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

Liver Unit, Instituto de Malalties Digestives, Hospital Clinic i Provincial, Instituto de Investigaciones Biomédicas August Pi Suñer, Barcelona, Spain.
Journal of Clinical Investigation (Impact Factor: 13.22). 02/2003; 111(2):197-208. DOI: 10.1172/JCI16010
Source: PubMed


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.

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    • "Uncovering the molecular mechanisms and interplay of key Garcia-Ruiz et al, JHEPAT-D-14-01606-R1 5 metabolic players may lead to the design of novel therapeutic combinations. Acid sphingomyelinase (ASMase), a specific generator of ceramide by sphingomyelin (SM) hydrolysis, mediates TNF/Fas-induced hepatocellular apoptosis and promotes liver fibrogenesis [14] [15] [16]. Recent evidence demonstrates that ASMase regulates key mechanisms involved in steatosis, fibrosis and lipotoxicity, including endoplasmic reticulum (ER) stress, autophagy and lysosomal membrane permeabilization (LMP), which contribute to ASH and NASH (Figure 1) [6] [17] [18]. "
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    ABSTRACT: Steatohepatitis (SH) is an intermediate stage of fatty liver disease and one of the most common causes of chronic liver disease worldwide that may progress to cirrhosis and liver cancer. SH encompasses alcoholic and nonalcoholic steatohepatitis, the latter being of particular concern due to its association with obesity and insulin resistance and a major cause of liver transplantation. The molecular mechanisms governing the transition from steatosis to SH are not fully understood. Here we discuss emerging data indicating that acid sphingomyelinase (ASMase), a specific mechanism of ceramide generation, is required for the activation of key pathways that regulate steatosis, fibrosis and lipotoxicity, including endoplasmic reticulum stress, autophagy and lysosomal membrane permeabilization. Moreover, ASMase modulates alterations of methionine cycle and phosphatidylcholine homeostasis, two crucial events involved in SH that regulate methylation reactions, antioxidant defense and membrane integrity. These new findings suggest that targeting ASMase in combination with restoration of methionine metabolism and phosphatidylcholine levels may be of utility in the treatment of SH.
    Full-text · Article · Oct 2014 · Journal of Hepatology
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    • "In particular, ceramides, which also accumulate in response to death receptor activation [98], promote direct mitochondriotoxic effects [99], stimulate ROS generation [100], and inhibit various anti-apoptotic signal transducers, including AKT [101]. Mice lacking acidic sphingomyelinase (aSMase) are significantly protected against the lethal hepatotoxic effects of D-galactosamine/LPS, TNFa and agonistic anti-FAS antibodies [102] [103]; and imipramine, a chemical aSMase inhibitor, has been shown to limit hepatic damage upon IR injury [104]. Along similar lines, myriocin, an inhibitor of de novo ceramide synthesis, appears to reduce body weight, enhances metabolism and ameliorates energy expenditure in genetically obese (ob/ob) mice as well as in animals exposed to a high-fat diet (HFD) [105]. "
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    ABSTRACT: Inflammation can be either beneficial or detrimental for the liver, depending on multiple factors. Mild (i.e., limited in intensity and destined to resolved) inflammatory responses have indeed been shown to exert consistent hepatoprotective effects, contributing to tissue repair and promoting the reestablishment of homeostasis. Conversely, excessive (i.e., disproportionate in intensity and permanent) inflammation may induce a massive loss of hepatocytes and hence exacerbate the severity of various hepatic conditions, including ischemia-reperfusion injury, systemic metabolic alterations (e.g., obesity, diabetes, non-alcoholic fatty liver disorders), alcoholic hepatitis, intoxication by xenobiotics and infection, de facto being associated with irreversible liver damage, fibrosis and carcinogenesis. Both liver-resident cells (e.g., Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells) and cells that are recruited in response to injury (e.g., monocytes, macrophages, dendritic cells, natural killer cells) emit pro-inflammatory signals including - but not limited to - cytokines, chemokines, lipid messengers and reactive oxygen species that contribute to the apoptotic or necrotic demise of hepatocytes. In turn, dying hepatocytes release damage-associated molecular patterns that - upon binding to evolutionary conserved pattern recognition receptors - activate cells of the innate immune system to further stimulate inflammatory responses, resulting in the establishment of a highly hepatotoxic feedforward cycle of inflammation and cell death. In this review, we discuss the cellular and molecular mechanisms that account for the most deleterious effect of hepatic inflammation at the cellular level, that is, the initiation of a massive cell death response among hepatocytes.
    Full-text · Article · Apr 2013 · Journal of Hepatology
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    • "It has been reported that ASM deficiency leads to Niemann-Pick disease in humans and that Asm gene (Asm is commonly used to represent mouse gene for ASM) knockout in mice resulted in the resistance to radiation [4] and other forms of stress-induced apoptosis [1]. Similarly, inhibition of ASM activity has also been shown to render cells and animals resistant to the apoptotic effects of diverse stimuli including Fas/CD95 [5], ischemia [6], radiation [7], chemotherapy [8] tumor necrosis factor-alpha (TNF-α) [9]. In addition, Asm knockout or Asm inhibition was shown to have protective action during the lung inflammation and fibrosis [10], cystic fibrosis [11]–[12], obesity and associated glomerular injury [13], liver fibrogenesis [14] and renal fibrosis [15]. "
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    ABSTRACT: Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs(+/-) and Asm(+/-) mice. Given that the homozygotes of Cbs(-/-/)Asm(-/-) mice could not survive for 3 weeks. Cbs(+/-/)Asm(+/+), Cbs(+/-/)Asm(+/-) and Cbs(+/-/)Asm(-/-) as well as their Cbs wild type littermates were used to study the role of Asm(-/-) under a background of Cbs(+/-) with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs(+/-)) mice with different copies of Asm gene compared to Cbs(+/+) mice with different Asm gene copies. Cbs(+/-/)Asm(+/+) mice had significantly increased renal Asm activity, ceramide production and O(2).(-) level compared to Cbs(+/+)/Asm(+/+), while Cbs(+/-/)Asm(-/-) mice showed significantly reduced renal Asm activity, ceramide production and O(2).(-) level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs(+/-/)Asm(-/-) mice compared to Cbs(+/-/)Asm(+/+) mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs(+/-/)Asm(-/-) mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs(+/-/)Asm(-/-) mice compared to Cbs(+/-/)Asm(+/+) mice. In in vitro studies of podocytes, hHcys-enhanced O(2).(-) production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or corresponding enzyme inhibition protects the podocytes and glomeruli from hHcys-induced oxidative stress and injury.
    Full-text · Article · Sep 2012 · PLoS ONE
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