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1.
Immunity ; 55(11): 2059-2073.e8, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36351375

RESUMO

T memory stem cells (TSCM) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell responses. TSCM cells can be expanded by Urolithin A (UA), which is produced by the commensal gut microbiome from foods rich in ellagitannins and is known to improve mitochondrial health. Oral UA administration to tumor-bearing mice conferred strong anti-tumor CD8+ T cell immunity, whereas ex vivo UA pre-treated T cells displayed improved anti-tumor function upon adoptive cell transfer. UA-induced TSCM formation depended on Pink1-mediated mitophagy triggering cytosolic release of the mitochondrial phosphatase Pgam5. Cytosolic Pgam5 dephosphorylated ß-catenin, which drove Wnt signaling and compensatory mitochondrial biogenesis. Collectively, we unravel a critical signaling pathway linking mitophagy to TSCM formation and suggest that the well-tolerated metabolic compound UA represents an attractive option to improve immune therapy.


Assuntos
Cumarínicos , Mitofagia , Camundongos , Animais , Cumarínicos/farmacologia , Via de Sinalização Wnt , Células-Tronco , Memória Imunológica
2.
EMBO Rep ; 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38937629

RESUMO

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.

3.
Gastroenterology ; 164(2): 214-227, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36402192

RESUMO

BACKGROUND & AIMS: Epigenetic processes regulating gene expression contribute markedly to epithelial cell plasticity in colorectal carcinogenesis. The lysine methyltransferase SUV420H2 comprises an important regulator of epithelial plasticity and is primarily responsible for trimethylation of H4K20 (H4K20me3). Loss of H4K20me3 has been suggested as a hallmark of human cancer due to its interaction with DNMT1. However, the role of Suv4-20h2 in colorectal cancer is unknown. METHODS: We examined the alterations in histone modifications in patient-derived colorectal cancer organoids. Patient-derived colorectal cancer organoids and mouse intestinal organoids were genetically manipulated for functional studies in patient-derived xenograft and orthotopic transplantation. Gene expression profiling, micrococcal nuclease assay, and chromatin immunoprecipitation were performed to understand epigenetic regulation of chromatin states and gene expression in patient-derived and mouse intestinal organoids. RESULTS: We found that reduced H4K20me3 levels occurred predominantly in right-sided patient-derived colorectal cancer organoids, which were associated with increased chromatin accessibility. Re-compaction of chromatin by methylstat, a histone demethylase inhibitor, resulted in reduced growth selectively in subcutaneously grown tumors derived from right-sided cancers. Using mouse intestinal organoids, we confirmed that Suv4-20h2-mediated H4K20me3 is required for maintaining heterochromatin compaction and to prevent R-loop formation. Cross-species comparison of Suv4-20h2-depleted murine organoids with right-sided colorectal cancer organoids revealed a large overlap of gene signatures involved in chromatin silencing, DNA methylation, and stemness/Wnt signaling. CONCLUSIONS: Loss of Suv4-20h2-mediated H4K20me3 drives right-sided colorectal tumorigenesis through an epigenetically controlled mechanism of chromatin compaction. Our findings unravel a conceptually novel approach for subtype-specific therapy of this aggressive form of colorectal cancer.


Assuntos
Neoplasias do Colo , Histona-Lisina N-Metiltransferase , Animais , Humanos , Camundongos , Transformação Celular Neoplásica/genética , Cromatina/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Epigênese Genética , Histonas/metabolismo , Xenoenxertos , Histona-Lisina N-Metiltransferase/metabolismo
4.
J Allergy Clin Immunol ; 152(4): 949-960, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37390900

RESUMO

BACKGROUND: The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. OBJECTIVES: This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics. METHODS: Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level. RESULTS: We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines. CONCLUSIONS: These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.


Assuntos
Actinas , Fosfatidilinositol 3-Quinases , Humanos , Animais , Camundongos , Morte Celular , Mutação , Movimento Celular/genética , Dano ao DNA , Proteínas , 1-Fosfatidilinositol 4-Quinase
5.
EMBO J ; 38(12)2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31036555

RESUMO

Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.


Assuntos
Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Citotoxicidade Imunológica , Modelos Biológicos , Organoides/patologia , Receptores de Antígenos Quiméricos/uso terapêutico , Técnicas de Cultura de Tecidos/métodos , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Terapia Genética/métodos , Células HEK293 , Humanos , Imunoterapia Adotiva/métodos , Cultura Primária de Células/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/uso terapêutico , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Alicerces Teciduais/química
6.
Nature ; 530(7590): 340-3, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26863187

RESUMO

Mammalian Wnt proteins are believed to act as short-range signals, yet have not been previously visualized in vivo. Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt. Wnt3 is produced specifically by Paneth cells. Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and 'plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.


