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1.
PLoS Genet ; 16(8): e1008976, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866141

RESUMO

Neural circuitry for mating and reproduction resides within the terminal segments of central nervous system (CNS) which express Hox paralogous group 9-13 (in vertebrates) or Abdominal-B (Abd-B) in Drosophila. Terminal neuroblasts (NBs) in A8-A10 segments of Drosophila larval CNS are subdivided into two groups based on expression of transcription factor Doublesex (Dsx). While the sex specific fate of Dsx-positive NBs is well investigated, the fate of Dsx-negative NBs is not known so far. Our studies with Dsx-negative NBs suggests that these cells, like their abdominal counterparts (in A3-A7 segments) use Hox, Grainyhead (Grh) and Notch to undergo cell death during larval development. This cell death also happens by transcriptionally activating RHG family of apoptotic genes through a common apoptotic enhancer in early to mid L3 stages. However, unlike abdominal NBs (in A3-A7 segments) which use increasing levels of resident Hox factor Abdominal-A (Abd-A) as an apoptosis trigger, Dsx-negative NBs (in A8-A10 segments) keep the levels of resident Hox factor Abd-B constant. These cells instead utilize increasing levels of the temporal transcription factor Grh and a rise in Notch activity to gain apoptotic competence. Biochemical and in vivo analysis suggest that Abdominal-A and Grh binding motifs in the common apoptotic enhancer also function as Abdominal-B and Grh binding motifs and maintains the enhancer activity in A8-A10 NBs. Finally, the deletion of this enhancer by the CRISPR-Cas9 method blocks the apoptosis of Dsx-negative NBs. These results highlight the fact that Hox dependent NB apoptosis in abdominal and terminal regions utilizes common molecular players (Hox, Grh and Notch), but seems to have evolved different molecular strategies to pattern CNS.


Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Receptores Notch/genética , Fatores de Transcrição/genética , Abdome/crescimento & desenvolvimento , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Células-Tronco Neurais/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética
2.
Arterioscler Thromb Vasc Biol ; 40(9): 2227-2243, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640901

RESUMO

OBJECTIVE: Perivascular adipose tissue (PVAT) surrounding arteries supports healthy vascular function. During obesity, PVAT loses its vasoprotective effect. We study pathological conversion of PVAT, which involves molecular changes in protein profiles and functional changes in adipocytes. Approach and Results: C57BL6/J mice were fed a 60% high-fat diet for 12 weeks or a cardioprotective 30% calorie-restricted diet for 5 weeks. Proteomic analysis identified PVAT as a molecularly distinct adipose depot, and novel markers for thermogenic adipocytes, such as GRP75 (stress-70 protein, mitochondrial), were identified. High-fat diet increased the similarity of protein signatures in PVAT and brown adipose, suggesting activation of a conserved whitening pathway. The whitening phenotype was characterized by suppression of UCP1 (uncoupling protein 1) and increased lipid deposition, leptin, and inflammation, and specifically in PVAT, elevated Notch signaling. Conversely, PVAT from calorie-restricted mice had decreased Notch signaling and less lipid. Using the Adipoq-Cre strain, we constitutively activated Notch1 signaling in adipocytes, which phenocopied the changes in PVAT caused by a high-fat diet, even on a standard diet. Preadipocytes from mouse PVAT expressed Sca1, CD140a, Notch1, and Notch2, but not CD105, showing differences compared with preadipocytes from other depots. Inhibition of Notch signaling during differentiation of PVAT-derived preadipocytes reduced lipid deposition and adipocyte marker expression. CONCLUSIONS: PVAT shares features with other adipose depots, but has a unique protein signature that is regulated by dietary stress. Increased Notch signaling in PVAT is sufficient to initiate the pathological conversion of PVAT by promoting adipogenesis and lipid accumulation and may thus prime the microenvironment for vascular disease.


