Dataset Information

Inflammatory and Proliferative Pathway Activation in Human Esophageal Myofibroblasts Treated with Acidic Bile Salts.

ABSTRACT: Subepithelial human esophageal myofibroblasts (HEMFs) in gastroesophageal reflux disease (GERD) are exposed to luminal contents via impaired squamous epithelium barrier integrity. The supernatant of HEMFs treated with acidic bile salts reflective of in vivo reflux increases squamous epithelial thickness. We aimed to identify the involved mechanisms using an unbiased approach. Acidic-bile-salt-treated primary HEMF cultures (n = 4) were submitted for RNA-Seq and analyzed with Partek Flow followed by Ingenuity Pathway Analysis (IPA). A total of 1165 molecules (579 downregulated, 586 upregulated) were differentially expressed, with most top regulated molecules either extracellular or in the plasma membrane. Increases in HEMF CXCL-8, IL-6, AREG, and EREG mRNA, and protein secretion were confirmed. Top identified canonical pathways were agranulocyte and granulocyte adhesion and diapedesis, PI3K/AKT signaling, CCR5 signaling in macrophages, and the STAT3 pathway. Top diseases and biological functions were cellular growth and development, hematopoiesis, immune cell trafficking, and cell-mediated response. The targets of the top upstream regulator ErbB2 included CXCL-8, IL-6, and AREG and the inhibition of CXCL-8 in the HEMF supernatant decreased squamous epithelial proliferation. Our work shows an inflammatory/immune cell and proliferative pathways activation in HEMFs in the GERD environment and identifies CXCL-8 as a HEMF-derived chemokine with paracrine proliferative effects on squamous epithelium.

SUBMITTER: Patankar M

PROVIDER: S-EPMC9498994 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Inflammatory and Proliferative Pathway Activation in Human Esophageal Myofibroblasts Treated with Acidic Bile Salts.

Patankar Madhura M Li Meng M Khalatbari Atousa A Castle Joshua D JD Hu Liping L Zhang Chunying C Shaker Anisa A

International journal of molecular sciences 20220908 18

Subepithelial human esophageal myofibroblasts (HEMFs) in gastroesophageal reflux disease (GERD) are exposed to luminal contents via impaired squamous epithelium barrier integrity. The supernatant of HEMFs treated with acidic bile salts reflective of in vivo reflux increases squamous epithelial thickness. We aimed to identify the involved mechanisms using an unbiased approach. Acidic-bile-salt-treated primary HEMF cultures (n = 4) were submitted for RNA-Seq and analyzed with Partek Flow followed ...[more]

PMID: 36142285

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Project description:BackgroundThe incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.MethodsReal-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.ResultsOur results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett's cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.ConclusionsIGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

Project description:Background & aimsMetaplastic glands buried under squamous epithelium are frequently detected in patients with Barrett esophagus (BE). This subsquamous intestinal metaplasia might be responsible for cancers that develop despite endoscopic surveillance and for metaplasia recurrences after endoscopic ablation. To determine whether reflux induces BE cells to undergo an epithelial-to-mesenchymal transition (EMT) that produces subsquamous intestinal metaplasia, we assessed EMT in BE cells exposed to acidic bile salts and in rat and human esophageal tissues.MethodsWe compared markers of EMT and cell motility in trans-well and 3-dimensional organotypic culture systems among dysplastic BE epithelial cell lines, nondysplastic telomerase-immortalized BE cell lines (BAR-T), and BAR-T cells exposed acutely or for 20 weeks to acidic bile salts. Vascular endothelial growth factor (VEGF) A was inhibited with a neutralizing antibody or CRISPR-Cas9n and VEGF receptor 2 was inhibited with SU1498 or shRNA, and cells were analyzed by immunohistochemistry, quantitative polymerase chain reaction, or immunoblotting for markers of VEGF signaling and EMT; cell motility was assessed by trans-well assay. We used immunohistochemistry and quantitative polymerase chain reaction to assess EMT markers in the columnar-lined esophagus of rats with surgically induced reflux esophagitis and in esophagectomy specimens from patients with BE.ResultsWe detected features of EMT (decreased cadherin 1 [CDH1]; increased fibronectin 1, vimentin, and matrix metalloproteinase 2; and increased motility) in dysplastic BE epithelial cell lines and in BAR-T cells exposed for 20 weeks, but not in unexposed BAR-T cells. Acute acidic bile salt exposure induced expression of zinc finger E-box binding homeobox 1 and 2 (ZEB1/2) in BAR-T cells, which decreased their expression of CDH1 and increased motility; inhibitors of VEGF signaling blocked these effects. Columnar-lined esophagus of rats with reflux esophagitis had increased expression of ZEB1/2 and decreased expression of CDH1 compared with controls. Dysplastic BE tissues also had significantly increased levels of ZEB1 and significantly decreased levels of CDH1 compared with nondysplastic BE tissues.ConclusionsIn BE cell lines, acidic bile salts induce EMT by VEGF signaling, which increases expression of ZEB1/2, repressors of CDH1. These observations suggest that reflux induces EMT in metaplastic BE tissues, which promotes development of subsquamous intestinal metaplasia.

Dataset Information

Inflammatory and Proliferative Pathway Activation in Human Esophageal Myofibroblasts Treated with Acidic Bile Salts.

Publications

Inflammatory and Proliferative Pathway Activation in Human Esophageal Myofibroblasts Treated with Acidic Bile Salts.

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