RESUMEN
Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing.
Asunto(s)
Adenina/análogos & derivados , Homólogo 4 de AlkB Lisina Desmetilasa/genética , Anomalías Craneofaciales/genética , ADN/genética , Metiltransferasas/genética , Proteínas Represoras/genética , Adenina/metabolismo , Homólogo 4 de AlkB Lisina Desmetilasa/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , ADN/metabolismo , Metilación de ADN , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Femenino , Silenciador del Gen , Genes Letales , Histonas/genética , Histonas/metabolismo , Endogamia , Masculino , Metiltransferasas/deficiencia , Ratones , Ratones Noqueados , Proteolisis , Proteínas Represoras/metabolismo , Transducción de Señal , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Transcripción Genética , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
BACKGROUND AND AIMS: Relative roles of HSCs and portal fibroblasts in alcoholic hepatitis (AH) are unknown. We aimed to identify subpopulations of collagen type 1 alpha 1 (Col1a1)-expressing cells in a mouse AH model by single-cell RNA sequencing (scRNA-seq) and filtering the cells with the HSC (lecithin retinol acyltransferase [Lrat]) and portal fibroblast (Thy-1 cell surface antigen [Thy1] and fibulin 2 [Fbln2]) markers and vitamin A (VitA) storage. APPROACH AND RESULTS: Col1a1-green fluorescent protein (GFP) mice underwent AH, CCl 4 , and bile duct ligation (BDL) procedures to have comparable F1-F2 liver fibrosis. Col1a1-expressing cells were sorted via FACS by VitA autofluorescence and GFP for single-cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1-Fbln2- activated HSCs were VitA-depleted (vs. ~13% in BDL and CCl 4 ). Supervised clustering identified a subset co-expressing Lrat and Fbln2 (Lrat+Fbln2+), which expanded 44-fold, 17-fold, and 1.3-fold in AH, BDL, and CCl 4 . Lrat+Fbln2+ cells had 3-15-times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+Fbln2- cells, but 2-4-times repressed HSC-selective genes. AH activated HSCs had up-regulated inflammatory (chemokine [C-X-C motif] ligand 2 [Cxcl2], chemokine [C-C motif] ligand 2), antimicrobial (Il-33, Zc3h12a), and antigen presentation (H2-Q6, H2-T23) genes versus BDL and CCl 4 . Computational deconvolution of AH versus normal human bulk-liver RNA-sequencing data supported an expansion of LRAT+FBLN2+ cells in AH; AH patient liver immunohistochemistry showed FBLN2 staining along fibrotic septa enriched with LRAT+ cells; and in situ hybridization confirmed co-expression of FBLN2 with CXCL2 and/or human leukocyte antigen E in patient AH. Finally, HSC tracing in Lrat-Cre;Rosa26mTmG mice detected GFP+FBLN2+ cells in AH. CONCLUSION: A highly profibrotic, inflammatory, and immunoregulatory Lrat+Fbln2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH.
Asunto(s)
Hepatitis Alcohólica , Ratones , Humanos , Animales , Hepatitis Alcohólica/patología , Ligandos , Células Estrelladas Hepáticas/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Aciltransferasas/metabolismo , Modelos Animales de EnfermedadRESUMEN
Regulatory T (Treg) cells are essential for maintenance of immune homeostasis. Here we found that hydrogen sulfide (H2S) was required for Foxp3(+) Treg cell differentiation and function and that H2S deficiency led to systemic autoimmune disease. H2S maintained expression of methylcytosine dioxygenases Tet1 and Tet2 by sulfhydrating nuclear transcription factor Y subunit beta (NFYB) to facilitate its binding to Tet1 and Tet2 promoters. Transforming growth factor-ß (TGF-ß)-activated Smad3 and interleukin-2 (IL-2)-activated Stat5 facilitated Tet1 and Tet2 binding to Foxp3. Tet1 and Tet2 catalyzed conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in Foxp3 to establish a Treg-cell-specific hypomethylation pattern and stable Foxp3 expression. Consequently, Tet1 and Tet2 deletion led to Foxp3 hypermethylation, impaired Treg cell differentiation and function, and autoimmune disease. Thus, H2S promotes Tet1 and Tet2 expression, which are recruited to Foxp3 by TGF-ß and IL-2 signaling to maintain Foxp3 demethylation and Treg-cell-associated immune homeostasis.
