Stratifin Is Necessary for Spasmolytic Polypeptide-Expressing Metaplasia Development After Acute Gastric Injury

Abstract

Background & aims: Chief cells can transdifferentiate into spasmolytic polypeptide-expressing metaplasia (SPEM), a metaplastic cell lineage, in response to acute injury after acid-secreting parietal cell loss in the stomach. Stratifin (SFN) acts as a multifunctional regulator, which can alter the function of multiple phosphoproteins. We have now examined how SFN contributes to the transdifferentiation of chief cells and the emergence of SPEM, as the initial metaplastic event in mucosal response to injury.

Methods: We performed single-cell RNA sequencing on transdifferentiating chief cells after a single dose of DMP-777 treatment to induce acute parietal cell atrophy in Mist1^CreERT2; LSL-tdTomato mice. We generated a Mist1^CreERT2; Sfn^flox/flox mouse model to examine the effects of SFN loss in the transdifferentiation of chief cells and SPEM development in response to acute injury. Histologic examination and immunostaining were performed in the mouse stomachs to assess cell lineage marker expression.

Results: The single-cell RNA sequencing showed the initial characteristics of transdifferentiation of chief cells in response to acute injury. SFN expression was increased in transdifferentiating chief cells and SPEM cells. We determined that SFN loss in mice impairs the transdifferentiation of chief cells into SPEM following acute oxyntic atrophy in part by modulating EGFR/ERK signaling after acute injury.

Conclusions: SFN is essential for the initiation of reprogramming of chief cells during transdifferentiation and SPEM development.

Keywords: 14-3-3σ; Gastric Chief Cells; Metaplasia; SPEM; Stratifin; Transdifferentiation.

Graphical abstract

Figure 1

scRNAseq defines the transdifferentiation of chief cells in the corpus and their transcriptional changes after acute gastric injury. (A) Schematic of mouse experiments. (B) Principal component analysis (PCA) plot of untreated and DMP-777-treated condition. (C) PCA plots of chief cell markers (Pgc and Gif) and SPEM cell markers (Gkn3 and Aqp5). (D) Bubble plot of chief cell markers (Pgc, Gif, and Rab3d), SPEM cell markers (Tff2, Gkn3, Aqp5, Wfdc2, Mcm2, Mcm3, Mcm7, and Sox9), and proliferating cell markers (Mki67 and Top2a). (E) Heatmap of up-regulated and down-regulated genes in chief cells and transdifferentiating (TD) chief cells. (F) Functional enrichment analysis of top 100 up-regulated genes in TD chief cells. (G) Bar plot of normalized expression of Sfn in the untreated or 1 day of DMP-777-treated conditions.

Figure 2

Up-regulation of Sfn in SPEM cell lineages. (A) Quantitative polymerase chain reaction analysis of Sfn mRNA levels in ImChief cells (n = 3) and ImSPEM cells (n = 3). Two-tailed unpaired t-test. (B) Immunoblots for SFN or β-actin in ImChief cells (n = 3) and ImSPEM cells (n = 3). (C) Quantitative polymerase chain reaction analysis of Sfn mRNA levels in untreated (n = 3) and L635-treated mice (n = 3). Two-tailed unpaired t-test. (D) Immunoblots for SFN or β-actin in untreated (n = 2) and 3 days of L635-treated mice (n = 2). (E) Immunostained sections from WT untreated (n = 3), DMP-777-treated mice killed after 5 (n = 3), 7 (n = 3), or 10 (n = 4) days of DMP-777 treatment for SFN (red), SPEM cell marker GSII-lectin (green), and parietal cell marker H⁺K⁺ATPase (white), with nuclear counterstain Hoechst 33342 (blue). (F) Immunostained sections from WT untreated (n = 3), L635-treated (n = 4) mice killed after 3 days of L635 treatment for SFN (red), SPEM cell marker CD44v9 (green), and parietal cell marker H⁺/K^+-ATPase (white), with nuclear counterstain Hoechst 33342 (blue). (G) Quantitation of positive cells for SFN. Data represent mean ± standard deviation (n = 9 images from 3 mice at 20x magnification in each group). Kruskal-Wallis test with 2-sided Dunn multiple comparison test. (H) Immunostained sections from Helicobacter felis–infected stomachs after 8 weeks postinfection (n = 3) for SFN (red) and SPEM cell marker CD44v9 (green) with nuclear counterstain Hoechst 33342 (blue). (I) Immunostained sections from human metaplastic regions for SFN (red) and SPEM cell marker CD44v9 (green) with nuclear counterstain Hoechst 33342 (blue). All panels show mean ± standard deviation. ∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001. ns, not significant. Scale bar = 100 μm.