Assuntos
Adipócitos Brancos/metabolismo , Adipogenia , Tecido Adiposo Branco/metabolismo , Lipogênese , Obesidade/metabolismo , Receptores Notch/metabolismo , Adipócitos Brancos/patologia , Tecido Adiposo Branco/patologia , Adiposidade , Animais , Ataxina-1/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Restrição Calórica , Dieta Hiperlipídica , Modelos Animais de Doenças , Endoglina/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Obesidade/patologia , Fenótipo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Proteômica , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Notch/genética , Transdução de Sinais
3.
PLoS Genet ; 16(6): e1008792, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579612

RESUMO

While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-neuronal phenotypes, functional studies evaluating these regions have focused on the molecular basis of neuronal defects. We report a systematic functional analysis of non-neuronal defects for homologs of 59 genes within ten pathogenic CNVs and 20 neurodevelopmental genes in Drosophila melanogaster. Using wing-specific knockdown of 136 RNA interference lines, we identified qualitative and quantitative phenotypes in 72/79 homologs, including 21 lines with severe wing defects and six lines with lethality. In fact, we found that 10/31 homologs of CNV genes also showed complete or partial lethality at larval or pupal stages with ubiquitous knockdown. Comparisons between eye and wing-specific knockdown of 37/45 homologs showed both neuronal and non-neuronal defects, but with no correlation in the severity of defects. We further observed disruptions in cell proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and KIF11/Klp61F. These findings were further supported by tissue-specific differences in expression patterns of human CNV genes, as well as connectivity of CNV genes to signaling pathway genes in brain, heart and kidney-specific networks. Our findings suggest that multiple genes within each CNV differentially affect both global and tissue-specific developmental processes within conserved pathways, and that their roles are not restricted to neuronal functions.


Assuntos
Variações do Número de Cópias de DNA , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Transtornos do Neurodesenvolvimento/genética , Animais , Olho Composto de Artrópodes/embriologia , Olho Composto de Artrópodes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Especificidade de Órgãos , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Asas de Animais/embriologia , Asas de Animais/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
4.
PLoS One ; 15(5): e0232981, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396580

RESUMO

Cell signaling pathways play key roles in coordinating cellular events in development. The Notch signaling pathway is highly conserved across all multicellular animals and is known to coordinate a multitude of diverse cellular events, including proliferation, differentiation, fate specification, and cell death. Specific functions of the pathway are, however, highly context-dependent and are not well characterized in post-traumatic regeneration. Here, we use a small-molecule inhibitor of the pathway (DAPT) to demonstrate that Notch signaling is required for proper arm regeneration in the brittle star Ophioderma brevispina, a highly regenerative member of the phylum Echinodermata. We also employ a transcriptome-wide gene expression analysis (RNA-seq) to characterize the downstream genes controlled by the Notch pathway in the brittle star regeneration. We demonstrate that arm regeneration involves an extensive cross-talk between the Notch pathway and other cell signaling pathways. In the regrowing arm, Notch regulates the composition of the extracellular matrix, cell migration, proliferation, and apoptosis, as well as components of the innate immune response. We also show for the first time that Notch signaling regulates the activity of several transposable elements. Our data also suggests that one of the possible mechanisms through which Notch sustains its activity in the regenerating tissues is via suppression of Neuralized1.


Assuntos
Equinodermos/fisiologia , Receptores Notch/fisiologia , Regeneração/fisiologia , Estruturas Animais/efeitos dos fármacos , Estruturas Animais/fisiologia , Animais , Elementos de DNA Transponíveis , Dipeptídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Equinodermos/efeitos dos fármacos , Equinodermos/genética , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Regeneração/efeitos dos fármacos , Regeneração/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transcriptoma/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(1): 49-55, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32314724

RESUMO

Objective To explore the change of the expression of microRNA-124-3p (miR-124-3p) in injured hippocampus of rats and investigate the role of miR-124-3p in neuranagenesis after traumatic brain injury (TBI). Methods The healthy male rats were randomly divided into a sham-operated group, TBI group, miR-124-3p agomir group and miR-124-3p antagomir group. TBI models were constructed by controlled cortical injury (CCI) device for all the groups except for the sham-operated group. The miR-124-3p agomir (1 nmol) was given to the miR-124-3p agomir group and miR-124-3p antagomir (1 nmol) to the miR-124-3p antagomir group via lateral ventricular injection, and equivalent solvent was given to the sham-operated group and TBI group after injury. The injured hippocampus of rats was collected at 12 hours, 1 day, 3, 7 days after injury. The real-time PCR and Western blot analysis were used to examine the expression of miR-124-3p and Delta-like 1 (DLL1) in the injured hippocampus. Immunofluorescence histochemistry was used to examine the expression levels of 5-bromodeoxyuridine (BrdU), neuronal nuclear antigen (NeuN) and nestin in the injured hippocampus. Bioinformatics software was used to predict and dual luciferase reporter assay to validate the regulatory relationship between miR-124-3p and DLL1. Results The miR-124-3p and DLL1 expression in the TBI group were significantly higher than those in the sham-operated group; compared with the TBI group, the miR-124-3p agomir group had significantly increased expression of miR-124-3p and significantly decreased expression of DLL1 in the injured hippocampus, and miR-124-3p antagomir group had significantly decreased expression of miR-124-3p and significantly increased expression of DLL1. Compared with the sham-operated group, the BrdU+NeuN+ cells and BrdU+nestin+ cells in the hippocampus significantly increased in the TBI group at 7 days after injury. The miR-124-3p agomir treatment increased the number of the BrdU+NeuN+ cells and BrdU+nestin+ cells, while the miR-124-3p antagomir treatment decreased the number of the BrdU+NeuN+ cells and BrdU+nestin+ cells. Bioinformatics analysis confirmed that DLL1 was a target of miR-124-3p. Conclusion High expression of miR-124-3p in the trauma region promotes the proliferation and differentiation of neural stem cells by targeting and inhibiting DLL1.


