Down-regulation of Txnip reduces ceramide-induced p38 MAPK and JNK phosphorylation and cell apoptosis. (A) After treatment with 20 μM C2-ceramide for 24 hours, vector control– and hTxnip siRNA-transfected Jurkat T cells were measured using PE-conjugated annexin V staining (red), followed by fluorescence microscopic observation. Hoechst 33342 (blue) was used for nuclear staining. The scale bar equals 40 μm. (B) Using flow cytometric analysis, the percentages of apoptotic cells (annexin V+) are shown (***P < .001 as compared with the vector control group). (C) After C2-ceramide treatment for 6 hours, the status of pp38 MAPK and pJNK in vector- and hTxnip siRNA-transfected Jurkat T cells were measured by flow cytometric analysis using pp38 MAPK and pJNK antibodies followed by rhodamine (red)–conjugated secondary antibody staining. The percentages of positive cells are shown (means ± SD of triplicate cultures; **P < .01 as compared with the vector control group). (D) A model for ceramide and etoposide-induced transcriptional apoptotic signaling which involves Txnip-regulated ASK1, p38 MAPK, and JNK activation. By microarray analysis, we show a novel role of ceramide on Txnip expression. Ceramide–up-regulated Txnip binds to thioredoxin and decreases the thioredoxin activity, which causes dissociation of thioredoxin from ASK1. ASK1 is activated via Akt-mediated dephosphorylation at serine 83 and phosphorylation at threonine 845 with unknown mechanism. The requirement of Txnip in ceramide-activated p38 MAPK and JNK was demonstrated using Txnip siRNA. Furthermore, Txnip-regulated p38 MAPK and JNK activation are, at least in part, involved in ceramide-induced cell apoptosis.
