The induction of programmed cell death, or apoptosis, involves activation of a signalling system, many elements of which remain unknown. The sphingomyelin pathway, initiated by hydrolysis of the phospholipid sphingomyelin in the cell membrane to generate the second messenger ceramide, is thought to mediate apoptosis in response to tumour-necrosis factor (TNF)-alpha, to Fas ligand and to X-rays. It is not known whether it plays a role in the stimulation of other forms of stress-induced apoptosis. Given that environmental stresses also stimulate a stress-activated protein kinase (SAPK/JNK), the sphingomyelin and SAPK/JNK signalling systems may be coordinated in induction of apoptosis. Here we report that ceramide initiates apoptosis through the SAPK cascade and provide evidence for a signalling mechanism that integrates cytokine- and stress-activated apoptosis.

About 50% of cancer patients receive radiation therapy. Here we investigated the hypothesis that tumor response to radiation is determined not only by tumor cell phenotype but also by microvascular sensitivity. MCA/129 fibrosarcomas and B16F1 melanomas grown in apoptosis-resistant acid sphingomyelinase (asmase)-deficient or Bax-deficient mice displayed markedly reduced baseline microvascular endothelial apoptosis and grew 200 to 400% faster than tumors on wild-type microvasculature. Thus, endothelial apoptosis is a homeostatic factor regulating angiogenesis-dependent tumor growth. Moreover, these tumors exhibited reduced endothelial apoptosis upon irradiation and, unlike tumors in wild-type mice, they were resistant to single-dose radiation up to 20 grays (Gy). These studies indicate that microvascular damage regulates tumor cell response to radiation at the clinically relevant dose range.

Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration.

Ceramide is a sphingosine-based lipid signaling molecule that regulates cellular differentiation, proliferation, and apoptosis. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus and cell-type specific. Ceramide action is determined within the context of other stimuli and by the subcellular topology of its production. Here, we discuss the pathways of ceramide generation and the interaction of ceramide with caspases and other apoptotic signaling cascades.

Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASMdeficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.Stimulation of a variety of surface receptors including the T-cell receptor/CD3 complex (1, 2), B-cell receptor (3), tumor necrosis factor receptor (TNF-R) 1 (4), CD2, CD44, L-selectin, or integrins (5) results in clustering of these receptors, which appears to be required for rapid and efficient receptor-mediated signaling. Recent studies on peptide antigen-induced signaling via the T-cell receptor/CD3 complex indicated that receptor aggregation rather than conformational changes of the intracellular part of the T-cell receptor/CD3 complex upon ligand binding is the predominant mechanism mediating signal transmission (1). Evidence suggests that many receptors aggregate in distinct cholesterol-and sphingolipid-rich membrane microdomains or rafts (6 -8). This notion is supported by the finding that disruption of rafts prevents clustering of many receptors including the B-cell receptor (9), CD48 (10), Fc␥ (11), or the TNF-R (12). Rafts seem to exist as preformed entities in the membrane of resting cells (13); whether receptor stimulation induces a biologically relevant modification of these rafts is unknown. Likewise, mechanisms mediating the trapping of activated receptor molecules within rafts require definition.In the present studies, we have investigated whether a hydrolysis of sphingomyelin to ceramide and, thus, a change in the composition of rafts contributes to clustering of receptor molecules. Ceramide is released by the activity of at least three forms of sphingomyelinases with an acidic, neutral, or basic pH optimum (14 Here, we suggest a novel mechanism for receptor clustering and show that ASM translocates from an intracellular compartment to the extracellular surface of the cell membrane upon stimulation via CD95. Translocated ASM localizes to sphingolipid-rich rafts and releases extracellularly orientated ceramide that mediates selective clustering of CD95 and constitutes an essential prerequisite for signaling. MATERIALS AND METHODSCells and Stimulation-Human ASM-or acid ceramidase (AC)-deficient lymphocytes or fibroblasts, respectively, were obtained from patients with Niemann Pick disease type A (NPDA) or Farber disease. JY and Jurkat were from ATCC. All lymphocytes were grown in phenol red-free RPMI 1640 supplemented with 10% fetal calf serum, 10 mM HEPES, pH 7.4, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 M non-essential ...

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The sphingomyelin pathway, which is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide, signals apoptosis for tumor necrosis factor alpha, Fas, and ionizing radiation. In the present studies, the anticancer drug daunorubicin also stimulated ceramide elevation and apoptosis in P388 and U937 cells. Cell-permeable analogs of ceramide, but not other lipid second messengers, mimicked daunorubicin in inducing apoptosis. Daunorubicin-stimulated ceramide elevation, however, did not result from sphingomyelin hydrolysis, but rather from de novo synthesis via activation of the enzyme ceramide synthase. An obligatory role for ceramide synthase was defined, since its natural specific inhibitor, fumonisin B1, blocked daunorubicin-induced ceramide elevation and apoptosis. These studies demonstrate that ceramide synthase activity can be regulated in eukaryotes and constitute definitive evidence for a requirement for ceramide elevation in the induction of apoptosis.