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1.
Cell ; 184(1): 149-168.e17, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33278357

RESUMO

COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.


Assuntos
COVID-19/imunologia , COVID-19/patologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Interferon gama/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Morte Celular , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/imunologia , Inflamação/patologia , Linfo-Histiocitose Hemofagocítica/induzido quimicamente , Masculino , Camundongos , Camundongos Transgênicos , Células THP-1
2.
Cell ; 183(6): 1665-1681.e18, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33188776

RESUMO

We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-µm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.


Assuntos
Código de Barras de DNA Taxonômico , Genômica , Especificidade de Órgãos/genética , Animais , Automação , Encéfalo/embriologia , Análise por Conglomerados , DNA Complementar/genética , Embrião de Mamíferos/metabolismo , Olho/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Microfluídica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única , Transcriptoma/genética
3.
Cell ; 183(2): 490-502.e18, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33002410

RESUMO

The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Células A549 , Animais , Criança , Pré-Escolar , Feminino , Mutação com Ganho de Função/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Células-Tronco Embrionárias Murinas , Mutação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Domínios de Homologia de src/genética
4.
Nat Immunol ; 22(4): 485-496, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33767426

RESUMO

Evasion of host immunity is a hallmark of cancer; however, mechanisms linking oncogenic mutations and immune escape are incompletely understood. Through loss-of-function screening of 1,001 tumor suppressor genes, we identified death-associated protein kinase 3 (DAPK3) as a previously unrecognized driver of anti-tumor immunity through the stimulator of interferon genes (STING) pathway of cytosolic DNA sensing. Loss of DAPK3 expression or kinase activity impaired STING activation and interferon (IFN)-ß-stimulated gene induction. DAPK3 deficiency in IFN-ß-producing tumors drove rapid growth and reduced infiltration of CD103+CD8α+ dendritic cells and cytotoxic lymphocytes, attenuating the response to cancer chemo-immunotherapy. Mechanistically, DAPK3 coordinated post-translational modification of STING. In unstimulated cells, DAPK3 inhibited STING K48-linked poly-ubiquitination and proteasome-mediated degradation. After cGAMP stimulation, DAPK3 was required for STING K63-linked poly-ubiquitination and STING-TANK-binding kinase 1 interaction. Comprehensive phospho-proteomics uncovered a DAPK3-specific phospho-site on the E3 ligase LMO7, critical for LMO7-STING interaction and STING K63-linked poly-ubiquitination. Thus, DAPK3 is an essential kinase for STING activation that drives tumor-intrinsic innate immunity and tumor immune surveillance.


Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Células Endoteliais da Veia Umbilical Humana/enzimologia , Imunidade Inata , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/enzimologia , Evasão Tumoral , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunidade Inata/efeitos dos fármacos , Interferon beta/genética , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Evasão Tumoral/efeitos dos fármacos , Ubiquitinação
5.
Cell ; 173(3): 762-775.e16, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29677517

RESUMO

Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.


Assuntos
Mecanotransdução Celular , Interferência de RNA , Receptores Acoplados a Proteínas G/fisiologia , Animais , Materiais Biocompatíveis , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração de Íons de Hidrogênio , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Resistência ao Cisalhamento , Estresse Mecânico , Resistência Vascular
6.
Cell ; 174(1): 143-155.e16, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29779947

RESUMO

Neisseria meningitidis, a bacterium responsible for meningitis and septicemia, proliferates and eventually fills the lumen of blood capillaries with multicellular aggregates. The impact of this aggregation process and its specific properties are unknown. We first show that aggregative properties are necessary for efficient infection and study their underlying physical mechanisms. Micropipette aspiration and single-cell tracking unravel unique features of an atypical fluidized phase, with single-cell diffusion exceeding that of isolated cells. A quantitative description of the bacterial pair interactions combined with active matter physics-based modeling show that this behavior relies on type IV pili active dynamics that mediate alternating phases of bacteria fast mutual approach, contact, and release. These peculiar fluid properties proved necessary to adjust to the geometry of capillaries upon bacterial proliferation. Intermittent attractive forces thus generate a fluidized phase that allows for efficient colonization of the blood capillary network during infection.


Assuntos
Aderência Bacteriana/fisiologia , Capilares/microbiologia , Fímbrias Bacterianas/fisiologia , Neisseria meningitidis/patogenicidade , Animais , Carga Bacteriana , Capilares/patologia , Endotélio/metabolismo , Endotélio/microbiologia , Endotélio/patologia , Feminino , Proteínas de Fímbrias/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos SCID , Microscopia Confocal , Neisseria meningitidis/fisiologia , Transplante de Pele , Tensão Superficial , Imagem com Lapso de Tempo , Transplante Heterólogo
7.
Cell ; 173(1): 117-129.e14, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29570992

RESUMO

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos Sulfúricos/deficiência , Sulfeto de Hidrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Aminoácidos Sulfúricos/metabolismo , Animais , Cistationina gama-Liase/metabolismo , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Condicionamento Físico Animal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética
8.
Cell ; 175(6): 1607-1619.e15, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30500539

RESUMO

In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.


