Sa1841 Increased Esophageal Transforming Growth Factor α Secretion in Patients With Barrett's...
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type mice had no visible nodules or irregularities at the squamo-columnar junction (SCJ) in response to DCA treatment. 14% of the K14-Cdx2 mice treated with DCA developed two nodules each at the SCJ, whereas none of the K14-Cdx2 mice receiving water alone developed nodules. In contrast, 70% of the IL-1β mice, and an equal number of Cdx2/IL- 1β mice developed nodular disease at the SCJ after DCA treatment. All DCA treatment- associated nodules were histologically consistent with metaplasia. However overall the dou- ble-transgenic mice had half as many nodules as IL-1β mice. Cdx2 did not affect IL-1β mRNA levels in the double transgenic mice, nor was the inflammation associated with IL- 1β expression reduced in the Cdx2/IL-1β mice compared to IL-1β mice, as determined by CD45R+ cell infiltration in the esophagi and forestomach. Moreover, cell proliferation rates were not altered by Cdx2 expression, as EdU incorporation was not significantly different. Lastly, the morphologic appearance of the nodules in all three lines, K14-Cdx2, L2-IL-1β, and K14-Cdx2/L2-IL-1β mice was similar, with abundant alcian blue expressing goblet cells detected, along with expression of other markers of intestinalization. Further molecular characterization of the metaplastic nodules by cDNA microarray profiling is underway. Conclusion: Ectopic expression of Cdx2 in the IL-1Beta mouse model for BE reduces the nodular intestinal metaplasia disease burden. These studies raise new questions as to the specific role of inflammation and intestinal transcription factors in the development of Barrett's esophagus in humans. Sa1839 Atonal Homolog 1 (ATOH1) Upregulates Gastric- and Intestinal-Type Protein Expression in Esophageal Squamous Cells, and Increases Proliferation in Barrett's Cells: Potential Roles for ATOH1 in Barrett's Pathogenesis and Carcinogenesis Yuji Tamagawa, Norihisa Ishimura, Anjana Tiwari, Peiguo Ding, Goichi Uno, Masahito Aimi, Naoki Oshima, Shunji Ishihara, Yoshikazu Kinoshita, Stuart J. Spechler, Rhonda F. Souza, David H. Wang Introduction: In the intestine, stem cell differentiation is mediated by the Notch receptor signaling pathway, which interacts with atonal homolog 1 (ATOH1), a basic helix-loop- helix transcription factor. Barrett's metaplasia is characterized by intestinal differentiation with goblet cells. Goblet cell differentiation occurs when there is low Notch activity and high ATOH1 activity. In earlier studies, we reported that bile acids suppressed Notch signaling in esophageal squamous epithelial cells by increasing ATOH1, which led to their expression of Cdx2 and MUC2 (intestinal-type proteins found in Barrett's metaplasia). In certain non-esophageal cells, ATOH1 has been shown to induce the expression of MUC5AC and MUC6 (gastric-type mucins also found in Barrett's metaplasia), and to regulate cell proliferation. In this study, we sought to determine whether ATOH1 regulates expression of MUC5AC and MUC6 in human esophageal squamous and Barrett's epithelial cells, and if ATOH1 regulates proliferation in Barrett's cells. Methods: We used human esophageal squamous epithelial cell lines (Het-1A, NES-B3T, and NES-B10T) and Barrett's epithelial cell lines (BAR-10T and CP-A). The expression of ATOH1, Cdx2, MUC5AC, MUC6, and MUC2 was examined using real-time PCR and Western blot under basal conditions. The expression of these genes also was determined by real-time PCR following treatment with a gamma-secretase inhibitor (GSI, which increases ATOH1), with siRNA-mediated knock- down of ATOH1, or with GSI in combination with siRNA-mediated knockdown of Cdx2. GSI effects on cell proliferation were determined by MTS assay. Results: Under basal conditions, expression levels of ATOH1, Cdx2 