RESUMO
Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1-RIPK3-pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3ß, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1-RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1-RIPK3-pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.
Assuntos
Hipocampo/enzimologia , Neurônios/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Inibidores de Caspase/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Imidazóis/farmacologia , Indóis/farmacologia , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Necrose , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oligopeptídeos/farmacologia , Estresse Oxidativo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaAssuntos
Imidazóis/química , Indóis/química , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Meia-Vida , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Células Jurkat , Camundongos , Microssomos Hepáticos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was â¼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.
Assuntos
Imidazóis/administração & dosagem , Imidazóis/química , Indóis/administração & dosagem , Indóis/química , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Tratamento Farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Especificidade da EspécieRESUMO
We have reported previously the development of small-molecule phosphatidylinositol-3,4,5-trisphosphate (PIP3) antagonists (PITs) that block pleckstrin homology (PH) domain interaction, including activation of Akt, and show anti-tumor potential. Here we show that the same molecules inhibit growth factor-induced actin remodeling, lamellipodia formation and, ultimately, cell migration and invasion, consistent with an important role of PIP3 in these processes. In vivo, a PIT-1 analog displays significant inhibition on tumor angiogenesis and metastasis. ADP ribosylation factor 6 (ARF6) was recently identified as an important mediator of cytoskeleton and cell motility, which is regulated by PIP3-dependent membrane translocation of the guanine nucleotide exchange factors (GEFs), such as ADP-ribosylation factor nucleotide binding site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1). We demonstrate that PITs inhibit PIP3/ARNO or GRP1 PH domain binding and membrane localization, resulting in the inhibition of ARF6 activation. Importantly, we show that expression of the constitutively active mutant of ARF6 attenuates inhibition of lamellipodia formation and cell migration by PITs, confirming that inhibition of ARF6 contributes to inhibition of these processes by PITs. Overall, our studies demonstrate the feasibility of developing specific small-molecule targeting PIP3 binding by PH domains as potential anticancer agents that can simultaneously interfere with cancer development at multiple points.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Movimento Celular , Neoplasias/patologia , Fator 6 de Ribosilação do ADP , Actinas/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/patologia , Fosfatos de Fosfatidilinositol/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Neoplasias Cutâneas/patologiaRESUMO
Caspases are a family of cysteine proteases homologous to the Caenorhabditis elegans programmed cell death gene product CED-3. Caspases and their distant relatives, meta- and paracaspases, have been found in phylogenetically distant nonmetazoan groups, including plants, fungi and prokaryotes. This review summarizes the current information on the mechanisms and functions of non-mammalian caspases and their relatives in apoptotic and nonapoptotic processes, and explores the possible evolutionary origin of the caspase family.
Assuntos
Apoptose/fisiologia , Caspases , Animais , Proteínas de Caenorhabditis elegans/genética , Caspases/classificação , Caspases/genética , Caspases/metabolismo , Evolução Molecular , HumanosRESUMO
The proapoptotic members of the Bcl-2 family have been proposed to participate in the formation of a channel that releases these apoptogenic factors when mitochondria receive apoptotic signals. A recent study provides the first direct, biophysical measurement of a potentially apoptosis-specific mitochondrial channel, which is regulated by Bcl-2 family members and may play a primary role in the release of the proapoptotic factors.
Assuntos
Apoptose/fisiologia , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Membranas Intracelulares/metabolismo , Modelos BiológicosRESUMO
To study the role of the BH3 domain in mediating pro-apoptotic and anti-apoptotic activities of Bcl-2 family members, we identified a series of novel small molecules (BH3Is) that inhibit the binding of the Bak BH3 peptide to Bcl-xL. NMR analyses revealed that BH3Is target the BH3-binding pocket of Bcl-xL. Inhibitors specifically block the BH3-domain-mediated heterodimerization between Bcl-2 family members in vitro and in vivo and induce apoptosis. Our results indicate that BH3-dependent heterodimerization is the key function of anti-apoptotic Bcl-2 family members and is required for the maintenance of cellular homeostasis.
Assuntos
Apoptose/fisiologia , Proteínas de Membrana/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Western Blotting , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Linhagem Celular , Separação Celular , Grupo dos Citocromos c/metabolismo , Dimerização , Citometria de Fluxo , Polarização de Fluorescência/métodos , Genes Reporter , Humanos , Marcação In Situ das Extremidades Cortadas , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína bcl-XRESUMO
How NGF promotes neuronal survival is unclear. New findings suggest that the mechanism involves induction of an anti-apoptotic protein called ITA.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Aviárias , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neurônios/patologia , Biossíntese de ProteínasRESUMO
The -195- to -500-bp region of the human elastin promoter has been shown to convey high activity in neonatal rat aortic smooth muscle cell and pulmonary fibroblast cell cultures. In addition, this region has been implicated in controlling the differential basal level of elastin transcription in these two cell types. The overall goal of this study was to define the positive element(s) within the -195- to - 500-bp region and to identify the trans-acting factors binding to this sequence. A combination of deletion and linker scan mutational analyses localizes the positive element between -401 and -415 bp. Gel shift analyses demonstrate that the positive element binds NF-1 family members. Co-transfection of a CTF1 expression vector in Drosophila Schneider cells shows the ability of an NF-1 family member to activate elastin promoter activity through this site. Comparative Western and Southwestern blot analyses of nuclear extracts isolated from SMC and lung fibroblasts lay the foundation for possible differential regulation of elastin transcriptional levels via cell specific expression of different NF-1 family members.