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1.
Cell Mol Life Sci ; 76(15): 3005-3018, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31006037

RESUMO

The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , RNA Longo não Codificante/metabolismo , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Animais , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Camundongos , Camundongos Transgênicos , Neuroglia/citologia , Neuroglia/metabolismo , Fragmentos de Peptídeos/farmacologia , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo
2.
Int J Mol Sci ; 18(4)2017 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-28338624

RESUMO

Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.


Assuntos
Caveolina 1/metabolismo , Núcleo Celular/metabolismo , Microdomínios da Membrana/metabolismo , Fator de Crescimento Neural/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Proteína de Ligação a CREB/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Células PC12 , Fosforilação/efeitos dos fármacos , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/química , Receptor trkA/imunologia , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/imunologia , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo
3.
Virology ; 492: 66-72, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26901486

RESUMO

BK polyomavirus (BKPyV) is a human pathogen that causes polyomavirus-associated nephropathy and hemorrhagic cystitis in transplant patients. Gangliosides and caveolin proteins have previously been reported to be required for BKPyV infection in animal cell models. Recent studies from our lab and others, however, have indicated that the identity of the cells used for infection studies can greatly influence the behavior of the virus. We therefore wished to re-examine BKPyV entry in a physiologically relevant primary cell culture model, human renal proximal tubule epithelial cells. Using siRNA knockdowns, we interfered with expression of UDP-glucose ceramide glucosyltransferase (UGCG), and the endocytic vesicle coat proteins caveolin 1, caveolin 2, and clathrin heavy chain. The results demonstrate that while BKPyV does require gangliosides for efficient infection, it can enter its natural host cells via a caveolin- and clathrin-independent pathway. The results emphasize the importance of studying viruses in a relevant cell culture model.


Assuntos
Vírus BK/efeitos dos fármacos , Caveolina 1/genética , Caveolina 2/genética , Cadeias Pesadas de Clatrina/genética , Células Epiteliais/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Vírus BK/genética , Vírus BK/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/metabolismo , Caveolina 2/antagonistas & inibidores , Caveolina 2/metabolismo , Linhagem Celular , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/metabolismo , Células Epiteliais/virologia , Gangliosídeo G(M1)/farmacologia , Gangliosídeos/farmacologia , Regulação da Expressão Gênica , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/virologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Internalização do Vírus/efeitos dos fármacos
4.
J Mol Cell Cardiol ; 72: 85-94, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24594319

RESUMO

Tumor necrosis factor-α (TNF-α) is an established pro-atherosclerotic factor, but the mechanism is not completely understood. We explored whether TNF-α could promote atherosclerosis by increasing the transcytosis of lipoproteins (e.g., LDL) across endothelial cells and how NF-κB and PPAR-γ were involved in this process. TNF-α significantly increased the transcytosis of LDL across human umbilical vein endothelial cells (HUVECs) and stimulated an increase of subendothelial retention of LDL in vascular walls. These effects of TNF-α were substantially blocked not only by transcytosis inhibitors, but also by NF-κB inhibitors and PPAR-γ inhibitors. In ApoE(-/-) mice, both NF-κB and PPAR-γ inhibitors alleviated the early atherosclerotic changes promoted by TNF-α. NF-κB and PPAR-γ inhibitors down-regulated the transcriptional activities of NF-κB and PPAR-γ induced by TNF-α. Furthermore, cross-binding activity assay revealed that NF-κB and PPAR-γ could form an active transcription factor complex containing both the NF-κB P65 subunit and PPAR-γ. The increased expressions of LDL transcytosis-related proteins (LDL receptor and caveolin-1, -2) stimulated by TNF-α were also blocked by both NF-κB inhibitors and PPAR-γ inhibitors. TNF-α promotes atherosclerosis by increasing the LDL transcytosis across endothelial cells and thereby facilitating LDL retention in vascular walls. In this process, NF-κB and PPAR-γ are activated coordinately to up-regulate the expression of transcytosis-related proteins. These observations suggest that inhibitors of either NF-κB or PPAR-γ can be used to target atherosclerosis.


