Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE212636 | GEO | 2022/11/01
REPOSITORIES: GEO
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Project description:Enteroendocrine cells (EECs) secrete hormones in response to ingested nutrients to control physiological processes such as appetite and insulin release. EEC hormones are synthesized as large proproteins that undergo proteolytic processing to generate bioactive peptides. Mutations in EEC-enriched proteases are associated with endocrinopathies. Due to the relative rarity of EECs and a paucity of in vitro models, intestinal prohormone processing remains challenging to assess. Here, human gut organoids in which EECs can efficiently be induced are subjected to CRISPR-Cas9-mediated modification of EEC-expressed endopeptidase and exopeptidase genes. We employ mass spectrometry-based analyses to monitor peptide processing and identify glucagon production in intestinal EECs, stimulated upon bone morphogenic protein (BMP) signaling. We map the substrates and products of major EECs endo- and exopeptidases. Our studies provide a comprehensive description of peptide hormones produced by human EECs and define the roles of specific proteases in their generation.
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Project description:Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models
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Project description:More than two decades of research has revealed a combination of proteases that determine cartilage degradation in osteoarthritis. These include metalloproteinases, which degrade the major macromolecules in cartilage, aggrecan and type II collagen, serine proteases, and cysteine proteases, for example cathepsin K. This review summarizes the function of proteases in osteoarthritis progression, as revealed by studies of genetically engineered mouse models. A brief overview of the biochemical characteristics and features of several important proteases is provided, with the objective of increasing understanding of their function. Published data reveal at least three enzymes to be major targets for osteoarthritis drug development: ADAMTS-5, MMP-13, and cathepsin K. In surgical models of osteoarthritis, mice lacking these enzymes are protected from cartilage damage and, to varying degrees, from bone changes. In-vivo studies targeting these proteases with selective small-molecule inhibitors have been performed for a variety of animal models. Mouse models will provide opportunities for future tests of the therapeutic effect of protease inhibitors, both on progression of structural damage to the joint and on associated pain.
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Project description:Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real-time at single-cell level using a novel knock-in allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L-cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR-modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1 and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.
2019-01-31 | GSE113561 | GEO
Project description:Enteroendocrine cells (EECs) alter hormone expression while migrating along the crypt-villus axis. We report this swich is regulated by a BMP morphogen gradient. Single cell RNA sequencing of control and BMP4 stimulated EECs was performed using organoids from different hormone reporter mice. We show that BMP4 stimulation can switch different subsets of EECs from their crypt to villus state.
2018-08-01 | GSE114988 | GEO
Project description:We generated knock-in mice expressing GFP under the control of the endogenous GIP (Glucose-dependent Insulinotropic Polypeptide) promoter that enable the isolation of a purified population of small intestine K cells. Using RNA-Seq, we comprehensively characterized the transcriptomes of GIP-GFP cells as well as the entire enteroendocrine lineage derived from Neurogenin3 (Ngn3)-expressing progenitors. We interrogated the whole transcriptome of FACS-isolated small intestine GIPGFP cells using high-throughput mRNA sequencing. We also obtained the global gene expression patterns of the entire enteroendocrine cell lineage as well as the non-enteroendocrine cell population, comprising enterocytes, goblet cells and Paneth cells. To achieve this, small intestine epithelial cells from male mice resulting from the breeding of Neurogenin3 (Ngn3)-Cre mice with ROSA26-LoxP-STOP-LoxP-tomato indicator mice were isolated based on Tomato fluorescence and negative staining for CD45. Due to the small cell numbers, we constructed each of the three RNA-Seq libraries (GIPGFP, Ngn3TOMATO, and Ngn3-) using a pool of equal amounts of individual RNA samples without RNA amplification.
2015-01-01 | E-GEOD-61250 | biostudies-arrayexpress
Project description:Despite major improvement in treatment of early stage localised prostate cancer, the distinction between indolent tumors and those that will become aggressive, as well as the lack of efficient therapies of advanced prostate cancer, remain major health problems. Genetically engineered mice (GEM) have been extensively used to investigate the molecular and cellular mechanisms underlying prostate tumor initiation and progression, and to evaluate new therapies. Moreover, the recent development of conditional somatic mutagenesis in the mouse prostate offers the possibility to generate new models that more faithfully reproduce the human disease, and thus should contribute to improve diagnosis and treatments. The strengths and weaknesses of various models will be discussed, as well as future opportunities.
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Project description:Enteroendocrine cells (EECs) are the source of a variety of gut hormones that control local intestinal functions such as gallbladder contraction, pancreatic enzyme secretion and intestinal motility, as well as peripheral nutrient metabolism and appetite. This study aimed to map human EECs isolated from organoids derived from duodenum and ileum using single-cell RNA sequencing.
2024-12-16 | GSE284419 | GEO
Project description:PurposeLung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed.Experimental designWe used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines.ResultsWe show that multiplex gene editing of mouse lung organoids using the CRISPR-Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell-mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti-PD-1 therapy.ConclusionsWe developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage-inducing therapies in the treatment of LSCC.
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Project description:Mouse duodenal enteroendocrine transcriptome
| PRJNA473085 | ENA