Asunto(s)
Colitis/inmunología , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Sulfuro de Hidrógeno/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Factor de Unión a CCAAT/metabolismo , Diferenciación Celular/genética , Colitis/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Dioxigenasas , Factores de Transcripción Forkhead/genética , Homeostasis/genética , Homeostasis/inmunología , Humanos , Interleucina-2/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Factor de Transcripción STAT5/metabolismo , Proteína smad3/metabolismo , Linfocitos T Reguladores/trasplante , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
BACKGROUND AND AIMS: The hepatic mitogen-activated protein kinase (MAPK) cascade leading to c-Jun N-terminal kinase (JNK) activation has been implicated in the pathogenesis of nonalcoholic fatty liver (NAFL)/NASH. In acute hepatotoxicity, we previously identified a pivotal role for mitochondrial SH3BP5 (SAB; SH3 homology associated BTK binding protein) as a target of JNK, which sustains its activation through promotion of reactive oxygen species production. Therefore, we assessed the role of hepatic SAB in experimental NASH and metabolic syndrome. APPROACH AND RESULTS: In mice fed high-fat, high-calorie, high-fructose (HFHC) diet, SAB expression progressively increased through a sustained JNK/activating transcription factor 2 (ATF2) activation loop. Inducible deletion of hepatic SAB markedly decreased sustained JNK activation and improved systemic energy expenditure at 8 weeks followed by decreased body fat at 16 weeks of HFHC diet. After 30 weeks, mice treated with control-antisense oligonucleotide (control-ASO) developed steatohepatitis and fibrosis, which was prevented by Sab-ASO treatment. Phosphorylated JNK (p-JNK) and phosphorylated ATF2 (p-ATF2) were markedly attenuated by Sab-ASO treatment. After 52 weeks of HFHC feeding, control N-acetylgalactosamine antisense oligonucleotide (GalNAc-Ctl-ASO) treated mice fed the HFHC diet exhibited progression of steatohepatitis and fibrosis, but GalNAc-Sab-ASO treatment from weeks 40 to 52 reversed these findings while decreasing hepatic SAB, p-ATF2, and p-JNK to chow-fed levels. CONCLUSIONS: Hepatic SAB expression increases in HFHC diet-fed mice. Deletion or knockdown of SAB inhibited sustained JNK activation and steatohepatitis, fibrosis, and systemic metabolic effects, suggesting that induction of hepatocyte Sab is an important driver of the interplay between the liver and the systemic metabolic consequences of overfeeding. In established NASH, hepatocyte-targeted GalNAc-Sab-ASO treatment reversed steatohepatitis and fibrosis.
