RESUMO
During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Lesões Encefálicas/metabolismo , Linhagem Celular , Dano ao DNA , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteólise , Transdução de Sinais , UbiquitinaçãoRESUMO
Our preliminary screening has shown that curcumin derivative BDMC33 [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] exerted promising nitric oxide inhibitory activity in activated macrophages. However, the molecular basis and mechanism for its pharmacological action is yet to be elucidated. The aim of this study was to investigate the anti-inflammatory properties of BDMC33 and elucidate its underlying mechanism action in macrophage cells. Our current study demonstrated that BDMC33 inhibits the secretion of major pro-inflammatory mediators in stimulated macrophages, and includes NO, TNF-α and IL-1ß through interference in both nuclear factor kappaB (NF-κB) and mitogen activator protein kinase (MAPK) signaling cascade in IFN-γ/LPS-stimulated macrophages. Moreover, BDMC33 also interrupted LPS signaling through inhibiting the surface expression of CD-14 accessory molecules. In addition, the inhibitory action of BDMC33 not only restricted the macrophages cell (RAW264.7), but also inhibited the secretion of NO and TNF-α in IFN-γ/LPS-challenged microglial cells (BV-2). The experimental data suggests the inflammatory action of BDMC33 on activated macrophage-like cellular systems, which could be used as a future therapeutic agent in the management of chronic inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Compostos de Benzilideno/farmacologia , Curcumina/análogos & derivados , Cicloexanonas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , NF-kappa B/antagonistas & inibidores , Animais , Linhagem Celular , Curcumina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Receptores de Lipopolissacarídeos/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
The increasing prevalence of neurodegenerative diseases has prompted investigation into innovative therapeutics over the last two decades. Non-steroidal anti-inflammatory drugs (NSAIDs) are among the therapeutic choices to control and suppress the symptoms of neurodegenerative diseases. However, NSAIDs-associated gastropathy has hampered their long term usage despite their clinical advancement. On the natural end of the treatment spectrum, our group has shown that cardamonin (2',4'-dihydroxy-6'-methoxychalcone) isolated from Alpinia rafflesiana exerts potential anti-inflammatory activity in activated macrophages. Therefore, we further explored the anti-inflammatory property of cardamonin as well as its underlying mechanism of action in IFN-γ/LPS-stimulated microglial cells. In this investigation, cardamonin shows promising anti-inflammatory activity in microglial cell line BV2 by inhibiting the secretion of pro-inflammatory mediators including nitric oxide (NO), prostaglandin E(2) (PGE(2)), tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). The inhibition of NO and PGE(2) by cardamonin are resulted from the reduced expression of inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2), respectively. Meanwhile the suppressive effects of cardamonin on TNF-α, IL-1ß and IL-6 were demonstrated at both protein and mRNA levels, thus indicating the interference of upstream signal transduction pathway. Our results also validate that cardamonin interrupts nuclear factor-kappa B (NF-κB) signalling pathway via attenuation of NF-κB DNA binding activity. Interestingly, cardamonin also showed a consistent suppressive effect on the cell surface expression of CD14. Taken together, our experimental data provide mechanistic insights for the anti-inflammatory actions of cardamonin in BV2 and thus suggest a possible therapeutic application of cardamonin for targeting neuroinflammatory disorders.