In oesophageal squamous cells, nitric oxide causes S-nitrosylation of Akt and blocks SOX2 (sex determining region Y-box 2) expression

Kiyotaka Asanuma, Xiaofang Huo, Agoston Agoston, Xi Zhang, Chunhua Yu, Edaire Cheng, Qiuyang Zhang, Kerry B. Dunbar, Thai H. Pham, David H. Wang, Katsunori Iijima, Tooru Shimosegawa, Robert D. Odze, Stuart J. Spechler, Rhonda F. Souza

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Objective Barrett's metaplasia might develop if GORD causes oesophageal squamous cells to convert into columnar cells. Acid and bile exposures upregulate columnar differentiation genes like CDX2 in oesophageal squamous cells, but it is not known if such exposures downregulate squamous differentiation genes like SOX2. In addition to acid and bile, patients with GORD also have high oesophageal concentrations of nitric oxide (NO). This study aims to determine how acid, bile salts and NO affect genes that influence oesophageal cell phenotype. Design Oesophageal squamous cells from patients with Barrett's oesophagus were exposed to acidic bile salts or NOC-9 (an NO donor). SOX2, p63 (squamous transcription factor) and CDX2 mRNAs were measured by quantitative RT-PCR. SOX2 and its regulatory Akt pathway proteins were evaluated by western blotting. S-nitrosylation by NO was blocked by dithiothreitol. Immunohistochemistry for SOX2 was performed on the oesophagus of rats with surgically induced GORD which were fed diets with and without nitrite supplementation. Results In oesophageal squamous cells, NO profoundly decreased SOX2 protein and caused a significantly greater decrease in SOX2 mRNA than did acidic bile salts. NO also decreased p63 and increased CDX2 expression. NO caused S-nitrosylation of Akt, blocking its phosphorylation. Akt pathway inhibition by LY294002 or Akt siRNA reduced SOX2 mRNA. Rats fed with nitrite-supplemented diets exhibited weaker SOX2 oesophageal staining than rats fed with normal diets. Conclusions In oesophageal squamous cells, NO blocks SOX2 expression through Akt S-nitrosylation. NO also increases CDX2 and decreases p63 expression. By triggering molecular events preventing squamous differentiation while promoting intestinal differentiation, NO might contribute to Barrett's pathogenesis.

Original languageEnglish (US)
Pages (from-to)1416-1426
Number of pages11
JournalGut
Volume65
Issue number9
DOIs
StatePublished - Sep 1 2016

Fingerprint

Nitric Oxide
Epithelial Cells
Bile Acids and Salts
Barrett Esophagus
Nitrites
Diet
Messenger RNA
Genes
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Nitric Oxide Donors
Dithiothreitol
Small Interfering RNA
Esophagus
Proteins
Up-Regulation
Down-Regulation
Western Blotting
Immunohistochemistry
Phosphorylation
Staining and Labeling

Keywords

  • Barrett's metaplasia
  • gastroesophageal reflux disease
  • nitric oxide

ASJC Scopus subject areas

  • Gastroenterology

Cite this

In oesophageal squamous cells, nitric oxide causes S-nitrosylation of Akt and blocks SOX2 (sex determining region Y-box 2) expression. / Asanuma, Kiyotaka; Huo, Xiaofang; Agoston, Agoston; Zhang, Xi; Yu, Chunhua; Cheng, Edaire; Zhang, Qiuyang; Dunbar, Kerry B.; Pham, Thai H.; Wang, David H.; Iijima, Katsunori; Shimosegawa, Tooru; Odze, Robert D.; Spechler, Stuart J.; Souza, Rhonda F.

In: Gut, Vol. 65, No. 9, 01.09.2016, p. 1416-1426.

Research output: Contribution to journalArticle

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title = "In oesophageal squamous cells, nitric oxide causes S-nitrosylation of Akt and blocks SOX2 (sex determining region Y-box 2) expression",
abstract = "Objective Barrett's metaplasia might develop if GORD causes oesophageal squamous cells to convert into columnar cells. Acid and bile exposures upregulate columnar differentiation genes like CDX2 in oesophageal squamous cells, but it is not known if such exposures downregulate squamous differentiation genes like SOX2. In addition to acid and bile, patients with GORD also have high oesophageal concentrations of nitric oxide (NO). This study aims to determine how acid, bile salts and NO affect genes that influence oesophageal cell phenotype. Design Oesophageal squamous cells from patients with Barrett's oesophagus were exposed to acidic bile salts or NOC-9 (an NO donor). SOX2, p63 (squamous transcription factor) and CDX2 mRNAs were measured by quantitative RT-PCR. SOX2 and its regulatory Akt pathway proteins were evaluated by western blotting. S-nitrosylation by NO was blocked by dithiothreitol. Immunohistochemistry for SOX2 was performed on the oesophagus of rats with surgically induced GORD which were fed diets with and without nitrite supplementation. Results In oesophageal squamous cells, NO profoundly decreased SOX2 protein and caused a significantly greater decrease in SOX2 mRNA than did acidic bile salts. NO also decreased p63 and increased CDX2 expression. NO caused S-nitrosylation of Akt, blocking its phosphorylation. Akt pathway inhibition by LY294002 or Akt siRNA reduced SOX2 mRNA. Rats fed with nitrite-supplemented diets exhibited weaker SOX2 oesophageal staining than rats fed with normal diets. Conclusions In oesophageal squamous cells, NO blocks SOX2 expression through Akt S-nitrosylation. NO also increases CDX2 and decreases p63 expression. By triggering molecular events preventing squamous differentiation while promoting intestinal differentiation, NO might contribute to Barrett's pathogenesis.",
keywords = "Barrett's metaplasia, gastroesophageal reflux disease, nitric oxide",
author = "Kiyotaka Asanuma and Xiaofang Huo and Agoston Agoston and Xi Zhang and Chunhua Yu and Edaire Cheng and Qiuyang Zhang and Dunbar, {Kerry B.} and Pham, {Thai H.} and Wang, {David H.} and Katsunori Iijima and Tooru Shimosegawa and Odze, {Robert D.} and Spechler, {Stuart J.} and Souza, {Rhonda F.}",
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T1 - In oesophageal squamous cells, nitric oxide causes S-nitrosylation of Akt and blocks SOX2 (sex determining region Y-box 2) expression

