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1.
Cell Mol Life Sci ; 78(7): 3743-3762, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33683377

RESUMO

Mutations in the intraflagellar transport-A (IFT-A) gene, THM1, have been identified in skeletal ciliopathies. Here, we report a genetic interaction between Thm1, and its paralog, Thm2, in postnatal skeletogenesis. THM2 localizes to primary cilia, but Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. However, by postnatal day 14, Thm2-/-; Thm1aln/+ mice exhibit small stature and small mandible. Radiography and microcomputed tomography reveal Thm2-/-; Thm1aln/+ tibia are less opaque and have reduced cortical and trabecular bone mineral density. In the mutant tibial growth plate, the proliferation zone is expanded and the hypertrophic zone is diminished, indicating impaired chondrocyte differentiation. Additionally, mutant growth plate chondrocytes show increased Hedgehog signaling. Yet deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog pathway, exacerbated the Thm2-/-; Thm1aln/+ small phenotype, and further revealed that Thm2-/-; Gli2+/- mice have small stature. In Thm2-/-; Thm1aln/+ primary osteoblasts, a Hedgehog signaling defect was not detected, but bone nodule formation was markedly impaired. This indicates a signaling pathway is altered, and we propose that this pathway may potentially interact with Gli2. Together, our data reveal that loss of Thm2 with one allele of Thm1, Gli2, or both, present new IFT mouse models of osteochondrodysplasia. Our data also suggest Thm2 as a modifier of Hedgehog signaling in postnatal skeletal development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Condrócitos/patologia , Condrogênese , Proteínas Hedgehog/metabolismo , Osteoblastos/patologia , Osteogênese , Animais , Animais Recém-Nascidos , Diferenciação Celular , Condrócitos/metabolismo , Cílios , Feminino , Proteínas Hedgehog/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Transdução de Sinais
2.
Int J Biol Macromol ; 171: 423-427, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33428955

RESUMO

Membrane-associated RING (really interesting new gene)-cysteine-histidine (CH) (MARCH) ubiquitin ligases belong to a RING finger domain E3 ligases family. So far, eleven members have been found in the MARCH family, which are MARCH 1 to 11. The members of the MARCH family are widely distributed and involve in a variety of cellular functions, including regulation of the immune system, transmembrane transport of proteins, protein stability, endoplasmic reticulum-related degradation, and endosome protein transport. Several seminal studies over the past decade have delineated that MARCH affects viral replication through various mechanisms by regulating the activity of signaling molecules and their expression in the antiviral innate immune responses. Here, we summarize the complex roles of MARCH ligases in the antiviral innate immune signaling pathway and its impact on viral replication in host immune defense systems. A better understanding of this interplay's molecular mechanisms is important concerning the development of new therapeutics targeting viral infections.


Assuntos
Imunidade Inata/fisiologia , Processamento de Proteína Pós-Traducional/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação/imunologia , Viroses/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Antivirais/farmacologia , DNA Viral/imunologia , Desenho de Fármacos , Interações Hospedeiro-Patógeno , Humanos , Receptores Imunológicos , Transdução de Sinais , Receptores Toll-Like/fisiologia , Viroses/imunologia , Replicação Viral/imunologia
3.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443153

RESUMO

The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3 polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1's functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3 as well as PI(3,4)P2 act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1's enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2 concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositóis/metabolismo , Fatores de Ribosilação do ADP/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Axônios/metabolismo , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais
4.
Nucleic Acids Res ; 49(2): 621-635, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33337475

RESUMO

The integration of retroviral reverse transcripts into the chromatin of the cells that they infect is required for virus replication. Retroviral integration has far-reaching consequences, from perpetuating deadly human diseases to molding metazoan evolution. The lentivirus human immunodeficiency virus 1 (HIV-1), which is the causative agent of the AIDS pandemic, efficiently infects interphase cells due to the active nuclear import of its preintegration complex (PIC). To enable integration, the PIC must navigate the densely-packed nuclear environment where the genome is organized into different chromatin states of varying accessibility in accordance with cellular needs. The HIV-1 capsid protein interacts with specific host factors to facilitate PIC nuclear import, while additional interactions of viral integrase, the enzyme responsible for viral DNA integration, with cellular nuclear proteins and nucleobases guide integration to specific chromosomal sites. HIV-1 integration favors transcriptionally active chromatin such as speckle-associated domains and disfavors heterochromatin including lamina-associated domains. In this review, we describe virus-host interactions that facilitate HIV-1 PIC nuclear import and integration site targeting, highlighting commonalities among factors that participate in both of these steps. We moreover discuss how the nuclear landscape influences HIV-1 integration site selection as well as the establishment of active versus latent virus infection.