Assuntos
Membrana Celular/metabolismo , Mucosa Intestinal/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt , Proteína Wnt3/metabolismo , Alelos , Animais , Adesão Celular , Divisão Celular , Difusão , Feminino , Receptores Frizzled/metabolismo , Técnicas de Introdução de Genes , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt3/genética
7.
J Biol Chem ; 295(14): 4526-4540, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32086379

RESUMO

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.


Assuntos
Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transdução de Sinais , Proteínas ras/antagonistas & inibidores , Proteínas ras/genética
8.
Gut ; 69(7): 1269-1282, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31685519

RESUMO

OBJECTIVE: Cancer-associated fibroblasts (CAFs) influence the tumour microenvironment and tumour growth. However, the role of CAFs in colorectal cancer (CRC) development is incompletely understood. DESIGN: We quantified phosphorylation of STAT3 (pSTAT3) expression in CAFs of human colon cancer tissue using a tissue microarray (TMA) of 375 patients, immunofluorescence staining and digital pathology. To investigate the functional role of CAFs in CRC, we took advantage of two murine models of colorectal neoplasia and advanced imaging technologies. In loss-of-function and gain-of-function experiments, using genetically modified mice with collagen type VI (COLVI)-specific signal transducer and activator of transcription 3 (STAT3) targeting, we evaluated STAT3 signalling in fibroblasts during colorectal tumour development. We performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblast subpopulations (COLVI+ vs COLVI-) on STAT3 activation (IL-6 vs IL-11). RESULTS: The analysis of pSTAT3 expression in CAFs of human TMAs revealed a negative correlation of increased stromal pSTAT3 expression with the survival of colon cancer patients. In the loss-of-function and gain-of-function approach, we found a critical role of STAT3 activation in fibroblasts in driving colorectal tumourigenesis in vivo. With different imaging technologies, we detected an expansion of activated fibroblasts in colorectal neoplasias. Comparative gene expression profiling of fibroblast subpopulations on STAT3 activation revealed the regulation of transcriptional patterns associated with angiogenesis. Finally, the blockade of proangiogenic signalling significantly reduced colorectal tumour growth in mice with constitutive STAT3 activation in COLVI+ fibroblasts. CONCLUSION: Altogether our work demonstrates a critical role of STAT3 activation in CAFs in CRC development.


Assuntos
Neoplasias Colorretais/etiologia , Interleucina-11/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Colo/metabolismo , Neoplasias Colorretais/diagnóstico , Fibroblastos/metabolismo , Humanos , Camundongos , Fosforilação , Prognóstico , Análise Serial de Tecidos , Transcriptoma
9.
Proc Natl Acad Sci U S A ; 114(7): 1643-1648, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28130546

RESUMO

Loss of tumor suppressor adenomatous polyposis coli (APC) activates ß-catenin to initiate colorectal tumorigenesis. However, ß-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Receptor gp130 de Citocina/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Idoso , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptor gp130 de Citocina/genética , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , beta Catenina/genética , beta Catenina/metabolismo
10.
Nat Methods ; 11(1): 106-12, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24292484

RESUMO

Although Lgr5(+) intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5(+) intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5(+) cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.


Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Colo/citologia , Intestino Delgado/citologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Cultivadas , Cromossomos/ultraestrutura , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Cariotipagem , Camundongos , Microscopia Confocal , Celulas de Paneth/citologia , Piridinas/química , Pirimidinas/química , Transdução de Sinais , Estômago/citologia , Ácido Valproico/química
11.
J Mol Cell Cardiol ; 97: 140-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27180262

RESUMO

Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an Hprt(Tbx18) misexpression allele. Myh6-Cre/+;Hprt(Tbx18/y) mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;Hprt(Tbx18/y) mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;Hprt(Tbx18/y) embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Miocárdio/metabolismo , Nó Sinoatrial/metabolismo , Proteínas com Domínio T/genética , Transcriptoma , Animais , Biomarcadores , Análise por Conglomerados , Feminino , Feto , Perfilação da Expressão Gênica , Genes Letais , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/patologia
12.
J Pathol ; 236(2): 155-64, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25712196

RESUMO

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Deleção de Genes , Mutação em Linhagem Germinativa/genética , Proteínas Supressoras de Tumor/genética , Polipose Adenomatosa do Colo/metabolismo , Adulto , Estudos de Casos e Controles , Cromossomos Humanos Par 9/genética , Neoplasias Colorretais/metabolismo , Variações do Número de Cópias de DNA/genética , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Fases de Leitura Aberta/genética , Proteínas Supressoras de Tumor/metabolismo
13.
PLoS Genet ; 9(1): e1003189, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23341776

