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Severe acute respiratory syndrome coronavirus 2 causes diffuse alveolar damage (DAD), lymphocyte infiltration in the lungs and a cytokine storm. In this study we examined inflammatory cell infiltrates and the expression of signal transducer and activator of transcription (STAT) 6 in the lungs of patients with coronavirus disease 2019 (COVID-19).Eighteen COVID-19 autopsy cases, 9 non-COVID cases with DAD, and 11 controls without lung diseases were included. Immunostainings for STAT6, CD3, CD4, CD8, CD68, and broad-spectrum keratins were performed.The average age of COVID-19 patients was 64.4±2.1 years. The disease duration was 7 to 53 days. The number of pneumocytes, macrophages or CD3+ T cells was significantly increased in the lungs of patients with COVID-19. Patients' age above 67 years, blood troponin levels0.2 ng/mL, platelet count100×109/L, lung macrophages130/high-power field (HPF), CD3+ T cells145/HPF, CD8+ T cells30/HPF, and CD8/CD4 ratio1 were associated with shorter survival duration after onset of symptoms. In addition, STAT6 staining was much stronger in pneumocytes and lymphocytes in the lungs of patients with COVID-19 than non-COVID DAD patients or controls.Older age, high blood troponin level and platelet count, more macrophages and fewer CD8+ T cells in the lungs of COVID-19 were associated with poorer outcome. STAT6 expression was increased in pneumocytes and lymphocytes in the lungs of patients with COVID-19, implying a role of STAT6 in cytokine storms.

Supplementary Table S3. Clinicopathologic features of patients with gastric cancer according to the expression status of TMEFF2.

Purpose: The role and clinical implication of the transmembrane protein with EGF and two follistatin motifs 2 (TMEFF2) in gastric cancer is poorly understood.Experimental Design: Gene expression profile analyses were performed and Gene Set Enrichment Analysis (GSEA) was used to explore its gene signatures. AGS and MKN45 cells were transfected with TMEFF2 or control plasmids and analyzed for gene expression patterns, proliferation, and apoptosis. TMEFF2 expression was knocked down with shRNAs, and the effects on genome stability were assessed. Interactions between TMEFF2 and SHP-1 were determined by mass spectrometry and immunoprecipitation assays.Results: Integrated analysis revealed that TMEFF2 expression was significantly decreased in gastric cancer cases and its expression was negatively correlated with the poor pathologic stage, large tumor size, and poor prognosis. GSEA in The Cancer Genome Atlas (TCGA) and Jilin datasets revealed that cell proliferation, apoptosis, and DNA damage–related genes were enriched in TMEFF2 lower expression patients. Gain of TMEFF2 function decreased cell proliferation by increasing of apoptosis and blocking of cell cycle in gastric cancer cells. The protein tyrosine phosphatase SHP-1 was identified as a binding partner of TMEEF2 and mediator of TMEFF2 function. TMEFF2 expression positively correlated with SHP-1, and a favorable prognosis was more likely in patients with gastric cancer with higher levels of both TMEFF2 and SHP-1.Conclusion: TMEFF2 acts as a tumor suppressor in gastric cancer through direct interaction with SHP-1 and can be a potential biomarker of carcinogenesis. Clin Cancer Res; 20(17); 4689–704. ©2014 AACR.

Supplementary Figure S1. Identification of TMEFF2 as a key regulator in gastric cancer progression and the expression of TMEFF2 decreased in gastric carcinogenesis. Supplementary Figure S2. Gene Set Enrichment Analysis (GSEA) in TMEFF2-higher/lower expression patients. Supplementary Figure S3. The mRNA levels of the top-score tumorigenesis genes were measured in gastric cells after transfection of TMEFF2 overexpression plasmid or siRNA Supplementary Figure S4. Functional impact of TMEFF2 Supplementary Figure S5. Cell proliferation assays were performed in MKN45 cells after overexpression of SHP-1WT/ΔSH2D1/ΔSH2D2/ΔPTPD or these constructs combined with TMEFF2. n = 3, non-parametric Mann-Whitney test. Supplementary Figure S6. Correlation of TMEFF2 and SHP-1 expression in human GC.

Programmed death-1 (PD1) expression has not been reported in gallbladder adenocarcinoma. In this study we examined PD1 expression in gallbladder cancer to explore the correlation between PD1 expression and the clinicopathologic parameters. We found that 98% (46/47) cases expressed programmed death-ligand 1 (PD-L1) with 85% cases being PD-L1 3+. PD1+ tumor-infiltrating lymphocytes (TILs) were present in 78.7% cases (37/47). The tumor size was significantly smaller and the stromal CD3+ TILs were significantly higher in tumors with PD1+ TILs than those with PD1- TILs. In the tumors with size of3 cm, stromal CD3+ TILs115/HPF or stromal CD8+ TILs45/HPF were associated with much better survival than those with stromal CD3+ TILs ≤115/HPF or stromal CD8+ TILs ≤45/HPF. In tumors with the size of 3 cm or larger, PD1+ TILs or stromal CD8+ TILs45/HPF carried a significantly poorer survival than PD1- tumors or stromal CD8+ TILs=45/HPF. No correlation was identified between PD1 expression and lymphovascular invasion, distant metastasis, pathologic tumor stage or prognostic stage. Multivariate survival analysis showed that tumor TNM stage and age were independent prognostic factors in gallbladder adenocarcinomas. We conclude that gallbladder adenocarcinomas may have high PD-L1 expression and PD1+ TILs. Smaller tumor size and greater amount of stromal CD3+ T cells were found in tumors with PD1+ TILs. In small tumors (3 cm), high stromal CD3+ TILs or high stromal CD8+ TILs were associated with better survival. However, in large tumors (≥3 cm), PD1+ TILs or high stromal CD8+ TILs carried a poorer survival. Our study implied that immune-based therapy including PD1/PD-L1 checkpoint blockade might be useful in gallbladder adenocarcinomas.