Assuntos
HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Integração Viral , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cromatina/genética , Cromatina/metabolismo , Citoplasma/metabolismo , Citoplasma/virologia , Proteínas do Citoesqueleto/metabolismo , Transcriptase Reversa do HIV/fisiologia , HIV-1/enzimologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Interfase , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Conformação Proteica , Domínios Proteicos , Fatores de Transcrição/deficiência , Fatores de Transcrição/fisiologia , Integração Viral/genética , Integração Viral/fisiologia , Latência Viral , Replicação Viral , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/fisiologia
5.
Proc Natl Acad Sci U S A ; 117(36): 22462-22472, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839311

RESUMO

Huntingtin-interacting protein family members are evolutionarily conserved from yeast to humans, and they are known to be key factors in clathrin-mediated endocytosis. Here we identified the Caenorhabditis elegans protein huntingtin-interacting protein-related 1 (HIPR-1) as a host factor essential for Orsay virus infection of C. elegans Ablation of HIPR-1 resulted in a greater than 10,000-fold reduction in viral RNA, which could be rescued by ectopic expression of HIPR-1. Viral RNA replication from an endogenous transgene replicon system was not affected by lack of HIPR-1, suggesting that HIPR-1 plays a role during an early, prereplication virus life-cycle stage. Ectopic expression of HIPR-1 mutants demonstrated that neither the clathrin light chain-binding domain nor the clathrin heavy chain-binding motif were needed for virus infection, whereas the inositol phospholipid-binding and F-actin-binding domains were essential. In human cell culture, deletion of the human HIP orthologs HIP1 and HIP1R led to decreased infection by Coxsackie B3 virus. Finally, ectopic expression of a chimeric HIPR-1 harboring the human HIP1 ANTH (AP180 N-terminal homology) domain rescued Orsay infection in C. elegans, demonstrating conservation of its function through evolution. Collectively, these findings further our knowledge of cellular factors impacting viral infection in C. elegans and humans.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Proteínas dos Microfilamentos/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/virologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Sequência Conservada/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Enterovirus Humano B/patogenicidade , Enterovirus Humano B/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Nodaviridae/patogenicidade , Nodaviridae/fisiologia , Domínios Proteicos/genética , Replicação Viral
6.
PLoS One ; 15(7): e0236744, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730309

RESUMO

Repeated exposures to environmental allergens in susceptible individuals drive the development of type 2 inflammatory conditions such as asthma, which have been traditionally considered to be mainly mediated by Th2 cells. However, emerging evidence suggest that a new innate cell type, group 2 innate lymphoid cells (ILC2), plays a central role in initiating and amplifying a type 2 response, even in the absence of adaptive immunity. At present, the regulatory mechanisms for controlling ILC2 activation remain poorly understood. Here we report that respiratory delivery of immunogenic extracellular RNA (exRNAs) derived from RNA- and DNA-virus infected cells, was able to activate a protective response against acute type 2 lung immunopathology and airway hyperresponsiveness (AHR) induced by IL-33 and a fungal allergen, A. flavus, in mice. Mechanistically, we found that the innate immune responses triggered by exRNAs had a potent suppressive effect in vivo on the proliferation and function of ILC2 without the involvement of adaptive immunity. We further provided the loss-of-function genetic evidence that the TLR3- and MAVS-mediated signaling axis is essential for the inhibitory effects of exRNAs in mouse lungs. Thus, our results indicate that the host detection of extracellular immunostimulatory RNAs generated during respiratory viral infections have an important function in the regulation of ILC2-driven acute lung inflammation.


