RESUMEN
Bleomycin, a chemotherapeutic agent, can cause pulmonary fibrosis in humans and is commonly used to induce experimental pulmonary fibrosis in rodents. In cell culture, bleomycin causes single- and double-stranded DNA breaks and produces reactive oxidative species, both of which require iron (Fe(2+)) and O(2). The mechanism of bleomycin-induced apoptosis is controversial due to its complexity. We investigated bleomycin apoptotic signaling events in primary pulmonary endothelial cells. Time course experiments revealed that bleomycin induced apoptosis within 4 h. Caspase-8, the initiator caspase for the extrinsic pathway, was activated within 2 h and preceded activation of the effector caspases-3 and -6 (4 h). Caspase-9, the initiator of the intrinsic pathway and release of cytochrome c from the mitochondria were not detected at these time points. Bleomycin induced the expression of Bcl-2 and Bcl-x(L), Bcl-2 family member proteins that protect cells from the mitochondria-dependent intrinsic apoptosis. Real-time quantitative RT-PCR results demonstrated that, at 4-8 h, bleomycin induced expression of TNF and TNF receptor family genes known to induce the extrinsic apoptotic pathway. Silencing of the death receptor adaptor protein Fas-associated death domain by short interfering RNA significantly reduced bleomycin-induced apoptosis. Apoptosis was also abrogated by caspase-8 inhibition, but only slightly reduced by caspase-3 inhibition. Together, these data suggest that bleomycin initiates apoptosis via the extrinsic pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Bleomicina/toxicidad , Pulmón/efectos de los fármacos , Pulmón/patología , Animales , Apoptosis/genética , Secuencia de Bases , Inhibidores de Caspasas , Caspasas/genética , Caspasas/metabolismo , Bovinos , Células Cultivadas , Cartilla de ADN/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Proteína de Dominio de Muerte Asociada a Fas/antagonistas & inhibidores , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Pulmón/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Hepatocyte growth factor (HGF) is upregulated in response to lung injury and has been implicated in tissue repair through its antiapoptotic and proliferative activities. Cyclooxygenase-2 (COX-2) is an inducible enzyme in the biosynthetic pathway of prostaglandins, and its activation has been shown to play a role in cell growth. Here, we report that HGF induces gene transcription of COX-2 in human bronchial epithelial cells (HBEpC). Treatment of HBEpC with HGF resulted in phosphorylation of the HGF receptor (c-Met), activation of Akt, and upregulation of COX-2 mRNA. Adenovirus-mediated gene transfer of a dominant negative (DN) Akt mutant revealed that HGF increased COX-2 mRNA in an Akt-dependent manner. COX-2 promoter analysis in luciferase reporter constructs showed that HGF regulation required the beta-catenin-responsive T cell factor-4 binding element (TBE). The HGF activation of the COX-2 gene transcription was blocked by DN mutant of beta-catenin or by inhibitors that blocked activation of Akt. Inhibition of p42/p44 MAPK pathway blocked HGF-mediated activation of beta-catenin gene transcription but not Akt activation, suggesting that p42/p44 MAPK acts in a parallel mechanism for beta-catenin activation. We also found that inhibition of COX-2 with NS-398 blocked HGF-induced growth in HBEpC. Together, the results show that the HGF increases COX-2 gene expression via an Akt-, MAPK-, and beta-catenin-dependent pathway in HBEpC.