RESUMO
Inflammation is often accompanied by hypoxia because of the high oxygen consumption of invading bacteria and immune cells. During resolution of inflammation, the formation of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), which is produced by macrophages, needs to be terminated. We show in RAW264.7 macrophages that TNF-alpha mRNA as well as intracellular and secreted TNF-alpha protein levels are reduced after prolonged incubations with lipopolysaccharide (LPS) under hypoxic conditions. The decrease in TNF-alpha was mediated by destabilization of TNF-alpha mRNA via a 3'-untranslated region-dependent mechanism. Specifically, the RNA-binding protein tristetraprolin (TTP) increased at mRNA and protein levels after 16-hour incubations with LPS under hypoxia. Interestingly, TTP accumulated in a dephosphorylated and active form, and this accumulation was attributable to reduced p38 mitogen-activated protein kinase activity under these conditions. Knockdown of TTP by small interfering RNA abolished destabilization of TNF-alpha mRNA. Prolonged incubations with LPS under hypoxia also reduced mRNA amounts and stability of other proinflammatory mediators such as macrophage inflammatory protein-2, interleukin-6, and granulocyte macrophage colony-stimulating factor. Therefore, we propose that hypoxia plays a key role during resolution of inflammation by activating posttranscriptional, TTP-dependent regulatory mechanisms.
Assuntos
Hipóxia/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Estabilidade de RNA/fisiologia , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regiões 3' não Traduzidas , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Hipóxia/genética , Imuno-Histoquímica , Inflamação/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Tristetraprolina/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Hypoxia and signaling via hypoxia-inducible factor-1 (HIF-1) is a key feature of solid tumors and is related to tumor progression as well as treatment failure. Although it is generally accepted that HIF-1 provokes tumor cell survival and induces chemoresistance under hypoxia, HIF-1-independent mechanisms operate as well. We present evidence that conditioned medium obtained from A549 cells, incubated for 24 h under hypoxia, protected naive A549 cells from etoposide-induced cell death. Lipid extracts generated from hypoxia-conditioned medium still rescued cells from apoptosis induced by etoposide. Specifically, the bioactive lipid sphingosine-1-phosphate (S1P) not only was essential for cell viability of A549 cells but also protected cells from apoptosis. We noticed an increase in sphingosine kinase 2 (SphK2) protein level and enzymatic activity under hypoxia, which correlated with the release of S1P into the medium. Knockdown of SphK2 using specific small interfering RNA relieved chemoresistance of A549 cells under hypoxia and conditioned medium obtained from SphK2 knockdown cells was only partially protective. Coincubations of conditioned medium with VPC23019, a S1P(1)/S1P(3) antagonist, reduced protection of conditioned medium, with the further notion that p42/44 mitogen-activated protein kinase transmits autocrine or paracrine survival signaling downstream of S1P(1)/S1P(3) receptors. Our data suggest that hypoxia activates SphK2 to promote the synthesis and release of S1P, which in turn binds to S1P(1)/S1P(3) receptors, thus activating p42/44 mitogen-activated protein kinase to convey autocrine or paracrine protection of A549 cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Morte Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Meios de Cultura , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Lisofosfolipídeos/biossíntese , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Interferente Pequeno/genética , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/biossíntese , Esfingosina/metabolismo , TransfecçãoRESUMO
Hypoxia inducible factor 1 is regulated by the appearance of the HIF-1alpha subunit. HIF-1alpha is subjected to proteasomal destruction or enhanced protein translation, which requires the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. We investigated how PI3K/Akt and glycogen synthase kinase 3beta (GSK3beta) affect HIF-1alpha in human RKO cells under prolonged periods of severe hypoxia/anoxia. 16- to 32-h lasting incubations attenuated Akt activity and decreased HIF-1alpha protein. This was reproduced by blocking PI3K with LY294002. GSK3beta inhibition by indirubins circumvented the effect of hypoxia/anoxia or LY294002 on HIF-1alpha. Ruling stability regulation of HIF-1alpha protein and/or enhanced transcription of HIF-1alpha mRNA via GSK3beta inhibition out is suggestive for translational modulation of HIF-1alpha under the influence of GSK3beta.