and MUC2 were higher in the Barrett's cells than in the squamous cell lines. In Het-1A squamous cells, GSI treatment (which increased expression of ATOH1) increased Cdx2, MUC2, MUC5AC, and MUC6 expression. In CP-A Barrett's cells, knockdown of ATOH1 by siRNA transfection significantly decreased MUC5AC and MUC6 expression. CP-A cells treated with the combination of GSI and Cdx2 knockdown by siRNA still showed significantly increased expression of MUC5AC and MUC6. BAR- 10T and CP-A cells treated with GSI showed increased expression of ATOH1, which was accompanied by a significant increase in cell growth. Conclusion: ATOH1 upregulates the expression of intestinal- and gastric-type proteins (Cdx2, MUC2, MUC5AC, and MUC6) in esophageal squamous cells, and ATOH1 functions as a transcriptional regulator of gastric- type mucins, independent of Cdx2, in Barrett's metaplastic cells. These findings support a potential role for ATOH1 in the pathogenesis of Barrett's esophagus. Our observation that increased ATOH1 expression was associated with increased proliferation in Barrett's cells also suggests a potential role for ATOH1 in the neoplastic progression of Barrett's metaplasia. Sa1840 APE1 Suppresses Acidic Bile Salts-Induced Cell Death Through Regulation of JNK/p38 Pathways in Esophageal Adenocarcinoma Jun Hong, Zheng Chen, DunFa Peng, Abbes Belkhiri, Wael El-Rifai Background: Over the last three decades the incidence of esophageal adenocarcinoma (EAC) has increased at an alarming rate in Western countries. The development of EAC has been associated with a precancerous lesion in the distal oesophagus, termed Barrett's esophagus (BE). Gastroesophageal reflux disease (GERD), a chronic exposure of the esophagus to acid and bile salts from the stomach, is a risk factor for BE and EAC. Apurinic/apyrimidinic endonuclease (APE1) is an enzyme involved in the DNA base excision repair. The aim of this study was to investigate the role of APE1 in acidic bile salts-induced DNA damage and elucidate the underlying mechanism in EAC. Methods and Results: Western blot and immunohistochemistry data indicated very low to undetectable APE1 protein expression in non-dysplastic Barrett's esophageal cell lines, but overexpressed in high-grade dysplastic and EAC cells. Similarly, APE1 expression was low in non-dysplastic Barrett's esophageal tissue, but high in EAC tissue. We investigated the regulation of APE1 by acidic bile salts (pH 4.0 and 5.5) in CPB and OE33 cells, and observed a transient induction of APE1 protein expression in response to acidic bile salts in a time-dependent manner. We examined the role of APE1 in acidic bile salts-induced DNA damage repair by comet and AP sites assays in CPB and OE33 cells. The data showed that the reconstitution APE1 expression decreased acidic bile salts-induced DNA damage, as indicated by comet and AP sites, in both cell lines. We further studied the role of APE1 in DNA damage repair by immunostaining of p-H2AX (double-strand DNA damage marker) and 8-OXO deoxyguanosine (oxidative DNA damage S-309 AGA Abstracts marker). Our data indicated that the reconstitution of APE1 in OE33 cells decreased p- H2AX (S139) and 8-OXO deoxyguanosine staining in response to acidic bile salts. Conversely, knockdown of APE1 in FLO-1 cells increased p-H2AX and 8-OXO deoxyguanosine staining in response to acidic bile salts. We examined the role of APE1 in regulating apoptosis by Annexin V staining. Our data indicated that the reconstitution of APE1 in OE33 cells protected against bile-salts induced DNA damage and cell death (P<0.01). In contrast, knockdown of APE1 in FLO-1 cells led to significant increases in DNA damage and cell death in response to acidic bile salts (P<0.01). Accordingly, Western blot analysis showed higher protein levels of cleaved caspase-3, cleaved PARP, and p-JNK (T183/Y185)/p-p38 (T180/Y182) in control