RESUMEN
Doxorubicin is the anthracycline with the widest spectrum of antitumor activity, and it has been shown that the antitumor activity is mediated in vivo by selective triggering of apoptosis in proliferating endothelial cells. We studied cultured human endothelial cells and observed that doxorubicin-induced apoptosis was mediated by p38 mitogen-activated protein kinase (MAPK). Doxorubicin-provoked apoptosis was significantly inhibited by expression of dominant negative p38 MAPK or pharmacological inhibition with SB203580. Furthermore, blocking phosphatidylinositol-3-kinase/Akt signaling significantly increased doxorubicin-induced caspase-3 activity and cell death, indicating that Akt is a survival factor in this system. Notably, we also found that doxorubicin-provoked apoptosis included p38 MAPK-mediated inhibition of Akt and Bad phosphorylation. Furthermore, doxorubicin-stimulated phosphorylation of Bad in cells expressing dominant negative p38 MAPK was impeded by the inhibition of PI3-K. In addition to the impact on Bad phosphorylation, doxorubicin-treatment caused p38 MAPK-dependent downregulation of Bcl-xL protein.
Asunto(s)
Apoptosis/efectos de los fármacos , Regulación hacia Abajo , Doxorrubicina/farmacología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína bcl-X/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
We recently reported that p38 MAPK regulates TNF-induced endothelial apoptosis via phosphorylation and downregulation of Bcl-xL. Here, we describe that such apoptosis includes p38 MAPK-mediated, protein phosphatase 2A (PP2A)-dependent, downregulation of the MEK-ERK pathway. Inhibition of PP2A with fostriecin or calyculin A significantly increased MEK phosphorylation, as did exposure to the p38 MAPK inhibitor SB203580. Inhibition of MEK potentiated TNF-induced caspase-3 activity and cell death, and both those events were suppressed by treatment with fostriecin or calyculin A. Immunoprecipitation experiments revealed an association between p38 MAPK, PP2A and MEK, and the results of a phosphatase assay suggested that PP2A is a downstream target of p38 MAPK. Importantly, phosphorylation of Bad at Ser-112 was found to be regulated by p38 MAPK and PP2A. In summary, the present findings indicate a novel p38 MAPK-mediated apoptosis pathway, involving activation of Bad via PP2A-dependent inhibition of the MEK-ERK pathway.
Asunto(s)
MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteína Letal Asociada a bcl/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Alquenos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Humanos , Imidazoles/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Toxinas Marinas , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Polienos , Proteína Fosfatasa 2 , Piridinas/farmacología , Pironas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína Letal Asociada a bcl/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.
Asunto(s)
Acetilcisteína/análogos & derivados , Apoptosis/fisiología , Células Endoteliales/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Proteína bcl-X , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Neutrophil apoptosis is essential for resolution of inflammatory reactions. Here, we studied the role of two apoptosis/survival-associated protein kinases in this process. We discovered a previously undetected early and transient inhibition of the activity of p38 mitogen-activated protein kinase (p38 MAPK) during both spontaneous and Fas-induced apoptosis. Pharmacological inhibition of this enzyme augmented the activation of caspases and the apoptotic response, which suggests that the p38 MAPK signals survival in neutrophils. Our finding that caspase-3 activity was initiated during the transient inhibition of p38 MAPK suggests that apoptosis is initiated during this inhibition. Furthermore, such transient inhibition was counteracted by granulocyte-macrophage colony-stimulating factor, which elicits survival. We also found that neither this inhibition of p38 MAPK nor the spontaneous apoptotic response depended on Fas. Instead, the early inhibition of p38 MAPK concurred with a Fas-induced activation of phosphatidylinositol 3-kinase, inhibition of which reduced apoptosis. Thus, the Fas-induced augmentation of spontaneous apoptosis can be explained by its activation of phosphatidylinositol 3-kinase. We conclude that p38 MAPK activity represents a survival signal that is inactivated transiently during both spontaneous and Fas-induced apoptosis, whereas Fas-induced phosphatidylinositol 3-kinase activity is a proapoptotic signal in isolated human neutrophils.