30, and 60 min), and assessment of MAPK activation was subsequently performed in cell lysates. Figure 2A demonstrates that p44/42 MAPK activation was first detected at 15 min and maximized by 60 min, while JNK activation was detected as early as 1 min and maximized by 5 min. Activation of p38 MAPK first appeared at 5 min and lasted for 30 min. Figure 2B shows the summarized data of relative density for MAPKs. These experiments suggest that exposure of HET-1A cells to acid results in transient early activation of JNK and prolonged activation of p44/42 MAPK if acid exposure is encountered in the esophagus. We confirmed that equal amounts of proteins were analyzed by stripping and reprobing the same blots with non-phospho-antibodies.
Figure 2C demonstrates the inhibition of these kinases in acid-activated HET-1A cells by specific pharmacological inhibitor and curcumin pretreatment. Phosphorylation of p44/42 MAPK was inhibited by PD-098059 (10 μM) pretreatment of HET-1A cells, p38 MAPK phosphorylation was attenuated by SB-203580 (5 μM), and JNK was inhibited by SP-600125 (10 μM). Curcumin (10 μM) was a potent inhibitor of all three MAPK family members (Fig. 2C).
Effect of pharmacological inhibitors on acid-induced IL-6 and IL-8 in HET-1A cells.
We then determined the effect of protein kinase inhibitors on the activation of specific signaling pathways in relation to IL-6 and IL-8 expression in acid-treated HET-1A cells. As shown above, acidic pH exposure resulted in increased expression of IL-6 and IL-8 at the mRNA and protein levels as well as their secretion. PD-098059 (10 μM), an inhibitor of MEK/p44/42 MAPK, blocked both IL-6 and IL-8 expression/secretion (Fig. 3). SP-600125 (10 μM), a specific JNK inhibitor, partially inhibited IL-6 expression/secretion in the acid-activated HET-1A cells but did not effect IL-8 expression/secretion (Fig. 3). However, SB-203580, a specific inhibitor of p38 MAPK (5 μM), did not block either IL-6 mRNA or protein expression, nor did it affect IL-6 secretion. In marked contrast, SB-203580 was a potent inhibitor of IL-8 expression and secretion (Fig. 3, A and C, respectively). In addition, a PKC inhibitor (1 μM chelerythrine) and a NF-κB inhibitor (18 μM SN-50) resulted in inhibition of IL-6 and IL-8 expression/secretion in acid-activated HET-1A cells (Fig. 3).
Curcumin inhibits IL-6 and IL-8 expression/secretion in HET-1A cells.
Next we examined the effect of curcumin on IL-6 and IL-8 gene expression with real-time PCR. Our results demonstrate that pretreatment of HET-1A cells with 10 μM curcumin abolished acidic pH induction of IL-6 and IL-8 mRNA expression (Fig. 4A). Consistent with the gene expression data, curcumin pretreatment of HET-1A cells inhibited IL-6 and IL-8 protein (not shown). Corresponding with the effect of curcumin on IL-6 and IL-8 mRNA and protein expression, pretreatment of HET-1A cells with curcumin also resulted in inhibition of both IL-6 and IL-8 secretion in culture media as determined by ELISA (Fig. 4B).
PKC activation in HET-1A cells following acidic pH exposure.
It has been shown that in epithelial cells PKC plays a key role in the regulation of NF-κB-dependent gene expression (25). To determine the involvement of PKC in HET-1A cells after acid stimulation, cytosolic and particulate fractions were examined. Using antibodies targeting specific PKC isoforms and Western blotting, we demonstrated that exposure of HET-1A cells to acid resulted in increased expression of PKCα and PKCδ (Fig. 5A). However, no alteration was observed in other PKC isoforms, because PKCλ was present in both control and acid-treated cells and no changes in β, ζ and ε PKC isoforms were detected (Fig. 5A). Summarized data of relative density for PKC isoforms is shown in Fig. 5B. Next we used a polyclonal antibody directed against all PKC isoforms (pan-PKC), which demonstrated that in resting, unstimulated HET-1A cells PKC is present in the cytosolic fraction. Exposure of HET-1A cells to pH 4.5 resulted in the translocation of PKC from the cytosolic fraction to the particulate fraction, and pretreatment of HET-1A cells with either 1 μM chelerythrine or 10 μM curcumin decreased this PKC translocation (Fig. 5C) as well as the IL-6 and IL-8 expression demonstrated in Fig. 3C and Fig. 4A. These data indicate that PKC plays a role in the regulation of acid induction of IL-6 and IL-8 in HET-1A cells.
NF-κB activation by acidic pH exposure in HET-1A cells.
