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CHAPTER 2 Literature Review
2.14 THE SPHINGOMYELIN PATHWAY, APOPTOSIS AND CERAMIDE
intracellular free sphingoid bases disrupts the regulatory mechanisms within cells.
Complex sphingolipids such as gangliosides, interact with growth factor receptors, the extracellular matrix, and neighbouring cells, whereas the backbones, So and other sphingoid bases, cerami des, and sphingosine-I-phosphate (So-I-phosphate); activate or inhibit protein kinases and phosphatases, ion transporters, and other regulatory machinery.
Tumour necrosis factor a, ll..,-~ and nerve growth factor (NGF), for example, induce SM hydrolysis to CER. Other agonists, such as platelet-derived growth factor (PDGF), trigger further hydrolysis of CER to So, and activate So kinase to form So-I-phosphate. These metabolites either stimulate or inhibit growth, and may be cytotoxic in some cases via induction of apoptosis, depending on which products are formed or added exogenously, the cellular levels, intracellular localization, and the cell type. Ceramide and So-1 phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival, their relative cellular level being proposed to be a rheostat that determines the fate of cells (Mandala et ai, 1998). Therefore, fumonisin disruption of sphingolipid metabolism and deregulation of lipid biosynthesis might impact many of these processes, and will likely have a variety of effects depending on cell type and other factors.
2.14.1 Ceramide in Apoptosis
Apoptosis is an inducible pre-programmed pathway of sequential biochemical events leading to activation of calcium and magnesium dependent endonucleases that cleave the nuclear chromatin at selective inter-nucleosomal linker sites (Wyllie, 1980; Vaux, 1993).
Bose et al. (1995) demonstrated that CER synthase activity can be regulated in eukaryotes and constitutes definitive evidence for a requirement for CER elevation in the induction of apoptosis. Signals generated at the membrane of affected cells activate neighbouring intact cells and infiltrating macrophages to phagocytose dying cells and disintegrating nuclei (Duvall et al., 1985).
The biosynthesis of sphingomyelin is approximately 10-fold more sensitive to inhibition by FB 1 than that of GSLs. This is also the case for other inhibitors of CER synthesis, such as 13-fluoroalanine (Medlock and Merrill, 1988; Merrill et al., 1993b). Although excessive amounts of CER are often toxic and resul ts in apoptosis of many cell types (Jarvis and Grant, 1998), a basal level of this messenger is required for cell proliferation and differentiation. Decreasing the steady-state CER leads to growth arrest and loss of differentiation (Spiegel and Merrill, 1996; Merrill et al., 1997). Therefore, a delicate balance in CER levels is needed for maintenance of proper cell function.
2.14.2 Ceramide metabolism and disease
Deregulation in intracellular CER levels also has been implicated in diabetes, Acquired Immune Deficiency Syndrome (AIDS) and autoimmunity (Mathias et al., 1998;
Sandhoff et aI., 1998). Increasing evidence points to the roles of disruption of these pathways in disease pathogenesis. For example, the primary mutation responsible for hereditary neuropathy has been mapped to the LCB 1 subunit of SPT, and it has been suggested to induce apoptosis of susceptible sensory neurons (Bejaoui et aI., 2001;
Dawkins et aI., 2001). Ceramide levels have been shown to be elevated in a number of neurodegenerative disorders such as Batten's disorder, and two of the genes responsible for subtypes of this disorder, CLN3 and protein palmitoyl thioesterase, have been shown to attenuate ceramide levels in response to apoptotic stimuli, suggesting a role for the CER pathway in mediating cell dysfunction and death in these disorders (Puranam et al., 1999;
Cho et aI., 2000).
The absence of CER in specialized areas of the brain like the nigro-striatal neurons is believed to result in the death of these neurons leading to Parkinson's disease (France-Lanord et aI., 1997; Hunot et al., 1997; Larocca et al., 1997). C2-ceramide has been shown to protect hippocampal neurons from death induced by amyloid b-peptide, suggesting that a basal level of CER is required for the delay of Alzheimer's disease (Goodman and Mattson, 1996; Mattson et aI., 1997). Increases in CER are therefore notably beneficial for delaying the onset ofneurodegenerative diseases.
This increase in CER may be manipulated to assist in limiting uncontrolled cell growth.
High amounts of CER usually lead to apoptosis, and this property can be used as a powerful weapon for killing tumour cells. Cerami de has potential for eliminating aberrant cells in circumstances when they are not desired. Given the pleotropic functions of CER in a plethora of signalling systems and its involvement in the aetiology of many diseases, it is possible that new approaches to treat diseases could be initiated by modulating CER levels, either through reduction in de novo CER synthesis or CER release through blockade of neutral SMase and acidic SMase activity (Spiegel and Merrill, 1996; Merrill et aI., 1997).
2.14.3 Ceramide synthase and fumonisins
All fumonisins of the "B" senes inhibit CER synthase at low or submicromolar concentrations (Wang et aI., 1991; Merrill et aI., 1993b; 1993c; 1996b). In assays ofFAl, less than 2% inhibition ofCER synthase at 10~M was found. Acetylation therefore appears to reduce the potency by more than 50 fold. Removal of the tricarballylic acid (TCA) side chains (Figure 2.4) reduces the potency in vitro by approximately ten fold (Merrill et aI., 1993c).
In order to determine whether fumonisins inhibit other enzymes aside from CER synthase, that interact with either long chain bases or fatty acyl-CoA's, FBI was tested with So kinase (the enzyme initiating sphingoid base disposal) and SPI (the regulatory enzyme for sphingoid base synthesis) (Wang et aI., 1991). Neither enzyme was inhibited by the levels of fumonisins that result In complete inhibition of CER synthase (Merrill et aI., 1993b; 1996b).
Norred et at. (1997) assessed the relative potency of analogues of FBI to inhibit CER synthase. Fumonisin B I, FB2, FB3, FB4, FC4 , and TA toxin (a structurally similar mycotoxin produced by the tomato pathogen, Alternaria alternata f sp. lycopersici) were approximately equipotent inhibitors. Hydrolysed FBI, FB2 and FB3 were only 30-40% as potent as the parent toxins. Fumonisin Al did not block CER synthase, suggesting that F Al is non-toxic. Inhibition of CER synthase by fumonisin analogues did not appear to be related to the Jipophilicity of the compounds. The ability of relatively high doses of other mycotoxins that bear no structural similarity to fumonisins, including aflatoxin B I, cyclopiazonic acid, beauvericin, T2, sterigmatocystin, luteoskyrin, verrucarin A, scirpentriol, and ZEA to block CER synthase was also determined. All of the toxins tested were negative in the bioassay with the exception of fumonisins, indicating that disruption of sphingolipid metabolism is a specific cytotoxic response (Norred et at., 1997).
Fumonisin BI inhibits CER synthase with either Sa or So as the substrate, and with a variety of fatty acyl-coenzyme As as the co-substrate (Wang et aI., 1991;
Merrill et aI., 1993b; 1993c). The inhibition is competitive with both sphingoid bases and fatty acyl-CoA (Merrill et aI., 1993c) which indicates that fumonisin may inhibit CER synthase by interacting with both the binding site for Sa or So, and the site for the fatty acyl-CoAs (Merrill et aI., 1996b). Ceramides also affect DNA synthesis. The inhibition of CER synthase by fumonisins causes Sa to accumulate, and at least a portion of Sa undergoes phosphorylation and cleavage (Merrill et aI., 1993c).