AU - Asanuma, Kiyotaka

AU - Huo, Xiaofang

AU - Agoston, Agoston

AU - Zhang, Xi

AU - Yu, Chunhua

AU - Cheng, Edaire

AU - Zhang, Qiuyang

AU - Dunbar, Kerry B.

AU - Pham, Thai H.

AU - Wang, David H.

AU - Iijima, Katsunori

AU - Shimosegawa, Tooru

AU - Odze, Robert D.

AU - Spechler, Stuart J.

AU - Souza, Rhonda F.

PY - 2016/9/1

Y1 - 2016/9/1

N2 - Objective Barrett's metaplasia might develop if GORD causes oesophageal squamous cells to convert into columnar cells. Acid and bile exposures upregulate columnar differentiation genes like CDX2 in oesophageal squamous cells, but it is not known if such exposures downregulate squamous differentiation genes like SOX2. In addition to acid and bile, patients with GORD also have high oesophageal concentrations of nitric oxide (NO). This study aims to determine how acid, bile salts and NO affect genes that influence oesophageal cell phenotype. Design Oesophageal squamous cells from patients with Barrett's oesophagus were exposed to acidic bile salts or NOC-9 (an NO donor). SOX2, p63 (squamous transcription factor) and CDX2 mRNAs were measured by quantitative RT-PCR. SOX2 and its regulatory Akt pathway proteins were evaluated by western blotting. S-nitrosylation by NO was blocked by dithiothreitol. Immunohistochemistry for SOX2 was performed on the oesophagus of rats with surgically induced GORD which were fed diets with and without nitrite supplementation. Results In oesophageal squamous cells, NO profoundly decreased SOX2 protein and caused a significantly greater decrease in SOX2 mRNA than did acidic bile salts. NO also decreased p63 and increased CDX2 expression. NO caused S-nitrosylation of Akt, blocking its phosphorylation. Akt pathway inhibition by LY294002 or Akt siRNA reduced SOX2 mRNA. Rats fed with nitrite-supplemented diets exhibited weaker SOX2 oesophageal staining than rats fed with normal diets. Conclusions In oesophageal squamous cells, NO blocks SOX2 expression through Akt S-nitrosylation. NO also increases CDX2 and decreases p63 expression. By triggering molecular events preventing squamous differentiation while promoting intestinal differentiation, NO might contribute to Barrett's pathogenesis.

AB - Objective Barrett's metaplasia might develop if GORD causes oesophageal squamous cells to convert into columnar cells. Acid and bile exposures upregulate columnar differentiation genes like CDX2 in oesophageal squamous cells, but it is not known if such exposures downregulate squamous differentiation genes like SOX2. In addition to acid and bile, patients with GORD also have high oesophageal concentrations of nitric oxide (NO). This study aims to determine how acid, bile salts and NO affect genes that influence oesophageal cell phenotype. Design Oesophageal squamous cells from patients with Barrett's oesophagus were exposed to acidic bile salts or NOC-9 (an NO donor). SOX2, p63 (squamous transcription factor) and CDX2 mRNAs were measured by quantitative RT-PCR. SOX2 and its regulatory Akt pathway proteins were evaluated by western blotting. S-nitrosylation by NO was blocked by dithiothreitol. Immunohistochemistry for SOX2 was performed on the oesophagus of rats with surgically induced GORD which were fed diets with and without nitrite supplementation. Results In oesophageal squamous cells, NO profoundly decreased SOX2 protein and caused a significantly greater decrease in SOX2 mRNA than did acidic bile salts. NO also decreased p63 and increased CDX2 expression. NO caused S-nitrosylation of Akt, blocking its phosphorylation. Akt pathway inhibition by LY294002 or Akt siRNA reduced SOX2 mRNA. Rats fed with nitrite-supplemented diets exhibited weaker SOX2 oesophageal staining than rats fed with normal diets. Conclusions In oesophageal squamous cells, NO blocks SOX2 expression through Akt S-nitrosylation. NO also increases CDX2 and decreases p63 expression. By triggering molecular events preventing squamous differentiation while promoting intestinal differentiation, NO might contribute to Barrett's pathogenesis.

KW - Barrett's metaplasia

KW - gastroesophageal reflux disease

KW - nitric oxide

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