Assuntos
Aterosclerose/genética , Lipoproteínas LDL/metabolismo , NF-kappa B/metabolismo , PPAR gama/metabolismo , Transcitose/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Anilidas/farmacologia , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Benzamidas/farmacologia , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Alcaloides de Cinchona/farmacologia , Filipina/farmacologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipoproteínas LDL/antagonistas & inibidores , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Nitrilos/farmacologia , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Prolina/análogos & derivados , Prolina/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de LDL/antagonistas & inibidores , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais , Sulfonas/farmacologia , Tiocarbamatos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
5.
Nat Commun ; 4: 2540, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24096474

RESUMO

Ca(2+) influx via voltage-dependent CaV1/CaV2 channels couples electrical signals to biological responses in excitable cells. CaV1/CaV2 channel blockers have broad biotechnological and therapeutic applications. Here we report a general method for developing novel genetically encoded calcium channel blockers inspired by Rem, a small G-protein that constitutively inhibits CaV1/CaV2 channels. We show that diverse cytosolic proteins (CaVß, 14-3-3, calmodulin and CaMKII) that bind pore-forming α1-subunits can be converted into calcium channel blockers with tunable selectivity, kinetics and potency, simply by anchoring them to the plasma membrane. We term this method 'channel inactivation induced by membrane-tethering of an associated protein' (ChIMP). ChIMP is potentially extendable to small-molecule drug discovery, as engineering FK506-binding protein into intracellular sites within CaV1.2-α1C permits heterodimerization-initiated channel inhibition with rapamycin. The results reveal a universal method for developing novel calcium channel blockers that may be extended to develop probes for a broad cohort of unrelated ion channels.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Caveolina 1/antagonistas & inibidores , Caveolina 2/antagonistas & inibidores , Proteínas de Ligação a Tacrolimo/genética , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Potenciais de Ação , Animais , Bloqueadores dos Canais de Cálcio/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Caveolina 1/química , Caveolina 1/metabolismo , Caveolina 2/química , Caveolina 2/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Descoberta de Drogas , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Camundongos , Mimetismo Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , Sirolimo/farmacologia , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo
6.
Biochim Biophys Acta ; 1833(10): 2176-89, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23665048

RESUMO

The role of caveolin-2 (cav-2), independently of caveolin-1 (cav-1) and caveolae, has remained elusive. Our data show that cav-2 exists in the plasma membrane (PM) in cells lacking cav-1 and forms homo-oligomeric complexes. Cav-2 did not interact with cavin-1 and cavin-2 in the PM. Rab6-GTP was required for the microtubule-dependent exocytic transport of cav-2 from the Golgi to the PM independently of cav-1. The cav-2-oligomerized noncaveolar microdomain was unaffected by cholesterol depletion and protected from shearing of silica-coated PM. Activation of insulin receptor (IR) was processed in the microdomain. Actin depolymerization affected the formation and sustenance of cav-2-oligomerized noncaveolar microdomain and attenuated IR recruitment to the microdomain thereby inhibiting IR signaling activation. Cav-2 shRNA stable cells and the cells ectopically expressing an oligomerization domain truncation mutant, cav-2∆47-86 exhibited retardation of IR signaling activation via the noncaveolar microdomain. Elevation in status of cav-2 expression rendered the noncaveolar activation of IR signaling in cav-1 down-regulated or/and cholesterol-depleted cells. Our findings reveal a novel homo-oligomeric cav-2 microdomain responsible for regulating activation of IR signaling in the PM.


Assuntos
Citoesqueleto de Actina/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Insulina/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Transporte Biológico , Western Blotting , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Células Cultivadas , Fibroblastos/citologia , Guanosina Trifosfato/metabolismo , Imunoprecipitação , Insulina/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Frações Subcelulares
7.
FEBS Lett ; 586(19): 3317-23, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22819829

RESUMO

Here we show that tyrosine phosphorylation of caveolin-2 (Cav-2) negatively regulates the anti-proliferative function of transforming growth factor beta (TGF-beta) in endothelial cells. In contrast to wild-type-Cav-2, retroviral re-expression of Y19/27F-Cav-2 in Cav-2 knockout endothelial cells did not affect anti-proliferative effect of TGF-beta compared to empty vector. Conversely, although less effective than wild-type, re-expression of S23/36A-Cav-2 reduced the effect of TGF-beta compared to empty vector. This differential effect of tyrosine and serine phosphorylation mutants of Cav-2 correlated with TGF-beta-induced Smad3 phosphorylation and transcriptional activation of plasminogen activator inhibitor-1. Thus tyrosine-phosphorylated Cav-2 counteracts anti-proliferative effect of TGF-beta in endothelial cells.