Autopsy training is required for board certification by the American Board of Pathology and may be affected by autopsy rate. It is unclear whether the COVID-19 pandemic has affected autopsy education and rate. Prior to the pandemic, our autopsy gross organ review lectures at the Brown University pathology residency program were in-person and used a detective style to discover the pathological lesions followed by an integrated discussion of anatomic and clinical pathology. During the COVID-19 pandemic, these lectures became remote and there was a noticeable impact on the involvement and responsiveness of the audience compared to in-person teaching. Certain qualities of face-to-face teaching can be difficult to be reproduced through virtual teaching, including the detective style to look for pathological lesions and the ability to palpate lesions from gross specimens. Our results showed that the autopsy case number increased during the COVID-19 pandemic, but the overall autopsy rate did not significantly change.

Coronavirus disease 2019 (COVID-19) may cause a hypercoagulability state and thrombotic complications. Multiorgan infarctions in young patients are very rare. Here we report a 35-year-old male patient with COVID-19 complicated by multiorgan infarctions. The patient had a past medical history of uncontrolled insulin-dependent diabetes mellitus and was admitted to the intensive care unit with progressive hypoxia in the setting of SARS-CoV-2 infection. The patient received prophylactic anticoagulant during the entire hospital course. During the hospitalization, the patient developed hypoxic respiratory arrest, diffuse anoxic brain injury and brain herniation. Postmortem examination demonstrated multiple infarctions and thromboses involving the heart, bilateral lungs, kidneys, and spleen. In conclusion, multiple organ infarctions may occur in young patients with COVID-19 despite prophylactic anticoagulation therapy.

Background Pulmonary tumor embolism (PTE) is difficult to detect before death, and it is unclear whether the discrepancy between antemortem clinical and postmortem diagnosis improves with the advance of the diagnostic technologies. In this study we determined the incidence of PTE and analyzed the discrepancy between antemortem clinical and postmortem diagnosis. Methods We performed a retrospective autopsy study on patients with the history of malignant solid tumors from 1990 to 2020 and reviewed all the slides of the patients with PTE. We also analyzed the discrepancies between antemortem clinical and postmortem diagnosis in 1999, 2009 and 2019 by using the Goldman criteria. Goldman category major 1 refers to cases in which an autopsy diagnosis was the direct cause of death and was not recognized clinically, but if it had been recognized, it may have changed treatment or prolonged survival. Results We found 20 (3%) cases with PTE out of the 658 autopsy cases with solid malignancies. Out of these 20 cases, urothelial carcinoma (30%, 6/20) and invasive ductal carcinoma of the breast (4/20, 20%) were the most common primary malignancies. Seven patients with shortness of breath died within 3–17 days (average 8.4±2.2 days) after onset of the symptoms. Pulmonary embolism was clinically suspected in seven out of twenty (35%, 7/20) patients before death, but only two patients (10, 2/20) were diagnosed by imaging studies before death. The rate of Goldman category major 1 was 13.2% (10/76) in 1999, 7.3% (4/55) in 2009 and 6.9% (8/116) in 2019. Although the rate of Goldman category major 1 appeared decreasing, the difference was not statistically significant. The autopsy rate was significantly higher in 2019 (8.4%, 116/1386) than in 2009 (4.4%, 55/1240). Conclusions The incidence of PTE is uncommon. Despite the advances of the radiological techniques, radiological imaging studies did not detect the majority of PTEs. The discrepancy between the antemortem clinical and the postmortem diagnosis has not improved significantly over the past 30 years, emphasizing the value of autopsy.

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is ubiquitously expressed and essential for the development and/or maintenance of multiple tissues and organs. SHP2 is involved in gastrointestinal (GI) epithelium development and homeostasis, but the underlying mechanisms remain elusive. While studying SHP2's role in skeletal development, we made osteoblast-specific SHP2 deficient mice using Osterix (Osx)-Cre as a driver to excise Ptpn11 floxed alleles. Phenotypic characterization of these SHP2 mutants unexpectedly revealed a critical role of SHP2 in GI biology. Mice lacking SHP2 in Osx+ cells developed a fatal GI pathology with dramatic villus hypoplasia. OSTERIX, an OB-specific zinc finger-containing transcription factor is for the first time found to be expressed in GI crypt cells, and SHP2 expression in the crypt Osx+ cells is critical for self-renewal and proliferation. Further, immunostaining revealed the colocalization of OSTERIX with OLFM4 and LGR5, two bona fide GI stem cell markers, at the crypt cells. Furthermore, OSTERIX expression is found to be associated with GI malignancies. Knockdown of SHP2 expression had no apparent influence on the relative numbers of enterocytes, goblet cells or Paneth cells. Given SHP2's key regulatory role in OB differentiation, our studies suggest that OSTERIX and SHP2 are indispensable for gut homeostasis, analogous to SOX9's dual role as a master regulator of cartilage and an important regulator of crypt stem cell biology. Our findings also provide a foundation for new avenues of inquiry into GI stem cell biology and of OSTERIX's therapeutic and diagnostic potential.

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is ubiquitously expressed and essential for the development and/or maintenance of multiple tissues and organs. SHP2 is involved in gastrointestinal (GI) epithelium development and homeostasis, but the underlying mechanisms remain elusive. While studying SHP2's role in skeletal development, we made osteoblast-specific SHP2 deficient mice using Osterix (Osx)-Cre as a driver to excise Ptpn11 floxed alleles. Phenotypic characterization of these SHP2 mutants unexpectedly revealed a critical role of SHP2 in GI biology. Mice lacking SHP2 in Osx

The pathogenesis of eosinophilic esophagitis (EoE) involves Th2-mediated eosinophil recruitment and degranulation into the esophagus. However, measuring serum Th2 cytokines, eosinophils, and eosinophil-derived products does not reliably distinguish EoE from control populations. Non-invasive methods to diagnose EoE are lacking. We evaluated the diagnostic value of a novel candidate biomarker of EoE: 15(S)-hydroxyeicosatetraenoic acid (HETE). We used immunoassay to measure 15(S)-HETE and cytokine profiles in patients undergoing endoscopy with known or suspected EoE. 31 subjects were enrolled, 16 with EoE, and 15 with an alternate diagnosis. 15(S)-HETE was elevated in the EoE group compared to non-EoE group. The sensitivity and specificity of 15(S)-HETE to be used as a non-invasive marker is 50% and 80%, respectively. 15(S)-HETE may aid in the diagnosis of EoE.

