RESUMO
Lung epithelial regeneration after acute injury requires coordination cellular coordination to pattern the morphologically complex alveolar gas exchange surface. During adult lung regeneration, Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells. Here, we report a refined primary murine alveolar organoid, which recapitulates critical aspects of in vivo regeneration. Paired scRNAseq and scATACseq followed by transcriptional regulatory network (TRN) analysis identified two AT1 transition states driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8+ state, and AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of progenitor state and uncontrolled growth of Krt8+ cells. Together, these data implicate dynamic epigenetic maintenance via Nkx2-1 as central to the control of facultative progenitor activity in AEPs.
Assuntos
Epigenômica , Pulmão , Animais , Camundongos , Diferenciação Celular , Células Epiteliais , Homeostase , Células-TroncoRESUMO
Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.
Assuntos
Carcinoma de Células Escamosas , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Feminino , Humanos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Transcriptoma , Epitélio/metabolismo , Queratinócitos/metabolismo , Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Proteínas Oncogênicas Virais/genéticaRESUMO
Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is driven by human papillomavirus (HPV) low-risk strains and is associated with significant morbidity. While previous studies of 2D cultures have shed light on disease pathogenesis and demonstrated the utility of personalized medicine approaches, monolayer cultures lack the 3D tissue architecture and physiology of stratified, sequentially differentiated mucosal epithelium important in RRP disease pathogenesis. Herein we describe the establishment of JoRRP-derived primary cell populations that retain HPV genomes and viral gene expression in culture. These were directly compared to cells from matched adjacent non-diseased tissue, given the known RRP patient-to-patient variability. JoRRP papilloma versus control cells displayed decreased growth at subconfluency, with a switch to increased growth after reaching confluency, suggesting relative resistance to cell-cell contact and/or differentiation. The same papilloma cells grown as 3D organotypic rafts harbored hyperproliferation as compared to controls, with increased numbers of proliferating basal cells and inappropriately replicating suprabasal cells, mimicking phenotypes in the patient biopsies from which they were derived. These complementary model systems provide novel opportunities to elucidate disease mechanisms at distinct stages in JoRRP progression and to identify diagnostic, prognostic and therapeutic factors to personalize patient management and treatment.
Assuntos
Alphapapillomavirus/genética , Alphapapillomavirus/isolamento & purificação , Células Epiteliais/virologia , Infecções por Papillomavirus/virologia , Infecções Respiratórias/patologia , Humanos , Técnicas de Cultura de Órgãos , Infecções por Papillomavirus/patologia , Fenótipo , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/virologia , Fatores de RiscoRESUMO
BACKGROUND AND AIMS: During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFßR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs. APPROACH AND RESULTS: We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100 ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl4 administration to rats every 3 days for 3 or 8 weeks, then challenged with LPS (5 mg/kg; IP). HSCs were isolated 24 hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFßR, transforming growth factor beta receptor 1 (TGFßR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFßR, αSMA, TGFßR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ. CONCLUSIONS: In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis.
Assuntos
Regulação da Expressão Gênica/imunologia , Células Estreladas do Fígado/imunologia , Lipídeo A/análogos & derivados , Cirrose Hepática Experimental/imunologia , Fígado/patologia , Animais , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Transdiferenciação Celular/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Regulação para Baixo , Inativação Gênica , Células Estreladas do Fígado/patologia , Humanos , Lipídeo A/imunologia , Lipídeo A/metabolismo , Fígado/citologia , Fígado/imunologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Camundongos , Camundongos Knockout , Miofibroblastos/imunologia , Miofibroblastos/patologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myb/metabolismo , Ratos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína Smad7/genética , Proteína Smad7/metabolismo , Regulação para Cima/imunologiaRESUMO
The metabolic requirements of hematopoietic stem cells (HSCs) change with their cell cycle activity. However, the underlying role of mitochondria remains ill-defined. Here we found that, after mitochondrial activation with replication, HSCs irreversibly remodel the mitochondrial network and that this network is not repaired after HSC re-entry into quiescence, contrary to hematopoietic progenitors. HSCs keep and accumulate dysfunctional mitochondria through asymmetric segregation during active division. Mechanistically, mitochondria aggregate and depolarize after stress because of loss of activity of the mitochondrial fission regulator Drp1 onto mitochondria. Genetic and pharmacological studies indicate that inactivation of Drp1 causes loss of HSC regenerative potential while maintaining HSC quiescence. Molecularly, HSCs carrying dysfunctional mitochondria can re-enter quiescence but fail to synchronize the transcriptional control of core cell cycle and metabolic components in subsequent division. Thus, loss of fidelity of mitochondrial morphology and segregation is one type of HSC divisional memory and drives HSC attrition.