Asunto(s)
Cirrosis Hepática/patología , Proteínas de la Membrana/metabolismo , Síndrome Metabólico/patología , Proteínas Mitocondriales/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Células Cultivadas , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Hepatocitos/patología , Humanos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Ratones , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Cultivo Primario de CélulasRESUMEN
BACKGROUND AND AIMS: Forkhead box M1 (FOXM1) and nuclear factor kappa B (NF-ĸB) are oncogenic drivers in liver cancer that positively regulate each other. We showed that methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor in the liver and inhibits NF-ĸB activity. Here, we examined the interplay between FOXM1/NF-κB and MAT1A in liver cancer. APPROACH AND RESULTS: We examined gene and protein expression, effects on promoter activities and binding of proteins to promoter regions, as well as effects of FOXM1 inhibitors T0901317 (T0) and forkhead domain inhibitory-6 (FDI-6) in vitro and in xenograft and syngeneic models of liver cancer. We found, in both hepatocellular carcinoma and cholangiocarcinoma, that an induction in FOXM1 and NF-κB expression is accompanied by a fall in MATα1 (protein encoded by MAT1A). The Cancer Genome Atlas data set confirmed the inverse correlation between FOXM1 and MAT1A. Interestingly, FOXM1 directly interacts with MATα1 and they negatively regulate each other. In contrast, FOXM1 positively regulates p50 and p65 expression through MATα1, given that the effect is lost in its absence. FOXM1, MATα1, and NF-κB all bind to the FOX binding sites in the FOXM1 and MAT1A promoters. However, binding of FOXM1 and NF-κB repressed MAT1A promoter activity, but activated the FOXM1 promoter. In contrast, binding of MATα1 repressed the FOXM1 promoter. MATα1 also binds and represses the NF-κB element in the presence of p65 or p50. Inhibiting FOXM1 with either T0 or FDI-6 inhibited liver cancer cell growth in vitro and in vivo. However, inhibiting FOXM1 had minimal effects in liver cancer cells that do not express MAT1A. CONCLUSIONS: We have found a crosstalk between FOXM1/NF-κB and MAT1A. Up-regulation in FOXM1 lowers MAT1A, but raises NF-κB, expression, and this is a feed-forward loop that enhances tumorigenesis.
Asunto(s)
Proteína Forkhead Box M1/metabolismo , Neoplasias Hepáticas/genética , Metionina Adenosiltransferasa/genética , FN-kappa B/genética , Proteínas Supresoras de Tumor/genética , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Retroalimentación Fisiológica/efectos de los fármacos , Proteína Forkhead Box M1/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hepatocitos , Humanos , Hidrocarburos Fluorados/administración & dosificación , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Masculino , Metionina Adenosiltransferasa/metabolismo , Ratones , Ratones Noqueados , Cultivo Primario de Células , Regiones Promotoras Genéticas/genética , Piridinas/administración & dosificación , S-Adenosilmetionina/metabolismo , Sulfonamidas/administración & dosificación , Tiofenos/administración & dosificación , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Kras mutations are associated with pancreatic ductal adenocarcinoma (PDAC). Although tobacco smoking, pancreatitis, and obesity are known environmental risk factors for PDAC, the contribution of moderate alcohol intake to PDAC remains elusive. In the present study, we tested whether a combination of risk factors or moderate alcohol intake induces PDAC development in mice. Control Pdx1Cre and Pdx1Cre;LSL-KrasG12D mutant mice were fed a Western alcohol diet containing high levels of cholesterol and saturated fat, 3.5% alcohol, and lipopolysaccharide for 5 mo. In addition, mice were treated with cerulein, for induction of pancreatitis, and nicotine every month. Treatment with all of these risk factors promoted development of advanced pancreatic neoplasia and PDAC in the Pdx1Cre;LSL-KrasG12D mice but not in the control Pdx1Cre mice. Moderate alcohol intake or Western diet feeding also significantly promoted advanced neoplasia and PDAC development in Pdx1Cre;LSL-KrasG12D mice compared with mice fed a regular chow. Alcohol, but not Western diet, increased tumor development in the liver in the Pdx1Cre;LSL-KrasG12D mice, but its origin remained elusive due to leakiness of Pdx1Cre in hepatocytes. RNA-seq analysis revealed that alcohol feeding increases expression of markers for tumors (Epcam, Krt19, Prom1, Wt1, and Wwtr1), stroma (Dcn, Fn1, and Tnc), and cytokines (Tgfb1 and Tnf) and decreases expression of Fgf21 and Il6 in the pancreatic tumor tissues. Immunostaining showed heterogeneous expression of nephronectin, S100 calcium-binding protein A6, and vascular cell adhesion molecule 1 in pancreatic tumors surrounded by podoplanin-positive stromal cells. Our data indicate that moderate alcohol drinking is a risk factor for development of PDAC.NEW & NOTEWORTHY Heavy alcohol intake has been suspected to be a risk factor of pancreatic ductal adenocarcinoma (PDAC) in humans. However, the contribution of moderate alcohol intake to PDAC development remains elusive. In the present study, we experimentally show that moderate alcohol feeding significantly induces advanced stages of pancreatic intraepithelial neoplasia development and invasive PDAC in Pdx1Cre;LSL-KrasG12D mutant mice. Our data indicate that moderate alcohol drinking is a risk factor for PDAC.