Defective TNF-α–mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

Abstract

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.

Critical role of acidic sphingomyelinase in murine hepatic ischemia-reperfusion injury

“…ASMase knockout mice (C57BL/6 strain) were propagated using heterozygous breeding pairs and genotyped as described previously. 16,26 Hepatic partial warm ischemia was performed during 90 minutes in wild-type or ASMase Ϫ/Ϫ (8-12 weeks) mice as described previously. 6 Animal survival was determined using a model of total hepatic ischemia in male wild-type mice (25-30 g), in which only the ischemic tissue is left in place.…”

“…14 Several SMases have been characterized, of which the neutral SMase (NSMase), with an optimum pH at 7.5, Mg 2ϩ dependent and membrane bound, and the acidic SMase (ASMase), with an optimum pH at 4.8, are of relevance in the signaling of diverse cell death stimuli. 11,[15][16][17][18] Ceramide levels are tightly regulated in cells, and hence, its potential participation in death signaling can be modulated by metabolic pathways that transform ceramide in other derivatives. For instance, ceramide can be metabolized into complex glycosphingolipids by its glucosylation in the cytosolic surface of the Golgi catalyzed by the rate-limiting enzyme glucosylceramide synthase (GCS).…”

“…25 However, their impact on ceramide regulation and contribution to hepatic I/R injury were not addressed. Because ceramide overgeneration by ASMase has been shown to contribute to TNFinduced hepatocellular cell death and liver injury, 16,26 the aims of this study were first to examine whether ASMase contributes to warm hepatic I/R injury, because this specific aim has not been previously reported, and second, to analyze the metabolic regulation of ceramide during I/R. We describe here a critical role for ceramide generation through ASMase activation in hepatic I/R injury, which may be of therapeutic relevance because its pharmacological inhibition or suppression by siRNA protects the liver from I/R-induced damage.…”

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo

“…Ceramide can be generated through several pathways such as its de novo synthesis from L-serine and palmitoyl coenzyme A or by sphingomyelin hydrolysis upon activation of sphingomyelinases (SMases) (Kolesnick, 2002;Garcia-Ruiz et al, 2003;Ogretmen and Hannun, 2004). However, the metabolism of ceramide can limit its accumulation, and hence its participation in death pathways.…”

The Ganglioside GD3 as the Greek Goddess Hecate: Several Faces Turned Towards as Many Directions

“…Indeed, in ASM knock out mice, hepatocytes are resistant to TNF-induced apoptosis but sensitive to exogenous human placental ASM (9). Moreover, after TNF stimulation, GD3 relocates to mitochondria in wild type but not in ASM-deficient hepatocytes.…”