Acid reflux may contribute to the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA). However, it is not clear whether the molecular changes present in BE patients are reversible after proton pump inhibitor (PPI) treatment. In this study we examined whether PPI treatment affects NOX5, microsomal prostaglandin E synthase (mPGES)-1 and inducible nitric oxide synthase (iNOS) expression. We found that NADPH oxidase 5 (NOX5), mPGES-1 and iNOS were significantly increased in BE mucosa. One-month PPI treatment significantly decreased NOX5, mPGES1 and iNOS. In BAR-T cells, NOX5 mRNA and p16 promoter methylation increased after pulsed acid treatment in a time-dependent manner. Four or eight-week-acid induced increase in NOX5 mRNA, NOX5 protein and p16 methylation may be reversible. Twelve-week acid treatment also significantly increased NOX5, mPGES1 and iNOS mRNA expression. However, twelve-week-acid-induced changes only partially restored or did not recover at all after the cells were cultured at pH 7.2 for 8 weeks. We conclude that NOX5, mPGES1 and iNOS may be reversible after PPI treatment. Short-term acid-induced increase in NOX5 expression and p16 methylation might be reversible, whereas long-term acid-induced changes only partially recovered 8 weeks after removal of acid treatment.

AIM To examined the bile acid receptor TGR5 expression in squamous mucosa, Barrett’s mucosa, dysplasia and esophageal adenocarcinoma (EA). METHODS Slides were stained with TGR5 antibody. The staining intensity was scored as 1+, 2+ and 3+. The extent of staining (percentage of cells staining) was scored as follows: 1+, 1%-10%, 2+, 11%-50%, 3+, 51%-100%. A combined score of intensity and extent was calculated and categorized as negative, weak, moderate and strong staining. TGR5 mRNA was measured by real time PCR. RESULTS We found that levels of TGR5 mRNA were significantly increased in Barrett’s dysplastic cell line CP-D and EA cell line SK-GT-4, when compared with Barrett’s cell line CP-A. Moderate to strong TGR5 staining was significantly higher in high-grade dysplasia and EA cases than in Barrett’s esophagus (BE) or in low-grade dysplasia. Moderate to strong staining was slightly higher in low-grade dysplasia than in BE mucosa, but there is no statistical significance. TGR5 staining had no significant difference between high-grade dysplasia and EA. In addition, TGR5 staining intensity was not associated with the clinical stage, the pathological stage and the status of lymph node metastasis. CONCLUSION We conclude that TGR5 immunostaining was much stronger in high-grade dysplasia and EA than in BE mucosa or low-grade dysplasia and that its staining intensity was not associated with the clinical stage, the pathological stage and the status of lymph node metastasis. TGR5 might be a potential marker for the progression from BE to high-grade dysplasia and EA.

Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid-induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA, and increased cell proliferation, effects that may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and OE33 and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increases in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-κB1 p50. Conversely, overexpression of NOX5-S, p50, or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFκB-Luc. An NF-κB binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA, and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-κB1 p50.

The polycomb group protein enhancer of zeste homologue 2 (EZH2), which has histone methyltransferase (HMT) activity, is overexpressed in malignant tumours. However, the role of EZH2 in colorectal cancer (CRC) invasion is little known. Here we investigated the clinical significance, biological effects, and mechanisms of EZH2 signalling. Knockdown of EZH2 significantly reduced cell invasion and secretion of matrix metalloproteinases 2/9 (MMP2/9) in in vitro studies. Knockdown of EZH2 dramatically increased overall survival and decreased metastasis of lung in in vivo studies. Conversely, overexpression of EZH2 significantly increased lung metastasis and shortened overall survival when compared with control tumours. EZH2-induced CRC cell invasion may depend on down-regulation of vitamin D receptor (VDR), which is considered to be a marker of CRC invasion. EZH2 regulates the histone trimethylation of lysine 27 (H3K27me3) in the VDR promoter. Moreover, we found that STAT3 directly binds to the EZH2 promoter and regulates VDR down-regulation in CRC cells. Significant inverse correlations were observed between the expression of EZH2 and pSTAT3 and that of VDR in CRC tissues compared with normal tissue in patients. We show the role of EZH2 in CRC metastasis and identify VDR as a target gene of EZH2. EZH2 expression may be directly regulated by STAT3, and STAT3 may play an important role in EZH2-mediated VDR down-regulation in CRC. This pathway may provide potential targets in aggressive CRC.

The mechanisms whereby bile acid reflux may accelerate the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett’s patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA.