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Carcinógenos/toxicidad , Carcinoma Ductal Pancreático/inducido químicamente , Depresores del Sistema Nervioso Central/toxicidad , Etanol/toxicidad , Neoplasias Pancreáticas/inducido químicamente , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Carcinoma Ductal Pancreático/patología , Ceruletida/farmacología , Citocinas/metabolismo , Dieta Occidental , Hepatocitos/metabolismo , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Neoplasias Hepáticas/inducido químicamente , Ratones , Mutación , Nicotina/farmacología , Neoplasias Pancreáticas/patología , Transactivadores/biosíntesis , Transactivadores/genéticaRESUMEN
Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH is largely unknown. To address this question, unbiased RNA sequencing and proteomic analyses were performed on livers of the recently developed AH mouse model, which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared to gene expression profiling data from AH patients. This cross-analysis has identified Casp11 (CASP4 in humans) as a commonly up-regulated gene known to be involved in the noncanonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients, but not in chronic ASH mice and healthy human livers. Gasdermin-D (GSDMD), which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and severity of AH in the mouse model. Conversely, the deficiency of interleukin-18, the key antimicrobial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Furthermore, hepatocyte-specific expression of constitutively active GSDMD worsens hepatocellular lytic death and polymorphonuclear leukocyte inflammation. CONCLUSION: These results implicate pyroptosis induced by the CASP11/4-GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737-1753).
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasas Iniciadoras/metabolismo , Caspasas/metabolismo , Hepatitis Alcohólica/metabolismo , Proteínas de Neoplasias/metabolismo , Piroptosis/genética , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica/métodos , Humanos , Immunoblotting/métodos , Péptidos y Proteínas de Señalización Intracelular , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión a Fosfato , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , TranscriptomaRESUMEN
We have investigated the regulation of genes and associated molecular pathways, genome-wide, in oral cells of electronic cigarette (e-cigs) users and cigarette smokers as compared to non-smokers. Interrogation of the oral transcriptome by RNA-sequencing (RNA-seq) analysis showed significant number of aberrantly expressed transcripts in both e-cig users (vapers) and smokers relative to non-smokers; however, smokers had ~50% more differentially expressed transcripts than vapers (1726 versus 1152). Whereas the deregulated transcripts in smokers were predominately from protein-coding genes (79% versus 53% in vapers), nearly 28% of the aberrantly expressed transcripts in vapers (versus 8% in smokers) belonged to regulatory non-coding RNAs, including long intergenic non-coding, antisense, small nucleolar and misc RNA (P < 0.0001). Molecular pathway and functional network analyses revealed that "cancer" was the top disease associated with the deregulated genes in both e-cig users and smokers (~62% versus 79%). Examination of the canonical pathways and networks modulated in either e-cig users or smokers identified the "Wnt/Ca⺠pathway" in vapers and the "integrin signaling pathway" in smokers as the most affected pathways. Amongst the overlapping functional pathways impacted in both e-cig users and smokers, the "Rho family GTPases signaling pathway" was the top disrupted pathway, although the number of affected targets was three times higher in smokers than vapers. In conclusion, we observed deregulation of critically important genes and associated molecular pathways in the oral epithelium of vapers that bears both resemblances and differences with that of smokers. Our findings have significant implications for public health and tobacco regulatory science.
Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Mucosa Bucal/metabolismo , Transducción de Señal , Biología Computacional/métodos , Femenino , Ontología de Genes , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Reproducibilidad de los Resultados , FumarRESUMEN
It has been suggested that multipotent stem cells with neural crest (NC) origin persist into adulthood in oral mucosa. However their exact localization and role in normal homeostasis is unknown. In this study, we discovered that Lgr5 is expressed in NC cells during embryonic development, which give rise to the dormant stem cells in the adult tongue and oral mucosa. Those Lgr5 positive oral stromal stem cells display properties of NC stem cells including clonal growth and multipotent differentiation. RNA sequencing revealed that adult Lgr5+ oral stromal stem cells express high number of neural crest related markers like Sox9, Twist1, Snai1, Myc, Ets1, Crabp1, Epha2, and Itgb1. Using lineage-tracing experiments, we show that these cells persist more than a year in the ventral tongue and some areas of the oral mucosa and give rise to stromal progeny. In vivo transplantation demonstrated that these cells reconstitute the stroma. Our studies show for the first time that Lgr5 is expressed in the NC cells at embryonic day 9.5 (E9.5) and is maintained during embryonic development and postnataly in the stroma of the ventral tongue, and some areas of the oral mucosa and that Lgr5+ cells participate in the maintenance of the stroma.
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Diferenciación Celular/genética , Desarrollo Embrionario/genética , Células Madre Multipotentes/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Animales , Linaje de la Célula/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Boca/citología , Boca/crecimiento & desarrollo , Células Madre Multipotentes/citología , Cresta Neural/citología , Cresta Neural/crecimiento & desarrollo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Regulation of adult stem cells (SCs) is fundamental for organ maintenance and tissue regeneration. On the body surface, different ectodermal organs exhibit distinctive modes of regeneration and the dynamics of their SC homeostasis remain to be unraveled. A slow cycling characteristic has been used to identify SCs in hair follicles and sweat glands; however, whether a quiescent population exists in continuously growing nails remains unknown. Using an in vivo label retaining cells (LRCs) system, we detected an unreported population of quiescent cells within the basal layer of the nail proximal fold, organized in a ring-like configuration around the nail root. These nail LRCs express the hair stem cell marker, keratin 15 (K15), and lineage tracing show that these K15-derived cells can contribute to both the nail structure and peri-nail epidermis, and more toward the latter. Thus, this stem cell population is bifunctional. Upon nail plucking injury, the homeostasis is tilted with these SCs dominantly delivering progeny to the nail matrix and differentiated nail plate, demonstrating their plasticity to adapt to wounding stimuli. Moreover, in vivo engraftment experiments established that transplanted nail LRCs can actively participate in functional nail regeneration. Transcriptional profiling of isolated nail LRCs revealed bone morphogenetic protein signaling favors nail differentiation over epidermal fate. Taken together, we have found a previously unidentified ring-configured population of bifunctional SCs, located at the interface between the nail appendage organ and adjacent epidermis, which physiologically display coordinated homeostatic dynamics but are capable of rediverting stem cell flow in response to injury.
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Ectodermo/citología , Pezuñas y Garras/citología , Regeneración , Células Madre/citología , Cicatrización de Heridas , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Epidérmicas , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis , Ratones , Ratones Noqueados , Transducción de Señal , Transcripción GenéticaRESUMEN
Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell-dermal papilla-based epithelial-mesenchymal interaction layer and the hair follicle-intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation.
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Células Madre Adultas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Redes Reguladoras de Genes , Folículo Piloso/citología , Folículo Piloso/metabolismo , Vía de Señalización Wnt , Células Madre Adultas/citología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/deficiencia , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Cabello/crecimiento & desarrollo , Folículo Piloso/efectos de los fármacos , Homeostasis/genética , Queratina-15/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Modelos Genéticos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Regiones Promotoras Genéticas , Transcriptoma , Proteínas Wnt/administración & dosificaciónRESUMEN
Adult stem cells (SCs) are important to maintain homeostasis of tissues including several mini-organs like hair follicles and sweat glands. However, the existence of stem cells in minor salivary glands (SGs) is largely unexplored. In vivo histone2B green fluorescent protein pulse chase strategy has allowed us to identify slow-cycling, label-retaining cells (LRCs) of minor SGs that preferentially localize in the basal layer of the lower excretory duct with a few in the acini. Engraftment of isolated SG LRC in vivo demonstrated their potential to differentiate into keratin 5 (basal layer marker) and keratin 8 (luminal layer marker)-positive structures. Transcriptional analysis revealed activation of TGFß1 target genes in SG LRC and BMP signaling in SG progenitors. We also provide evidence that minor SGSCs are sensitive to tobacco-derived tumor-inducing agent and give rise to tumors resembling low grade adenoma. Our data highlight for the first time the existence of minor SG LRCs with stem cells characteristic and emphasize the role of transforming growth factor beta (TGFß) pathway in their maintenance.