Assuntos
Lesões Encefálicas Traumáticas/genética , Proteínas de Membrana/antagonistas & inibidores , MicroRNAs/genética , Células-Tronco Neurais/citologia , Receptores Notch/genética , Animais , Lesões Encefálicas Traumáticas/patologia , Diferenciação Celular , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
6.
PLoS Biol ; 18(4): e3000696, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275659

RESUMO

It is well known that various developmental signals play diverse roles in hematopoietic stem and progenitor cell (HSPC) production; however, how these signaling pathways are orchestrated remains incompletely understood. Here, we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium (HE) specification by endocytic trafficking of Notch ligands and receptor. We further show that the interaction between Rab5c and Appl1 in the endosome is required for the survival of HE in the ventral wall of the dorsal aorta through AKT signaling. Interestingly, Rab5c overactivation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Notch/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Endocitose , Endotélio/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Receptores Notch/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/genética
7.
Science ; 367(6483): 1264-1269, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165588

RESUMO

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.


Assuntos
Genes Supressores de Tumor , Predisposição Genética para Doença , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Supressoras de Tumor/genética , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Sistemas CRISPR-Cas , Feminino , Testes Genéticos , Células HEK293 , Humanos , Proteínas com Domínio LIM/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Receptores Notch/genética , Transdução de Sinais/genética
8.
Artigo em Inglês | MEDLINE | ID: mdl-32186932

RESUMO

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.


Assuntos
Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.5/genética , Hipertensão Arterial Pulmonar/genética , Receptores de Detecção de Cálcio/genética , Molécula 2 de Interação Estromal/genética , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estrenos/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Proteína Jagged-1/genética , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Pirrolidinonas/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única
9.
PLoS Genet ; 16(3): e1008470, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203506

RESUMO

Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Expressão Gênica/genética , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proto-Oncogenes/genética , Animais , Membrana Basal/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Proteínas Proto-Oncogênicas c-fos/genética , Receptores Notch/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética
10.
PLoS Genet ; 16(3): e1008650, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196486

RESUMO

Stem cell systems are essential for the development and maintenance of polarized tissues. Intercellular signaling pathways control stem cell systems, where niche cells signal stem cells to maintain the stem cell fate/self-renewal and inhibit differentiation. In the C. elegans germline, GLP-1 Notch signaling specifies the stem cell fate, employing the sequence-specific DNA binding protein LAG-1 to implement the transcriptional response. We undertook a comprehensive genome-wide approach to identify transcriptional targets of GLP-1 signaling. We expected primary response target genes to be evident at the intersection of genes identified as directly bound by LAG-1, from ChIP-seq experiments, with genes identified as requiring GLP-1 signaling for RNA accumulation, from RNA-seq analysis. Furthermore, we performed a time-course transcriptomics analysis following auxin inducible degradation of LAG-1 to distinguish between genes whose RNA level was a primary or secondary response of GLP-1 signaling. Surprisingly, only lst-1 and sygl-1, the two known target genes of GLP-1 in the germline, fulfilled these criteria, indicating that these two genes are the primary response targets of GLP-1 Notch and may be the sole germline GLP-1 signaling protein-coding transcriptional targets for mediating the stem cell fate. In addition, three secondary response genes were identified based on their timing following loss of LAG-1, their lack of a LAG-1 ChIP-seq peak and that their glp-1 dependent mRNA accumulation could be explained by a requirement for lst-1 and sygl-1 activity. Moreover, our analysis also suggests that the function of the primary response genes lst-1 and sygl-1 can account for the glp-1 dependent peak protein accumulation of FBF-2, which promotes the stem cell fate and, in part, for the spatial restriction of elevated LAG-1 accumulation to the stem cell region.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptores Notch/metabolismo , Células-Tronco Germinativas Adultas/citologia , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular/fisiologia , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Células Germinativas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores Notch/genética , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
J Mol Biol ; 432(7): 2030-2041, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32061928