Assuntos
Armadilhas Extracelulares/imunologia , Imunidade Inata/imunologia , Linfócitos/imunologia , Pneumonia/imunologia , RNA/imunologia , Hipersensibilidade Respiratória/imunologia , Imunidade Adaptativa , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Citocinas/metabolismo , Armadilhas Extracelulares/metabolismo , Interleucina-33/imunologia , Interleucina-33/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/metabolismo , Pneumonia/patologia , RNA/metabolismo , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Transdução de Sinais , Células Th2/imunologia , Células Th2/metabolismo , Receptor 3 Toll-Like/fisiologia
7.
J Neurosci ; 40(27): 5214-5227, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32467358

RESUMO

The limitation of plasticity in the adult brain impedes functional recovery later in life from brain injury or disease. This pressing clinical issue may be resolved by enhancing plasticity in the adult brain. One strategy for triggering robust plasticity in adulthood is to reproduce one of the hallmark physiological events of experience-dependent plasticity observed during the juvenile critical period: to rapidly reduce the activity of parvalbumin (PV)-expressing interneurons and disinhibit local excitatory neurons. This may be achieved through the enhancement of local inhibitory inputs, particularly those of somatostatin (SST)-expressing interneurons. However, to date the means for manipulating SST interneurons for enhancing cortical plasticity in the adult brain are not known. We show that SST interneuron-selective overexpression of Lypd6, an endogenous nicotinic signaling modulator, enhances ocular dominance plasticity in the adult primary visual cortex (V1). Lypd6 overexpression mediates a rapid experience-dependent increase in the visually evoked activity of SST interneurons as well as a simultaneous reduction in PV interneuron activity and disinhibition of excitatory neurons. Recapitulating this transient activation of SST interneurons using chemogenetics similarly enhanced V1 plasticity. Notably, we show that SST-selective Lypd6 overexpression restores visual acuity in amblyopic mice that underwent early long-term monocular deprivation. Our data in both male and female mice reveal selective modulation of SST interneurons and a putative downstream circuit mechanism as an effective method for enhancing experience-dependent cortical plasticity as well as functional recovery in adulthood.SIGNIFICANCE STATEMENT The decline of cortical plasticity after closure of juvenile critical period consolidates neural circuits and behavior, but this limits functional recovery from brain diseases and dysfunctions in later life. Here we show that activation of cortical somatostatin (SST) interneurons by Lypd6, an endogenous modulator of nicotinic acetylcholine receptors, enhances experience-dependent plasticity and recovery from amblyopia in adulthood. This manipulation triggers rapid reduction of PV interneuron activity and disinhibition of excitatory neurons, which are known hallmarks of cortical plasticity during juvenile critical periods. Our study demonstrates modulation of SST interneurons by Lypd6 to achieve robust levels of cortical plasticity in the adult brain and may provide promising targets for restoring brain function in the event of brain trauma or disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Ligadas por GPI/fisiologia , Interneurônios/fisiologia , Plasticidade Neuronal/fisiologia , Somatostatina/fisiologia , Córtex Visual/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Dominância Ocular/genética , Potenciais Evocados Visuais/genética , Potenciais Evocados Visuais/fisiologia , Feminino , Proteínas Ligadas por GPI/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Plasticidade Neuronal/genética , Fosfatidilinositóis/farmacologia , Receptores Nicotínicos/genética , Recuperação de Função Fisiológica/genética , Visão Monocular/genética , Visão Monocular/fisiologia , Acuidade Visual/genética
8.
J Neurosci ; 40(20): 3915-3932, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32341094