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Células Madre Adultas/citología , Separación Celular/métodos , Glándulas Salivales Menores/citología , Animales , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here, we identified neuronal differentiation features of MD cells that sense the local and systemic environment and secrete angiogenic, growth, and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models, and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors, including CCN1, as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ-specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue-regenerative therapeutic strategies.
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Diferenciación Celular , Regeneración , Animales , Ratones , Humanos , Riñón/metabolismo , Neuronas/metabolismo , Neuronas/patología , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/genética , MasculinoRESUMEN
QUESTION: How can a library-based bioinformatics support program be implemented and expanded to continuously support the growing and changing needs of the research community? SETTING: A program at a health sciences library serving a large academic medical center with a strong research focus is described. METHODS: The bioinformatics service program was established at the Norris Medical Library in 2005. As part of program development, the library assessed users' bioinformatics needs, acquired additional funds, established and expanded service offerings, and explored additional roles in promoting on-campus collaboration. RESULTS: Personnel and software have increased along with the number of registered software users and use of the provided services. CONCLUSION: With strategic efforts and persistent advocacy within the broader university environment, library-based bioinformatics service programs can become a key part of an institution's comprehensive solution to researchers' ever-increasing bioinformatics needs.
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Biología Computacional/organización & administración , Bibliotecas Médicas/organización & administración , Servicios de Biblioteca/organización & administración , Bibliotecólogos , Evaluación de Necesidades , Rol Profesional , Desarrollo de Programa , Programas InformáticosRESUMEN
Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.
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Bacteriófagos , Guanosina Pentafosfato , Bacterias/metabolismo , Bacterias/virología , Bacteriófagos/fisiología , Fosfatos de Dinucleósidos/metabolismo , Inmunidad , Nucleótidos , Nucleotidiltransferasas/metabolismoRESUMEN
A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Células Neoplásicas Circulantes , Ratones , Animales , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Línea Celular Tumoral , Tretinoina/uso terapéuticoRESUMEN
Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.
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Hepatocellular carcinoma (HCC) is the 3rd most deadly malignancy. Activated hepatic stellate cells (aHSC) give rise to cancer-associated fibroblasts in HCC and are considered a potential therapeutic target. Here we report that selective ablation of stearoyl CoA desaturase-2 (Scd2) in aHSC globally suppresses nuclear CTNNB1 and YAP1 in tumors and tumor microenvironment and prevents liver tumorigenesis in male mice. Tumor suppression is associated with reduced leukotriene B4 receptor 2 (LTB4R2) and its high affinity oxylipin ligand, 12-hydroxyheptadecatrienoic acid (12-HHTrE). Genetic or pharmacological inhibition of LTB4R2 recapitulates CTNNB1 and YAP1 inactivation and tumor suppression in culture and in vivo. Single cell RNA sequencing identifies a subset of tumor-associated aHSC expressing Cyp1b1 but no other 12-HHTrE biosynthetic genes. aHSC release 12-HHTrE in a manner dependent on SCD and CYP1B1 and their conditioned medium reproduces the LTB4R2-mediated tumor-promoting effects of 12-HHTrE in HCC cells. CYP1B1-expressing aHSC are detected in proximity of LTB4R2-positive HCC cells and the growth of patient HCC organoids is blunted by LTB4R2 antagonism or knockdown. Collectively, our findings suggest aHSC-initiated 12-HHTrE-LTB4R2-CTNNB1-YAP1 pathway as a potential HCC therapeutic target.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Masculino , Ratones , beta Catenina/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/metabolismo , Ácido Graso Desaturasas , Células Estrelladas Hepáticas/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores de Leucotrieno B4/genética , Receptores de Leucotrieno B4/metabolismo , Microambiente TumoralRESUMEN
