GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia.

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.

Helicobacter pylori antibiotic eradication coupled with a chemically defined diet in INS-GAS mice triggers dysbiosis and vitamin K deficiency resulting in gastric hemorrhage.

Infection with Helicobacter pylori causes chronic inflammation and is a risk factor for gastric cancer. Antibiotic treatment or increased dietary folate prevents gastric carcinogenesis in male INS-GAS mice. To determine potential synergistic effects, H. pylori-infected male INS-GAS mice were fed an amino acid defined (AAD) diet with increased folate and were treated with antibiotics after 18 weeks of H. pylori infection. Antibiotic therapy decreased gastric pathology, but dietary folate had no effect. However, the combination of antibiotics and the AAD diet induced anemia, gastric hemorrhage, and mortality. Clinical presentation suggested hypovitaminosis K potentially caused by dietary deficiency and dysbiosis. Based on current dietary guidelines, the AAD diet was deficient in vitamin K. Phylloquinone administered subcutaneously and via a reformulated diet led to clinical improvement with no subsequent mortalities and increased hepatic vitamin K levels. We characterized the microbiome and menaquinone profiles of antibiotic-treated and antibiotic-free mice. Antibiotic treatment decreased the abundance of menaquinone producers within orders Bacteroidales and Verrucomicrobiales. PICRUSt predicted decreases in canonical menaquinone biosynthesis genes, menA and menD. Reduction of menA from Akkermansia muciniphila, Bacteroides uniformis, and Muribaculum intestinale were confirmed in antibiotic-treated mice. The fecal menaquinone profile of antibiotic-treated mice had reduced MK5 and MK6 and increased MK7 and MK11 compared to antibiotic-free mice. Loss of menaquinone-producing microbes due to antibiotics altered the enteric production of vitamin K. This study highlights the role of diet and the microbiome in maintaining vitamin K homeostasis.

Krüppel-like factor 4 regulates intestinal epithelial cell morphology and polarity.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells.

Folic acid increases global DNA methylation and reduces inflammation to prevent Helicobacter-associated gastric cancer in mice.

BACKGROUND & AIMS: Previous studies have suggested that dietary folic acid (FA) can protect against certain types of cancers. However, the findings have varied, and the mechanisms by which FA exerts chemopreventive effects remain to be clarified. We examined the effects of FA supplementation on DNA methylation, gene expression, and gastric dysplasia in a transgenic mouse model that is etiologically and histologically well matched with human gastric cancers. METHODS: Hypergastrinemic mice infected with Helicobacter felis were studied at multiple stages of gastric dysplasia and early cancer with FA supplementation initiated both at weaning and later in life. Global DNA methylation was assessed by a methylation sensitive cytosine incorporation assay, bisulfite pyrosequencing of B1 repetitive elements, and immunohistochemistry with anti-5-methylcytosine. We also profiled gene expression in the same tissues. RESULTS: We found a decrease in global DNA methylation and tissue folate and an increase in serum homocysteine with progression of gastric dysplasia. FA supplementation prevented this loss of global DNA methylation and markedly reduced gastric dysplasia and mucosal inflammation. FA protected against the loss of global DNA methylation both in the dysplastic gastric epithelial cells and in gastric stromal myofibroblasts. In addition, FA supplementation had an anti-inflammatory effect, as indicated by expression profiling and immunohistochemistry for lymphocyte markers. CONCLUSIONS: We conclude that FA supplementation is chemopreventive in this model of Helicobacter-associated gastric cancer. The beneficial effect of FA is likely due to its ability to prevent global loss of methylation and suppress inflammation.

Vitamin C supplementation does not protect L-gulono-gamma-lactone oxidase-deficient mice from Helicobacter pylori-induced gastritis and gastric premalignancy.

In human studies, low vitamin C intake has been associated with more severe Helicobacter pylori gastritis and a higher incidence of gastric cancer. However, vitamin C supplementation has not been definitively shown to protect against gastric cancer. Using vitamin C-deficient B6.129P2-Gulo(tm1Umc/mmcd) (gulo(-/-)) mice lacking L-gulono-gamma-lactone oxidase, we compared gastric lesions and Th1 immune responses in H. pylori-infected gulo(-/-) mice supplemented with low (33 mg/L) or high (3,300 mg/L) vitamin C in drinking water for 16 or 32 weeks. Vitamin C levels in plasma and gastric tissue correlated with the vitamin C supplementation levels in gulo(-/-) mice. H. pylori infection resulted in comparable gastritis and premalignant lesions in wildtype C57BL/6 and gulo(-/-) mice supplemented with high vitamin C, but lesions were less severe in gulo(-/-) mice supplemented with low vitamin C at 32 weeks post infection. The reduced gastric lesions in infected gulo(-/-) mice supplemented with low vitamin C correlated with reduced Th1-associated IgG2c, gastric IFN-gamma and TNF-alpha mRNA and higher H. pylori colonization levels. These results in the H. pylori-infected gulo(-/-) mouse model suggest that although supplementation with a high level of vitamin C achieved physiologically normal vitamin C levels in plasma and gastric tissue, this dose of vitamin C did not protect gulo(-/-) mice from H. pylori-induced premalignant gastric lesions. In addition, less severe gastric lesions in H.pylori infected gulo(-/-) mice supplemented with low vitamin C correlated with an attenuated Th1 inflammatory response.

Yin yang 1 (YY1) represses histidine decarboxylase gene expression with SREBP-1a in part through an upstream Sp1 site.

Histidine decarboxylase (HDC) is the enzyme that converts histidine to histamine, a bioamine that plays an important role in many physiological aspects including allergic responses, inflammation, neurotransmission, and gastric acid secretion. In previous studies, we demonstrated that Kruppel-like factor 4 represses HDC promoter activity in a gastric cell line through both an upstream Sp1-binding GC box (GGGCGG sequence) and downstream gastrin-responsive elements. In the current study, Yin Yang 1 (YY1), a pleiotropic transcriptional factor, was also shown in cotransfection assays to repress HDC promoter activity through the upstream GC box. DNA affinity purification assay demonstrated that YY1 was pulled down specifically by the upstream GC box. In addition, sterol-responsive element-binding protein 1a (SREBP-1a), a transcriptional factor that binds YY1, represses the HDC promoter. Interestingly, deletion analysis and cotransfection assays indicated that mutation of the upstream GC box or truncation of downstream gastrin-responsive elements in the HDC promoter disrupted the inhibitory effect of YY1 and SREBP-1a in an identical fashion. Furthermore, quantitative real-time PCR analysis indicated that gastrin treatment downregulated SREBP-1a gene expression and reduced the DNA binding activity of SREBP in EMSAs. Taken together, these results suggest that YY1 and SREBP-1a form a complex to inhibit HDC gene expression through both the upstream GC box and downstream gastrin-responsive elements and gastrin-induced activation of HDC gene expression is mediated at least partly through downregulation of transcriptional repressors such as SREBPs.