Suppression of heat shock protein-70 by ceramide in heat shock-induced HL-60 cell apoptosis.

Ceramide has emerged as a mediator of cell growth, differentiation, and apoptosis in many biological systems. Many kinds of stresses are reported to induce apoptosis with an increase of ceramide generation. Here we showed that the intracellular ceramide levels increased in parallel with heat shock (HS)-induced apoptosis in an intensity- and time-dependent manner, and synthetic N-acetylsphingosine (C(2)-ceramide) synergistically enhanced HS-induced apoptosis in HL-60 cells. In order to know the role of ceramide generation in HS-induced apoptosis, we examined the effects of C(2)-ceramide on the levels of mRNA and protein of heat shock proteins (HSPs). The increase of HSP-70 mRNA levels 1-2 h after HS at 42 degrees C for 30 min was suppressed by C(2)-ceramide in a dose-dependent manner. In comparison with HSP-70, the levels of HSP-60 and -90 mRNAs were faintly suppressed by C(2)-ceramide. Similarly, the increase in the protein levels of HSP-70 was significantly suppressed 4-8 h after HS by C(2)-ceramide in a dose-dependent manner. Additionally, in 293 cells, which are constitutively overexpressing HSP-70 gene, the levels of HSP-70 mRNA were suppressed by C(2)-ceramide in parallel with the increase of apoptotic cells. We next examined the mechanisms by which C(2)-ceramide suppressed HS-increased HSP-70 expression. The treatment with C(2)-ceramide did not affect both an activation of a nuclear transcription factor for HSP-70, heat shock factor-1, and an increased transcriptional rate of HSP-70 by HS, but increased the rates of HSP-70 mRNA degradation. In summary, ceramide may efficiently induce HS-induced apoptosis by suppressing anti-apoptotic HSP-70 through a post-transcriptional regulation.