We have shown that NADPH oxidase (NOX)5-S may mediate the acid-induced decrease in cell apoptosis. However, mechanisms of NOX5-S–dependent decrease in cell apoptosis are not fully understood. In this study, we found that silencer-of-death domain (SODD) was significantly increased in esophageal adenocarcinoma (EA) tissues, EA cell lines FLO and OE33, and a dysplastic cell line CP-B. Strong SODD immunostaining was significantly higher in low-grade dysplasia (66.7%), high-grade dysplasia (81.2%), and EA (71.2%) than in Barrett’s mucosa (10.5%). Acid treatment significantly increased SODD protein and mRNA expression and promoter activity in FLO cells, an increase that was significantly decreased by the knockdown of NOX5-S and nuclear factor κB (NF-κB)1 p50 with their small interfering RNAs. Similarly, acid-induced increase of SODD mRNA was blocked by knockdown of NOX5-S and p50 in a BE cell line CP-A. Overexpression of NOX5-S significantly increased SODD protein expression in FLO cells. Moreover, overexpression of NOX5-S or p50 significantly increased the SODD promoter activity and decreased the caspase 9 activity or apoptosis. NOX5-S overexpression-induced increase in SODD promoter activity was significantly decreased by knockdown of p50. In addition, acid treatment significantly decreased the caspase 9 activity, a decrease that was significantly inhibited by knockdown of SODD. Furthermore, chromatin immunoprecipitation assay showed that NF-κB1 p50 bound to SODD genomic DNA containing a NF-κB–binding element GGGGACACCCT. This binding element was further confirmed by a gel mobility shift assay. We conclude that acid-induced increase in SODD expression and decrease in cell apoptosis may depend on the activation of NOX5-S and NF-κB1 p50 in FLO cells.

The following on progression to adenocarcinoma and markers of Barrett's esophagus includes commentariess on the expression of claudin 4 in Barrett's adenocarcinoma; the role of acid and bile salts; the role of insulin-like growth factor; the value of reactive oxygen species; the importance of abnormal methylation; genetic alterations in stromal cells and genomic changes in the epithelial cells; the value of confocal laser endomicroscopy for the subsurface analysis of the mucosa; indications for statins as adjuvant chemotherapeutic agent; the sequence of molecular events in malignant progression in Barrett's mucosa; and the value of the macroscopic markers and of p53 mutations.

The following on esophageal disease provides updated information the mucosal defense against acid and acid-pepsin injury; the roles of platelet activating factor, mast cells, proinflammatory cytokines, and chemokines in inflammation; differences and similarities in erosive and nonerosive esophagitis; acid and vanilloid receptors in esophageal mucosa; and bile acid receptors in esophageal epithelium.

We have shown that a novel NADPH oxidase isoform, NOX5-S, is the major isoform of NADPH oxidases in an esophageal adenocarcinoma (EA) cell line, FLO, and is overexpressed in Barrett's mucosa with high-grade dysplasia. NOX5-S is responsible for acid-induced reactive oxygen species production. In this study, we found that mRNA levels of NOX5-S were significantly higher in FLO EA cells than in the normal human esophageal squamous cell line HET-1A or in a Barrett cell line, BAR-T. The mRNA levels of NOX5-S were also significantly increased in EA tissues. The data suggest that NOX5-S may be important in the development of EA. Mechanisms of functional regulation of NOX5-S are not fully understood. We show that small G protein Rac1 was present in HET-1A cells, BAR-T cells, and EA cell lines FLO and OE33. Rac1 protein levels were significantly higher in FLO and OE33 cells than in HET-1A or BAR-T cells. Knockdown of Rac1 with Rac1 small interfering RNA significantly decreased acid-induced increase in H2O2 production in FLO EA cells. Overexpression of constitutively active Rac1 significantly increased H2O2 production, an increase that was blocked by knockdown of NOX5-S. By immunofluorescence staining and immunoprecipitation, we found that NOX5-S was present in the cytosol of FLO EA cells and colocalized with Rac1 and SERCA1/2 Ca2+-ATPase which is located in the endoplasmic reticulum membrane. We conclude that Rac1 may be important in activation of NOX5-S in FLO EA cells.

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

Background and aimsMechanisms of the progression from Barrett's oesophagus to oesophageal adenocarcinoma (OA) are not fully understood. Bile acids may have an important role in this progression. This study aimed at examining the role of NADPH oxidase NOX5-S and a novel bile acid receptor TGR5 in taurodeoxycholic acid (TDCA)-induced increase in cell proliferation.MethodsHuman Barrett's cell line BAR-T and OA cell line FLO were transfected by the Lipofectamine 2000 or Amaxa-Nucleofector-System. mRNAs were measured by real-time PCR. H2O2 was measured by a fluorescent assay. Cell proliferation was determined by measurement of thymidine incorporation.ResultsNOX5-S was present in FLO cells. TDCA significantly increased NOX5-S expression, H2O2 production and thymidine incorporation in FLO and BAR-T cells. This increase in thymidine incorporation was significantly reduced by knockdown of NOX5-S. TGR5 mRNA and protein levels were significantly higher in OA tissues than in normal oesophageal mucosa or Barrett's mucosa. Knockdown of TGR5 markedly inhibited TDCA-induced increase in NOX5-S expression, H2O2 production and thymidine incorporation in FLO and BAR-T cells. Overexpression of TGR5 significantly enhanced the effects of TDCA in FLO cells. TGR5 receptors were coupled with Gαq and Gαi3 proteins, but only Gαq mediated TDCA-induced increase in NOX5-S expression, H2O2 production and thymidine incorporation in FLO cells.ConclusionsTDCA-induced increase in cell proliferation depends on upregulation of NOX5-S expression in BAR-T and FLO cells. TDCA-induced NOX5-S expression may be mediated by activation of the TGR5 receptor and Gαq protein. These data may provide potential targets to prevent and/or treat Barrett's OA.

