T cell-derived interferon-γ programs stem cell death in immune-mediated intestinal damage.

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.

Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts.

Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.

Sphingosine 1-phosphate as a therapeutic agent.

The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P), formed by activation of sphingosine kinase in response to diverse stimuli, is an important lipid mediator that has novel dual actions - both inside and outside of cells. S1P is the ligand for a family of five G protein-coupled receptors. Activation of these GPCRs by S1P or dihydro-S1P regulates diverse processes, including cell migration, angiogenesis, vascular maturation, heart development, and neurite retraction. There is also abundant evidence that S1P can function as a second messenger important for regulation of calcium homeostasis, cell growth, and suppression of apoptosis. In many cases, the intracellular level of S1P and ceramide, another important sphingolipid metabolite associated with cell death and cell growth arrest, coordinately determine cell fate. Changes in S1P and ceramide have been implicated in a number of pathological conditions in which apoptosis plays an important role. Importantly, radiation-induced oocyte loss in adult female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with S1P. Understanding the biosynthesis, metabolism and functions of S1P can uncover new targets for the pharmaceutical and therapeutic applications of S1P.

jkk-1 and mek-1 regulate body movement coordination and response to heavy metals through jnk-1 in Caenorhabditis elegans.

Although in vitro evidence suggests two c-Jun N-terminal kinase (JNK) kinases, MKK4 and MKK7, transactivate JNK, in vivo confirmation is incomplete. In fact, JNK deficiency may differ from the composite deficiency of MKK4 and MKK7 in Drosophila and mice. Recently, the Caenorhabditis elegans homolog of human JNK, jnk-1, and two MKK-7s, mek-1 and jkk-1, were cloned. Here we characterize jnk-1, which encodes two isoforms JNK-1 alpha and JNK-1 beta. A null allele, jnk-1(gk7), yielded worms with defective body movement coordination and modest mechanosensory deficits. Similarly to jkk-1 mutants, elimination of GABAergic signals suppressed the jnk-1(gk7) locomotion defect. Like mek-1 nulls, jnk-1(gk7) showed copper and cadmium hypersensitivity. Conditional expression of JNK-1 isoforms rescued these defects, suggesting that they are not due to developmental errors. While jkk-1 or mek-1 inactivation mimicked jnk-1(gk7) locomotion and heavy metal stress defects, respectively, mkk-4 inactivation did not, but rather yielded defective egg laying. Our results delineate at least two different JNK pathways through jkk-1 and mek-1 in C.elegans, and define interaction between MKK7, but not MKK4, and JNK.

Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.

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.

Ceramide enables fas to cap and kill.

Recent studies suggest that trimerization of Fas is insufficient for apoptosis induction and indicate that super-aggregation of trimerized Fas might be prerequisite. For many cell surface receptors, cross-linking by multivalent ligands or antibodies induces their lateral segregation within the plasma membrane and co-localization into "caps" on one pole of the cell. In this study, we show that capping of Fas is essential for optimal function and that capping is ceramide-dependent. In Jurkat T lymphocytes and in primary cultures of hepatocytes, ceramide elevation was detected as early as 15-30 s and peaked at 1 min after CH-11 and Jo2 anti-Fas antibody treatment, respectively. Capping was detected 30 s after Fas ligation, peaked at 2 min, and was maintained at a lower level for as long as 30 min in both cell types. Ceramide generation appeared essential for capping. Acid sphingomyelinase -/- hepatocytes were defective in Jo2-induced ceramide generation, capping, and apoptosis, and nanomolar concentrations of C(16)-ceramide restored these events. To further explore the role of ceramide in capping of Fas, we employed FLAG-tagged soluble Fas ligand (sFasL), which binds trimerized Fas but is unable to induce capping or apoptosis in Jurkat cells. Cross-linking of sFasL with M2 anti-FLAG antibody induced both events. Pretreatment of cells with natural C(16)-ceramide bypassed the necessity for forced antibody cross-linking and enabled sFasL to cap and kill. The presence of intact sphingolipid-enriched membrane domains may be essential for Fas capping since their disruption with cholesterol-depleting agents abrogated capping and prevented apoptosis. These data suggest that capping is a ceramide-dependent event required for optimal Fas signaling in some cells.

CD95 signaling via ceramide-rich membrane rafts.

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 ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.

Kinase suppressor of Ras signals through Thr269 of c-Raf-1.

We recently established a two-stage in vitro assay for KSR kinase activity in which KSR never comes in contact with any recombinant kinase other than c-Raf-1 and defined the epidermal growth factor (EGF) as a potent activator of KSR kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). That study, however, did not address the mechanism of c-Raf-1 stimulation by activated KSR. Here we show that phosphorylation of c-Raf-1 on Thr(269) by KSR is necessary for optimal activation in response to EGF stimulation. In vitro, KSR specifically phosphorylated c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay. Using purified wild-type and mutant c-Raf-1 proteins, we demonstrate that Thr(269) is the major c-Raf-1 site phosphorylated by KSR in vitro and that phosphorylation of this site is essential for c-Raf-1 activation by KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, as kinase-inactive c-Raf-1(K375M) served as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulated KSR activation and induced Thr(269) phosphorylation and activation of c-Raf-1. Low dose EGF did not induce serine or tyrosine phosphorylation of c-Raf-1. High dose EGF (10-100 ng/ml) induced no additional Thr(269) phosphorylation, but rather increased c-Raf-1 phosphorylation on serine residues and Tyr(340)/Tyr(341). A Raf-1 mutant with valine substituted for Thr(269) was unresponsive to low dose EGF, but was serine- and Tyr(340)/Tyr(341)-phosphorylated and partially activated at high dose EGF. This study shows that Thr(269) is the major c-Raf-1 site phosphorylated by KSR. Furthermore, phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo.

