RESUMEN
We investigated the effects of tumor necrosis factor-α (TNF-α) exposure on mitogen-activated protein kinase signaling in human microvascular endothelial cells. TNF-α caused a significant suppression of a dual specificity phosphatase, DUSP4, that regulates ERK1/2 activation. Thus, we hypothesized that suppression of DUSP4 enhances cell survival by increasing ERK1/2 signaling in response to growth factor stimulation. In support of this concept, TNF-α pre-exposure increased growth factor-mediated ERK1/2 activation, whereas overexpression of DUSP4 with an adenovirus decreased ERK1/2 compared to an empty adenovirus control. Overexpression of DUSP4 also significantly decreased cell viability, lessened recovery in an in vitro wound healing assay, and decreased DNA synthesis. Pharmacological inhibition of NFκB activation or a dominant negative construct of the inhibitor of κB significantly lessened TNF-α-mediated suppression of DUSP4 expression by 70-84% and attenuated ERK activation, implicating NFκB-dependent pathways in the TNF-α-mediated suppression of DUSP4 that contributes to ERK1/2 signaling. Taken together, our findings show that DUSP4 attenuates ERK signaling and reduces cell viability, suggesting that the novel crosstalk between NFκB and MAPK pathways contributes to cell survival.
Asunto(s)
Fosfatasas de Especificidad Dual/antagonistas & inhibidores , Células Endoteliales/citología , Células Endoteliales/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/antagonistas & inhibidores , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fosfatasas de Especificidad Dual/metabolismo , Células Endoteliales/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Microvasos/citología , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
To establish a homing signal in the lung to recruit circulating stem cells for tissue repair, we formulated a nanoparticle, SDF-1α NP, by complexing SDF-1α with dextran sulfate and chitosan. The data show that SDF-1α was barely released from the nanoparticles over an extended period of time in vitro (3% in 7 days at 37 °C); however, incorporated SDF-1α exhibited full chemotactic activity and receptor activation compared to its free form. The nanoparticles were not endocytosed after incubation with Jurkat cells. When aerosolized into the lungs of rats, SDF-1α NP displayed a greater retention time compared to free SDF-1α (64 vs 2% remaining at 16 h). In a rat model of monocrotaline-induced lung injury, SDF-1α NP, but not free form SDF-1α, was found to reduce pulmonary hypertension. These data suggest that the nanoparticle formulation protected SDF-1α from rapid clearance in the lung and sustained its biological function in vivo.