Mitochondrial cytochrome c release mediates ceramide-induced activator protein 2 activation and gene expression in keratinocytes

The intracellular signaling pathway(s) through which second messenger ceramides induce gene expression in human cells has not yet been characterized. In the present study, ceramide-induced expression of intercellular adhesion molecule-1 (ICAM-1), which requires activation of transcription factor activator protein 2 (AP-2), was found to be mediated through a mitochondrial pathway. Inhibitors of mitochondrial electron transport chain (e.g. rotenone, thenoyltrifluoroacetone, and antimycin A) reduced ceramide-induced ICAM-1 expression. Stimulation of human keratinocytes with cell-permeant ceramides at concentrations that did not induce apoptosis ( no activation of caspases 3, 8, and 9 and no nucleosomal fragmentation) but caused AP-2 activation and ICAM-1 induction released cytochrome c (cyt c) from mitochondria into the cytoplasm of cells. This cyt c release was an indispensable prerequisite for effective ceramide signaling, because its inhibition by modulating the mitochondrial megachannel with bonkrekic acid or carboxyatractyloside prevented ceramide-induced AP-2 activation and ICAM-1 expression. Analysis of the interaction between cyt c and AP-2 revealed that cyt c oxidized AP-2 and that this redox regulation greatly enhanced the DNA binding capacity of AP-2. Mitochondria thus have a previously unrecognized function in signaling ceramide-induced transcription factor activation and gene regulation.