RESUMO

Vertebrate organ development relies on the precise spatiotemporal orchestration of proliferation rates and differentiation patterns in adjacent tissue compartments. The underlying integration of patterning and cell cycle control during organogenesis is insufficiently understood. Here, we have investigated the function of the patterning T-box transcription factor gene Tbx2 in lung development. We show that lungs of Tbx2-deficient mice are markedly hypoplastic and exhibit reduced branching morphogenesis. Mesenchymal proliferation was severely decreased, while mesenchymal differentiation into fibrocytes was prematurely induced. In the epithelial compartment, proliferation was reduced and differentiation of alveolar epithelial cells type 1 was compromised. Prior to the observed cellular changes, canonical Wnt signaling was downregulated, and Cdkn1a (p21) and Cdkn1b (p27) (two members of the Cip/Kip family of cell cycle inhibitors) were strongly induced in the Tbx2-deficient lung mesenchyme. Deletion of both Cdkn1a and Cdkn1b rescued, to a large degree, the growth deficits of Tbx2-deficient lungs. Prolongation of Tbx2 expression into adulthood led to hyperproliferation and maintenance of mesenchymal progenitor cells, with branching morphogenesis remaining unaffected. Expression of Cdkn1a and Cdkn1b was ablated from the lung mesenchyme in this gain-of-function setting. We further show by ChIP experiments that Tbx2 directly binds to Cdkn1a and Cdkn1b loci in vivo, defining these two genes as direct targets of Tbx2 repressive activity in the lung mesenchyme. We conclude that Tbx2-mediated regulation of Cdkn1a and Cdkn1b represents a crucial node in the network integrating patterning information and cell cycle regulation that underlies growth, differentiation, and branching morphogenesis of this organ.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Pulmão , Proteínas com Domínio T , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Mesoderma , Camundongos , Morfogênese , Transdução de Sinais , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética
14.
PLoS Genet ; 9(4): e1003467, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23633963

RESUMO

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Botões de Extremidades/crescimento & desenvolvimento , Proteínas com Domínio T/genética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Citocinas , Epitélio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Botões de Extremidades/metabolismo , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Fenótipo , Transdução de Sinais , Proteínas com Domínio T/metabolismo
16.
Development ; 139(17): 3099-108, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22833126

RESUMO

Smooth muscle cells (SMCs) are a key component of many visceral organs, including the ureter, yet the molecular pathways that regulate their development from mesenchymal precursors are insufficiently understood. Here, we identified epithelial Wnt7b and Wnt9b as possible ligands of Fzd1-mediated ß-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated ureteric mesenchyme. Mice with a conditional deletion of Ctnnb1 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional obstruction of the ureter. Histological analysis revealed that the layer of undifferentiated mesenchymal cells directly adjacent to the ureteric epithelium did not undergo characteristic cell shape changes, exhibited reduced proliferation and failed to differentiate into SMCs. Molecular markers for prospective SMCs were lost, whereas markers of the outer layer of the ureteric mesenchyme fated to become adventitial fibroblasts were expanded to the inner layer. Conditional misexpression of a stabilized form of Ctnnb1 in the prospective ureteric mesenchyme resulted in the formation of a large domain of cells that exhibited histological and molecular features of prospective SMCs and differentiated along this lineage. Our analysis suggests that Wnt signals from the ureteric epithelium pattern the ureteric mesenchyme in a radial fashion by suppressing adventitial fibroblast differentiation and initiating smooth muscle precursor development in the innermost layer of mesenchymal cells.


Assuntos
Hipoxantina Fosforribosiltransferase/genética , Mioblastos de Músculo Liso/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Ureter/embriologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Diferenciação Celular/fisiologia , Cruzamentos Genéticos , Fluorescência , Técnicas de Introdução de Genes , Hibridização In Situ , Camundongos , Mioblastos de Músculo Liso/metabolismo , Ureter/citologia , Ureter/metabolismo , beta Catenina/deficiência
17.
J Allergy Clin Immunol ; 134(6): 1354-1364.e6, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25174867

