Cellular and Molecular Gastroenterology and Hepatology最新文献

Background and aims: The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically engineered mouse models that allow temporal and spatial activation of oncogenic KRAS (KRAS^G12D). One of the most commonly used models involves targeted insertion of a cre-recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS (KRAS^G12D). This study aims to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRAS^G12D before and after induction of pancreatic injury.

Methods: We performed morphological and molecular analysis of three genetically engineered mouse models that express a tamoxifen-inducible cre-recombinase to activate Kras^G12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a or Mist1/Bhlha15, or expressed within a BAC-derived Elastase transgene. Histological and RNA-seq analyses were used to delineate differences between the models.

Results: Up to two months after tamoxifen induction of KRAS^G12D, morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in widespread PanIN lesions in Ptf1a^creERT pancreata within seven days and maintained for at least five weeks post-injury, which was not seen in the models with two functional Ptf1a alleles. RNA-seq analysis prior to injury induction suggested Ptf1a^creERT and Mist1^creERT mice have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the models.

Conclusions: These findings suggest Ptf1a haploinsufficiency in Ptf1a^creERT mouse models promotes KRAS^G12D priming of genes for promotion of PDAC.

Background & aims: TGFβ1 induces plasma membrane (PM) accumulation of glucose transporter 1 (Glut1) required for glycolysis of hepatic stellate cells (HSCs) and HSC activation. This study aimed to understand how Glut1 is anchored/docked onto the PM of HSCs.

Methods: HSC expression of protein kinase M zeta isoform (PKMζ) was detected by RT-PCR, Western blotting, and immunofluorescence. PKMζ level was manipulated by shRNA or overexpression; HSC activation was assessed by cell expression of activation markers; PM Glut1, glucose uptake, and glycolysis of HSCs were analyzed by biotinylation, 2-NBDG-based assay, and Seahorse Glycolysis Stress Test. Phospho-mutants of vasodilator-stimulated phosphorylated protein (VASP) were created by site-directed mutagenesis. TGFβ transcriptome was obtained by RNA sequencing. Single-cell RNA sequencing datasets and immunofluorescence were leveraged to analyze PKMζ expression in cancer-associated fibroblasts (CAFs) of colorectal liver metastases. Function of HSC PKMζ was determined by tumor/HSC co-implantation study.

Results: Primary human and murine HSCs express PKMζ, but not full-length PKCζ. PKMζ knockdown suppresses whereas PKMζ overexpression potentiates PM accumulation of Glut1, glycolysis, and HSC activation induced by TGFβ1. Mechanistically, PKMζ binds to and induces VASP phosphorylation at serines 157 and 239 facilitating anchoring/docking of Glut1 onto the PM of HSCs. PKMζ expression is increased in the CAFs of murine and patient colorectal liver metastases compared to quiescent HSC. Targeting PKMζ suppresses transcriptome, CAF activation of HSCs, and colorectal tumor growth in mice.

Conclusions: Since HSCs are also a major contributor of liver fibrosis, our data highlight PKMζ and VASP as targets to inhibit metabolic reprogramming, HSC activation, liver fibrosis, and the pro-metastatic microenvironment of the liver.

The onset of colorectal cancer (CRC) in patients under 50 continues to rapidly increase. This study highlights the epidemiological changes, risk factors, clinical characteristics, and molecular profiles prevalent in early-onset colorectal cancer (EO-CRC) patients, and identifies key areas for future research. It has been noted that only a small fraction of EO-CRC cases is attributed to known hereditary mutations and fit the canonical pathway of late onset colorectal cancer (LOCRC) development. To highlight this, we review the genetic and epigenetic modifications specific to EO-CRC. We also discuss the synergetic effect of single nucleotide polymorphisms (SNPs) and environmental factors on the early onset of CRC. Additionally, we discuss the potential of non-invasive biomarker assays to enhance early detection, screening, diagnosis, and prognostic outcome predictions.

Background & aims: Cystic fibrosis (CF) is an autosomal recessive genetic disorder, affecting multiple organ systems. CF intestinal disease develops early, manifesting as intestinal bacterial overgrowth/dysbiosis, neutrophilic inflammation and obstruction. As unresolvable infection and inflammation reflect host immune deficiency, we sought to determine if the CF-affected immune system plays any significant role in CF intestinal disease pathogenesis.