Assuntos
Caveolina 2/química , Caveolina 2/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Substituição de Aminoácidos , Animais , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serpina E2/genética , Proteína Smad3/metabolismo , Ativação Transcricional/efeitos dos fármacos , Tirosina/química
8.
Int J Oncol ; 38(5): 1395-402, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21373752

RESUMO

We investigated whether altering caveolin-2 (cav-2) expression affects the proliferation of cancer cells. Cav-2 was not detected in HepG2, SH-SY5Y and LN-CaP cells, and the loss of cav-2 expression was not restored by 5-aza-2'-deoxycytidine treatment. In contrast, C6, HeLa, A549, MCF7 and PC3M cells expressed cav-2. Effects of re-expression of exogenous cav-2 in HepG2, SH-SY5Y and LN-CaP cells, and siRNA-mediated down-regulation of endogenous cav-2 in C6, HeLa, A549, MCF7 and PC3M cells on cancer proliferation were examined by MTT assay, colony formation assay and flow cytometric analysis. Cav-2 transfection in HepG2 hepatocellular carcinoma cells and knockdown in C6 glioma cells caused reduction in cell proliferation and growth with retarded entry into the S phase. Cav-2 re-expression in SH-SY5Y neuroblastoma cells and depletion in HeLa epithelial cervical cancer and A549 lung adenocarcinoma cells promoted cancer cell proliferation. Luciferase reporter assay showed that transcriptional activation of Elk-1 and STAT3 was significantly decreased in cav-2-transfected HepG2 hepatocellular carcinoma and down-regulated C6 glioma cells. Our data suggest that cav-2 acts as a modulator of cancer progression.


Assuntos
Caveolina 2/fisiologia , Proliferação de Células , Neoplasias/patologia , Animais , Caveolina 2/antagonistas & inibidores , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glioma/patologia , Células Hep G2 , Humanos , Neuroblastoma/patologia , Ratos , Fator de Transcrição STAT3/fisiologia , Proteínas Elk-1 do Domínio ets/fisiologia
9.
Biochim Biophys Acta ; 1793(7): 1325-33, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19427337

RESUMO

The regulatory function of caveolin-2 in signal transducer and activator of transcription 3 (STAT3) signaling by insulin was investigated. Insulin-induced increase in phosphorylation of STAT3 was reduced by caveolin-2 siRNA. Mutagenesis studies identified that phosphorylation of tyrosines 19 and 27 on caveolin-2 is required for the STAT3 activation. Caveolin-2 Y27A mutation decreased insulin-induced phosphorylation of STAT3 interacting with caveolin-2. pY27-Caveolin-2 was required for nuclear translocation of pY705-STAT3 in response to insulin. In contrast, caveolin-2 Y19A mutation influenced neither the phosphorylation of STAT3 nor nuclear translocation of pY705-STAT3. pY19-Caveolin-2, however, was essential for insulin-induced DNA binding of pS727-STAT3 and STAT3-targeted gene induction in the nucleus. Finally, insulin-induced transcriptional activation of STAT3 depended on phosphorylation of both 19 and 27 tyrosines. Together, our data reveal that phosphotyrosine-caveolin-2 is a novel regulator for transcriptional activation of STAT3 in response to insulin.


Assuntos
Caveolina 2/metabolismo , Fibroblastos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fator de Transcrição STAT3/genética , Ativação Transcricional/efeitos dos fármacos , Animais , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Immunoblotting , Imunoprecipitação , Luciferases , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Tirosina/metabolismo
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