The gene repertoire regulating vertebrate biomineralization is poorly understood. Dental enamel, the most highly mineralized tissue in mammals, differs from other calcifying systems in that the formative cells (ameloblasts) lack remodeling activity and largely degrade and resorb the initial extracellular matrix. Enamel mineralization requires that ameloblasts undergo a profound functional switch from matrix-secreting to maturational (calcium transport, protein resorption) roles as mineralization progresses. During the maturation stage, extracellular pH decreases markedly, placing high demands on ameloblasts to regulate acidic environments present around the growing hydroxyapatite crystals. To identify the genetic events driving enamel mineralization, we conducted genome-wide transcript profiling of the developing enamel organ from rat incisors and highlight over 300 genes differentially expressed during maturation. Using multiple bioinformatics analyses, we identified groups of maturation-associated genes whose functions are linked to key mineralization processes including pH regulation, calcium handling, and matrix turnover. Subsequent qPCR and Western blot analyses revealed that a number of solute carrier (SLC) gene family members were up-regulated during maturation, including the novel protein Slc24a4 involved in calcium handling as well as other proteins of similar function (Stim1). By providing the first global overview of the cellular machinery required for enamel maturation, this study provide a strong foundation for improving basic understanding of biomineralization and its practical applications in healthcare.
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Amelogénesis/fisiología , Esmalte Dental/química , Esmalte Dental/metabolismo , Perfilación de la Expresión Génica/métodos , Genoma , Calcificación de Dientes/genética , Ameloblastos/metabolismo , Animales , Calcio/metabolismo , Matriz Extracelular/metabolismo , Expresión Génica , Humanos , Incisivo/anatomía & histología , Incisivo/metabolismo , Ratas , Ratas WistarRESUMEN
Monoamine oxidase B (MAO B) oxidizes trace amine phenylethylamine (PEA), and neurotransmitters serotonin and dopamine in the brain. We reported previously that PEA levels increased significantly in all brain regions, but serotonin and dopamine levels were unchanged in MAO B knockout (KO) mice. PEA and dopamine are both synthesized from phenylalanine by aromatic L-amino acid decarboxylase in dopaminergic neurons in the striatum. A high concentration of PEA in the striatum may cause dopaminergic neuronal death in the absence of MAO B. We isolated the RNA from brain tissue of MAO B KO mice (2-month old) and age-matched wild type (WT) male mice and analyzed the altered genes by Affymetrix microarray. Differentially expressed genes (DEGs) in MAO B KO compared to WT mice were analyzed by Partek Genomics Suite, followed by Ingenuity Pathway Analysis (IPA) to assess their functional relationships. DEGs in MAO B KO mice are involved in brain inflammation and the genesis of GABAnergic neurons. The significant DEGs include four brain injury or inflammation genes (upregulated: Ido1, TSPO, AVP, Tdo2), five gamma-aminobutyric acid (GABA) receptors (down-regulated: GABRA2, GABRA3, GABRB1, GABRB3, GABRG3), five transcription factors related to adult neurogenesis (upregulated: Wnt7b, Hes5; down-regulated: Pax6, Tcf4, Dtna). Altered brain injury and inflammation genes in MAO B knockout mice are involved in various neurological disorders: attention deficit hyperactive disorder, panic disorder, obsessive compulsive disorder, autism, amyotrophic lateral sclerosis, Parkinson's diseases, Alzheimer's disease, bipolar affective disorder. Many were commonly involved in these disorders, indicating that there are overlapping molecular pathways.