RESUMO

BACKGROUND: Inflammatory bowel disease (IBD) is one of the most common chronic gastrointestinal diseases, but the underlying molecular mechanisms remain largely unknown. Studies of monogenic diseases can provide insight into the pathogenesis of IBD. OBJECTIVE: We thought to determine the underlying molecular causes of IBD occurring in 2 unrelated families in association with an immune deficiency. METHODS: We performed genetic linkage analysis and candidate gene sequencing on 13 patients from a large consanguineous family affected by early-onset IBD, progressive immune deficiency, and, in some cases, autoimmunity and alopecia, a condition we named enteropathy-lymphocytopenia-alopecia. The candidate gene was also sequenced in an unrelated patient with a similar phenotype. We performed histologic analysis of patients' intestinal biopsy specimens and carried out functional assays on PBMCs. Gut organoids derived from a patient's biopsy specimen were analyzed. RESULTS: We identified biallelic missense mutations in tetratricopeptide repeat domain 7A (TTC7A) in all patients from both families. The resulting TTC7A depletion modified the proliferation, adhesion, and migratory capacities of lymphocytes through inappropriate activation of the RhoA signaling pathway. Normal function was restored by wild-type TTC7A expression or addition of a RhoA kinase inhibitor. The growth and polarity of gut epithelial organoids were also found to be dependent on the RhoA signaling pathway. CONCLUSIONS: We show that TTC7A regulates the actin cytoskeleton dynamics in lymphocytes through the RhoA signaling pathway and is required in both lymphocytes and epithelial cells for maintaining equilibrium between cell proliferation, migration, polarization, and cell death. Our study highlights variability in the phenotypic expression resulting from TTC7A deficiency and outlines that impairment of both epithelial cells and lymphocytes cooperatively causes IBD.


Assuntos
Alopecia , Doenças Inflamatórias Intestinais , Linfopenia , Proteínas/genética , Proteínas/imunologia , Adolescente , Adulto , Alopecia/genética , Alopecia/imunologia , Alopecia/patologia , Criança , Pré-Escolar , Colo/patologia , Duodeno/patologia , Feminino , Humanos , Lactente , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Antro Pilórico/patologia , Adulto Jovem , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/imunologia , Proteína rhoA de Ligação ao GTP/imunologia
18.
Nat Methods ; 9(1): 81-3, 2011 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-22138822

RESUMO

The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.


Assuntos
Mucosa Intestinal/citologia , Organoides/citologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Integrases/metabolismo , Camundongos , Organoides/metabolismo , Receptores Notch/fisiologia , Retroviridae/genética , Células-Tronco/virologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Transdução Genética/métodos
19.
Nat Commun ; 15(1): 4096, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750019

RESUMO

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.


Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Succinatos , Animais , Humanos , Terapia Viral Oncolítica/métodos , Succinatos/farmacologia , Camundongos , Linhagem Celular Tumoral , Interferon Tipo I/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias do Colo/terapia , Neoplasias do Colo/imunologia , Neoplasias do Colo/tratamento farmacológico , Antivirais/farmacologia , NF-kappa B/metabolismo , Quinase I-kappa B/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Inflamação/tratamento farmacológico , Feminino , Vírus da Estomatite Vesicular Indiana/fisiologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
20.
Gastroenterology ; 143(6): 1518-1529.e7, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22922422

RESUMO

BACKGROUND & AIMS: Wnt signaling regulates multiple aspects of intestinal physiology, including stem cell maintenance. Paneth cells support stem cells by secreting Wnt, but little is known about the exact sources and primary functions of individual Wnt family members. METHODS: We analyzed intestinal tissues and cultured epithelial cells from adult mice with conditional deletion of Wnt3 (Vil-CreERT2;Wnt3fl/fl mice). We also analyzed intestinal tissues and cells from Atoh1 mutant mice, which lack secretory cells. RESULTS: Unexpectedly, Wnt3 was dispensable for maintenance of intestinal stem cells in mice, indicating a redundancy of Wnt signals. By contrast, cultured crypt organoids required Paneth cell-derived Wnt3. Addition of exogenous Wnt, or coculture with mesenchymal cells, restored growth of Vil-CreERT2;Wnt3fl/fl crypt organoids. Intestinal organoids from Atoh1 mutant mice did not grow or form Paneth cells; addition of Wnt3 allowed growth in the absence of Paneth cells. Wnt signaling had a synergistic effect with the Lgr4/5 ligand R-spondin to induce formation of Paneth cells. Mosaic expression of Wnt3 in organoids using a retroviral vector promoted differentiation of Paneth cells in a cell-autonomous manner. CONCLUSIONS: Wnt is part of a signaling loop that affects homeostasis of intestinal stem and Paneth cells in mice. Wnt3 signaling is required for growth and development of organoid cultures, whereas nonepithelial Wnt signals could provide a secondary physiological source of Wnt.


Assuntos
Diferenciação Celular/fisiologia , Intestino Delgado/citologia , Celulas de Paneth/citologia , Células-Tronco/citologia , Proteína Wnt3/fisiologia , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Homeostase/fisiologia , Técnicas In Vitro , Intestino Delgado/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Animais , Celulas de Paneth/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia
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