Assuntos
Antígenos de Diferenciação/biossíntese , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Hepatócitos/metabolismo , Esferoides Celulares/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular Transformada , Células Hep G2 , Hepatócitos/transplante , Células Endoteliais da Veia Umbilical Humana , Humanos , Fígado/lesões , Fígado/metabolismo , Camundongos Knockout , Esferoides Celulares/transplante , Fatores de Tempo
9.
Immunity ; 54(9): 1989-2004.e9, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34363750

RESUMO

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.


Assuntos
Autofagia/fisiologia , Células Endoteliais/fisiologia , Infiltração de Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Animais , Quimiotaxia de Leucócito/fisiologia , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Junções Intercelulares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia
10.
Nature ; 629(8012): 660-668, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38693258

RESUMO

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células Endoteliais , Isquemia , Mitocôndrias , Mitofagia , Animais , Humanos , Masculino , Camundongos , Autofagossomos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Metabolismo Energético , Células Endoteliais da Veia Umbilical Humana/metabolismo , Isquemia/metabolismo , Isquemia/terapia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Mitocôndrias/metabolismo , Mitocôndrias/transplante , Proteínas Quinases/deficiência , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos
11.
Mol Cell ; 81(19): 4091-4103.e9, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34348091

RESUMO

We describe PROPER-seq (protein-protein interaction sequencing) to map protein-protein interactions (PPIs) en masse. PROPER-seq first converts transcriptomes of input cells into RNA-barcoded protein libraries, in which all interacting protein pairs are captured through nucleotide barcode ligation, recorded as chimeric DNA sequences, and decoded at once by sequencing and mapping. We applied PROPER-seq to human embryonic kidney cells, T lymphocytes, and endothelial cells and identified 210,518 human PPIs (collected in the PROPER v.1.0 database). Among these, 1,365 and 2,480 PPIs are supported by published co-immunoprecipitation (coIP) and affinity purification-mass spectrometry (AP-MS) data, 17,638 PPIs are predicted by the prePPI algorithm without previous experimental validation, and 100 PPIs overlap human synthetic lethal gene pairs. In addition, four previously uncharacterized interaction partners with poly(ADP-ribose) polymerase 1 (PARP1) (a critical protein in DNA repair) known as XPO1, MATR3, IPO5, and LEO1 are validated in vivo. PROPER-seq presents a time-effective technology to map PPIs at the transcriptome scale, and PROPER v.1.0 provides a rich resource for studying PPIs.


Assuntos
Biologia Computacional , Perfilação da Expressão Gênica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteínas/genética , Proteínas/metabolismo , RNA-Seq , Transcriptoma , Bases de Dados Genéticas , Feminino , Genes Letais , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células Jurkat , Carioferinas/genética , Carioferinas/metabolismo , Rim/metabolismo , Masculino , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Software , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína Exportina 1
12.
Mol Cell ; 81(15): 3187-3204.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34157307

RESUMO

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.


Assuntos
Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Endopeptidases/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Poliubiquitina/metabolismo , Adulto , Animais , Endopeptidases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Fator 2 de Diferenciação de Crescimento/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Mutantes , Mutação , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Telangiectasia Hemorrágica Hereditária , Ubiquitina-Proteína Ligases/metabolismo
13.
EMBO J ; 43(15): 3175-3191, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38886581

RESUMO

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell-cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease.


Assuntos
Junções Intercelulares , Mecanotransdução Celular , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Animais , Humanos , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Junções Intercelulares/metabolismo , Junções Intercelulares/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Estresse Mecânico , Peixe-Zebra/metabolismo , Peixe-Zebra/genética
14.
Cell ; 155(3): 606-20, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24243018

RESUMO

Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and interchromosomal contact. This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.


Assuntos
Cromossomos , Regulação da Expressão Gênica , Técnicas Genéticas , Análise de Célula Única , Transcrição Gênica , Cromossomos/química , Desoxirribonucleases/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Hibridização in Situ Fluorescente , Proteínas Repressoras/genética , Fator de Necrose Tumoral alfa/metabolismo
15.
Cell ; 150(3): 563-74, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22863009

RESUMO

Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.


Assuntos
Genes Supressores de Tumor , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tristetraprolina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estabilidade de RNA , RNA Mensageiro/química
16.
Mol Cell ; 76(4): 562-573.e4, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31543423

RESUMO

Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.


