RESUMEN
BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.
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Adenocarcinoma/metabolismo , Adenoma/metabolismo , Transformación Celular Neoplásica/metabolismo , Daño del ADN , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenoma/tratamiento farmacológico , Adenoma/genética , Adenoma/patología , Animales , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Bases de Datos Genéticas , Estrés del Retículo Endoplásmico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Neoplasias Intestinales/tratamiento farmacológico , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Inhibidores mTOR/farmacología , Ratones Noqueados , Transducción de Señal , Sirolimus/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.
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Factor de Transcripción Activador 6/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Células Epiteliales/patología , Íleon/metabolismo , Íleon/patología , Enfermedades Inflamatorias del Intestino/metabolismo , Animales , Autofagia , Células CACO-2 , Técnicas de Cultivo de Célula , Chaperón BiP del Retículo Endoplásmico , Células HEK293 , Humanos , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Ratones , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.
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Transformación Celular Neoplásica/metabolismo , Células Epiteliales/enzimología , Inestabilidad Genómica , Neoplasias Intestinales/prevención & control , Intestino Delgado/enzimología , Ribonucleasa H/metabolismo , Animales , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Colitis/inducido químicamente , Colitis/enzimología , Colitis/genética , Colitis/patología , Daño del ADN , Sulfato de Dextran , Modelos Animales de Enfermedad , Células Epiteliales/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Intestino Delgado/patología , Masculino , Ratones Noqueados , Fenotipo , Ribonucleasa H/deficiencia , Ribonucleasa H/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
INTRODUCTION: Patients with inflammatory bowel disease (IBD) are predisposed to the reactivation of viral infections such as cytomegalovirus (CMV). Clinical discrimination of disease flares and colonic CMV reactivation is difficult in patients with established diagnosis of IBD, and there are no reliable noninvasive diagnostic tools yet. Furthermore, the influence of novel therapeutics including biologicals and Janus kinase inhibitors on the risk of CMV colitis is unclear. The goal of this study was to identify risk factors and clinical determinants of CMV colitis that could serve as minimally invasive markers both for active CMV colitis and relapse. METHODS: To this end, a retrospective analysis of 376 patients with suspected or confirmed CMV colitis 2016-2023 was performed. RESULTS: Previous administration of systemic steroids increased the odds of CMV colitis to OR 4.6. Biologicals did not change the incidence of CMV colitis but decreased the OR of a relapse to 0.13. Clinical parameters such as severely bloody diarrhea, intense microscopic ulcerative damage, and decreased serum tryptophan correlated with detection of CMV. Importantly, persistent decrease of tryptophan was observed in patients with CMV relapse. Furthermore, tryptophan degradation through the kynurenine pathway was increased in CMV-positive patients. DISCUSSION: Taken together, we identify decreased serum tryptophan as a novel potential minimally invasive marker to aid identification of IBD patients with active CMV colitis and at high risk for relapse.
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Infecciones por Citomegalovirus , Citomegalovirus , Mucosa Intestinal , Triptófano , Humanos , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/virología , Infecciones por Citomegalovirus/complicaciones , Masculino , Femenino , Estudios Retrospectivos , Persona de Mediana Edad , Adulto , Mucosa Intestinal/patología , Mucosa Intestinal/virología , Triptófano/sangre , Triptófano/metabolismo , Citomegalovirus/aislamiento & purificación , Citomegalovirus/inmunología , Factores de Riesgo , Colon/patología , Colon/virología , Colitis Ulcerosa/complicaciones , Colitis Ulcerosa/sangre , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/virología , Colitis Ulcerosa/tratamiento farmacológico , Colitis/virología , Colitis/sangre , Colitis/diagnóstico , Colitis/complicaciones , Biomarcadores/sangre , Recurrencia , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Anciano , Colonoscopía , Activación ViralRESUMEN
BACKGROUND: Chronic inflammatory diseases (CIDs) are systems disorders that affect diverse organs including the intestine, joints and skin. The essential amino acid tryptophan (Trp) can be broken down to various bioactive derivatives important for immune regulation. Increased Trp catabolism has been observed in some CIDs, so we aimed to characterise the specificity and extent of Trp degradation as a systems phenomenon across CIDs. METHODS: We used high performance liquid chromatography and targeted mass spectrometry to assess the serum and stool levels of Trp and Trp derivatives. Our retrospective study incorporates both cross-sectional and longitudinal components, as we have included a healthy population as a reference and there are also multiple observations per patient over time. FINDINGS: We found reduced serum Trp levels across the majority of CIDs, and a prevailing negative relationship between Trp and systemic inflammatory marker C-reactive protein (CRP). Notably, serum Trp was low in several CIDs even in the absence of measurable systemic inflammation. Increases in the kynurenine-to-Trp ratio (Kyn:Trp) suggest that these changes result from increased degradation along the kynurenine pathway. INTERPRETATION: Increases in Kyn:Trp indicate the kynurenine pathway as a major route for CID-related Trp metabolism disruption and the specificity of the network changes indicates excessive Trp degradation relative to other proteogenic amino acids. Our results suggest that increased Trp catabolism is a common metabolic occurrence in CIDs that may directly affect systemic immunity. FUNDING: This work was supported by the DFG Cluster of Excellence 2167 "Precision medicine in chronic inflammation" (KA, SSchr, PR, BH, SWa), the BMBF (e:Med Juniorverbund "Try-IBD" 01ZX1915A and 01ZX2215, the e:Med Network iTREAT 01ZX2202A, and GUIDE-IBD 031L0188A), EKFS (2020_EKCS.11, KA), DFG RU5042 (PR, KA), and Innovative Medicines Initiative 2 Joint Undertakings ("Taxonomy, Treatments, Targets and Remission", 831434, "ImmUniverse", 853995, "BIOMAP", 821511).
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Enfermedades Inflamatorias del Intestino , Triptófano , Humanos , Triptófano/metabolismo , Quinurenina , Estudios Retrospectivos , Estudios Transversales , Inflamación/metabolismo , Enfermedad CrónicaRESUMEN
Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.
RESUMEN
A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling, which is augmented in Atg16l1 ΔIEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo, IL-22 treatment in Atg16l1 ΔIEC and Atg16l1 ΔIEC/Xbp1 ΔIEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22-induced ileal inflammation in Atg16l1 ΔIEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium.