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Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.BioRxiv : the Preprint Server For... Apr 2024Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of...
Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of -knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of , a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.
PubMed: 38659853
DOI: 10.1101/2024.04.12.589235 -
Journal of Proteome Research May 2024Alcohol consumption perturbs the gut immune barrier and ultimately results in alcoholic liver diseases, but little is known about how immune-related cells in the gut are...
Alcohol consumption perturbs the gut immune barrier and ultimately results in alcoholic liver diseases, but little is known about how immune-related cells in the gut are perturbed in this process. In this study, we employed laser capture microdissection and a label-free proteomics approach to investigate the consequences of alcohol exposure to the proteomes of crypts and villi in the proximal small intestine. Intestinal tissues from alcohol-fed and pair-fed mice were microdissected to selectively capture cells in the crypts and villi regions, followed by one-pot protein digestion and data-independent LC-MS/MS analysis. We successfully identified over 3000 proteins from each of the crypt or villi regions equivalent to ∼3000 cells. Analysis of alcohol-treated tissues indicated an enhanced alcohol metabolism and reduced levels of α-defensins in crypts, alongside increased lipid metabolism and apoptosis in villi. Immunofluorescence imaging further corroborated the proteomic findings. Our work provides a detailed profiling of the proteomic changes in the compartments of the mouse small intestine and aids in molecular-level understanding of alcohol-induced tissue damage.
Topics: Animals; Intestine, Small; Proteomics; Mice; Ethanol; Tandem Mass Spectrometry; Proteome; Laser Capture Microdissection; Chromatography, Liquid; Intestinal Mucosa; Mice, Inbred C57BL; Male; Apoptosis; Lipid Metabolism
PubMed: 38655769
DOI: 10.1021/acs.jproteome.4c00037 -
FASEB Journal : Official Publication of... Apr 2024Intestinal barrier dysfunction usually occurred in acute pancreatitis (AP) but the mechanism remains unclear. In this study, RNA sequencing of ileum in...
Intestinal barrier dysfunction usually occurred in acute pancreatitis (AP) but the mechanism remains unclear. In this study, RNA sequencing of ileum in L-arginine-induced AP mice demonstrated that phosphoenolpyruvate kinase 1 (Pck1) was significantly up-regulated. Increased Pck1 expression in intestinal epithelial cells (IECs) was further validated in ileum of AP mice and duodenum of AP patients. In AP mice, level of Pck1 was positively correlated with pancreatic and ileal histopathological scores, serum amylase activity, and intestinal permeability (serum diamine oxidase (DAO), D-lactate, and endotoxin). In AP patients, level of Pck1 had a positive correlation with Ranson scores, white blood cell count and C-reactive protein. Inhibition of Pck1 by 3-Mercaptopicolinic acid hydrochloride (3-MPA) alleviated pancreatic and ileal injuries in AP mice. AP + 3-MPA mice showed improved intestinal permeability, including less epithelial apoptosis, increased tight junction proteins (TJPs) expression, decreased serum DAO, D-lactate, endotoxin, and FITC-Dextran levels, and reduced bacteria translocation. Lysozyme secreted by Paneth cells and mucin2 (MUC2) secretion in goblet cells were also partly restored in AP + 3-MPA mice. Meanwhile, inhibition of Pck1 improved intestinal immune response during AP, including elevation of M2/M1 macrophages ratio and secretory immunoglobulin A (sIgA) and reduction in neutrophils infiltration. In vitro, administration of 3-MPA dramatically ameliorated inflammation and injuries of epithelial cells in enteroids treated by LPS. In conclusion, inhibition of Pck1 in IECs might alleviate AP via modulating intestinal homeostasis.
Topics: Animals; Mice; Epithelial Cells; Homeostasis; Intestinal Mucosa; Mice, Inbred C57BL; Pancreatitis; Phosphoenolpyruvate Carboxykinase (GTP); Picolinic Acids
PubMed: 38651689
DOI: 10.1096/fj.202400039R -
Ecotoxicology and Environmental Safety Jun 2024The intricate architecture of the intestinal epithelium, crucial for nutrient absorption, is constantly threatened by environmental factors. The epithelium undergoes...
The intricate architecture of the intestinal epithelium, crucial for nutrient absorption, is constantly threatened by environmental factors. The epithelium undergoes rapid turnover, which is essential for maintaining homeostasis, under the control of intestinal stem cells (ISCs). The central regulator, Wnt/β-catenin signaling plays a key role in intestinal integrity and turnover. Despite its significance, the impact of environmental factors on this pathway has been largely overlooked. This study, for the first time, investigates the influence of Cd on the intestinal Wnt signaling pathway using a mouse model. In this study, male BALB/c mice were administered an environmentally relevant Cd dose (0.98 mg/kg) through oral gavage to investigate the intestinal disruption and Wnt signaling pathway. Various studies, including histopathology, immunohistochemistry, RT-PCR, western blotting, ELISA, intestinal permeability assay, and flow cytometry, were conducted to study Cd-induced changes in the intestine. The canonical Wnt signaling pathway experienced significant downregulation as a result of sub-chronic Cd exposure, which caused extensive damage throughout the small intestine. Increased intestinal permeability and a skewed immune response were also observed. To confirm that Wnt signaling downregulation is the key driver of Cd-induced gastrointestinal toxicity, mice were co-exposed to LiCl (a recognized Wnt activator) and Cd. The results clearly showed that the harmful effects of Cd could be reversed, which is strong evidence that Cd mostly damages the intestine through the Wnt/β-catenin signalling axis. In conclusion, this research advances the current understanding of the role of Wnt/β catenin signaling in gastrointestinal toxicity caused by diverse environmental pollutants.