Assuntos
Células-Tronco Hematopoéticas , Mitocôndrias , Ciclo Celular , Divisão Celular , Autorrenovação Celular , Células-Tronco Hematopoéticas/metabolismoRESUMO
In the version of this Article originally published, in ref. 34 the first author's name was spelled incorrectly. The correct reference is: Rodón, L. et al. Active CREB1 promotes a malignant TGFß2 autocrine loop in glioblastoma. Cancer Discov. 10, 1230-1241 (2014). This has now been amended in all online versions of the Article.
RESUMO
In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.
RESUMO
Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias Encefálicas/enzimologia , Proliferação de Células , Metabolismo Energético , Glioblastoma/enzimologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Animais , Antineoplásicos/farmacologia , Apoptose , Autofagia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Fator de Transcrição de Proteínas de Ligação GA/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Carga Tumoral , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL-17A)-green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL-17 receptor A-/- mice were used to examine T-lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus-induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4+ lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL-17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c+ dendritic cells diminished hepatic IL-17A production and ameliorated intrahepatic bile duct injury. Recombinant IL-17A induced expression of chemokine (C-C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C-C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL-17A signaling was associated with down-regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6Chi macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL-17 receptor A, and the prevalence of IL-17A+ cells was positively correlated with the degree of CD68+ macrophage infiltration at diagnosis. Hepatic CD4+ lymphocytes were chief producers of IL-17A in patients with progressive disease undergoing liver transplantation. CONCLUSION: These findings identify the dendritic cell-T helper 17-macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174-188).
Assuntos
Atresia Biliar/imunologia , Células Dendríticas/fisiologia , Macrófagos/fisiologia , Células Th17/fisiologia , Animais , Ductos Biliares Intra-Hepáticos/citologia , Progressão da Doença , Células Epiteliais/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene-deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.
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Alérgenos/imunologia , Aspergillus fumigatus/imunologia , Asma/imunologia , Células Epiteliais/imunologia , Cinesinas/imunologia , Mucosa Respiratória/imunologia , Células Th2/imunologia , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/patologia , Cinesinas/genética , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Mucosa Respiratória/patologia , Células Th2/patologiaRESUMO
Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Infiltração de Neutrófilos/fisiologia , Neutrófilos/fisiologia , Proteínas rap de Ligação ao GTP/fisiologia , Actinas/metabolismo , Animais , Antígeno CD11b/metabolismo , Quimiotaxia de Leucócito/imunologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/fisiopatologia , Transtornos Leucocíticos/imunologia , Transtornos Leucocíticos/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Migração Transendotelial e Transepitelial/imunologia , Migração Transendotelial e Transepitelial/fisiologia , Proteínas rap de Ligação ao GTP/deficiência , Proteínas rap de Ligação ao GTP/genéticaRESUMO
In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.
Assuntos
Junções Intercelulares/fisiologia , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Complexos Multiproteicos/metabolismo , Tubo Neural/embriologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Caderinas/metabolismo , Primers do DNA/genética , Imunofluorescência , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Hibridização In Situ , Proteínas de Membrana/fisiologia , Morfolinos , Proteínas de Xenopus/fisiologia , beta Catenina/metabolismoRESUMO
Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.
Assuntos
Papillomavirus Bovino 1/patogenicidade , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , HumanosRESUMO
Since the three main pathways (the Wnt, VegT and BMP pathways) involved in organizer and axis formation in the Xenopus embryo are now characterized, the challenge is to understand their interactions. Here three comparisons were made. Firstly, we made a systematic comparison of the expression of zygotic genes in sibling wild-type, VegT-depleted (VegT(-)), beta-catenin-depleted (beta-catenin(-)) and double depleted (VegT(-)/beta-catenin(-)) embryos and placed early zygotic genes into specific groups. In the first group some organizer genes, including chordin, noggin and cerberus, required the activity of both the Wnt pathway and the VegT pathway to be expressed. A second group including Xnr1, 2, 4 and Xlim1 were initiated by the VegT pathway but their dorsoventral pattern and amount of their expression was regulated by the Wnt pathway. Secondly, we compared the roles of the Wnt and VegT pathways in producing dorsal signals. Explant co-culture experiments showed that the Wnt pathway did not cause the release of a dorsal signal from the vegetal mass independent from the VegT pathway. Finally we compared the extent to which inhibiting Smad 1 phosphorylation in one area of VegT(-), or beta-catenin(-) embryos would rescue organizer and axis formation. We found that BMP inhibition with cm-BMP7 mRNA had no rescuing effects on VegT(-) embryos, while cm-BMP7 and noggin mRNA caused a complete rescue of the trunk, but not of the anterior pattern in beta-catenin(-) embryos.