RESUMO

AIMS: Several signaling pathways contribute to endothelial-mesenchymal transitions and vascular calcification, including bone morphogenetic protein (BMP) and transforming growth factor (TGF) ß signaling. The transcription factor homeobox D3 (Hoxd3) is known to regulate an invasive endothelial phenotype, and the aim of the study is to determine if HOXD3 modulates BMP and TGFß signaling in the endothelium. METHODS AND RESEARCH: We report that the endothelium with high BMP activity due to the loss of BMP inhibitor matrix Gla protein (MGP) shows induction of Hoxd3. HOXD3 is part of a BMP-triggered cascade. When activated by BMP9, activin receptor-like kinase (ALK) 1 induces HOXD3 expression. Hoxd3 promoter is a direct target of phosphorylated (p) SMAD1, a mediator of BMP signaling. High BMP activity further results in enhanced TGFß signaling due to induction of TGFß1 and its receptor, ALK5. This is mediated by HOXD3, which directly targets the Tgfb1 promoter. Finally, TGFß1 and BMP9 stimulate the expression of MGP, which limits the enhanced ALK1 induction by counteracting BMP4. The cascade of BMP9-HOXD3-TGFß also affects Notch signaling and angiogenesis through induction of Notch ligand Jagged 2 and suppression of Notch ligand delta-like 4 (Dll4). CONCLUSION: The results suggest that HOXD3 is a novel link between BMP9/ALK1 and TGFß1/ALK5 signaling. TRANSLATIONAL PERSPECTIVE: BMP and TGFß signaling are instrumental in vascular disease such as vascular calcification and atherosclerosis. This study demonstrated a novel type of cross talk between endothelial BMP and TGFß signaling as mediated by HOXD3. The results provide a possible therapeutic approach to control dysfunctional BMP and TGFß signaling by regulating HOXD3.


Assuntos
Receptores de Activinas Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Neovascularização Fisiológica , Proteínas/fisiologia , Receptores Notch/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Receptores de Activinas Tipo II/genética , Animais , Proteínas de Ligação a DNA/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Fator 2 de Diferenciação de Crescimento/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Receptores Notch/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética
12.
Adv Exp Med Biol ; 1227: 9-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32072496

RESUMO

Notch signaling is an evolutionarily conserved pathway associated with the development and differentiation of all metazoans. It is needed for proper germ layer formation and segmentation of the embryo and controls the timing and duration of differentiation events in a dynamic manner. Perturbations of Notch signaling result in blockades of developmental cascades, developmental anomalies, and cancers. An in-depth understanding of Notch signaling is thus required to comprehend the basis of development and cancer, and can be further exploited to understand and direct the outcomes of targeted cellular differentiation into desired cell types and complex tissues from pluripotent or adult stem and progenitor cells. In this chapter, we briefly summarize the molecular, evolutionary, and developmental basis of Notch signaling. We will focus on understanding the basics of Notch signaling and its signaling control mechanisms, its developmental outcomes and perturbations leading to developmental defects, as well as have a brief look at mutations of the Notch signaling pathway causing human hereditary disorders or cancers.


Assuntos
Desenvolvimento Embrionário , Neoplasias/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Humanos , Neoplasias/patologia , Receptores Notch/genética , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismo
13.
Mol Med Rep ; 21(2): 583-588, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31974606

RESUMO

Diabetes mellitus can exacerbate renal ischemia­reperfusion (I/R) injury (RI/RI) in diabetic rats. Previous studies have shown that Notch signaling is involved in renal disorders. The aim of the present study was to evaluate the protective effect of the Notch inhibitor γ­secretase N­[N­(3,5­difluorophenacetyl)­L­alanyl]­S­phenylglycine t­butyl ester (DAPT) on RI/RI in a streptozocin (STZ)­induced diabetic rat model. STZ­induced diabetic rats were randomly grouped for different treatments. Cisplatin was used to trigger the Notch signaling pathway and the animals were preconditioned with DAPT to block the signaling pathway. Renal function, oxidative stress and inflammatory factors were examined. DAPT­treated diabetic rats demonstrated mitigated renal injury and function, antioxidative activity was significantly improved and HIF­1a was upregulated. Notch inhibitor DAPT is a potential therapeutic target to improve the outcome of RI/RI in STZ­induced diabetic rats in part via the regulation of anti­oxidation and HIF­1a.