Natural ceramide reverses Fas resistance of acid sphingomyelinase(-/-) hepatocytes.

The role of the second messenger ceramide in Fas-mediated death requires clarification. To address this issue, we generated hepatocytes from paired acid sphingomyelinase (ASMase; asmase)(+/+) and asmase(-/-) mice. asmase(-/-) hepatocytes, derived from 8-week-old mice, manifested normal sphingomyelin content and normal morphological, biochemical, and biologic features. Nonetheless, ASMase-deficient hepatocytes did not display rapid ceramide elevation or apoptosis in response to Jo2 anti-Fas antibody. asmase(-/-) hepatocytes were not inherently resistant to apoptosis because staurosporine, which did not induce early ceramide elevation, stimulated a normal apoptotic response. The addition of low nanomolar quantities of natural C16-ceramide, which by itself did not induce apoptosis, completely restored the apoptotic response to anti-Fas in asmase(-/-) hepatocytes. Other sphingolipids did not replace natural ceramide and restore Fas sensitivity. Overcoming resistance to Fas in asmase(-/-) hepatocytes by natural ceramide is evidence that it is the lack of ceramide and not ASMase which determines the apoptotic phenotype. The ability of natural ceramide to rescue the phenotype without reversing the genotype provides evidence that ceramide is obligate for Fas induction of apoptosis in hepatocytes.

Cell autonomous apoptosis defects in acid sphingomyelinase knockout fibroblasts.

A body of evidence suggests that stress-induced sphingomyelin hydrolysis to the second messenger ceramide initiates apoptosis in some cells. Although studies using lymphoblasts from Niemann-Pick disease patients or acid sphingomyelinase (ASMase)-deficient mice have provided genetic support for this hypothesis, these models have not been universally accepted as definitive. Here, we show that mouse embryonic fibroblasts (MEFs) prepared from asmase mice manifest cell autonomous defects in apoptosis in response to several stresses. In particular, asmase(-/-) MEFs failed to generate ceramide and were totally resistant to radiation-induced apoptosis but remained sensitive to staurosporine, which did not induce ceramide. asmase(-/-) MEFs were also partially resistant to tumor necrosis factor alpha/ actinomycin D and serum withdrawal. Thus, resistance to apoptosis in asmase(-/-) MEFs was not global but rather stress type specific. Most importantly, the sensitivity to stress could be restored in the asmase(-/-) MEFs by administration of natural ceramide. Overcoming apoptosis resistance by natural ceramide is evidence that it is the lack of ceramide, not ASMase, that determines apoptosis sensitivity. The ability to rescue the apoptotic phenotype without reversing the genotype by the product of the enzymatic deficiency provides proof that ceramide is obligate for apoptosis induction in response to some stresses.

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

CD95-mediated apoptosis in vivo involves acid sphingomyelinase.

Acid sphingomyelinase (ASM) is reported to have an essential function in stress-induced apoptosis although the physiological function of ASM in receptor-triggered apoptosis is unknown. Here, we delineate a pivotal role for ASM in CD95-triggered apoptosis of peripheral lymphocytes or hepatocytes in vivo. We employed intravenous injection of anti-CD4 antibodies or phytohemagglutinin that was previously shown to result in apoptosis of peripheral blood lymphocytes or hepatocytes via the endogenous CD95/CD95 ligand system. Our results demonstrate a high susceptibility in normal mice whereas ASM knock-out mice fail to immunodeplete T cells or develop autoimmune-like hepatitis. Likewise, ASM-deficient mice or hepatocytes and splenocytes ex vivo manifest resistance to anti-CD95 treatment. These results provide in vivo evidence for an important physiological function of ASM in CD95-induced apoptosis.

Ceramide mediates radiation-induced death of endothelium.

The sphingomyelin (SM) pathway is an ubiquitous, evolutionarily conserved signaling system, analogous to conventional systems such as the cAMP and phosphoinositide pathways. Ceramide is generated from SM by the action of a neutral or acid SMase, or by de novo synthesis coordinated through the enzyme ceramide synthase. Once generated, ceramide may serve as a second messenger in signaling responses to physiologic or environmental stimuli, or may be converted to a variety of structural or effector molecules. In the radiation response, ceramide serves as a second messenger in initiating apoptosis, while some of its metabolites block apoptosis. In certain cells, such as endothelial, lymphoid and haematopoietic cells, ceramide mediates apoptosis while in others ceramide may serve only as a co-signal for or play no role in the death response. Regulated ceramide metabolism may determine the balance between pro- and anti-apoptotic signals, and hence, the intensity of the apoptotic response, thus constituting a mechanism of radiation sensitivity or resistance. This paradigm may offer new opportunities for modulation of the radiation effects in the treatment of cancer. Chemical modifiers of ceramide metabolism may be useful to enhance the therapeutic effects or reduce the toxicity of radiation treatment.