Gastroenterology

Gastroenterology

Volume 158, Issue 6, May 2020, Pages 1650-1666.e15
Gastroenterology

Original Research
Basic and Translational—Alimentary Tract
GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia

https://doi.org/10.1053/j.gastro.2020.01.046Get rights and content

Background & Aims

Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells.

Methods

We performed transcriptome analysis of Mist1-CreERT–traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-KrasG12D, LSL-KrasG12D, and LSL-HrasG12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)–dependent cell competition.

Results

We identified GPR30, the G-protein–coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30+ chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl+ clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion.

Conclusions

In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells.

Section snippets

Mice

Mist1-CreERT,13 Lgr5-DTR-GFP,24 Lox-STOP-Lox(LSL)-KrasG12D,13 LSL-HrasG12V-IRES-GFP,25 and LSL-GFP26 mice were previously described. R26 transgenic mice, Kitl-GFP mice, and Gpr30-knockout mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Gpr30-rtTA mice and TetO-KrasG12D mice were generated as described in the Supplementary Methods and Supplementary Figure 2. Mouse and in vitro culture experiments were repeated at least twice, with at least 2 biological replicates per group. All

Gpr30 Is Specifically Expressed in Gastric Chief Cells

To identify chief cell–specific genes, we performed transcriptome analyses using fluorescence-activated sorted cells isolated from Mist1-CreERT;R26-TdTomato mice with or without depletion of Lgr5+ chief cells (see Supplementary Methods). TdTomato+ Mist1-expressing cells were sorted from mice 2 days after tamoxifen induction and were designated as the chief cell–enriched group, whereas TdTomato+ cells sorted from induced Mist1-CreERT;Lgr5-DTR-EGFP;R26-TdTomato mice treated with diphtheria toxin

Discussion

Chief cells attracted considerable attention after a number of studies suggested they might be the cellular origin of cancer and precancerous metaplasia.7,12,23 However, this proposed model of chief cell transdifferentiation/dedifferentiation rested largely on 2 major observations: namely, lineage-tracing events in CreERT mice and coexpression of neck cell markers with chief cell markers on immunostaining. There are several critical limitations to lineage-tracing experiments, including the

CRediT Authorship Contributions

Masahiro Hata, MD (Data curation: Lead; Formal analysis: Lead; Validation: Lead;

Visualization: Lead). Hiroto Kinoshita, MD, PhD (Data curation: Lead; Formal analysis: Lead;

Visualization: Lead). Yoku Hayakawa, MD, PhD (Conceptualization: Lead; Data curation: Lead; Formal analysis: Lead; Funding acquisition: Lead; Investigation: Lead; Methodology: Lead;

Supervision: Lead; Validation: Supporting; Visualization: Supporting; Writing – original

draft: Lead; Writing – review & editing: Lead). Mitsuru

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    Conflicts of interest The authors disclose no conflicts.

    Funding Yoku Hayakawa is supported by the KAKENHI Grant-in-Aid for Scientific Research (17K09347 and 17H05081), Project for Cancer Research and Therapeutic Evolution (P-CREATE) from Japan Agency for Medical Research and development (AMED), Pharmacological Research Foundation, research grant of Bristol-Myers Squibb, Kowa Life Science Foundation, Senshin Medical Research Foundation, Yokoyama Clinical Pharmacological Research Foundation, Kanae Foundation of the Promotion of Medical Science, Inoue Science Research Award, and Takeda Science Foundation Visionary Research Grant, Princess Takamatsu Cancer Research Fund, and Advanced Research and Development Programs for Medical Innovation. Mitsuru Konishi is supported by the research grant from the Institute for Adult Diseases, Asahi Life Foundation. Timothy C. Wang received the National Institutes of Health grants (R35CA210088 and 5U01DK103155-04).

    Author names in bold designate shared co-first authorship.

    Authors share co-first authorship.

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