We have shown that NADPH oxidase NOX5-S is overexpressed in Barrett's esophageal adenocarcinoma (EA) cells and may contribute to the progression from Barrett's esophagus (BE) to EA presumably by increasing cell proliferation and decreasing apoptosis (Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W. J Biol Chem 281: 20368–20382, 2006). The mechanism(s) of NOX5-S overexpression in EA, however, is not fully understood. In SEG1 EA cells we found that acid treatment significantly increased platelet-activating factor (PAF) production, which in turn markedly increased NOX5-S expression and hydrogen peroxide (H2O2) production. Knockdown of NOX5-S by NOX5-S small interfering RNA (siRNA) blocked PAF-dependent H2O2production. PAF-dependent induction of NOX5-S expression and H2O2production were significantly decreased by the MAPK kinase 1 inhibitor PD-98059, by the cytosolic phospholipase A2(cPLA2) inhibitor AACOCF3, and by STAT5 downregulation with STAT5 siRNA. PAF significantly increased the phosphorylation of ERK1/2 MAPK, cPLA2, and STAT5. Using inhibitors, we demonstrated that PAF-induced STAT5 phosphorylation depends on activation of ERK1/2 MAPK and cPLA2, whereas PAF-induced cPLA2phosphorylation was associated with activation of ERK1/2 MAPK. Given that STAT5 bound to the c-sis-inducible element (TTCTGGTAA) of the NOX5-S promoter, overexpression of STAT5 significantly increased NOX5-S promoter activity. We conclude that acid-induced NOX5-S expression and H2O2production is mediated in part by production of PAF in SEG1 EA cells, and that PAF-induced increase in NOX5-S expression depends on sequential activation of ERK MAP kinases, cPLA2, and STAT5 in these cells.

Mechanisms of the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

Gastroesophageal reflux disease complicated by Barrett esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not known. We found that NOX1 and NOX5-S were the major isoforms of NADPH oxidase in SEG1-EA cells. The expression of NOX5-S mRNA was significantly higher in these cells than in esophageal squamous epithelial cells. NOX5 mRNA was also significantly higher in Barrett tissues with high grade dysplasia than without dysplasia. Pulsed acid treatment significantly increased H(2)O(2) production in both SEG1-EA cells and BE mucosa, which was blocked by the NADPH oxidase inhibitor apocynin. In SEG1 cells, acid treatment increased mRNA expression of NOX5-S, but not NOX1, and knockdown of NOX5 by NOX5 small interfering RNA abolished acid-induced H(2)O(2) production. In addition, acid treatment increased intracellular Ca(2+) and phosphorylation of cAMP-response element-binding protein (CREB). Acid-induced NOX5-S expression and H(2)O(2) production were significantly inhibited by removal of extracellular Ca(2+) and by knockdown of CREB using CREB small interfering RNA. Two novel CREB-binding elements TGACGAGA and TGACGCTG were identified in the NOX5-S gene promoter. Overexpression of CREB significantly increased NOX5-S promoter activity. Knockdown of NOX5 significantly decreased [(3)H]thymidine incorporation, which was restored by 10(-13) M H(2)O(2). Knockdown of NOX5 also significantly decreased retinoblastoma protein phosphorylation and increased cell apoptosis and caspase-9 expression. In conclusion, in SEG1 EA cells NOX5-S is overexpressed and mediates acid-induced H(2)O(2) production. Acid-induced NOX5-S expression depends on an increase in intracellular Ca(2+) and activation of CREB. NOX5-S contributes to increased cell proliferation and decreased apoptosis.

In a human in vitro model of esophagitis, we investigated the genesis of esophagitis-associated dysmotility by examining HCl-induced production of inflammatory mediators in the mucosa and investigating their effect on esophageal circular muscle. Muscularis propria was removed from organ donors' esophagi, leaving the mucosal tube intact. The tube was tied at both ends, forming a sac, and filled with HCl at pH 4. After 3 h of incubation, the supernatant surrounding the sac was analyzed or applied to circular muscle strips. HCl alone did not affect circular muscle contraction in response to electrical field stimulation (EFS), but supernatant of HCl-treated mucosa abolished contraction. The inhibition was reversed by the platelet-activating factor (PAF) antagonist CV3988 [(+/-)-3-(N-octadecylcarbamoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate], whereas the PAF analog 2-O-methyl platelet-activating factor C-16 (PAF-16) inhibited EFS-induced contraction and acetylcholine (ACh) release in circular muscle strips. The hydrogen peroxide scavenger catalase reversed the inhibition in contraction, to the same extent as CV3988. We therefore measured PAF and hydrogen peroxide (H(2)O(2)) in mucosa, mucosa supernatant, and circular muscle. HCl increased PAF and interleukin (IL)-1beta (but not IL-6, prostaglandin E(2), or H(2)O(2)) in mucosa, and only PAF was released into the supernatant, presumably to affect circular muscle. In circular muscle, exogenous PAF induced sequential formation of IL-6, H(2)O(2), IL-1beta, and PAF. Release of PAF by the mucosa inhibits ACh release from circular muscle layer neurons and initiates sequential formation of inflammatory mediators in muscle, resulting in production of PAF by the muscle itself, possibly initiating in a self-sustaining cycle.

Human colonic circular muscle produces spontaneous phasic contractions that are reduced in ulcerative colitis. How the spontaneous phasic contractions develop and why they decrease in ulcerative colitis are not known. We found that spontaneous phasic contractions of normal sigmoid circular muscle strips were significantly reduced by 90-min incubation with tetrodotoxin (10–5 M), which blocked neurokinin A release in basal conditions and in response to electrical stimulation. In addition, spontaneous contraction of human sigmoid colon was significantly decreased by the NK2 receptor antagonists MEN10376 (Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2) and NK2ra (Bz-Ala-Ala-d-Trp-Phe-d-Pro-Pro-Nle-NH2) but not by atropine or by the NK1 antagonist FK888 ( N 2-[(4 R )-4-hydroxyl-1-(1-methyl-1 H -indol-3-yl)carbonyl-l-prolyl]- N -methyl- N -phenylmethyl-3-(2-naphthyl)-l-alaninamide), suggesting that NK2 receptors are involved in their development. The spontaneous phasic contractions were abolished by thapsigargin and cyclopiazonic acid and significantly decreased by the protein kinase C inhibitor chelerythrine and by the calmodulin inhibitor CGS9343B (1,3-dihydro-1-[1-[(4-methyl-4 H ,6 H -pyrrolo[1,2- a ]-[4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2 H -benzimidazol-2-one (1:1) maleate), suggesting that spontaneous phasic contractions may be mediated by Ca2+ release from intracellular stores and by a protein kinase C- and calmodulin-dependent pathway. In strips from patients with ulcerative colitis, spontaneous contractions were significantly reduced, and this reduction was partially restored by the hydrogen peroxide scavenger catalase. Neurokinin A release, however, was not affected. We conclude that spontaneous phasic contractions of human sigmoid circular smooth muscle may be mediated by activation of NK2 receptors, calcium release from intracellular stores, and activation of calmodulin and protein kinase C. In ulcerative colitis patients, spontaneous phasic contractions are decreased, and this decrease may be in part due to overproduction of hydrogen peroxide affecting sigmoid circular muscle.