RESUMO

Loss of sensory hair cells causes permanent hearing and balance deficits in humans and other mammals, but for nonmammals such deficits are temporary. Nonmammals recover hearing and balance sensitivity after supporting cells proliferate and differentiate into replacement hair cells. Evidence of mechanical differences between those sensory epithelia and their supporting cells prompted us to investigate whether the capacity to activate YAP, an effector in the mechanosensitive Hippo pathway, correlates with regenerative capacity in acceleration-sensing utricles of chickens and mice of both sexes. After hair cell ablation, YAP accumulated in supporting cell nuclei in chicken utricles and promoted regenerative proliferation, but YAP remained cytoplasmic and little proliferation occurred in mouse utricles. YAP localization in supporting cells was also more sensitive to shape change and inhibition of MST1/2 in chicken utricles than in mouse utricles. Genetic manipulations showed that in vivo expression of the YAP-S127A variant caused robust proliferation of neonatal mouse supporting cells, which produced progeny that expressed hair cell markers, but proliferative responses declined postnatally. Expression of YAP-5SA, which more effectively evades inhibitory phosphorylation, resulted in TEAD-dependent proliferation of striolar supporting cells, even in adult utricles. Conditional deletion of LATS1/2 kinases abolished the inhibitory phosphorylation of endogenous YAP and led to striolar proliferation in adult mouse utricles. The findings suggest that damage overcomes inhibitory Hippo signaling and facilitates regenerative proliferation in nonmammalian utricles, whereas constitutive LATS1/2 kinase activity suppresses YAP-TEAD signaling in mammalian utricles and contributes to maintaining the proliferative quiescence that appears to underlie the permanence of sensory deficits.SIGNIFICANCE STATEMENT Loud sounds, ototoxic drugs, infections, and aging kill sensory hair cells in the ear, causing irreversible hearing loss and balance deficits for millions. In nonmammals, damage evokes shape changes in supporting cells, which can divide and regenerate hair cells. Such shape changes are limited in mammalian ears, where supporting cells develop E-cadherin-rich apical junctions reinforced by robust F-actin bands, and the cells fail to divide. Here, we find that damage readily activates YAP in supporting cells within balance epithelia of chickens, but not mice. Deleting LATS kinases or expressing YAP variants that evade LATS-mediated inhibitory phosphorylation induces proliferation in supporting cells of adult mice. YAP signaling eventually may be harnessed to overcome proliferative quiescence that limits regeneration in mammalian ears.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ciclo Celular/fisiologia , Células Ciliadas Auditivas/fisiologia , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/genética , Proliferação de Células , Embrião de Galinha , Galinhas , Deleção de Genes , Variação Genética , Perda Auditiva/genética , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Estimulador Tireóideo de Ação Prolongada , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Sáculo e Utrículo/efeitos dos fármacos , Especificidade da Espécie , Proteínas Supressoras de Tumor/genética
9.
Oncogene ; 39(22): 4375-4389, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32313226

RESUMO

Hippo signaling functions to limit cellular growth, but the aberrant nuclear accumulation of its downstream YAP1 leads to carcinogenesis. YAP1/TEAD complex activates the oncogenic downstream transcription, such as CTGF and c-Myc. How YAP1 is protected in the cytoplasm from ubiquitin-mediated degradation remains elusive. In this study, a member of Angiomotin (Motin) family, AMOTL1 (Angiomotin Like 1), was screened out as the only one to promote YAP1 nuclear accumulation by several clinical cohorts, which was further confirmed by the cellular functional assays. The interaction between YAP1 and AMOTL1 was suggested by co-immunoprecipitation and immunofluorescent staining. The clinical significance of the AMOTL1-YAP1-CTGF axis in gastric cancer (GC) was analyzed by multiple clinical cohorts. Moreover, the therapeutic effect of targeting the oncogenic axis was appraised by drug-sensitivity tests and xenograft-formation assays. The upregulation of AMOTL1 is associated with unfavorable clinical outcomes of GC, and knocking down AMOTL1 impairs its oncogenic properties. The cytoplasmic interaction between AMOTL1 and YAP1 protects each other from ubiquitin-mediated degradation. AMOTL1 promotes YAP1 translocation into the nuclei to activate the downstream expression, such as CTGF. Knocking down AMOTL1, YAP1, and CTGF enhances the therapeutic efficacies of the first-line anticancer drugs. Taken together, AMOTL1 plays an oncogenic role in gastric carcinogenesis through interacting with YAP1 and promoting its nuclear accumulation. A combination of AMOTL1, YAP1, and CTGF expression might serve as a surrogate of Hippo activation status. The co-activation of the AMOTL1/YAP1-CTGF axis is associated with poor clinical outcomes of GC patients, and targeting this oncogenic axis may enhance the chemotherapeutic effects.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transformação Celular Neoplásica/genética , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Gástricas/genética , Fatores de Transcrição/fisiologia , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fator de Crescimento do Tecido Conjuntivo/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Proteínas de Membrana/genética , Camundongos , Proteínas de Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Fatores de Transcrição/genética , Verteporfina/farmacologia , Verteporfina/uso terapêutico
10.
Invest Ophthalmol Vis Sci ; 61(4): 1, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32271890

RESUMO

Purpose: Purpose The role of endothelial Yes-associated protein 1 (YAP) in the pathogenesis of retinal angiogenesis and the astrocyte network in the mouse oxygen-induced retinopathy (OIR) model is unknown. Methods: For in vivo studies, OIR was induced in conditional endothelial YAP knockout mice and their wild-type littermates. Retinal vascularization and the astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro experiments were performed in astrocytes cultured with human microvascular endothelial cell-1-conditioned medium to analyze the mechanisms underlying the effect of endothelial YAP on astrocytes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Astrócitos/patologia , Proteínas de Ciclo Celular/fisiologia , Células Endoteliais/metabolismo , Fator Inibidor de Leucemia/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados , Citoplasma/metabolismo , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxigênio/toxicidade , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia , Vasos Retinianos/citologia , Retinopatia da Prematuridade/induzido quimicamente , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia
11.
Hum Cell ; 33(3): 768-779, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32166565

RESUMO

Non-catalytic region of tyrosine kinase adaptor protein 1 (Nck1) is crucial for the progression of cancers. However, little is known on the role of Nck1 in the progression of ovarian carcinoma (OC). Here, we show that Nck1 expression is up-regulated in 176 OC tissues, compared with non-carcinoma ovarian tissues, and the up-regulated Nck1 expression is associated with the aggressiveness of OC and shorter overall and disease-free survival in this population. Higher Nck1 expression was an independent risk factor for poor prognosis of OC. Furthermore, Nck1 silencing by short hairpin RNA (shRNA) technology significantly inhibited the proliferation, migration and invasion of OC cells in vitro and the growth and metastasis of implanted OC tumors in vivo. Human kinase phosphorylation array indicated that Nck1 silencing significantly reduced the relative levels of 11 kinase expression and phosphorylation in OC cells, particularly for decreased levels of p70S6 kinase (p70S6K) and protein kinase B (AKT) expression in SKOV3 cells. Actually, Nck1 silencing significantly decreased PI3K and AKT expression, and reduced AKT and p70S6K phosphorylation while Nck1 over-expression had opposite effects in OC cells. Therefore, our data indicate that Nck1 promotes the progression of OC by enhancing the PI3k/AKT/p70S6K signaling in OC. Our findings suggest that Nck1 expression may be valuable for evaluating the prognosis of OC and as a target for design of new therapies for OC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Carcinoma/genética , Proteínas Oncogênicas/fisiologia , Neoplasias Ovarianas/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/fisiologia , Carcinoma/terapia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Terapia de Alvo Molecular , Neoplasias Ovarianas/terapia , Prognóstico , Transdução de Sinais/genética
12.
Development ; 147(6)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179574

RESUMO

Precise temporal coordination of signaling processes is pivotal for cellular differentiation during embryonic development. A vast number of secreted molecules are produced and released by cells and tissues, and travel in the extracellular space. Whether they induce a signaling pathway and instruct cell fate, however, depends on a complex network of regulatory mechanisms, which are often not well understood. The conserved bilateral left-right asymmetrically formed habenulae of the zebrafish are an excellent model for investigating how signaling control facilitates the generation of defined neuronal populations. Wnt signaling is required for habenular neuron type specification, asymmetry and axonal connectivity. The temporal regulation of this pathway and the players involved have, however, have remained unclear. We find that tightly regulated temporal restriction of Wnt signaling activity in habenular precursor cells is crucial for the diversity and asymmetry of habenular neuron populations. We suggest a feedback mechanism whereby the tumor suppressor Wnt inhibitory factor Wif1 controls the Wnt dynamics in the environment of habenular precursor cells. This mechanism might be common to other cell types, including tumor cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Padronização Corporal/genética , Habenula/embriologia , Neurogênese/genética , Neurônios/fisiologia , Proteínas Repressoras/fisiologia , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Encéfalo/embriologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Dominância Cerebral/genética , Embrião não Mamífero , Habenula/metabolismo , Neurogênese/fisiologia , Neurônios/citologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
Science ; 367(6482)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32054698