Activation of NF-κB by proinflammatory cytokines has been reported (9, 25). We examined acid-mediated activation of NF-κB with various techniques. The activation of NF-κB and nuclear protein DNA binding was confirmed with a nonradioactive, end-labeled biotinylated double-stranded NF-κB oligonucleotide binding assay. In these experiments, the acid-activated HET-1A cell nuclear extract bound the oligonucleotide, and this complex was detected with the Chemiluminescent Nucleic Acid Detection Module (Fig. 6A). We used nuclear protein from microvascular endothelial cells after TNF-α/LPS activation as a positive control. Pretreatment of HET-1A cells with curcumin, PD-098059, and SP-600125 resulted in inhibition of NF-κB activity, but, in marked contrast, SB-203580 did not inhibit NF-κB activity. The role of PKC in acidic pH-induced NF-κB activation was investigated with chelerythrine (PKC inhibitor). As demonstrated in Fig. 6A, chelerythrine effectively inhibited NF-κB nuclear protein DNA binding in HET-1A cells. These findings demonstrate that NF-κB undergoes activation in HET-1A cells exposed to acid and will bind to nuclear DNA during epithelial activation. Next we investigated the ability of acid to promote the nuclear translocation of NF-κB. We used antibodies against the p65 subunit of human NF-κB and demonstrated its immunoreactivity in HET-1A cell nuclear protein after acid stimulation (Fig. 6B). Pretreatment of HET-1A cells with SN-50, chelerythrine, and curcumin resulted in inhibition of NF-κB activation. Finally, we performed immunofluorescence staining of NF-κB p65 subunit in HET-1A cells (Fig. 6C), which demonstrated nuclear translocation following acid exposure.
Enhanced IL-6 production by mucosal biopsy.
Next we investigated the IL-6 production in organ culture media from proximal and distal esophageal mucosal biopsy. Figure 7A demonstrates that IL-6 production was significantly enhanced in distal biopsy specimens from the patients with esophagitis compared with control specimens. No detectable change in level of IL-6 production in proximal biopsy specimens was observed.
Enhanced interleukin production by HEEC, EPC1-hTERT, and EPC2-hTERT cells.
In final sets of experiments, we utilized a primary human esophageal epithelial cell line (HEEC) and two telomerase-induced immortalized human esophageal epithelial cell lines containing functional p53 gene, EPC1-hTERT, and EPC2-hTERT (1, 13, 33), to investigate the effect of acid (pH 4.5) on these cells. As shown in Fig. 7B, exposure of EPC1-hTERT and EPC2-hTERT cells to acid followed by overnight incubation in neutral medium increased the secretion of IL-8 in culture medium compared with control cells. Similarly, acidic exposure markedly increased the level of both IL-6 and IL-8 mRNA in HEEC, EPC1-hTERT, and EPC2-hTERT cell lines (Fig. 7C) compared with control cells. Moreover, 1 μM curcumin pretreatment of the cells exerted an inhibitory affect on cytokine production (not shown).
In the present study, we characterized the effect of acidic pH exposure on HET-1A cells, defining a role for activation of PKC, NF-κB, and MAPK family members as well as confirming increased expression and production of the proinflammatory cytokines IL-6 and IL-8. These findings show that HET-1A cells, a relevant human esophageal epithelial cell population, are sensitive to acid stimulation, demonstrating inflammatory activation. We focused on long-term (60 min) acid exposure, which also represents a physiological stimulus, because patients with GERD undergoing ambulatory esophageal pH monitoring will commonly demonstrate up to 1 h of acidic esophageal refluxate during sleep (6). Using this strategy to model acidic pH stimulation, we demonstrated that activation of both PKC and NF-κB as well as MAPKs plays a role in the regulation and expression of IL-6 and IL-8 in HET-1A cells. This acidic pH activation correlated with altered cellular function in HET-1A cells involving inflammatory (i.e., induction of IL-6 and IL-8 via MAPK, PKC, and NF-κB) activation. Our studies demonstrate that the MAPK cascades are involved in the regulation of IL-6 and IL-8 expression in acid-activated HET-1A cells, suggesting a role for classic stress signaling pathways in esophageal adaptation to acid exposure during esophagitis and GERD.
Esophagitis and GERD are presently believed to result from the prolonged contact of acidic gastric refluxate with the esophageal mucosal lining, which is typically encountered nocturnally. Twenty-four-hour ambulatory pH monitoring in healthy control subjects and patients with GERD, which records the exposure of the esophageal lumen to gastric refluxate, has demonstrated prolonged acid retention in the esophagus during sleep (6, 8). We hypothesized that the damaged esophageal mucosal surfaces would allow for acid activation of the esophageal epithelium, and we chose to model this phenomenon in vitro with HET-1A cells, the relevant esophageal epithelial population.
Investigation into the cellular and molecular mechanisms that follow acid exposure has also focused on reparative and proliferative effects on the esophageal epithelium. Of particular interest has been the characterization of acid exposure and its relationship to metaplastic intestinal transformation of the squamous epithelium, Barrett's esophagus, which is a precursor lesion of esophageal adenocarcinoma, the most dreaded complication of GERD. Investigation has demonstrated that acidic pH promotes cell growth in explants of Barrett's tissue maintained in organ culture (10, 11, 23). Furthermore, Souza and colleagues (31) have characterized the activation of MAPK pathways in response to acid in SEG-1 cells, a lung carcinoma cell line, showing that transient exposure of these transformed epithelial cells to acidic pH resulted in cell proliferation and decreased apoptosis, which corresponded with activation of p38 MAPK and ERK and a delayed activation of JNK. In these studies, increased epithelial proliferation in response to acidic pH was abolished by inhibition of p38 MAPK or JNK, while inhibition of apoptosis was linked to ERK activity. Souza et al. correlated these in vitro studies with biopsies taken from patients before and after esophageal acid perfusion, demonstrating the activation of MAPK pathways in response to acid.
