c-Jun NH2-terminal kinase activation is essential for up-regulation of LC3 during ceramide-induced autophagy in human nasopharyngeal carcinoma cells

Abstract

Background: Autophagy is a dynamic catabolic process characterized by the formation of double membrane vacuoles termed autophagosomes. LC3, a homologue of yeast Atg8, takes part in autophagosome formation, but the exact regulation mechanism of LC3 still needs to be elucidated.

Methods: Ceramide-induced autophagy was determined by detecting LC3 expression with Western blotting and confocal microscopy in human nasopharyngeal carcinoma cell lines CNE2 and SUNE1. The activation of JNK pathway was assessed by Western blotting for phospho-specific forms of JNK and c-Jun. The JNK activity specific inhibitor, SP600125, and siRNA directed against JNK were used to block JNK/c-Jun pathway. ChIP and luciferase reporter analysis were applied to determine whether c-Jun was involved in the regulation of LC3 transcription.

Results: Ceramide-treated cells exhibited the characteristics of autophagy and JNK pathway activation. Inhibition of JNK pathway could block the ceramide-induced autophagy and the up-regulation of LC3 expression. Transcription factor c-Jun was involved in LC3 transcription regulation in response to ceramide treatment.

Conclusions: Ceramide could induce autophagy in human nasopharyngeal carcinoma cells, and activation of JNK pathway was involved in ceramide-induced autophagy and LC3 expression.

Figure 1

Autophagy induced by ceramide in CNE2 and SUNE1 cells. (A) YFP-LC3 expression and localization in CNE2 and SUNE1 cells treated with DMSO (control) or 20 μM ceramide for 12 h. Representative immunofluorescence pictures are shown at the original magnification ×1000. (B) Ceramide dose and time-dependently induced the formation of LC3-II, a marker for autophagy. SUNE1 cells were treated with ceramide in the indicated concentrations for 24 h or treated with 20 μM ceramide for the indicated times. Lysates were analyzed by immunoblotting with LC3 antibody.

Figure 2

The effect of ceramide on JNK and c-Jun phosphorylation and up-regulation of LC3 expression. SUNE1 or CNE2 cells were treated with various concentrations of ceramide for 24 h or with 20 μM ceramide for the indicated periods. (A)The expression levels of JNK, phospho-JNK, c-Jun and phospho-c-Jun protein were analyzed with immunoblotting. (B)The expression of LC3 mRNA was detected by RT-PCR analysis.

Figure 3

Specific inhibitor SP600125 or siRNA directed JNK blocked ceramide-induced autophagy and up-regulation of LC3 expression. (A) SUNE1 cells were treated with 20 μM ceramide for 24 h in the absence or presence of SP600125 or JNK1/2 siRNA. Lysates were analyzed by immunoblotting. (B) Autophagosome formation was visualized using YFP-LC3 expressing and observed under a confocal microscope. Representative immunofluorescence pictures are shown at the original magnification × 1000. (C) The expression of LC3 mRNA was examined by RT-PCR analysis.

Figure 4

c-Jun was directly involved in LC3 transcription in response to ceramide treatment. (A) CNE2 cells were treated with 20 μM ceramide for 24 h in the absence or presence of c-Jun siRNA. Then LC3, c-Jun or phospho-c-Jun protein expression were analyzed with immunoblotting. LC3 mRNA was examined by RT-PCR analysis. Pepstatin A is an inhibitor of acid proteases (aspartyl peptidases). (B) Sequential analysis of the core region of LC3 promoter was analyzed by TESS. CNE2 cells were treated with or without ceramide 20 μM for 12 h, then ChIP analysis was to detect the binding of c-Jun to LC3 promoter in vivo according to the manufacturer's instructionand. (C) CNE2 cells were transfected with LC3 (-1993/+7)-luc or LC3(-1993/+7)-MUT-luc; empty vector was co-transfected as negative control; PCMV-RL was co-transfected as internal control. 16 h after transfection, the cells were treated with or without ceramide 20 μM for 12 h. Luciferase activities were detected as described in Material and Methods. (p < 0.05)