We have previously shown that sigmoid circular muscle cells from patients with ulcerative colitis (UC) exhibit reduced contraction and Ca2+signaling in response to the neurotransmitter neurokinin A (NKA) and that IL-1β and H2O2may contribute to these reduced responses in UC. In addition, we have found that nitric oxide (NO) levels were significantly increased in UC circular muscle. To establish the site of origin for IL-1β, H2O2, and NO, we assembled an in vitro system in which normal or UC mucosa were sealed between two chambers filled with oxygenated Krebs solution. Because the mucosa consists of full-thickness mucosa and submucosa, it is expected that whatever is released into the undernatant from the submucosal side may diffuse to the circular muscle layer in the intact colon. Treatment of normal sigmoid circular muscle cells for 2 h with undernatants collected from the UC submucosal side (UCS) significantly decreased contraction induced by NKA and thapsigargin and the NKA- and caffeine-induced Ca2+signal in Ca2+-free medium. In addition, UC mucosa released into the undernatant on its submucosal side significantly more H2O2, IL-1β, and NO than normal mucosa. The reduction in contraction and Ca2+signal induced by UCS was partially reversed by pretreatment with an IL-1β antibody or with catalase. The NO scavenger hemoglobin partially prevented UCS-induced reduction in contraction and Ca2+signaling in response to NKA but not the reduced response to thapsigargin or caffeine. Sodium nitroprusside inhibited NKA but not the caffeine-induced Ca2+signal. We conclude that in UC the mucosa releases IL-1β, H2O2, and NO, which may contribute to the impaired Ca2+release and altered sigmoid muscle contractility.

ACh is a neurotransmitter in cat esophageal circular muscle, as atropine nearly abolishes contraction of in vitro circular muscle strips in response to electric field stimulation (EFS) ( 5 , 12 ). Experimental esophagitis reduced EFS- but not ACh-induced contraction of esophageal circular muscle, suggesting that esophagitis impairs neurotransmitter release. Because IL-1β and IL-6 are produced in esophagitis and reproduce these changes in normal esophageal muscle ( 12 ), we examined the role of IL-1β and IL-6 in this motor dysfunction. IL-1β, IL-6 ( 12 ), H2O2, PGE2, and platelet-activating factor (PAF) were elevated in esophagitis specimens. Normal muscle incubated (2 h) in IL-1β and IL-6 had increases in H2O2, PGE2, and PAF levels. H2O2contributed to increased PGE2and PAF, as the increase was partially (60–80%) reversed by the H2O2scavenger catalase. EFS-induced [3H]ACh release from muscle strips significantly (42%) decreased in esophagitis and after 2 h incubation in PGE2and in PAF C-16. Similarly, EFS-induced but not ACh-induced muscle contraction decreased in esophagitis and after incubation in PGE2and PAF C-16. Finally, in normal muscle strips treated with IL-1β electrical field stimulation (EFS)-induced contraction was partially restored by indomethacin or by the PAF antagonist CV3988 and was completely restored by the combination of CV3988 and indomethacin, whereas in strips treated with IL-6, EFS-induced contraction was partially restored by the PAF antagonist CV3988 and not affected by indomethacin. We conclude that IL-1β-induced production of H2O2causes formation of PGE2and PAF that inhibit ACh release from esophageal cholinergic neurons without affecting ACh-induced contraction of esophageal circular muscle. IL-6 causes production of H2O2, PAF, and other unidentified inflammatory mediators.

Contraction of esophageal (Eso) and lower esophageal sphincter (LES) circular muscle depends on distinct signal-transduction pathways. ACh-induced contraction of Eso muscle is linked to phosphatidylcholine metabolism, production of diacylglycerol and arachidonic acid (AA), and activation of the Ca2+-insensitive PKCε. Although PKCε does not require Ca2+for activation, either influx of extracellular Ca2+or release of Ca2+from stores is needed to activate the phospholipases responsible for hydrolysis of membrane phospholipids and production of second messengers, which activate PKCε. In contrast, the LES uses two distinct intracellular pathways: 1) a PKC-dependent pathway activated by low doses of agonists or during maintenance of spontaneous tone, and 2) a Ca2+-calmodulin-myosin light chain kinase (MLCK)-dependent pathway activated in response to maximally effective doses of agonists during the initial phase of contraction. The Ca2+levels, released by agonist-induced activity of phospholipase C, determine which contractile pathway is activated in the LES. The Ca2+-calmodulin-MLCK-dependent contractile pathway has been well characterized in a variety of smooth muscles. The steps linking activation of PKC to myosin light chain (MLC20) phosphorylation and contraction, however, have not been clearly defined for LES, Eso, or other smooth muscles. In addition, in LES circular muscle, a low-molecular weight pancreatic-like phospholipase A2 (group I PLA2) causes production of AA, which is metabolized to prostaglandins and thromboxanes. These AA metabolites act on receptors linked to heterotrimeric G proteins to induce activation of phospholipases and production of second messengers to maintain contraction of LES circular muscle. We have examined the signal-transduction pathways activated by PGF2αand by thromboxane analogs during the initial contractile phase and found that these pathways are the same as those activated by other agonists. In response to low doses of agonists or during maintenance of tone, presumably due to low levels of calcium release, a PKC-dependent pathway is activated, whereas at high doses of PGF2αand thromboxane analogs, in the initial phase of contraction, calmodulin is activated, PKC activity is reduced, and contraction is mediated, in part, through a Ca2+-calmodulin-MLCK-dependent pathway. The PKC-dependent signaling pathways activated by PGF2αand by thromboxanes during sustained LES contraction, however, remain to be examined, but preliminary data indicate that a distinct PKC-dependent pathway may be activated during maintenance of tonic contraction, which is different from the one activated during the initial contractile response. The initial contractile response to low levels of agonists depends on activation of Gq. Sustained contraction in response to PGF2αmay involve activation of the monomeric G protein RhoA, because the contraction is inhibited by the RhoA-kinase antagonist Y27632. This shift in signal-transduction pathways between initial and sustained contraction has been recently reported in intestinal smooth muscle.

Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high Ca2+concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca2+levels. The link between activation of the Ca2+-independent protein kinase Cε (PKCε) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281: G467–G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKCε-dependent, Ca2+-independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKCε, but not antibodies to PKCβII, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKCε from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKCε, MEK, ERK1/2, and ILK.

We have shown that neurokinin A-induced contraction of human sigmoid circular muscle (HSCM) is reduced in patients with ulcerative colitis and that interleukin (IL)-1beta may play a role in this change. We now examine changes in the signal transduction pathway mediating neurokinin A-induced contraction of HSCM and explore the role of IL-1beta and of H(2)O(2) in these changes. In Fura 2-AM-loaded ulcerative colitis HSCM cells, neurokinin A- and caffeine-induced peak Ca(2+) increase and cell shortening were significantly reduced. In normal cells, neurokinin A-induced contraction was decreased by protein kinase C inhibitor chelerythrine and by calmodulin inhibitor CGS9343B [1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate]. In ulcerative colitis muscle cells, contraction was inhibited only by chelerythrine but not by CGS9343B. IL-1beta treatment of normal HSCM strips and cells reproduced the changes observed in ulcerative colitis. IL-1beta-induced reduction in caffeine-induced peak Ca(2+) increase and contraction was reversed by catalase, suggesting a role of H(2)O(2). IL-1beta-induced H(2)O(2) production was inhibited by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) and by cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3 (arachidonyltrifluoromethyl ketone), but neither by p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] nor by nuclear factor-kappaB (NF-kappaB) inhibitory peptide NF-kappaB SN50 (H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). IL-1beta significantly increased the phosphorylation of extracellular signal-regulated kinase 1 (ERK1)/ERK2 MAPKs and cPLA(2) and IL-1beta-induced cPLA(2) phosphorylation was blocked by PD98059. We conclude that Ca(2+) stores of HSCM cells may be reduced in ulcerative colitis and that the signal transduction pathway of neurokinin A-induced contraction switches from calmodulin- and protein kinase C-dependent in normal cells to protein kinase C-dependent in ulcerative colitis cells. IL-1beta reproduces these changes, possibly by production of H(2)O(2) via sequential activation of MAPKs (ERK1/ERK2) and cPLA(2).

Ulcerative colitis (UC) affects colonic motor function, but the mechanism responsible for this motor dysfunction is not well understood. We have shown that neurokinin A (NKA) may be an endogenous neurotransmitter mediating contraction of human sigmoid colonic circular muscle (HSCCM). To elucidate factors responsible for UC motor dysfunction, we examined the role of hydrogen peroxide (H2O2) in the decrease of NKA-induced response of HSCCM. As previously demonstrated, NKA-induced contraction or Ca2+increase of normal muscle cells is mediated by release of Ca2+from intracellular stores, because it was not affected by incubation in Ca2+-free medium (CFM) containing 200 μM BAPTA. In UC, however, CFM reduced both cell contraction and NKA-induced Ca2+increase, suggesting reduced Ca2+release from intracellular stores. In normal Ca2+medium, NKA and KCl caused normal Ca2+signal in UC cells but reduced cell shortening. The decreased Ca2+signal and contraction in response to NKA or thapsigargin were partly recovered in the presence of H2O2scavenger catalase, suggesting involvement of H2O2in UC-induced dysmotility. H2O2levels were higher in UC than in normal HSCCM, and enzymatically isolated UC muscle cells contained much higher levels of H2O2than normal cells, which were significantly reduced by catalase. H2O2treatment of normal cells in CFM reproduced the reduction of NKA-induced Ca2+release observed in UC cells. In addition, H2O2caused a measurable, direct release of Ca2+from intracellular stores. We conclude that H2O2may contribute to reduction of NKA-induced Ca2+release from intracellular Ca2+stores in UC and contribute to the observed colonic motor dysfunction.

Lower esophageal sphincter (LES) tone depends on PGF2αand thromboxane A2acting on receptors linked to Gi3and Gqto activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PGF2αsignal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PGF2α-induced contraction was inhibited by antibodies against the α-subunit of G13and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [35S]GTPγS-binding assay confirmed that G13, RhoA, and ARF1 were activated by PGF2α. Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PGF2α-induced contraction was inhibited by antibodies against the α-subunit of Gqand Gi3, and a [35S]GTPγS-binding assay confirmed that Gqand Gi3were activated by PGF2α. PGF2α-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PGF2αconcentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PGF2αactivates a PLD- and protein kinase C (PKC)-dependent pathway through G13, RhoA, and ARF1. In LES, PGF2αreceptors are coupled to Gqand Gi3, activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF2αactivates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.