Assuntos
Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Dipeptídeos/uso terapêutico , Receptores Notch/antagonistas & inibidores , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Nitrogênio da Ureia Sanguínea , Cisplatino , Creatinina/sangue , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mediadores da Inflamação/metabolismo , Rim/patologia , Rim/fisiopatologia , Masculino , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores Notch/genética , Receptores Notch/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Estreptozocina
14.
Sci Adv ; 6(1): eaay3566, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911948

RESUMO

In this study, we investigated the roles of Epac1 in pathological angiogenesis and its potential as a novel therapeutic target for the treatment of vasoproliferative diseases. Genetic deletion of Epac1 ameliorated pathological angiogenesis in mouse models of oxygen-induced retinopathy (OIR) and carotid artery ligation. Moreover, genetic deletion or pharmacological inhibition of Epac1 suppressed microvessel sprouting from ex vivo aortic ring explants. Mechanistic studies revealed that Epac1 acted as a previously unidentified inhibitor of the γ-secretase/Notch signaling pathway via interacting with γ-secretase and regulating its intracellular trafficking while enhancing vascular endothelial growth factor signaling to promote pathological angiogenesis. Pharmacological administration of an Epac-specific inhibitor suppressed OIR-induced neovascularization in wild-type mice, recapitulating the phenotype of genetic Epac1 knockout. Our results demonstrate that Epac1 signaling is critical for the progression of pathological angiogenesis but not for physiological angiogenesis and that the newly developed Epac-specific inhibitors are effective in combating proliferative retinopathy.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Neovascularização Patológica/genética , Neovascularização Retiniana/genética , Animais , Movimento Celular/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Neovascularização Patológica/patologia , Receptores Notch/genética , Neovascularização Retiniana/patologia , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genética
15.
Environ Pollut ; 259: 113839, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31918133

RESUMO

Exposure to Aluminum oxide nanoparticles (Al2O3 NPs) has been associated with pulmonary inflammation in recent years; however, the underlying mechanism that causes adverse effects remains unclear. In the present study, we characterized microRNA (miRNA) expression profiling in human bronchial epithelial (HBE) cells exposed to Al2O3 NPs by miRNA microarray. Among the differentially expressed miRNAs, miR-297, a homologous miRNA in Homo sapiens and Mus musculus, was significantly up-regulated following exposure to Al2O3 NPs, compared with that in control. On combined bioinformatic analysis, proteomics analysis, and mRNA microarray, NF-κB-activating protein (NKAP) was found to be a target gene of miR-297 and it was significantly down-regulated in Al2O3 NPs-exposed HBE cells and murine lungs, compared with that in control. Meanwhile, inflammatory cytokines, including IL-1ß and TNF-α, were significantly increased in bronchoalveolar lavage fluid (BALF) from mice exposed to Al2O3 NPs. Then we set up a mouse model with intranasal instillation of antagomiR-297 to further confirm that inhibition of miR-297 expression can rescue pulmonary inflammation via Notch pathway suppression. Collectively, our findings suggested that up-regulation of miR-297 expression was an upstream driver of Notch pathway activation, which might be the underlying mechanism involved in lung inflammation induced by exposure to Al2O3 NPs.


Assuntos
Óxido de Alumínio , Células Epiteliais , Inflamação , Nanopartículas , Regulação para Cima , Óxido de Alumínio/toxicidade , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Inflamação/induzido quimicamente , Pulmão/efeitos dos fármacos , Camundongos , MicroRNAs , Nanopartículas/toxicidade , Pneumonia , Receptores Notch/genética , Ativação Transcricional/efeitos dos fármacos
16.
J Vis Exp ; (155)2020 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-31984955

RESUMO

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to interference with proper development and may even result in initiation or progression of cancers in certain cases. Because this pathway serves complex and versatile functions, it can be studied extensively through many different approaches. Of these, bioinformatics provides an undeniably cost-efficient, approachable, and user-friendly method of study. Bioinformatics is a useful way to extract smaller pieces of information from large-scale datasets. Through the implementation of various bioinformatics approaches, researchers can quickly, reliably, and efficiently interpret these large datasets, yielding insightful applications and scientific discoveries. Here, a protocol is presented for integration of bioinformatics approaches to investigate the role of Notch signaling in ovarian cancer. Furthermore, bioinformatics findings are validated through experimentation.


