Activin A-mediated epithelial de-differentiation contributes to injury repair in an in vitro gastrointestinal reflux model

Abstract

Reflux esophagitis is a result of esophageal exposure to acid and bile during episodes of gastroesophageal reflux. Aside from chemical injury to the esophageal epithelium, it has been shown that acid and bile induce cytokine-mediated injury by stimulating the release of pro-inflammatory cytokines. During the repair and healing process following reflux injury, the squamous esophageal cells are replaced with a columnar epithelium causing Barrett's metaplasia, which predisposes patients to esophageal adenocarcinoma. We identified a novel player in gastroesophageal reflux injury, the TGFβ family member Activin A (ActA), which is a known regulator of inflammation and tissue repair. In this study, we show that in response to bile salt and acidified media (pH 4) exposure, emulating the milieu to which the distal esophagus is exposed during gastroesophageal reflux, long-term treated, tolerant esophageal keratinocytes exhibit increased ActA secretion and a pro-inflammatory cytokine signature. Furthermore, we noted increased motility and expression of the stem cell markers SOX9, LGR5 and DCLK1 supporting the notion that repair mechanisms were activated in the bile salt/acid-tolerant keratinocytes. Additionally, these experiments demonstrated that de-differentiation as characterized by the induction of YAP1, FOXO3 and KRT17 was altered by ActA/TGFβ signaling. Collectively, our results suggest a pivotal role for ActA in the inflammatory GERD environment by modulating esophageal tissue repair and de-differentiation.

Keywords: Barrett’s esophagus; Gastroesophageal reflux; Inflammation; Stem cell markers; TGFβ signaling.

Fig. 1:

pH 4-bile salt-tolerant cell populations emerge upon initial induction of apoptosis. Cell lysates were collected 5 hours upon repeated treatment with pH4-acidified and bile salt containing media, and analyzed for the cleavage of PARP and Caspase 9 using specific antibodies by Western blotting. β-actin was used as a loading control. The signal intensity was calculated in each lane, normalized according to β-actin and compared to untreated control.

Fig. 2:

Inflammatory signaling is activated upon long-term pH4-bile salt exposure of esophageal epithelial cells. A. Activation of inflammatory signaling pathways such as STAT1/3 is shown by Western Blot analysis with phospho-specific antibodies, pY701 and pY 705, compared to total protein control and NF-κB p65 using cell lysates from long-term treated EPC-hTERT cells and controls. The signal intensity was calculated in each lane normalized according to β-actin and compared to untreated control. Pro-inflammatory cytokines relevant to Barrett’s tumorigenesis are upregulated as shown B. using a cytokine antibody array and C. validated by qRT-PCR using RNA isolates from pooled independent long-term treated EPC-hTERT cell lines compared to no treatment controls, unpaired t-test. D. Selective upregulation of COX-2 (PTGS2) after long-term combination treatment; the signal intensity was calculated in each lane normalized according to β-actin, and compared to untreated control. E. Comparison of IL1β and PTGS2 arbitrary expression values in human patients was based on a publicly available GEO dataset (accession number GDS1321), N=normal, BE= Barrett’s esophagus; unpaired t-test. Statistical significance: ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Fig. 3:

Activin A expression and secretion is induced in response to bile/acid treatment in esophageal epithelial cells. A. ActA concentration in conditioned media is increased with bile salt-containing pH4 media treatments compared to media only and media from control untreated cells (collected at the indicated time points) as determined by ELISA. n=4. B. The ActA gene, INHBA, and SMAD3 are upregulated as shown by qRT-PCR from RNA isolated from controls compared to bile acid treatments. C. Downstream canonical SMAD-mediated and non-canonical ERK1/2- induced signaling is activated based on phosphorylation of pSMAD2/3 on residues Ser423/425 and pERK1/2 (Thr202/Tyr204) shown in Western Blot. The signal intensity was calculated in each lane normalized according to β-actin, and compared to untreated control. Unpaired t-test, statistical significance: *** p ≤ 0.001, **** p ≤ 0.0001

Fig. 4:

(A-B) The repair of bile/acid injury is associated with an Activin A-dependent increase in cell invasion and associated with (C-E) Increased expression of stem cell markers. A. Motility over a basement membrane (BM) is increased upon bile acid exposure, comparable to the Barrett’s esophageal cell line BART. B. The increased motility is ActA dependent as shown using recombinant ActA and the TGFβ/ActA chemical compound inhibitor A83-01 (one-way ANOVA). Stem cell markers, such as SOX9, LGR5 and DCLK1, were upregulated in an ActA-dependent bile acid-induced manner as shown by C. qRT-PCR (n= 3, one-way ANOVA) and D. Western Blot for SOX9 using RNA isolates from long-term treated EPC-hTERT cells compared to no treatment controls. E. Comparison of SOX9, LGR5 and DCLK1 expression was based on a publicly available GEO dataset (accession number GDS1321). N=normal, BE= Barrett’s esophagus; unpaired t-test. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Fig. 5:

Long term pH4-bile acid treatments results in the de-differentiation of esophageal epithelial cells. A. Expression of potential de-differentiation markers, YAP1, FOXO3, KRT19 and KRT17, was increased in long-term pH4 and bile salt-treated esophageal cells, and suppressed in the presence of the A83-01 inhibitor for al markers other than KRT19 as shown by qRT-PCR, n=3, statistical analysis using one-way ANOVA. B. Expression of KRT8, KRT18, KRT19 and KRT17 was evaluated based on a publicly available GEO dataset (accession number GDS1321). N=normal, BE= Barrett’s esophagus; unpaired t-test. Statistical significance: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Fig. 6:

In situ expression of select markers is elevated upon long-term bile/acid exposure. A. Increased signal for LGR5, KRT8, KRT19 and ACVR1B was detected using double immunofluorescence staining of organotypic reconstruct cultures for long-term treated tolerant and control cells were grown on an extracellular matrix deposited by human esophageal fibroblasts. KRT14 signal is lost in KRT8-positive cells, arrows. H&E is hematoxylin & eosin staining of the paraffin embedded organotypic culture tissue. B. Double immunofluorescence with antibody against LGR5 and ACVR1B showed an increase in pH4+bile salt-exposed cells grown suspended 2% Matrigel to induce spheroid formation. The number of KRT8-positive, KRT14-negative cells is increased in tolerant-cells compared to control.

Fig. 7:

Model. Increased ActA expression and secretion as a result of bile and acid epithelial injury induce stem cell activation and de-differentiation to facilitate tissue repair.