Direct Inhibition of Mitochondrial Respiratory Chain Complex III by Cell-permeable Ceramide

Abstract

Ceramide is a lipid second messenger that mediates the effects of tumor necrosis factor ␣ and other agents on cell growth and differentiation. Ceramide is believed to act via activation of protein phosphatase, prolinedirected protein kinase, or protein kinase C. Tumor necrosis factor ␣-induced common pathway of apoptosis is associated with an early impairment of mitochondria. Herein, we demonstrate that ceramide can directly inhibit mitochondrial respiratory chain function. In isolated mitochondria, a rapid decline of mitochondrial oxidative phosphorylation occurs in the presence of Nacetylsphingosine (C 2 -ceramide), a synthetic cell-permeable ceramide analog. An investigation of the site of ceramide action revealed that the activity of respiratory chain complex III is reduced by C 2 -ceramide with halfmaximum effect at 5-7 M. In contrast, N-acetylsphinganine (C 2 -dihydroceramide), which lacks a functionally critical double bond and is ineffective in cells, did not alter mitochondrial respiration or complex III activity. We suggest that these in vitro observations may set the stage for identifying a novel mechanism of regulation of mitochondrial function in vivo.The pro-inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) 1 elicits a wide variety of cellular responses including profound alterations in transcriptional programs, perturbation of mitochondrial function, and apoptosis in a number of cell types (1, 2). Strong evidence supports a pivotal role for TNF-␣ in the genesis of septic shock (2), and it also has been implicated in ischemia reperfusion injury of heart (3). A recent report demonstrates that a physiologically relevant concentration of TNF-␣ induced apoptosis in rat cardiomyocytes as quantified by single cell microgel electrophoresis of nuclei and in situ 3Ј nick end labeling assay (4). Mitochondria are considered an early target in TNF-␣-induced cytotoxicity because they appear swollen with a reduced number of cristae, in association with profound inhibition of mitochondrial respiration (1,5,6). A growing body of evidence suggests that treatment of cells with TNF-␣ results in an electron transport inhibition at the level of complex III (1, 6) followed by an increased generation of oxygen radicals in mitochondria (7-9). However, the mechanism of TNF-␣-induced inhibition of mitochondrial respiration has not been elucidated.The sphingomyelin pathway has been implicated as a major signaling mechanism mediating the action of a number of extracellular agents (such as TNF-␣, Fas ligands, and chemotherapeutic agents) causing the activation of sphingomyelinases that cleave membrane sphingomyelin resulting in the formation of ceramide (10, 11). Synthetic cell-permeable ceramide analogs have been shown to mimic many TNF-␣-induced cell responses (10 -13). In malignant and nonmalignant cell lines, ceramides specifically induce apoptosis that involves activation of interleukin-1␤-converting enzyme-like proteases, whereas closely related dihydro-analogs are inactive (10). The intracellular targets for ceramid...

Inducible resistance to Fas-mediated apoptosis in B cells

“…Thus, Bcl-xL appears to account for at least a portion of sIg-induced Fas-resistance because, expression was upregulated coordinately with induction of Fas-resistance (in both murine and human B cells, reference [69]), and, because isolated overexpression diminished B cell susceptibility to Fas killing separate and apart from any other potential effects of sIg engagement (although in connection with this last point it should be mentioned that the capacity of Bcl-xL to inhibit Fas-mediated apoptosis in B cell lines has been questioned, reference [70]). Further, the reduction in susceptibility to Th1-induced cytotoxicity produced by Bcl-xL suggests that Fas death signaling in B cells involves mitochondrial cytochrome c release; this notion is supported by our observation that inducibly Fas-resistant B cells are protected against apoptosis produced by C2-ceramide, the cytotoxicity of which has been associated with mitochondrial damage [71][72][73][74][75][ [71][72][73][74][75].…”

Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency

“…22,23 The direct effect of ceramide on mitochondria was reproduced with GD3 ganglioside that leads to a marked loss of mitochondrial DCm and apoptosis. 24 These studies led us to assume that the mitochondrial dysfunction that we observed was not an artefact but was secondary to the GM3 synthase deficiency.…”

Isc1p Plays a Key Role in Hydrogen Peroxide Resistance and Chronological Lifespan through Modulation of Iron Levels and Apoptosis

“…In Aspergillus nidulans, LCBs induce apoptosis associated with the accumulation of ROS, although ROS scavenging does not impair the induction of apoptosis (Cheng et al, 2003). In mammalian cells, ceramide has also been shown to increase the production of ROS, by directly inhibiting the mitochondrial complex III (Gudz et al, 1997) and via inhibition of Bcl2 mediated by the ceramide-activated protein phosphatase 2A (Ruvolo et al, 1999). The present study showed that Isc1p, the budding yeast neutral sphingomyelinase homologue, plays a key role in oxidative stress resistance and chronological lifespan.…”