Topics: Animals; Male; Mice; beta Catenin; Cadmium; Inflammation; Intestinal Mucosa; Intestines; Mice, Inbred BALB C; Wnt Signaling Pathway
PubMed: 38640798
DOI: 10.1016/j.ecoenv.2024.116337 -
International Journal of Biological... 2024The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the...
The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell (IEC)-specific knockout () of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and reduces mTORC1 signaling. Surprisingly, adult intestinal crypts have increased cell proliferation but reduced mature Paneth cells. Goblet cells, the other major secretory cell type in the small intestine, are increased in the crypts but reduced in the villi. Analyses with scRNA-seq and electron microscopy have revealed dedifferentiation of Paneth cells in mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. Thus, SLC7A5 likely regulates secretory cell differentiation to affect stem cell niche and indirectly regulate cell proliferation.
Topics: Animals; Mice; Amino Acid Transport Systems; Cell Differentiation; Cell Proliferation; Large Neutral Amino Acid-Transporter 1; Mechanistic Target of Rapamycin Complex 1
PubMed: 38617535
DOI: 10.7150/ijbs.94297 -
BioRxiv : the Preprint Server For... Apr 2024Obesity has been associated with dysbiosis, but innate mechanisms linking intestinal epithelial cell subsets and obesity remain poorly understood. Using mice lacking...
Obesity has been associated with dysbiosis, but innate mechanisms linking intestinal epithelial cell subsets and obesity remain poorly understood. Using mice lacking Paneth cells (Sox9 mice), small intestinal epithelial cells specialized in the production of antimicrobial products and cytokines, we show that dysbiosis alone does not induce obesity or metabolic disorders. Loss of Paneth cells reduced ILC3 and increased ILC2 numbers in the intestinal lamina propria. High-fat diet (HFD) induced higher weight gain and more severe metabolic disorders in Sox9 mice. Further, HFD enhances the number of ILC1 in the intestinal lamina propria of Sox9 mice and increases intestinal permeability and the accumulation of immune cells (inflammatory macrophages and T cells, and B cells) in abdominal fat tissues of obese Sox9 . Transplantation of fecal materials from Sox9 mice in germ-free mice before HFD further confirmed the regulatory role of Paneth cells for gut ILC subsets and the development of obesity.
PubMed: 38617293
DOI: 10.1101/2024.03.29.587349 -
Nature Communications Apr 2024Epithelial barrier dysfunction and crypt destruction are hallmarks of inflammatory bowel disease (IBD). Intestinal stem cells (ISCs) residing in the crypts play a...
Epithelial barrier dysfunction and crypt destruction are hallmarks of inflammatory bowel disease (IBD). Intestinal stem cells (ISCs) residing in the crypts play a crucial role in the continuous self-renewal and rapid recovery of intestinal epithelial cells (IECs). However, how ISCs are dysregulated in IBD remains poorly understood. Here, we observe reduced DHX9 protein levels in IBD patients, and mice with conditional DHX9 depletion in the intestinal epithelium (Dhx9) exhibit an increased susceptibility to experimental colitis. Notably, Dhx9 mice display a significant reduction in the numbers of ISCs and Paneth cells. Further investigation using ISC-specific or Paneth cell-specific Dhx9-deficient mice demonstrates the involvement of ISC-expressed DHX9 in maintaining epithelial homeostasis. Mechanistically, DHX9 deficiency leads to abnormal R-loop accumulation, resulting in genomic instability and the cGAS-STING-mediated inflammatory response, which together impair ISC function and contribute to the pathogenesis of IBD. Collectively, our findings highlight R-loop-mediated genomic instability in ISCs as a risk factor in IBD.
Topics: Animals; Humans; Mice; DEAD-box RNA Helicases; Epithelial Cells; Homeostasis; Inflammatory Bowel Diseases; Intestinal Mucosa; Neoplasm Proteins; Paneth Cells; R-Loop Structures; Stem Cells
PubMed: 38594251
DOI: 10.1038/s41467-024-47235-2 -
American Journal of Physiology.... Jun 2024Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via...
Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA in regulating intestinal epithelial regeneration. Control mice () and mice with an inducible, epithelial cell-specific deletion of in the small intestine () were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in mice with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of in Lgr5+-stem cells or ). Irradiation of organoids resulted in fewer numbers of organoids retaining Lgr5+-derived progenitor cells compared with organoids. Finally, we observed that impaired regeneration in mice was associated with reduced numbers of Paneth cells and decreased expression of Yes-associated protein (YAP), a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche. We used mice lacking expression of the lysophosphatidic acid receptor 5 (LPA) in intestinal epithelial cells and intestinal organoids to show that the LPA receptor protects intestinal stem cells and progenitors from radiation-induced injury. We show that LPA induces YAP signaling and regulates Paneth cells.
Topics: Animals; Mice; Apoptosis; Cell Proliferation; Intestinal Mucosa; Intestine, Small; Lysophospholipids; Mice, Knockout; Organoids; Radiation Injuries, Experimental; Receptors, Lysophosphatidic Acid; Regeneration; Signal Transduction; Stem Cells; YAP-Signaling Proteins
PubMed: 38593468
DOI: 10.1152/ajpgi.00269.2023 -
Cell & Bioscience Apr 2024The prevalence of Crohn's disease (CD), a subtype of inflammatory bowel disease (IBD), is increasing worldwide. The pathogenesis of CD is hypothesized to be related to... (Review)
Review
The prevalence of Crohn's disease (CD), a subtype of inflammatory bowel disease (IBD), is increasing worldwide. The pathogenesis of CD is hypothesized to be related to environmental, genetic, immunological, and bacterial factors. Current studies have indicated that intestinal epithelial cells, including columnar, Paneth, M, tuft, and goblet cells dysfunctions, are strongly associated with these pathogenic factors. In particular, goblet cells dysfunctions have been shown to be related to CD pathogenesis by direct or indirect ways, according to the emerging studies. The mucus barrier was established with the help of mucins secreted by goblet cells. Not only do the mucins mediate the mucus barrier permeability and bacterium selection, but also, they are closely linked with the endothelial reticulum stress during the synthesis process. Goblet cells also play a vital role in immune response. It was indicated that goblet cells take part in the antigen presentation and cytokines secretion process. Disrupted goblet cells related immune process were widely discovered in CD patients. Meanwhile, dysbiosis of commensal and pathogenic microbiota can induce myriad immune responses through mucus and goblet cell-associated antigen passage. Microbiome dysbiosis lead to inflammatory reaction against pathogenic bacteria and abnormal tolerogenic response. All these three pathways, including the loss of mucus barrier function, abnormal immune reaction, and microbiome dysbiosis, may have independent or cooperative effect on the CD pathogenesis. However, many of the specific mechanisms underlying these pathways remain unclear. Based on the current understandings of goblet cell's role in CD pathogenesis, substances including butyrate, PPARγagonist, Farnesoid X receptor agonist, nuclear factor-Kappa B, nitrate, cytokines mediators, dietary and nutrient therapies were all found to have potential therapeutic effects on CD by regulating the goblet cells mediated pathways. Several monoclonal antibodies already in use for the treatment of CD in the clinical settings were also found to have some goblet cells related therapeutic targets. In this review, we introduce the disease-related functions of goblet cells, their relationship with CD, their possible mechanisms, and current CD treatments targeting goblet cells.
PubMed: 38561835
DOI: 10.1186/s13578-024-01220-w -
Mucosal Immunology Mar 2024Studies have reported the occurrence of gastrointestinal (GI) symptoms, primarily diarrhea, in COVID-19. However, the pathobiology regarding COVID-19 in the GI tract...
Studies have reported the occurrence of gastrointestinal (GI) symptoms, primarily diarrhea, in COVID-19. However, the pathobiology regarding COVID-19 in the GI tract remains limited. This work aimed to evaluate SARS-CoV-2 Spike protein interaction with gut lumen in different experimental approaches. Here, we present a novel experimental model with the inoculation of viral protein in the murine jejunal lumen, in vitro approach with human enterocytes, and molecular docking analysis. Spike protein led to increased intestinal fluid accompanied by Cl secretion, followed by intestinal edema, leukocyte infiltration, reduced glutathione levels, and increased cytokine levels [interleukin (IL)-6, tumor necrosis factor-α, IL-1β, IL-10], indicating inflammation. Additionally, the viral epitope caused disruption in the mucosal histoarchitecture with impairment in Paneth and goblet cells, including decreased lysozyme and mucin, respectively. Upregulation of toll-like receptor 2 and toll-like receptor 4 gene expression suggested potential activation of local innate immunity. Moreover, this experimental model exhibited reduced contractile responses in jejunal smooth muscle. In barrier function, there was a decrease in transepithelial electrical resistance and alterations in the expression of tight junction proteins in the murine jejunal epithelium. Additionally, paracellular intestinal permeability increased in human enterocytes. Finally, in silico data revealed that the Spike protein interacts with cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride conductance (CaCC), inferring its role in the secretory effect. Taken together, all the events observed point to gut impairment, affecting the mucosal barrier to the innermost layers, establishing a successful experimental model for studying COVID-19 in the GI context.
PubMed: 38555027
DOI: 10.1016/j.mucimm.2024.03.009