Purpose: The role and clinical implication of the transmembrane protein with EGF and two follistatin motifs 2 (TMEFF2) in gastric cancer is poorly understood. Experimental Design: Gene expression profile analyses were performed and Gene Set Enrichment Analysis (GSEA) was used to explore its gene signatures. AGS and MKN45 cells were transfected with TMEFF2 or control plasmids and analyzed for gene expression patterns, proliferation, and apoptosis. TMEFF2 expression was knocked down with shRNAs, and the effects on genome stability were assessed. Interactions between TMEFF2 and SHP-1 were determined by mass spectrometry and immunoprecipitation assays. Results: Integrated analysis revealed that TMEFF2 expression was significantly decreased in gastric cancer cases and its expression was negatively correlated with the poor pathologic stage, large tumor size, and poor prognosis. GSEA in The Cancer Genome Atlas (TCGA) and Jilin datasets revealed that cell proliferation, apoptosis, and DNA damage–related genes were enriched in TMEFF2 lower expression patients. Gain of TMEFF2 function decreased cell proliferation by increasing of apoptosis and blocking of cell cycle in gastric cancer cells. The protein tyrosine phosphatase SHP-1 was identified as a binding partner of TMEEF2 and mediator of TMEFF2 function. TMEFF2 expression positively correlated with SHP-1, and a favorable prognosis was more likely in patients with gastric cancer with higher levels of both TMEFF2 and SHP-1. Conclusion: TMEFF2 acts as a tumor suppressor in gastric cancer through direct interaction with SHP-1 and can be a potential biomarker of carcinogenesis. Clin Cancer Res; 20(17); 4689–704. ©2014 AACR.

Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA.

C9orf140 is a newly identified and characterized gene which is associated with cell proliferation and tumorigenicity. Expression of C9orf140 is upregulated in human gastric cancer and colorectal cancer (CRC); however, little is known about its role in CRC progression. We have investigated the clinical significance, biological effects and mechanisms of C9orf140 signaling. We found that the expression of C9orf140 is dramatically increased in a subset of CRC and correlates significantly with vascular invasion and lymph node metastasis. Our finding showed that knockdown of C9orf140 significantly reduced cell proliferation and invasion in vitro and dramatically increased overall survival and decreased lung metastasis in vivo. Conversely, overexpression of C9orf140 significantly increased lung metastasis and shortened overall survival when compared with control tumors. C9orf140-induced CRC cell invasion may depend on promoting the epithelial-mesenchymal transition progression. STAT5 may directly interact with the enhancer of zeste homolog 2 (EZH2) and β-catenin to enhance C9orf140 gene transactivation. Furthermore, C9orf140 may participate in cell invasion which is induced by STAT5, EZH2 or β-catenin activation. We describe the role of C9orf140 in CRC progression and find that C9orf140 overexpression may be regulated by STAT5, EZH2 and β-catenin interaction.

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+influx and activation of protein kinase Cε (PKCε). PKCε, however, is known to be Ca2+independent. To determine where Ca2+is needed in this PKCε-mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle cells permeabilized by saponin. Ca2+(0.2–1.0 μM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCε (but not by PKCβII or PKCγ antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPβs. Addition of 1 μM Ca2+to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). Ca2+-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by AACOCF3(cytosolic phospholipase A2inhibitor). These data suggest that Ca2+may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCε, which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPγS augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular Ca2+and G protein activation), producing DAG and AA, and activating PKCε-dependent mechanisms to cause contraction.

Background & Aims: In cat spontaneous lower esophageal sphincter (LES), tone is maintained by the activity of group I secreted phospholipase A2 (sPLA2-I) that produces arachidonic acid. Arachidonic acid metabolites activate G proteins linked to phospholipases, producing second messengers and activation of a protein kinase C–dependent pathway to maintain tone. We examined the role of sPLA2-I in the maintenance of tone in human LES samples obtained from organ donors. Methods: In vitro LES tone and sPLA2-I–induced contraction of enzymatically isolated LES smooth muscle cells were measured in the absence or presence of inhibitors. Cell permeabilization by saponin allowed use of G-protein antibodies. Results: In vitro LES tone was reduced by inhibitors of sPLA2-I, by indomethacin, by the phosphatidylcholine-specific phospholipase C inhibitor D609, and by the protein kinase C inhibitor chelerythrine. sPLA2-I–induced contraction of isolated LES smooth muscle cells was reduced by indomethacin, pertussis toxin, Gi3 antibodies, D609, and by chelerythrine. Conclusions: Human LES tone is maintained by the activity of sPLA2-I that produces arachidonic acid and metabolites and activation of Gi3-linked receptors and of phosphatidylcholine-specific phospholipase C, resulting in production of diacylglycerol, activation of PKC, and maintenance of tone through a protein kinase C–dependent contractile pathway. GASTROENTEROLOGY 2000;119:1243-1252

Background & Aims: Because tachykinins have been identified as neurotransmitters in the guinea pig colon and human ileum, we examined a possible role of tachykinin receptors and neurokinin (NK) A in neurally induced contraction of human sigmoid colon circular muscle. Methods: Muscle strips were stimulated electrically for 10 seconds. Single cells were isolated by enzymatic digestion and permeabilized by saponin. [ 35 S]GTPγS binding was assayed with or without NKA for 5 minutes. Intracellular Ca 2+ was measured using Fura 2. Results: In the presence of 100 μmol/L L-NNA, 100 μmol/L atropine did not affect electrical field stimulation (EFS)-induced contraction. A peptide NK 2 -receptor antagonist (NK-2ra) but not an NK 1 antagonist FK888 (1 μmol/L) eliminated EFS-induced contraction. NKA-induced contraction in muscle strips and single cells was virtually abolished by NK-2ra, but not by FK888. In permeabilized cells, contraction was blocked by Gq-protein antibodies, but not by other G-protein antibodies, suggesting that NKA activates Gq, which was confirmed by a [ 35 S]GTPγS binding assay. NKA-induced contraction and increase in cytosolic Ca 2+ were abolished by depletion of intracellular Ca 2+ stores. Conclusions: Tachykinins may be the main excitatory neurotransmitters in human sigmoid circular muscle. NKA activates Gq-linked NK 2 receptors, which cause Ca 2+ release, followed by contraction. GASTROENTEROLOGY 2000;119:51-61