than APE1-overexpressing OE33 cells in response to acidic bile salts. Conversely, knockdown of APE1 in FLO-1 cells reversed these effects. Conclusions: Collectively, our data suggest that APE1 promotes cell survival in response to acidic bile salts through enhancing DNA damage repair. This ultimately suppresses DNA damage response kinases, reducing the JNK/p38 pro-apoptotic pathways in EAC. Sa1841 Increased Esophageal Transforming Growth Factor α Secretion in Patients With Barrett's Esophagus: Repair of Excessive Injury or Promotion of Cancerogenesis? Marek Majewski, Tomasz Skoczylas, Grzegorz Wallner, Jerzy Sarosiek Gastroesophageal reflux disease represents a spectrum of clinical manifestations, starting from negative endoscopically reflux disease (NERD), sometimes presenting also as reflux esophagitis (RE) and in 3% to 15% ending on Barrett' esophagus (BE). Although the mean value of the esophageal acid exposure is greater in BE than in NERD, individual values in both groups overlap greatly. It is well established that the esophageal mucosal integrity depends upon the balance between aggressive factors and protective mechanisms (Majewski M. et al. Clin. Gastroenterol. & Hepatol. 5:430-8, 2007). Mucosal integrity peptides are pivotal in maintaining mucosal strength resisting the injury by the luminal chemical or physical insults. The leading agent within the family of these integrity peptide growth factors is transforming growth factor α (TGFα), secreted by the esophageal submucosal glands that remain under control of serotonergic (5HT-4) pathway in humans. Mounting evidence supports the role of TGFα in malignant transformation. If so, the rate of TGFα secretion, in patients with BE should be significantly higher than in patients with NERD. Aim: To explore the rate of secretion of esophageal TGFα in patients with BE and to compare with corresponding values recorded in patients with NERD. Subjects & Methods: The study was approved by IRB and conducted in 10 patients (4F, mean age of 40) with NERD and positive 24h esophageal pH monitoring test and in 10 patients (3F, mean age of 49) with long segment (over 3cm) of BE. Esophageal secretion was explored using the esophageal perfusion catheter (Wilson Cook Med. NC) during the mucosal challenge with initial NaCl, HCl/Pepsin and final NaCl, mimicking the natural gastroesophageal reflux scenario. TGFα in esophageal secretions was measured using a commercially available RIA kit (Biomed. Technol.Stoughton, MA). Statistical analysis of results (Mean ±SEM) was performed using Σ-Stat (SyStat Inc., CA). Results: The rate of TGFα secretion in patients with BE was 85% higher in basal conditions (initial saline) than in NERD (0.61 ± 0.08 vs. 0.33 ± 0.10 ng/ min, p<0.05). The rate, however, of TGFα secretion in patients with BE during mucosal exposure to HCl/pepsin (pH 2.1) became 140% higher than in NERD (0.60 ± 0.09 vs. 0.25 ± 0.10 ng/min, p<0.01) and TGFα secretion in BE remained increased by 256% during exposure to the final saline (0.57 ± 0.06 vs. 0.16 ± 0.07 ng/min, p<0.001). Conclusions: 1. Significantly higher rate of esophageal TGFα secretion in patients with BE, especially during mucosal exposure to HCl/pepsin, mimicking the natural gastroesophageal reflux scenario reflects excessive response of the intestinal metaplasia epithelium to the luminal challenge. 