RESUMO

Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/fisiologia , Oogênese/genética , Proteínas Repressoras/fisiologia , Diferenciação Sexual/genética , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Feto/citologia , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Oócitos/citologia , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/genética , Processos de Determinação Sexual , Transdução de Sinais , Fatores de Transcrição/genética , Transcriptoma , Tretinoína/fisiologia
14.
Dev Cell ; 52(4): 429-445.e10, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32032549

RESUMO

The mechanisms regulating meiotic initiation in mammals are enigmatic. It is known that retinoic acid (RA) signaling plays a pivotal role during meiotic initiation. STRA8, which is expressed in response to RA, is thought to be a key factor promoting meiotic initiation. However, the specific role of STRA8 in meiotic initiation has remained elusive. Here, we identified MEIOSIN as a germ-cell-specific factor that associates with STRA8. MEIOSIN, like STRA8, is expressed in response to RA and plays an essential role in meiotic initiation in both males and females. Functional analyses revealed that MEIOSIN acts as a transcription factor together with STRA8, and that both factors are critical for driving meiotic gene activation. Furthermore, temporally restricted expression of MEIOSIN leads to meiotic entry decision during spermatogenesis. The present study demonstrates that MEIOSIN, in collaboration with STRA8, plays a central role in regulating the mitosis to meiosis germ cell fate decision in mammals.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Ciclo Celular , Regulação da Expressão Gênica , Células Germinativas/fisiologia , Meiose , Mitose , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Feminino , Células Germinativas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Espermatogênese
15.
Exp Cell Res ; 389(2): 111912, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32084391

RESUMO

Ischemic stroke leads to neuronal cell death and induces a cascade of inflammatory signals that results in secondary brain damage. Although constant efforts to develop therapeutic strategies and to reveal the molecular mechanism resulting in the physiopathology of this disease, much still remains unclear. Membrane-bound Toll-like receptors (TLRs) and cytosolic nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) are two major families of pattern recognition receptors that initiate pro-inflammatory signaling pathways. In the present study, we explored the role of NLRP10 in regulating inflammatory responses in acute ischemic stroke using the wild type (WT) and NLRP10 knockout (KO) mice by inducing middle cerebral artery occlusion/reperfusion (MCAO) injuries. The study first showed that NLRP10 was over-expressed in the ischemic penumbra of WT mice. Then, the brain infarct volume was significantly decreased, and the moving activity was improved post-MCAO in mice with NLRP10 knockout. Apoptosis was also alleviated by NLRP10-knockout, as evidenced by the decreased number of TUNEL-staining cells. Further, NLRP10 deficiency attenuated the activation of glia cells in hippocampus of mice with MCAO operation. NLRP10 inhibition ameliorated the levels of inflammatory factors in peripheral blood serum and hippocampus of mice after stroke. The activation of toll-like receptor (TLR)-4/nuclear factor-κB (NF-κB) signaling pathways was markedly suppressed by NLRP10 ablation in mice after MCAO treatment. Importantly, inflammasome, including NLRP12, ASC and Caspase-1, induced by MCAO in hippocampus of mice was clearly impeded by the loss of NLRP10. The results above were mainly verified in LPS-incubated astrocytes in the absence of NLRP10. Correspondingly, in LPS-treated astrocytes, NLRP10 knockout-reduced inflammation via impairing TLR-4/NF-κB and NLRP12/ASC/Caspase-1 pathways was evidently restored by over-expressing NLRP10. Therefore, the results above indicated an essential role of NLRP10 in regulating ischemic stroke, presenting NLRP10 as a promising target to protect human against stroke.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Lesões Encefálicas/prevenção & controle , Isquemia Encefálica/complicações , Inflamação/prevenção & controle , Substâncias Protetoras , Traumatismo por Reperfusão/complicações , Acidente Vascular Cerebral/complicações , Animais , Apoptose , Lesões Encefálicas/etiologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Caspase 1/metabolismo , Infarto da Artéria Cerebral Média , Inflamassomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptores Toll-Like/metabolismo
16.
Transplantation ; 104(9): 1869-1878, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32058468

