Activation of ERK1/2 and cPLA(2) by the p55 TNF receptor occurs independently of FAN.

The generation of proinflammatory eicosanoids in response to tumor necrosis factor (TNF) involves the activation of cytosolic phospholipase A(2) (cPLA(2)), presumably by phosphorylation through extracellular signal-regulated kinases (ERK). Earlier results had suggested that a pathway involving the p55 TNF receptor (TNF-R55), neutral sphingomyelinase (N-SMase), and c-Raf-1 activates ERK and cPLA(2). We have previously shown that a cytoplasmic region of TNF-R55 distinct from the death domain regulates the activation of N-SMase through binding of the adapter protein FAN. Analysis of embryonal fibroblasts from FAN knockout mice revealed that TNF-induced activation of both ERK and cPLA(2) occurs without involvement of FAN. Furthermore, we provide evidence that the TNF-dependent activation of ERK and cPLA(2) requires the intact death domain of TNF-R55. Finally, we demonstrate that in murine fibroblasts cPLA(2) is phosphorylated in response to TNF solely by ERK, but not by p38 mitogen-activated protein kinase, suggesting a signaling pathway from TNF-R55 via the death domain to ERK and cPLA(2).

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain.

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.

CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway.

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.

Impaired neutral sphingomyelinase activation and cutaneous barrier repair in FAN-deficient mice.

The WD-40 repeat protein FAN binds to a distinct domain of the p55 receptor for tumor necrosis factor (TNF) and signals the activation of neutral sphingomyelinase (N-SMase). To analyze the physiological role of FAN in vivo, we generated FAN-deficient mice by targeted gene disruption. Mice lacking a functional FAN protein do not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs appeared to be unaltered. An essential role of FAN in the TNF-induced activation of N-SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal-regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN-deficient mice show delayed kinetics of recovery after cutaneous barrier disruption suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN-deficient mice did not reproduce the phenotype of beige mice.

Caspase-mediated inhibition of human cytosolic phospholipase A2 during apoptosis.

Activation of cytosolic phospholipase A2 (cPLA2) is an essential step in the initiation of the cascade of enzymatic reactions leading to the generation of proinflammatory lipid mediators. Hence, the regulation of cPLA2 is a key event in the induction of inflammatory responses. cPLA2 is activated, in part, by apoptotic stimuli such as TNF or Fas ligand. Apoptosis, however, does not provoke an inflammatory response. Here, we demonstrate that cPLA2 is cleaved by caspase-3 and/or a related caspase in HeLa cells undergoing apoptosis. Mutation of a predicted caspase-3 cleavage site abolishes cPLA2 processing both in vitro and in intact cells. The 70-kDa cleavage product of cPLA2 itself has no catalytic function, while inhibition of cleavage results in an increased enzymatic activity. Additionally, overexpression of the 70-kDa fragment appears to produce a dominant negative effect on endogenous cPLA2 activity. In HeLa cells, cPLA2 activity was dispensable for the course of apoptosis. We cannot rule out, however, that cPLA2 activity is involved in the induction of apoptosis in other cell types. Taken together, our results suggest that the enzymatic activity of cPLA2 is specifically inhibited by caspase-mediated cleavage during apoptosis. The inactivation of cPLA2 represents a previously unrecognized mechanism for avoiding an inflammatory reaction against apoptotic cells.

Distinct adapter proteins mediate acid versus neutral sphingomyelinase activation through the p55 receptor for tumor necrosis factor.

Ceramide, generated by the enzymatic function of sphingomyelinases (SMases) has emerged as an important signaling pathway transducing diverse biological effects of various cytokine receptors. The 55-kDa receptor for tumor necrosis factor (TNF-R55) activates two types of SMases through distinct cytoplasmic domains. The death domain that is responsible for the initiation of the apoptotic pathway also signals for the activation of an acid SMase (A-SMase). The adapter protein TRADD binds to TNF-R55 in a ligand-dependent manner and serves as anchor for the subsequent recruitment of other proteins into the signaling complex that directly lead to cell death or nuclear factor-kappaB (NF-kappaB) induction. Notably, the two pro-apoptotic adapter proteins TRADD and FADD are also involved in the activation of A-SMase. In contrast, the NF-kappaB-inducing adapters TRAF2 and RIP do not signal for A-SMase. Thus, activation of A-SMase appears to belong to signals leading to TNF-induced cell death. A second signaling domain (NSD) is located upstream of the death domain and directly links the TNF-R55 to the activation of a neutral SMase (N-SMase). A novel adapter protein, FAN, has been identified that specifically binds to the NSD. FAN contains five WD repeats at its carboxy terminus, while it shows significant sequence homology with the mouse beige protein and its human homolog, the CHS protein, in the center portion of the protein. Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, whereas truncated mutants of FAN produced dominant negative effects. FAN, however, did not interfere with any of the TNF responses signaled for by the death domain. Taken together, our data suggest that distinct cytoplasmic domains of TNF-R55 initiate independent signaling pathways by binding different adapter proteins.

TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase.

Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or Fas. Here we describe the TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF receptor-associated proteins TRADD and FADD. Overexpression of TRADD and FADD in 293 cells did not change basal activity of A-SMase but enhanced TNF-induced stimulation of A-SMase. Other TNF R55-associated proteins like TRAF2 and RIP, which were reported to mediate TNF R55-mediated activation of nuclear factor kappaB, did not affect activation of A-SMase. Caspase inhibitors markedly reduced A-SMase activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase. Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) did not change A-SMase activity, suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like protease distinct from caspase-8/a or -10/b.