2. The role of this enhancing TGFα secretion phenomenon in the development of dysplasia and subsequent adenocarcinoma substantiates further investigation. Sa1842 In Esophageal Epithelial Cells From Barrett's Patients, Protein Kinase a Phosphorylates SOX9, Enhancing Its Binding to Target Gene Promoters: A Potential Role for SOX9 in Barrett's Pathogenesis Anjana Tiwari, Peiguo Ding, Yuji Tamagawa, Edaire Cheng, Qiuyang Zhang, Thai H. Pham, Stuart J. Spechler, Rhonda F. Souza, David H. Wang Introduction: Barrett's esophagus results from a phenotypic switch from squamous to colum- nar epithelium in the distal esophagus in response to gastroesophageal reflux disease (GERD). The molecular mechanisms underlying this phenotypic switch are not known, but probably involve GERD-induced alterations in the expression of key developmental transcription factors. In earlier studies, we demonstrated that the transcription factor SOX9 is expressed in Barrett's metaplasia, but not in normal esophageal squamous epithelium, and that SOX9 induces expression of columnar cytokeratins (CK) 8 and 18. Thus, SOX9 might be involved in Barrett's pathogenesis. In non-esophageal cells, protein kinase A (PKA) has been shown to phosphorylate SOX9 at serines (Ser) 64 and 181, thereby enhancing SOX9 binding to promoter regions of its target genes. We sought to determine whether SOX9 is phosphorylated in esophageal epithelial cells and, if so, the mechanisms regulating this phosphorylation. Methods: We used telomerase-immortalized, non-neoplastic esophageal squamous (NES- B3T and NES-B10T) and metaplastic columnar (BAR-10T) epithelial cells derived from Barrett's patients. Western blotting was used to assess total and phospho-SOX9 expression following electroporation with wild-type SOX9 or transfection with SOX9 siRNA. Chromatin immunoprecipitation (ChIP) was performed with a total SOX9 antibody and with primers for CK8 and CK18. PKA activity was induced with forskolin or inhibited with a cAMP- dependent protein kinase inhibitor (PKI). We mutated both PKA-specific phosphorylation sites of SOX9 (Ser64 and Ser181) to determine the effect of phosphorylation on DNA binding (using a ChIP assay), and on nuclear translocation (using immunofluorescence and Western blotting). Results: The squamous and the metaplastic columnar esophageal cell lines all expressed cytokeratins 8/18 and phosphorylated SOX9. Electroporation with SOX9 led to increased total and phospho-SOX9 and increased CK8/18 expression, while siRNA knock- down of SOX9 led to decreased total and phospho-SOX9 and decreased CK8/18 expression. Forskolin (the PKA inducer) increased SOX9 phosphorylation while PKI (the PKA inhibitor) AGA Abstracts
Transcript of Sa1841 Increased Esophageal Transforming Growth Factor α Secretion in Patients With Barrett's...
type mice had no visible nodules or irregularities at the squamo-columnar junction (SCJ)in response to DCA treatment. 14% of the K14-Cdx2 mice treated with DCA developedtwo nodules each at the SCJ, whereas none of the K14-Cdx2 mice receiving water alonedeveloped nodules. In contrast, 70% of the IL-1β mice, and an equal number of Cdx2/IL-1β mice developed nodular disease at the SCJ after DCA treatment. All DCA treatment-associated nodules were histologically consistent with metaplasia. However overall the dou-ble-transgenic mice had half as many nodules as IL-1β mice. Cdx2 did not affect IL-1βmRNA levels in the double transgenic mice, nor was the inflammation associated with IL-1β expression reduced in the Cdx2/IL-1β mice compared to IL-1β mice, as determined byCD45R+ cell infiltration in the esophagi and forestomach. Moreover, cell proliferation rateswere not altered by Cdx2 expression, as EdU incorporation was not significantly different.Lastly, the morphologic appearance of the nodules in all three lines, K14-Cdx2, L2-IL-1β,and K14-Cdx2/L2-IL-1β mice was similar, with abundant alcian blue expressing goblet cellsdetected, along with expression of other markers of intestinalization. Further molecularcharacterization of the metaplastic nodules by cDNA microarray profiling is underway.Conclusion: Ectopic expression of Cdx2 in the IL-1Beta mouse model for BE reduces thenodular intestinal metaplasia disease burden. These studies raise new questions as to thespecific role of inflammation and intestinal transcription factors in the development ofBarrett's esophagus in humans.