RESUMO

BACKGROUND: Triple progressive thermopreconditioning (3PTP) may induce high Hsp-70 expression to maintain cardiac function. We suggest that 3PTP may reduce myocardial ischemia/reperfusion (I/R) injury during organ transplantation through Bag3/Hsp-70 mediated defense mechanisms. METHODS: Male Wistar rats were divided into sham control group and 72 h after 3PTP in a 42°C water bath (3PTP) group. Rats underwent 60 min of ischemia by occlusion of the left anterior descending coronary artery followed by 240 min reperfusion. Hemodynamic parameters, including the electrocardiogram, microcirculation, heart rate, left ventricular end-diastolic pressure, maximal rate of rise (+dp/dt), and fall (-dp/dt) in the left ventricular pressure for index of contraction and relaxation were determined. Myocardial infarct size was evaluated by the Evans blue-2,3,5-triphenyltetrazolium chloride method. 3PTP-induced protective mechanisms were determined by Western blot and immunohistochemistry. RESULTS: Cardiac I/R depressed cardiac microcirculation, induced S-T segment elevation, and R-R and P-R interval elongation increased infarct size associated with erythrocyte extravasation, leukocytes and macrophage/monocyte infiltration, granulocyte colony-stimulating factor, poly(ADP-ribose) polymerase 1 stain, and transferase-mediated dUTP-biotin nick end labeling positive cells. However, 3PTP evoked significant cardioprotection against I/R injury, characterized by the increased +dp/dt value and the decreased elevated left ventricular end-diastolic pressure, erythrocyte extravasation, leukocyte and macrophage/monocyte infiltration, granulocyte colony-stimulating factor expression, poly(ADP-ribose) polymerase 1 expression, transferase-mediated dUTP-biotin nick end labeling positive cells, and fragmentation and infarct area. In addition, 3PTP increased Hsp-70 and Bag3 expression and decreased Bax/Bcl-2 ratio, but did not affect the Beclin-1 and LC3-II/LC3-I ratio in the heart with I/R injury. CONCLUSIONS: 3PTP therapies may through Bag3 upregulation alleviate I/R injury-induced left ventricular structural deterioration and dysfunction.


Assuntos
Ventrículos do Coração/patologia , Precondicionamento Isquêmico Miocárdico , Contração Miocárdica/fisiologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Disfunção Ventricular Esquerda/prevenção & controle , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Masculino , Microcirculação , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Ratos , Ratos Wistar
17.
Arterioscler Thromb Vasc Biol ; 40(4): 973-985, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31996024

RESUMO

OBJECTIVE: STAP1, encoding for STAP1 (signal transducing adaptor family member 1), has been reported as a candidate gene associated with familial hypercholesterolemia. Unlike established familial hypercholesterolemia genes, expression of STAP1 is absent in liver but mainly observed in immune cells. In this study, we set out to validate STAP1 as a familial hypercholesterolemia gene. Approach and Results: A whole-body Stap1 knockout mouse model (Stap1-/-) was generated and characterized, without showing changes in plasma lipid levels compared with controls. In follow-up studies, bone marrow from Stap1-/- mice was transplanted to Ldlr-/- mice, which did not show significant changes in plasma lipid levels or atherosclerotic lesions. To functionally assess whether STAP1 expression in B cells can affect hepatic function, HepG2 cells were cocultured with peripheral blood mononuclear cells isolated from heterozygotes carriers of STAP1 variants and controls. The peripheral blood mononuclear cells from STAP1 variant carriers and controls showed similar LDLR mRNA and protein levels. Also, LDL (low-density lipoprotein) uptake by HepG2 cells did not differ upon coculturing with peripheral blood mononuclear cells isolated from either STAP1 variant carriers or controls. In addition, plasma lipid profiles of 39 carriers and 71 family controls showed no differences in plasma LDL cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, and lipoprotein(a) levels. Similarly, B-cell populations did not differ in a group of 10 STAP1 variant carriers and 10 age- and sex-matched controls. Furthermore, recent data from the UK Biobank do not show association between STAP1 rare gene variants and LDL cholesterol. CONCLUSIONS: Our combined studies in mouse models and carriers of STAP1 variants indicate that STAP1 is not a familial hypercholesterolemia gene.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , LDL-Colesterol/sangue , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/genética , Animais , Aterosclerose/sangue , Aterosclerose/genética , Linfócitos B/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Lipídeos/sangue , Linfócitos/imunologia , Masculino , Camundongos Knockout , Monócitos/imunologia
18.
PLoS Biol ; 18(1): e3000591, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929526

