Dieter Adam

Ceramide mediates caspase-independent programmed cell death

Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase‐dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase‐independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase‐independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor‐interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase‐independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl‐2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.—Thon, L., Möhlig, H., Mathieu, S., Lange, R., Bulanova, E., Winoto‐Morbach, S., Schütze, S., Bulfone‐Paus, S., Adam, D. Ceramide mediates caspase‐independent programmed cell death. FASEB J. 19, 1945–1956 (2005)

The Polycomb group protein EED couples TNF receptor 1 to neutral sphingomyelinase

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer’s, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the “missing link” that completes one of the last unresolved signaling pathways of TNF-R1.

Toso regulates the balance between apoptotic and nonapoptotic death receptor signaling by facilitating RIP1 ubiquitination

The regulation of cellular survival and apoptosis is of critical importance for the immune system to maintain immune homeostasis and to establish tolerance. Here, we demonstrate that the immune specific cell surface molecule Toso exhibits antiapoptotic effects on death receptor signaling by a novel regulatory mechanism involving the adaptor kinase RIP1. The antiapoptotic function of Toso depends on RIP1 ubiquitination and involves the recruitment of the death adaptor FADD to a Toso/RIP1 protein complex. In response to CD95L and TNFα, Toso promotes the activation of MAPK and NF-κB signaling pathways. Because of this relative augmentation of survival versus apoptotic signals, Toso raises the threshold for death receptor-mediated apoptosis. Our analysis of Toso-deficient mice revealed that Toso is essential for TNFα-mediated liver damage. Furthermore, the antiapoptotic function of Toso could be blocked by a Toso-specific monoclonal antibody, opening up new therapeutic prospects for the treatment of immune disorders and hematologic malignancies.

MaxiK Blockade Selectively Inhibits the Lipopolysaccharide-Induced IκB-α/NF-κB Signaling Pathway in Macrophages

Macrophages have a pivotal function in innate immunity against bacterial infections. They are present in all body compartments and able to detect invading microorganisms with high sensitivity. LPS (endotoxin) of Gram-negative bacteria is among the most potent stimuli for macrophages and initiates a wide panel of cellular activation responses. The release of mediators such as TNF-α and ILs is essential for the initiation of a proinflammatory antibacterial response. Here, we show that blockade of the large-conductance Ca2+-activated potassium channel MaxiK (BK) inhibited cytokine production from LPS-stimulated macrophages at the transcriptional level. This inhibitory effect of channel blockade was specific to stimulation with LPS and affected neither stimulation of macrophages with the cytokine TNF-α nor LPS-induced activation of cells that do not express MaxiK. Investigation of the upstream intracellular signaling pathways induced by LPS revealed that the blockade of MaxiK selectively inhibited signaling pathways leading to the activation of the transcription factor NF-κB and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor IκB-α is critically involved in the observed inhibition of NF-κB translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-κB and subsequent cytokine production is not restricted to in vitro-generated monocyte-derived macrophages but also can be observed in primary cells. Thus, MaxiK appears to be a central molecule in the NF-κB-dependent inflammatory response of macrophages to bacterial LPS.

Cross-linking of surface immunoglobulin activates src-related tyrosine kinases in WEHI 231 cells

Crosslinking of sIgM on the B cell line WEHI 231 with anti-sIgM antibody induces protein tyrosine phosphorylation, implicating protein tyrosine kinases (PTKs) in sIg-mediated signal transduction. We have analyzed this cell line for members of the src family of PTKs and have evaluated whether these PTKs might be involved in the process of sIgM-mediated signaling. Our results show that Blk, Lyn, Lck, and Hck are detectable in WEHI 231 cells. Addition of antibodies to sIgM were found to variably stimulate the activities of Blk, Lyn, Lck, and Hck as measured by immune-complex protein kinase assays. Autophosphorylation of these src PTKs, as assessed by reaction with anti-phosphotyrosine antibodies, increased over the time course of sIgM-mediated activation. Co-immunoprecipitation studies to investigate the potential physical interaction of src PTKs with the sIgM receptor complex revealed that, under digitonin and Brij 96 lysis conditions Lyn, Lck, Hck, but not Blk associated with sIgM.

TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

During apoptosis induction by TNF, the extrinsic and intrinsic apoptosis pathways converge at the lysosomal-mitochondrial interface. Earlier studies showed that the lysosomal aspartic protease Cathepsin D (CtsD) cleaves Bid to tBid, resulting in the amplification of the initial apoptotic cascade via mitochondrial outer membrane permeabilization (MOMP). The goal of this study was to identify further targets for CtsD that might be involved in activation upon death receptor ligation. Using a proteomics screen, we identified the heat shock protein 90 (HSP90) to be cleaved by CtsD after stimulation of U937 or other cell lines with TNF, FasL and TRAIL. HSP90 cleavage corresponded to apoptosis sensitivity of the cell lines to the different stimuli. After mutation of the cleavage site, HSP90 partially prevented apoptosis induction in U937 and Jurkat cells. Overexpression of the cleavage fragments in U937 and Jurkat cells showed no effect on apoptosis, excluding a direct pro-apoptotic function of these fragments. Pharmacological inhibition of HSP90 with 17AAG boosted ligand mediated apoptosis by enhancing Bid cleavage and caspase-9 activation. Together, we demonstrated that HSP90 plays an anti-apoptotic role in death receptor signalling and that CtsD-mediated cleavage of HSP90 sensitizes cells for apoptosis. These findings identify HSP90 as a potential target for cancer therapy in combination with death ligands (e.g. TNF or TRAIL).

Expression of non-secreted IL-4 is associated with HDAC inhibitor-induced cell death, histone acetylation and c-Jun regulation in human gamma/delta T-cells

Previously, the expression of a non-secreted IL-4 variant (IL-4δ13) has been described in association with apoptosis and age-dependent Th2 T-cell polarization. Signaling pathways involved in this process have so far not been studied. Here we report the induction of IL-4δ13 expression in human γδ T-cells upon treatment with a sublethal dose of histone deacetylase (HDACi) inhibitor valproic acid (VPA). Induction of IL-4δ13 was associated with increased cytoplasmic IL-4Rα and decreased IL-4 expression, while mRNA for mature IL-4 was concomitantly down-regulated. Importantly, only the simultaneous combination of apoptosis and necroptosis inhibitors prevented IL-4δ13 expression and completely abrogated VPA-induced global histone H3K9 acetylation mark. Further, our work reveals a novel involvement of transcription factor c-Jun in the signaling network of IL-4, HDAC1, caspase-3 and mixed lineage kinase domain-like protein (MLKL). This study provides novel insights into the effects of epigenetic modulator VPA on human γδ T-cell differentiation.