Sa1839
Atonal Homolog 1 (ATOH1) Upregulates Gastric- and Intestinal-Type ProteinExpression in Esophageal Squamous Cells, and Increases Proliferation inBarrett's Cells: Potential Roles for ATOH1 in Barrett's Pathogenesis andCarcinogenesisYuji Tamagawa, Norihisa Ishimura, Anjana Tiwari, Peiguo Ding, Goichi Uno, MasahitoAimi, Naoki Oshima, Shunji Ishihara, Yoshikazu Kinoshita, Stuart J. Spechler, Rhonda F.Souza, David H. Wang
Introduction: In the intestine, stem cell differentiation is mediated by the Notch receptorsignaling pathway, which interacts with atonal homolog 1 (ATOH1), a basic helix-loop-helix transcription factor. Barrett's metaplasia is characterized by intestinal differentiationwith goblet cells. Goblet cell differentiation occurs when there is low Notch activity andhigh ATOH1 activity. In earlier studies, we reported that bile acids suppressed Notchsignaling in esophageal squamous epithelial cells by increasing ATOH1, which led to theirexpression of Cdx2 and MUC2 (intestinal-type proteins found in Barrett's metaplasia). Incertain non-esophageal cells, ATOH1 has been shown to induce the expression of MUC5ACand MUC6 (gastric-type mucins also found in Barrett's metaplasia), and to regulate cellproliferation. In this study, we sought to determine whether ATOH1 regulates expressionof MUC5AC and MUC6 in human esophageal squamous and Barrett's epithelial cells, andif ATOH1 regulates proliferation in Barrett's cells. Methods: We used human esophagealsquamous epithelial cell lines (Het-1A, NES-B3T, and NES-B10T) and Barrett's epithelialcell lines (BAR-10T and CP-A). The expression of ATOH1, Cdx2, MUC5AC, MUC6, andMUC2 was examined using real-time PCR and Western blot under basal conditions. Theexpression of these genes also was determined by real-time PCR following treatment witha gamma-secretase inhibitor (GSI, which increases ATOH1), with siRNA-mediated knock-down of ATOH1, or with GSI in combination with siRNA-mediated knockdown of Cdx2.GSI effects on cell proliferation were determined by MTS assay. Results: Under basalconditions, expression levels of ATOH1, Cdx2 and MUC2 were higher in the Barrett's cellsthan in the squamous cell lines. In Het-1A squamous cells, GSI treatment (which increasedexpression of ATOH1) increased Cdx2, MUC2, MUC5AC, and MUC6 expression. In CP-ABarrett's cells, knockdown of ATOH1 by siRNA transfection significantly decreased MUC5ACand MUC6 expression. CP-A cells treated with the combination of GSI and Cdx2 knockdownby siRNA still showed significantly increased expression of MUC5AC and MUC6. BAR-10T and CP-A cells treated with GSI showed increased expression of ATOH1, which wasaccompanied by a significant increase in cell growth. Conclusion: ATOH1 upregulates theexpression of intestinal- and gastric-type proteins (Cdx2, MUC2, MUC5AC, and MUC6) inesophageal squamous cells, and ATOH1 functions as a transcriptional regulator of gastric-type mucins, independent of Cdx2, in Barrett's metaplastic cells. These findings support apotential role for ATOH1 in the pathogenesis of Barrett's esophagus. Our observation thatincreased ATOH1 expression was associated with increased proliferation in Barrett's cellsalso suggests a potential role for ATOH1 in the neoplastic progression of Barrett's metaplasia.