Assuntos
Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular , Mitógenos/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/metabolismo
17.
Proc Natl Acad Sci U S A ; 121(38): e2405474121, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39255000

RESUMO

Endometriosis negatively impacts the health-related quality of life of 190 million women worldwide. Novel advances in nonhormonal treatments for this debilitating condition are desperately needed. Macrophages play a vital role in the pathophysiology of endometriosis and represent a promising therapeutic target. In the current study, we revealed the full transcriptomic complexity of endometriosis-associated macrophage subpopulations using single-cell analyses in a preclinical mouse model of experimental endometriosis. We have identified two key lesion-resident populations that resemble i) tumor-associated macrophages (characterized by expression of Folr2, Mrc1, Gas6, and Ccl8+) that promoted expression of Col1a1 and Tgfb1 in human endometrial stromal cells and increased angiogenic meshes in human umbilical vein endothelial cells, and ii) scar-associated macrophages (Mmp12, Cd9, Spp1, Trem2+) that exhibited a phenotype associated with fibrosis and matrix remodeling. We also described a population of proresolving large peritoneal macrophages that align with a lipid-associated macrophage phenotype (Apoe, Saa3, Pid1) concomitant with altered lipid metabolism and cholesterol efflux. Gain of function experiments using an Apoe mimetic resulted in decreased lesion size and fibrosis, and modification of peritoneal macrophage populations in the preclinical model. Using cross-species analysis of mouse and human single-cell datasets, we determined the concordance of peritoneal and lesion-resident macrophage subpopulations, identifying key similarities and differences in transcriptomic phenotypes. Ultimately, we envisage that these findings will inform the design and use of specific macrophage-targeted therapies and open broad avenues for the treatment of endometriosis.


Assuntos
Endometriose , Macrófagos , Análise de Célula Única , Feminino , Análise de Célula Única/métodos , Animais , Humanos , Endometriose/metabolismo , Endometriose/patologia , Endometriose/genética , Camundongos , Macrófagos/metabolismo , Fenótipo , Endométrio/metabolismo , Endométrio/patologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/metabolismo , Transcriptoma
18.
J Cell Sci ; 137(8)2024 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-38563084

RESUMO

Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The spatiotemporal activation of the transcriptional co-factors YAP (herein referring to YAP1) and TAZ (also known WWTR1), collectively denoted YAP/TAZ, is crucial to allow for efficient collective endothelial migration in angiogenesis. The focal adhesion protein deleted-in-liver-cancer-1 (DLC1) was recently described as a transcriptional downstream target of YAP/TAZ in endothelial cells. In this study, we uncover a negative feedback loop between DLC1 expression and YAP activity during collective migration and sprouting angiogenesis. In particular, our study demonstrates that signaling via the RhoGAP domain of DLC1 reduces nuclear localization of YAP and its transcriptional activity. Moreover, the RhoGAP activity of DLC1 is essential for YAP-mediated cellular processes, including the regulation of focal adhesion turnover, traction forces, and sprouting angiogenesis. We show that DLC1 restricts intracellular cytoskeletal tension by inhibiting Rho signaling at the basal adhesion plane, consequently reducing nuclear YAP localization. Collectively, these findings underscore the significance of DLC1 expression levels and its function in mitigating intracellular tension as a pivotal mechanotransductive feedback mechanism that finely tunes YAP activity throughout the process of sprouting angiogenesis.


Assuntos
Adesões Focais , Proteínas Ativadoras de GTPase , Mecanotransdução Celular , Proteínas Supressoras de Tumor , Proteínas de Sinalização YAP , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Movimento Celular , Retroalimentação Fisiológica , Adesões Focais/metabolismo , Adesões Focais/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mecanotransdução Celular/genética , Neovascularização Fisiológica , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAP/metabolismo
19.
J Cell Sci ; 137(13)2024 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-38881365

RESUMO

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.


Assuntos
Matriz Extracelular , Adesões Focais , Quinases da Família src , Adesões Focais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Quinases da Família src/metabolismo , Quinases da Família src/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Transdução de Sinais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Metaloproteinases da Matriz/metabolismo
20.
J Cell Sci ; 137(14)2024 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-38940198

RESUMO

TMEM16F (also known as ANO6), a Ca2+-activated lipid scramblase (CaPLSase) that dynamically disrupts lipid asymmetry, plays a crucial role in various physiological and pathological processes, such as blood coagulation, neurodegeneration, cell-cell fusion and viral infection. However, the mechanisms through which it regulates these processes remain largely elusive. Using endothelial cell-mediated angiogenesis as a model, here we report a previously unknown intracellular signaling function of TMEM16F. We demonstrate that TMEM16F deficiency impairs developmental retinal angiogenesis in mice and disrupts angiogenic processes in vitro. Biochemical analyses indicate that the absence of TMEM16F enhances the plasma membrane association of activated Src kinase. This in turn increases VE-cadherin phosphorylation and downregulation, accompanied by suppressed angiogenesis. Our findings not only highlight the role of intracellular signaling by TMEM16F in endothelial cells but also open new avenues for exploring the regulatory mechanisms for membrane lipid asymmetry and their implications in disease pathogenesis.


Assuntos
Anoctaminas , Células Endoteliais , Transdução de Sinais , Animais , Anoctaminas/metabolismo , Anoctaminas/genética , Camundongos , Humanos , Células Endoteliais/metabolismo , Quinases da Família src/metabolismo , Quinases da Família src/genética , Neovascularização Fisiológica , Fosforilação , Caderinas/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Membrana Celular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Angiogênese , Proteínas de Transferência de Fosfolipídeos
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