Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial ‘miniguts’), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role of circadian rhythms in GI stem cell dynamics, epithelial homeostasis and disease.
BackgroundIndividuals with persistent Helicobacter pylori (H. pylori) infection are known to have an increased risk of gastric adenocarcinoma development. The circadian clock has been shown to be intimately involved in various gastrointestinal functions and clock components BMAL1 and PER2 have been found to be disrupted in colorectal cancer tissues. Aged individuals have a dampened expression of such components which may further increase their susceptibility to gastric cancer development.HypothesisPersistent infection of H. pylori induces an unregulated proliferation of gastric epithelial cells via activation of BMAL1 leading to a predisposition to gastric cancer development.MethodsGastric organoids (hFGOs) were generated from either primary gastric tissue derived from aged (> 55 years of age) and young (14–20 years) individuals. Gastric organoids were then infected with H. pylori. BMAL1 and PER2 expression was then quantified over a period of 24 hours. Proliferation and expression of gastric cancer markers were quantified by qRT‐PCR.ResultsH. pylori infection correlated with a significant upregulation of cytoplasmic BMAL1 expression within the gastric fundus of infected individuals. Robust circadian rhythmicity of clock genes Bmal1 and Per2 were observed in human hFGOs. Phosphorylation (inactivation) of GSK‐3b showed circadian changes in hFGOs over 52 hours. H. pylori infection of hFGOs resulted in sustained GSK‐3b phosphorylation, increased BMAL1 expression and epithelial cell hyperproliferation when compared to uninfected controls. Organoids derived from elderly patients exhibited a dampened rhythmicity of of clock genes Bmal1 and Per2. Dampened rhythmicity correlated with increased gastric cancer biomarkers of intestinal metaplasia including Cdx1, Cdx2 and MUC2.ConclusionsChronic H. pylori infection induces a hyperproliferative response in gastric epithelial cells by inactivating GSK‐3β leading to the activation of BMAL1:CLOCK. This may be a plausible mechanism that predisposes individuals with persistent H. pylori infection to gastric cancer development and also heightens this risk in aged individuals.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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