RESUMO
Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.
Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Mutação , Receptores Purinérgicos P2Y2/metabolismo , Trifosfato de Adenosina/genética , Adulto , Terapia Antirretroviral de Alta Atividade/métodos , Membrana Celular/genética , Conexinas/genética , Conexinas/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Purinérgicos P2Y2/genética , Transdução de Sinais , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies.
Assuntos
Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Cádmio/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Etoposídeo/farmacologia , Citometria de Fluxo , Imunofluorescência , Camundongos , Mitomicina/farmacologia , Mitoxantrona/farmacologia , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Tapsigargina/farmacologiaRESUMO
Fusogenic HIV-1 isolates induce the fusion of infected and bystander cells. Such syncytia can be found as "multinucleated giant cells" in the brain from HIV-1-infected individuals, as well as in lymphoid tissues. Syncytia elicited by the HIV-1 envelope glycoprotein (Env) manifest the aggregation of PML in discrete nuclear bodies and the recruitment of TopBP1, NBS1 and ATM to DNA damage foci containing phosphorylated ATM and histone H2AX ("-H2AX). This DNA damage response then culminates in p53-dependent activation of the mitochondrial pathway of apoptosis. Here, we show that Env-elicited syncytia also manifest activating phosphorylations of the checkpoint kinases 1 and 2 (Chk1 and Chk2), and both Chk1 and Chk2 colocalize with "-H2AX foci. However, only the siRNA-mediated knockdown of Chk2, not the depletion of Chk1, inhibits mitochondrial outer membrane permeabilization and subsequent syncytial apoptosis. Depletion of PML, TopBP1, NBS1 or ATM inhibit the activating phosphorylation of Chk2. Altogether, these results indicate that Chk2 (but not Chk1) participates in the DNA damage-elicited pro-apoptotic cascade that leads to the demise of Env-elicited syncytia.
Assuntos
Apoptose/fisiologia , Produtos do Gene env/metabolismo , Células Gigantes/fisiologia , HIV-1/ultraestrutura , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Produtos do Gene env/genética , Células Gigantes/citologia , HIV-1/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.