Abstract
The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) deprivation was shown to be accompanied by down-regulation of CREB activity (). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells.
Highlights
From the Departments of §Biology and ‡Human and General Physiology and the ¶Luigi Galvani Interdepartmental Center, University of Bologna, 40126 Bologna, Italy
We provide evidence that NO protects SK-N-BE neuroblastoma cells from serum deprivation-induced apoptosis by activating the transcription factor CREB
Using an array of different methods, we show here that the overexpression of nNOS protects neuroblastoma cells from serum withdrawal-induced apoptosis
Summary
Cell Culture—The human neuroblastoma SK-N-BE cells [22] were seeded at a density of 105 cells/cm in plastic culture plates and grown to confluency in RPMI 1640 medium (Invitrogen) containing 10% heatinactivated fetal calf serum (Invitrogen), 2 mM glutamine, 100 units/ml penicillin, and 50 g/ml streptomycin (Sigma) at 37 °C in a 5% CO2containing humidified atmosphere. Luciferase Measurement—After serum withdrawal, when the plasmids with a luciferase reporter were used, cells were washed twice with ice-cold phosphate-buffered saline and lysed by incubation in 50 mM Tris-MES (pH 7.8), 1 mM dithiothreitol, and 1% Triton X-100 for 5 min on ice. The lysate was cleared of cellular debris by centrifugation [24]. Cells were washed twice with PBS and fixed for 10 min with ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Cells were stained with 2 g/ml Hoechst dye 33342 for 10 min at room temperature, observed, and photographed with the fluorescence microscope. Visualization of Apoptotic Cells through TUNEL—After fixation in paraformaldehyde, cells were washed with PBS and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate, washed again with PBS, and incubated for 60 min at 37 °C in the dark with the in situ cell death fluorescence detection kit (Roche Molecular Biochemicals). Differences were considered significant starting from p Ͻ 0.01 value
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