Sa1840
APE1 Suppresses Acidic Bile Salts-Induced Cell Death Through Regulation ofJNK/p38 Pathways in Esophageal AdenocarcinomaJun Hong, Zheng Chen, DunFa Peng, Abbes Belkhiri, Wael El-Rifai
Background: Over the last three decades the incidence of esophageal adenocarcinoma (EAC)has increased at an alarming rate in Western countries. The development of EAC has beenassociated with a precancerous lesion in the distal oesophagus, termed Barrett's esophagus(BE). Gastroesophageal reflux disease (GERD), a chronic exposure of the esophagus to acidand bile salts from the stomach, is a risk factor for BE and EAC. Apurinic/apyrimidinicendonuclease (APE1) is an enzyme involved in the DNA base excision repair. The aim ofthis study was to investigate the role of APE1 in acidic bile salts-induced DNA damageand elucidate the underlying mechanism in EAC. Methods and Results: Western blot andimmunohistochemistry data indicated very low to undetectable APE1 protein expression innon-dysplastic Barrett's esophageal cell lines, but overexpressed in high-grade dysplasticand EAC cells. Similarly, APE1 expression was low in non-dysplastic Barrett's esophagealtissue, but high in EAC tissue. We investigated the regulation of APE1 by acidic bile salts(pH 4.0 and 5.5) in CPB and OE33 cells, and observed a transient induction of APE1 proteinexpression in response to acidic bile salts in a time-dependent manner. We examined therole of APE1 in acidic bile salts-induced DNA damage repair by comet and AP sites assaysin CPB and OE33 cells. The data showed that the reconstitution APE1 expression decreasedacidic bile salts-induced DNA damage, as indicated by comet and AP sites, in both cell lines.We further studied the role of APE1 in DNA damage repair by immunostaining of p-H2AX(double-strand DNA damage marker) and 8-OXO deoxyguanosine (oxidative DNA damage
S-309 AGA Abstracts
marker). Our data indicated that the reconstitution of APE1 in OE33 cells decreased p-H2AX (S139) and 8-OXO deoxyguanosine staining in response to acidic bile salts. Conversely,knockdown of APE1 in FLO-1 cells increased p-H2AX and 8-OXO deoxyguanosine stainingin response to acidic bile salts. We examined the role of APE1 in regulating apoptosis byAnnexin V staining. Our data indicated that the reconstitution of APE1 in OE33 cellsprotected against bile-salts induced DNA damage and cell death (P<0.01). In contrast,knockdown of APE1 in FLO-1 cells led to significant increases in DNA damage and celldeath in response to acidic bile salts (P<0.01). Accordingly, Western blot analysis showedhigher protein levels of cleaved caspase-3, cleaved PARP, and p-JNK (T183/Y185)/p-p38(T180/Y182) in control than APE1-overexpressing OE33 cells in response to acidic bilesalts. Conversely, knockdown of APE1 in FLO-1 cells reversed these effects. Conclusions:Collectively, our data suggest that APE1 promotes cell survival in response to acidic bilesalts through enhancing DNA damage repair. This ultimately suppresses DNA damageresponse kinases, reducing the JNK/p38 pro-apoptotic pathways in EAC.
Sa1841
Increased Esophageal Transforming Growth Factor α Secretion in PatientsWith Barrett's Esophagus: Repair of Excessive Injury or Promotion ofCancerogenesis?Marek Majewski, Tomasz Skoczylas, Grzegorz Wallner, Jerzy Sarosiek
Gastroesophageal reflux disease represents a spectrum of clinical manifestations, startingfrom negative endoscopically reflux disease (NERD), sometimes presenting also as refluxesophagitis (RE) and in 3% to 15% ending on Barrett' esophagus (BE). Although the meanvalue of the esophageal acid exposure is greater in BE than in NERD, individual values inboth groups overlap greatly. It is well established that the esophageal mucosal integritydepends upon the balance between aggressive factors and protective mechanisms (MajewskiM. et al. Clin. Gastroenterol. & Hepatol. 