CD95 Stimulation and ceramide have a different impact on the morphology and physiology of mitochondria and endoplasmic reticulum.

Stimulation with C2 ceramide (1) or with antibodies to the death receptor CD95/Fas/APO-1 (2) induces apoptosis in a wide variety of cell types. Both stimuli cause cytochrome c release, caspase activation and DNA fragmentation. Less known are the effects of these apoptotic agents on organelles such as mitochondria and endoplasmic reticulum (ER). We studied the modifications occurring to mitochondria and ER in HeLa cells during the onset of apoptosis, showing that ceramide and CD95 stimulation have a different impact on morphology and physiology of mitochondria and ER. In C2 ceramide-stimulated cells, mitochondria were fragmented, spherical and smaller than in control cells; these changes occurred as early as 30 min from the stimulation and were not detected in anti-CD95 treated cells where the mitochondrial network did not apparently change its morphology even after several hours. This could be due to a direct damaging effect of ceramide on mitochondrial structures but also to the fact that ceramide itself induces a rapid Ca2+ release from the ER (3). In addition, ceramide induced a dramatic reduction in the mitochondrial Ca2+ response upon stimulation of the ER with histamine, while on the contrary, anti-CD95-stimulated HeLa cells showed a histamine-induced Ca2+ response similar to that of unstimulated control cells. The reduction in mitochondrial Ca2+ response in cells treated with C2 ceramide did not depend on a diminished capacity of mitochondria to take-up Ca2+ released by the ER but primarily on the fact that [Ca2+]ER was lower in ceramide-treated cells. Morphological changes occurring to the ER during ceramide treatment are under investigation. These findings suggest that Ca2+ is involved in the apoptotic pathway of C2 ceramide and show that different apoptotic stimuli have a different impact on the morphology and physiology of mitochondria and ER. Bibliography: 1) Okazaki T, Kondo T, Kitano T, Tashima M. Diversity and complexity of ceramide signalling in apoptosis. Cell Signal 1998, 10:685-92. 2) Sharma K, Wang RX, Zhang LY, Yin DL, Luo XY, Solomon JC, Jiang RF, Markos K, Davidson W, Scott DW, Shi YF. Death the Fas way: regulation and pathophysiology of CD95 and its ligand. Pharmacol Ther 2000, 88:333-47. 3) Pinton P, Ferrari D, Rapizzi E, Di Virgilio F, Pozzan T, Rizzuto R. The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action. EMBO 2001, 20:2690 2701.