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

Ceramide has emerged as a mediator of cell growth, differentiation, and apo ptosis in many biological systems. Many kinds of stresses are reported to i nduce apoptosis with an increase of ceramide generation. Here we showed tha t the intracellular ceramide levels increased in parallel with heat shock ( HS)-induced apoptosis in an intensity- and time-dependent manner, and synth etic N-acetylsphingosine (C-2-ceramide) synergistically enhanced MS-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 le vels of mRNA and protein of heat shock proteins (HSPs). The increase of MSP -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 le vels of HSP-60 and -90 mRNAs were faintly suppressed by C-2-ceramide. Simil arly, the increase in the protein levels of HSP-70 was significantly suppre ssed 4-8 h after HS by C-2-ceramide in a dose-dependent manner. Additionall y, in 293 cells, which are constitutively overexpressing MSP-70 gene, the l evels of HSP-70 mRNA were suppressed by C-2-ceramide in parallel with the i ncrease of apoptotic cells. We next examined the mechanisms by which C-2-ce ramide suppressed MS-increased MSP-70 expression. The treatment with C-2-ce ramide did not affect both an activation of a nuclear transcription factor for HSP-70, heat shock factor-1, and an increased transcriptional rate of H SP-70 by MS, but increased the rates of MSP-70 mRNA degradation. In summary , ceramide may efficiently induce MS-induced apoptosis by suppressing anti- apoptotic MSP-70 through a post-transcriptional regulation.