5:430-8, 2007). Mucosal integrity peptides arepivotal in maintaining mucosal strength resisting the injury by the luminal chemical orphysical insults. The leading agent within the family of these integrity peptide growth factorsis transforming growth factor α (TGFα), secreted by the esophageal submucosal glands thatremain under control of serotonergic (5HT-4) pathway in humans. Mounting evidencesupports the role of TGFα in malignant transformation. If so, the rate of TGFα secretion,in patients with BE should be significantly higher than in patients with NERD. Aim: Toexplore the rate of secretion of esophageal TGFα in patients with BE and to compare withcorresponding values recorded in patients with NERD. Subjects & Methods: The studywas approved by IRB and conducted in 10 patients (4F, mean age of 40) with NERD andpositive 24h esophageal pH monitoring test and in 10 patients (3F, mean age of 49) withlong segment (over 3cm) of BE. Esophageal secretion was explored using the esophagealperfusion catheter (Wilson Cook Med. NC) during the mucosal challenge with initial NaCl,HCl/Pepsin and final NaCl, mimicking the natural gastroesophageal reflux scenario. TGFαin esophageal secretions was measured using a commercially available RIA kit (Biomed.Technol.Stoughton, MA). Statistical analysis of results (Mean ±SEM) was performed usingΣ-Stat (SyStat Inc., CA). Results: The rate of TGFα secretion in patients with BE was 85%higher in basal conditions (initial saline) than in NERD (0.61 ± 0.08 vs. 0.33 ± 0.10 ng/min, p<0.05). The rate, however, of TGFα secretion in patients with BE during mucosalexposure to HCl/pepsin (pH 2.1) became 140% higher than in NERD (0.60 ± 0.09 vs. 0.25± 0.10 ng/min, p<0.01) and TGFα secretion in BE remained increased by 256% duringexposure to the final saline (0.57 ± 0.06 vs. 0.16 ± 0.07 ng/min, p<0.001). Conclusions:1. Significantly higher rate of esophageal TGFα secretion in patients with BE, especiallyduring mucosal exposure to HCl/pepsin, mimicking the natural gastroesophageal refluxscenario reflects excessive response of the intestinal metaplasia epithelium to the luminalchallenge. 2. The role of this enhancing TGFα secretion phenomenon in the developmentof dysplasia and subsequent adenocarcinoma substantiates further investigation.
Sa1842
In Esophageal Epithelial Cells From Barrett's Patients, Protein Kinase aPhosphorylates SOX9, Enhancing Its Binding to Target Gene Promoters: APotential Role for SOX9 in Barrett's PathogenesisAnjana Tiwari, Peiguo Ding, Yuji Tamagawa, Edaire Cheng, Qiuyang Zhang, Thai H.Pham, Stuart J. Spechler, Rhonda F. Souza, David H. Wang
Introduction: Barrett's esophagus results from a phenotypic switch from squamous to colum-nar epithelium in the distal esophagus in response to gastroesophageal reflux disease (GERD).The molecular mechanisms underlying this phenotypic switch are not known, but probablyinvolve GERD-induced alterations in the expression of key developmental transcriptionfactors. In earlier studies, we demonstrated that the transcription factor SOX9 is expressedin Barrett's metaplasia, but not in normal esophageal squamous epithelium, and that SOX9induces expression of columnar cytokeratins (CK) 8 and 18. Thus, SOX9 might be involvedin Barrett's pathogenesis. In non-esophageal cells, protein kinase A (PKA) has been shownto phosphorylate SOX9 at serines (Ser) 64 and 181, thereby enhancing SOX9 binding topromoter regions of its target genes. We sought to determine whether SOX9 is phosphorylatedin esophageal epithelial cells and, if so, the mechanisms regulating this phosphorylation.Methods: We used telomerase-immortalized, non-neoplastic esophageal squamous (NES-B3T and NES-B10T) and metaplastic columnar (BAR-10T) epithelial cells derived fromBarrett's patients. Western blotting was used to assess total and phospho-SOX9 expressionfollowing electroporation with wild-type SOX9 or transfection with SOX9 siRNA. Chromatinimmunoprecipitation (ChIP) was performed with a total SOX9 antibody and with primersfor CK8 and CK18. PKA activity was induced with forskolin or inhibited with a cAMP-dependent protein kinase inhibitor (PKI). We mutated both PKA-specific phosphorylationsites of SOX9 (Ser64 and Ser181) to determine the effect of phosphorylation on DNA binding(using a ChIP assay), and on nuclear translocation (using immunofluorescence and Westernblotting). Results: The squamous and the metaplastic columnar esophageal cell lines allexpressed cytokeratins 8/18 and phosphorylated SOX9. Electroporation with SOX9 led toincreased total and phospho-SOX9 and increased CK8/18 expression, while siRNA knock-down of SOX9 led to decreased total and phospho-SOX9 and decreased CK8/18 expression.Forskolin (the PKA inducer) increased SOX9 phosphorylation while PKI (the PKA inhibitor)
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