Methods: CF and sibling wild-type (WT) mice underwent reciprocal bone marrow transplantation. After immune reconstitution, their mortality, intestinal transit, fecal inflammatory markers, and mucosal immune cell composition were assessed. Moreover, reciprocal neutrophil transfusion was conducted to determine if neutrophil function affects intestinal movement. Furthermore, expression of induced nitric oxide synthase (iNOS) and production of nitric oxide (NO) in CF and WT neutrophils were compared. Lastly, specific iNOS inhibitor 1400W was tested to prevent CF intestinal obstruction.

Results: Immune restoration in CF mice reversed the intestinal neutrophilic inflammation, improved the intestinal dysmotility, and rescued the mice from mortality. Transfusion of WT neutrophils into CF mice ameliorated the retarded bowel movement. CF neutrophils expressed significantly more iNOS and produced significantly more NO. Pharmaceutical blocking of iNOS significantly improved intestinal transit and survival of CF mice.

Conclusion: CF immune defect plays a critical role in CF intestinal disease development. Activation of iNOS in inflammatory cells produces excessive NO, slows the bowel movement, and facilitates intestinal paralysis and obstruction in CF. Thus, normalization of the CF immune system may offer a novel therapy to treat CF intestinal disease.

The intestinal epithelium forms the boundary between the intestinal immune system in the lamina propria and the outside world, the intestinal lumen, which contains a diverse array of microbial and environmental antigens. Composed of specialized cells, this epithelial monolayer has an exceptional turnover rate. Differentiated epithelial cells are released into the intestinal lumen within a few days, at the villus tip, a process that requires strict regulation. Dysfunction of the epithelial barrier increases the intestinal permeability and paves the way for luminal antigens to pass into the intestinal serosa. Stem cells at the bottom of Lieberkühn crypts provide a constant supply of mature epithelial cells. Differentiated intestinal epithelial cells exhibit a diverse array of mechanisms that enable communication with surrounding cells, fortification against microorganisms, and orchestration of nutrient absorption and hormonal balance. Furthermore, tight junctions regulate paracellular permeability properties, and their disruption can lead to an impairment of the intestinal barrier, allowing inflammation to develop or further progress. Intestinal epithelial cells provide a communication platform through which they maintain homeostasis with a spectrum of entities including immune cells, neuronal cells, and connective tissue cells. This homeostasis can be disrupted in disease, such as inflammatory bowel disease. Patients suffering from inflammatory bowel disease show an impaired gut barrier, dysregulated cellular communication, and aberrant proliferation and demise of cells. This review summarizes the individual cellular and molecular mechanisms pivotal for upholding the integrity of the intestinal epithelial barrier and shows how these can be disrupted in diseases, such as inflammatory bowel disease.

Background & aims: Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC; however, its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cells (iPSCs) model.

Methods: Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps).

Results: The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation(H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function.

Conclusions: Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Background & aims: The interactions between human milk and the regulation of innate immune homeostasis in newborns, and their impact on intestinal health, are not fully understood. This study aimed to explore the role of peptides in human milk extracellular vesicles (EVs) in this process.

Methods: A comprehensive screening of peptides within human milk EVs was performed, leading to the identification of a beta-casein-derived peptide (CASB_135-150). The effects of CASB_135-150 on intestinal injury were evaluated in a rat necrotizing enterocolitis (NEC) model. Immunofluorescence analysis was used to determine its distribution, and its impact on NF-κB signaling and inflammation was studied in bone marrow-derived macrophages (BMDMs) and intestinal macrophages. Protein-protein interaction (PPI) analysis, single-cell RNA-seq (scRNA-seq), and co-immunoprecipitation (co-IP) experiments were conducted to explore the mechanism underlying CASB_135-150 function.

Results: CASB_135-150 significantly mitigated intestinal injury in the rat NEC model. Immunofluorescence analysis revealed that CASB_135-150 could target intestinal macrophages and rapidly inhibited NF-κB signaling and reduced inflammation. ScRNA-seq analyses indicated a strong association between FHL2 and NEC development, and co-IP confirmed the interaction between CASB_135-150 and FHL2. CASB_135-150 disrupted the FHL2/TRAF6 complex, reducing TRAF6 protein levels. Mutation of key amino acids in CASB_135-150 disrupted its interaction with FHL2 and abolished its ability to inhibit NF-κB signaling, which also prevented its protective effect in vivo. RNA-seq of intestinal tissue further highlighted the impact of CASB_135-150 on the NF-κB signaling pathway.

Conclusions: Our study identifies CASB_135-150, a novel peptide in human milk EVs, that rapidly regulates macrophage inflammatory responses and protects against NEC-induced intestinal injury. These findings provide new insights into the role of human milk in modulating the infant immune system and intestinal health.