Figure 3

Sfn KO does not affect chief cell maintenance. (A) Schematic for mouse experiments. L635 was administered to WT or Sfn KO mice for 3 days to induce acute gastric injury. (B) Hematoxylin and eosin–stained images from untreated or L635-treated stomach tissues in WT or Sfn KO mice. (C) Immunostained sections from WT untreated or Sfn KO untreated mice for chief cell marker MIST1 (red) and GIF (green), and epithelial membrane marker ECAD (white), with nuclear counterstain Hoechst 33342 (blue). (D) Quantitation of MIST1-positive cells per 20x field of images. Each dot indicates the MIST1-positive cells per x20 field of images. (E) Immunostained sections from WT or Sfn KO mice for SFN (red) and parietal cell marker H⁺/K⁺ATPase (red) with nuclear counterstain Hoechst 33342 (blue) after 3 days of L635 treatment. Scale bars: 100 μm (B and C). All panels show mean ± standard deviation. ns, not significant. n ≥ 3 mice per condition.

Figure 4

Sfn is indispensable for transdifferentiating chief cells into SPEM cells after acute gastric injury. (A) Immunostained sections from WT or Sfn KO mice for SFN (red) and SPEM cell marker GSII-lectin (green) with nuclear counterstain Hoechst 33342 (blue) in untreated or 3 days of L635 treatment. (B) Profiling of the expression of SFN across the corpus mucosal height (geometric mean value, n = 5 images from 3 mice per condition). (C) Profiling of the expression of GSII-lectin across the corpus mucosal height (geometric mean value, n = 5 images from 3 mice per condition).

Figure 5

Sfn KO inhibits reprogramming into proliferative SPEM cells. (A) Immunostained sections from WT or Sfn KO mice for proliferation marker Ki-67 (red) and SPEM cell marker AQP5 (green) with nuclear counterstain Hoechst 33342 (blue) after 3 days of L635 treatment. (B) Profiling of the expression of Ki-67 across the corpus mucosal height (geometric mean value, n = 5 images from 3 mice per condition). (C) Profiling of the expression of AQP5 across the corpus mucosal height (geometric mean value, n = 5 images from 3 mice per condition).

Figure 6

SOX9 expression does not increase in Sfn KO. (A) Immunostained sections from WT or Sfn KO mice for chief cell marker MIST1 (red), SPEM cell marker SOX9, and epithelial cell membrane marker ECAD (blue) in untreated or 3 days of L635 treatment. (B) Profiling of the expression of SOX9 across the corpus mucosal height (geometric mean value, n = 6 images from 3 mice per condition). (C) Immunostained sections from WT or Sfn KO mice for SFN (red) and SPEM cell marker SOX9 (green) with nuclear counterstain Hoechst 33342 (blue) after 3 days of L635 treatment. n ≥ 3 mice per condition.

Figure 7

The EGFR/ERK signaling pathway is regulated by Sfn. (A) Immunoblots for phosphorylated ERK (pERK), tyrosine 1068-phosphorylated EGFR (pEGFR), tyrosine 705-phosphorylated STAT3 (pSTAT3), serine 729-phosphorylated BRAF (pBRAF), serine 240/244-phosphorylated S6 (pS6), serine 473-phosphorylated AKT (pAKT), or β-actin (b-actin) loading control for corpus mucosal tissues. (B, C) Quantitation of immunoblots for phosphorylated proteins in WT untreated (n = 5), Sfn KO untreated (n = 5), WT mice sacrificed after 3 days of L635 treatment (n = 4), and Sfn KO mice sacrificed after 3 days of L635 treatment (n = 4). All panels show mean ± standard deviation. ∗P < .05, ∗∗∗P < .001. ns, not significant. (D) Immunostained sections from WT or Sfn KO mice for SPEM cell marker SOX9 (red), phosphorylated EGFR (green), and E-cadherin (ECAD, grayscale) with nuclear counterstain Hoechst 33342 (blue) after 3 days of L635 treatment. Scale bar = 100 μm.