Assuntos
Biologia Computacional/métodos , Neoplasias Ovarianas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptores Notch/genética , Transdução de Sinais/genética
17.
Dev Growth Differ ; 62(1): 15-34, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31943162

RESUMO

Notch signaling is involved in the development of almost all organ systems and is required post-developmentally to modulate tissue homeostasis. Rare variants in Notch signaling pathway genes are found in patients with rare Mendelian disorders, while unique or recurrent somatic mutations in a similar set of genes are identified in cancer. The human genome contains four genes that encode Notch receptors, NOTCH1-4, all of which are linked to genetic diseases and cancer. Although some mutations have been classified as clear loss- or gain-of-function alleles based on cellular or rodent based assay systems, the functional consequence of many variants/mutations in human Notch receptors remain unknown. In this review, I will first provide an overview of the domain structure of Notch receptors and discuss how each module is known to regulate Notch signaling activity in vivo using the Drosophila Notch receptor as an example. Next, I will introduce some interesting mutant alleles that have been isolated in the fly Notch gene over the past > 100 years of research and discuss how studies of these mutations have facilitated the understanding of Notch biology. By identifying unique alleles of the fly Notch gene through forward genetic screens, mapping their molecular lesions and characterizing their phenotypes in depth, one can begin to unravel new mechanistic insights into how different domains of Notch fine-tune signaling output. Such information can be useful in deciphering the functional consequences of rare variants/mutations in human Notch receptors, which in turn can influence disease management and therapy.


Assuntos
Proteínas de Drosophila , Mutação de Sentido Incorreto , Receptores Notch , Substituição de Aminoácidos , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Domínios Proteicos , Receptores Notch/química , Receptores Notch/genética , Receptores Notch/metabolismo , Relação Estrutura-Atividade
19.
Dev Cell ; 52(3): 294-308.e3, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31978324

RESUMO

The ability of XIST to dosage compensate a trisomic autosome presents unique experimental opportunities and potentially transformative therapeutic prospects. However, it is currently thought that XIST requires the natural context surrounding pluripotency to initiate chromosome silencing. Here, we demonstrate that XIST RNA induced in differentiated neural cells can trigger chromosome-wide silencing of chromosome 21 in Down syndrome patient-derived cells. Use of this tightly controlled system revealed a deficiency in differentiation of trisomic neural stem cells to neurons, correctible by inducing XIST at different stages of neurogenesis. Single-cell transcriptomics and other analyses strongly implicate elevated Notch signaling due to trisomy 21, thereby promoting neural stem cell cycling that delays terminal differentiation. These findings have significance for illuminating the epigenetic plasticity of cells during development, the understanding of how human trisomy 21 effects Down syndrome neurobiology, and the translational potential of XIST, a unique non-coding RNA.


Assuntos
Diferenciação Celular , Síndrome de Down/patologia , Inativação Gênica , Células-Tronco Neurais/patologia , Neurogênese , Neurônios/patologia , RNA Longo não Codificante/metabolismo , Células Cultivadas , Compensação de Dosagem (Genética) , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Inativação do Cromossomo X
20.
Development ; 147(3)2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31988187

RESUMO

The strength of Notch signaling contributes to pleiotropic actions of Notch; however, we do not yet have a full understanding of the molecular regulation of Notch-signaling strength. We have investigated the mode of Notch activation in binary fate specification in the Drosophila spermathecal linage, where Notch is asymmetrically activated across three divisions to specify different cell fates. Using clonal analysis, we show that Delta (Dl) serves as the ligand for Notch in the first and second divisions. Dl and Serrate (Ser) function redundantly in the third division. Compared with the third division, cell-fate decision in the second division requires a lower level of Suppressor of Hairless protein, and, consequently, a lower level of Notch signaling. Several Notch endosomal trafficking regulators differentially regulate Notch signaling between the second and third divisions. Here, we demonstrate that cell differentiation in spermathecae involves different Notch-activation modes, Notch-signaling strengths and Notch-trafficking regulations. Thus, the Drosophila spermathecal lineage is an exciting model for probing the molecular mechanisms that modulate the Notch signaling pathway.


Assuntos
Linhagem da Célula/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Genitália/citologia , Receptores Notch/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Drosophila/genética , Proteínas de Drosophila/genética , Endossomos/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Ligantes , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores Notch/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais
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