Classic mediators of inflammation including cytokines and chemokines have been implicated in the pathophysiology of esophageal inflammation. Increased IL-1β and IL-6 expression has been demonstrated in cat esophageal tissues during experimental esophagitis (2, 3). Increased expression of various cytokines and chemokines including IL-1β, IL-6, IL-8, IL-10, IFN-γ, and monocyte chemoattractant protein-1 in mucosal biopsy specimens of patients with GERD has been shown (5, 18). With the use of an organ culture system, increased IL-6 secretion from mucosal biopsies of the lower esophagus in patients with esophagitis has been reported (29).
Generating and maintaining primary cultures of epithelial cells from human esophageal explants in sufficient numbers to allow for sophisticated, mechanistic experiments has proven difficult, and thus we utilized HET-1A cells for the investigation of IL-6 and IL-8 expression. When epithelial events in GERD were modeled in HET-1A cells by the addition of acidified culture medium, the level of both IL-6 and IL-8 secretion was high.
It has been shown that exposure to the gastric refluxate stimulates esophageal cells to release proinflammatory cytokines, which in turn mediate the esophageal dysmotility associated with GERD-induced esophagitis (29). Our data indicate that the HET-1A cells produce both IL-6, a cytokine that is known to affect muscle cell contractility, and IL-8, which plays an important role in the pathogenesis of autoimmune and inflammatory diseases. Thus acid activation of the epithelium will potentially result in production of cytokines and chemokines, which will exert effects in esophageal cell populations, ultimately mediating the pathogenesis of GERD.
Pretreatment of HET-1A cells with pharmacological inhibitors of NF-κB (SN-50), p44/42 MAPK (PD-098059), and PKC (chelerythrine) for 30 min before acidic exposure abolished both IL-6 and IL-8 expression/secretion and NF-κB activity. In contrast, JNK (SP-600125) and p38 MAPK (SB-203580) inhibitors demonstrated differential inhibitory patterns on IL-6 and IL-8 expression/secretion. The JNK inhibitor SP-600125 was a potent inhibitor of IL-6 but did not block IL-8 expression/secretion from the esophageal epithelium in response to acid. In marked contrast, SB-203580, a selective p38 MAPK inhibitor, exerted an inhibitory effect on IL-8 but not IL-6 expression/secretion. Moreover, SB-203580 did not block NF-κB activation in HET-1A cells after acid exposure. These findings suggest that NF-κB, MAPKs, and PKC activity play an important role in acid-induced IL-6 and IL-8 expression, which may contribute to subsequent inflammation. The inhibitory effect of chelerythrine, a potent PKC inhibitor, on NF-κB activity and IL-6 and IL-8 expression demonstrates that PKC activity is essential for this cytokine/chemokine expression in HET-1A cells exposed to acid.
One of the more interesting findings in our study was the demonstration that the natural product curcumin proved to be a potent inhibitor of acid activation in HET-1A cells. Curcumin has already demonstrated a beneficial effect on lower gastrointestinal inflammation in both animal models and human inflammatory bowel disease in a randomized crossover clinical trial (12). Our findings with inhibition of intracellular activation following acid exposure in an esophageal epithelial cell line suggest that this compound may also possess therapeutic potential in the treatment of acid injury in GERD as well as Barrett's esophagus, and further work in this area is warranted.
Furthermore, to confirm that induction of IL-6 and IL-8 observed in HET-1A cells represents a specific physiological response of esophageal epithelial cells to acid exposure, we investigated the effect of acid exposure on one primary cell line (HEEC) and two telomerase-induced immortalized human esophageal epithelial cell lines with functionally intact p53 gene (EPC1-hTERT and EPC2-hTERT) and demonstrated enhanced expression of both IL-6 and IL-8 in these cell lines. Thus IL induction in HET-1A cells by acid is a specific physiological response.
In conclusion, we demonstrate activation of intracellular signaling mechanisms that were linked to enhanced IL-6 and IL-8 expression in HET-1A esophageal epithelial cells exposed to acid. In addition to the MAPK activation, PKC and NF-κB activity played an important role in the regulation of IL-6 and IL-8 expression in acid-activated HET-1A cells. Finally, the natural anti-inflammatory product curcumin exerted a potent inhibitory effect on acid-mediated activation of esophageal epithelial cells, which also warrants further investigation. These findings support a potential role for the targeting of stress signaling pathways in patients suffering from severe esophageal inflammation, and clinical studies evaluating the use of the natural product curcumin can be envisioned.
This work was supported by the Disease Center of the Medical College of Wisconsin (P. Rafiee, D. G. Binion).
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.
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