Abstract
Ceramide functions as an important second messenger in apoptosis signaling pathways. In this report, we show that treatment of NT-2 neuronal precursor cells with hypoxia/reoxygenation (H/R) resulted in ceramide up-regulation. This elevation in ceramide was primarily due to the actions of acid sphingomyelinase and ceramide synthase LASS 5, demonstrating the action of the salvage pathway. Hypoxia/reoxygenation treatment led to Bax translocation from the cytoplasm to mitochondria and cytochrome c release from mitochondria. Down-regulation of either acid sphingomyelinase or LASS 5-attenuated ceramide accumulation and H/R-induced Bax translocation to mitochondria. Overall, we have demonstrated that ceramide up-regulation following H/R is pertinent to Bax activation to promote cell death.
Highlights
The pro-apoptotic protein Bax is a member of the Bcl-2 family that plays an important role in apoptosis regulation [1]
We have demonstrated that ceramide upregulation following H/R is pertinent to Bax activation to promote cell death
When these cells were subjected to H/R treatment, there was a shift in the fluorescence pattern of GFP-Bax from a diffuse cytoplasmic state to a punctate membrane-bound state
Summary
Materials—NT-2 human neuronal precursor cell line was obtained from ATCC. Fetal bovine serum, Dulbecco’s modified Eagle’s medium, tissue culture supplements, SuperScript firststrand synthesis system, caspase-3 assay kit, and the Quant-iT RiboGreen RNA assay kit were from Invitrogen. To develop stable clones of NT-2 cells expressing GFP-Bax, NT-2 cells (in a 10-cm plate) were transfected with 12 g of C3-EGFP-Bax [10] with the FuGENE transfection reagent. GFP-Bax-stable NT-2 cells were plated onto either 6-well (for Bax localization analysis) or 10-cm (for activity assays or lipid analyses) plates. Quantitative Real-time PCR Analysis—RNA was extracted from NT-2 cells using the Qiagen RNeasy kit according to the manufacturer’s protocol. The cells were collected and subjected to in vitro acid and neutral sphingomyelinase enzymatic assays using [14C]sphingomyelin according to the previously described protocol [40, 45]. Caspase-3 Activity Assay—At various time points post hypoxia, cells were harvested, and caspase-3 activity was determined using the EnzChek Caspase-3 assay kit 2 (Molecular Probes) according to the manufacturer’s instructions. A p value of 0.05 or less is considered as statistically significant and marked with an asterisk
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