RESUMO

A major challenge for cancer immunotherapy is sustaining T-cell activation and recruitment in immunosuppressive solid tumors. Here, we report that the levels of the Hippo pathway effector Yes-associated protein (Yap) are sharply induced upon the activation of cluster of differentiation 4 (CD4)-positive and cluster of differentiation 8 (CD8)-positive T cells and that Yap functions as an immunosuppressive factor and inhibitor of effector differentiation. Loss of Yap in T cells results in enhanced T-cell activation, differentiation, and function, which translates in vivo to an improved ability for T cells to infiltrate and repress tumors. Gene expression analyses of tumor-infiltrating T cells following Yap deletion implicates Yap as a mediator of global T-cell responses in the tumor microenvironment and as a negative regulator of T-cell tumor infiltration and patient survival in diverse human cancers. Collectively, our results indicate that Yap plays critical roles in T-cell biology and suggest that Yap inhibition improves T-cell responses in cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ciclo Celular/fisiologia , Quimiotaxia de Leucócito/genética , Linfócitos T/fisiologia , Microambiente Tumoral/imunologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proliferação de Células/genética , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Imunoterapia Adotiva , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Microambiente Tumoral/genética
19.
Exp Eye Res ; 191: 107917, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31923414

RESUMO

The transparent and refractive properties of the ocular lens are dependent on its precise cellular structure, supported by the regulation of lens cellular processes of proliferation and differentiation that are essential throughout life. The ERK/MAPK-signalling pathway plays a crucial role in regulating lens cell proliferation and differentiation, and in turn is regulated by inhibitory molecules including the Spred family of proteins to modulate and attenuate the impact of growth factor stimulation. Given Spreds are strongly and distinctly expressed in lens, along with their established inhibitory role in a range of different tissues, we investigated the role these antagonists play in regulating lens cell proliferation and differentiation, and their contribution to lens structure and growth. Using established mice lines deficient for either or both Spred 1 and Spred 2, we demonstrate their role in regulating lens development by negatively regulating ERK1/2 activity. Mice deficient for both Spred 1 and Spred 2 have impaired lens and eye development, displaying irregular lens epithelial and fibre cell activity as a result of increased levels of phosphorylated ERK1/2. While Spred 1 and Spred 2 do not appear to be necessary for induction and early stages of lens morphogenesis (prior to E11.5), nor for the formation of the primary fibre cells, they are required for the continuous embryonic growth and differentiation of the lens.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Olho/embriologia , Cristalino/embriologia , Morfogênese/fisiologia , Proteínas Repressoras/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Técnicas de Genotipagem , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Reação em Cadeia da Polimerase
20.
Am J Pathol ; 190(2): 333-346, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31837290

RESUMO

Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-nephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/metabolismo , Efrina-B1/metabolismo , Glomérulos Renais/citologia , Proteínas de Membrana/metabolismo , Síndrome Nefrótica/patologia , Podócitos/citologia , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Diafragma , Efrina-B1/genética , Células HEK293 , Humanos , Glomérulos Renais/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Síndrome Nefrótica/metabolismo , Fosforilação , Podócitos/metabolismo , Ratos , Ratos Wistar
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