Perturbation of membrane microdomains reduces mitogenic signaling and increases susceptibility to apoptosis after T cell receptor stimulation

Abstract

Acid sphingomyelinase-deficient (asmase 7/7 ) mice generated by gene targeting abundantly store sphingomyelin in the reticuloendothelial system of liver, spleen, bone marrow, and in brain. Liver cells of asmase 7/7 mice accumulate sphingomyelin and glycosphingolipids in purified lipid bilayers of microsomes, Golgi, and the plasma membrane, but cholesterol is depleted in the plasma membrane. Detergent-insoluble glycolipid-enriched membrane microdomains (GEM) can be isolated from hepatocytes, embryonic fibroblasts, and splenocytes of wild-type, but not of asmase 7/7 mice, by sucrose gradient density centrifugation. Lck and other Src-family kinases are reduced in isopycnic fractions of asmase 7/7 splenocytes compared to GEMcontaining fractions of wild-type cells. The proliferation of asmase 7/7 T lymphocytes is reduced, whereas their susceptibility to Fas-induced apoptosis is increased after T cell receptor (TCR) stimulation. TNF receptor I signaling remains unimpaired. The perturbation of GEM impairs tyrosine phosphorylation and, consequently, mitogenic signaling of the TCR. Reduced MAPK activity-dependent FLICE-like inhibitory protein (FLIP) expression in asmase 7/7 T lymphocytes increases their sensitivity towards Fasmediated apoptosis. Cell Death and Differentiation (2000) 7, 413 ± 424.

Cell Autonomous Apoptosis Defects in Acid Sphingomyelinase Knockout Fibroblasts

“…Furthermore, these ASMase-deficient mouse lines were developed using nonisogenic stem cell clones (22,57,58). MEFs derived from their mice displayed dramatic reduction in caveolin content and resistance to isolation of caveolae (56). Further, hepatocytes displayed a 2-fold increase in plasma membrane SM content accompanied by reduced signaling through tyrosine kinases in T lymphocytes, lymphopenia, the absence of proliferation in response to anti-CD3, reduced expression of the antiapoptotic adaptor FLIP, and a paradoxic increase in apoptosis of anti-CD3 pretreated splenocytes upon activation of CD95 (56).…”

“…MEFs derived from their mice displayed dramatic reduction in caveolin content and resistance to isolation of caveolae (56). Further, hepatocytes displayed a 2-fold increase in plasma membrane SM content accompanied by reduced signaling through tyrosine kinases in T lymphocytes, lymphopenia, the absence of proliferation in response to anti-CD3, reduced expression of the antiapoptotic adaptor FLIP, and a paradoxic increase in apoptosis of anti-CD3 pretreated splenocytes upon activation of CD95 (56). The conclusion of these studies was that disruption of membrane microdomains in response to altered sphingolipid metabolism has significant impact on signaling.…”

Phenotype of acid sphingomyelinase deficiency knockout mice

“…1 Analyses of 8-week-old asmase 7/7 did not show hepatic or spleenic accumulation of sphingomyelin, lymphopenia, or alterations of proliferatory and apoptotic responses of T cells in comparison to wild-type mice, which in the author's view, differ from our results recently published in this journal. 2 The crucial point in the comparison of the two studies is that the experiments described by Lozano et al are not comparable to ours, although studying several of the parameters described in our publication, for the following reasons. A major characteristic of both the human Niemann-Pick disease (NPD) 3 and, likewise, aSMase deficiency in mice generated by gene targeting, is the age-dependent accumulation of sphingomyelin in lysosomes and subcellular membranes of asmase 7/7 cells.…”

“…The experiments described in our publication have been performed with mice from our asmase 7/7 mouse line 4 of at least 12 ± 16 weeks of age and older which have developed the histological and clinical manifestations of NPD: the alterations of the lipid composition of hepatocyte, splenocyte and fibroblast cell membranes as well as the consequences for cellular signal transduction pathways which parallel the increasing incorporation of sphingomyelin and the changes of the complex lipid composition of the bilayer of plasmamembranes. 2 Young asmase 7/7 mice are phenotypically indistinguishable from wild-type mice 4,5 and MEFs derived from asmase 7/7 mice initially divide normally. With increasing number of passages, MEFs severely accumulate sphingomyelin, show a prolonged cell cycle, and eventually die after the sixth or seventh passage 1,2 (M Nix and W Stoffel, unpublished observations).…”

“…The authors also address in the letter our studies described in Cell Death and Differentiation. 2 Lozano et al 1 report that mouse embryonic fibroblasts (MEFs) derived from acid sphingomyelinase (aSMase)-deficient mice display a normal sphingomyelin content, normal morphology and doubling times, and a morphology and distribution of caveolae comparable to wild-type cells. 1 Analyses of 8-week-old asmase 7/7 did not show hepatic or spleenic accumulation of sphingomyelin, lymphopenia, or alterations of proliferatory and apoptotic responses of T cells in comparison to wild-type mice, which in the author's view, differ from our results recently published in this journal.…”