The mechanisms responsible for thyrocyte destruction in Hashimoto's thyroiditis (HT) are poorly understood. Thyrocytes from HT glands, but not from nonautoimmune thyroids, expressed Fas. Interleukin-1beta (IL-1beta), abundantly produced in HT glands, induced Fas expression in normal thyrocytes, and cross-linking of Fas resulted in massive thyrocyte apoptosis. The ligand for Fas (FasL) was shown to be constitutively expressed both in normal and HT thyrocytes and was able to kill Fas-sensitive targets. Exposure to IL-1beta induced thyrocyte apoptosis, which was prevented by antibodies that block Fas, suggesting that IL-1beta-induced Fas expression serves as a limiting factor for thyrocyte destruction. Thus, Fas-FasL interactions among HT thyrocytes may contribute to clinical hypothyroidism.
Gangliosides participate in development and tissue differentiation. Cross-linking of the apoptosis-inducing CD95 protein (also called Fas or APO-1) in lymphoid and myeloid tumor cells triggered GD3 ganglioside synthesis and transient accumulation. CD95-induced GD3 accumulation depended on integral receptor "death domains" and on activation of a family of cysteine proteases called caspases. Cell-permeating ceramides, which are potent inducers of apoptosis, also triggered GD3 synthesis. GD3 disrupted mitochondrial transmembrane potential (DeltaPsim), and induced apoptosis, in a caspase-independent fashion. Transient overexpression of the GD3 synthase gene directly triggered apoptosis. Pharmacological inhibition of GD3 synthesis and exposure to GD3 synthase antisense oligodeoxynucleotides prevented CD95-induced apoptosis. Thus, GD3 ganglioside mediates the propagation of CD95-generated apoptotic signals in hematopoietic cells.
SummaryIntraceilular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed ceil surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monodonal antibody was produced by immunizing mice with human CD95-transfected L ceils. Crosslinkiug of CD95 with DX2 resulted in the activation of a sphingomydinase (SMase) in promydocytic U937 ceils, as well as in other human tumor ceil lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomydin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labded SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hy&olysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to ceil-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be rdevant in defining the biochemical nature of intraceilular messengers leading to apoptotic cell death.
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