Manfred Neumann

Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation

ABSTRACT Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-κB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-κB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

Anoxia-Induced Up-Regulation of Interleukin-8 in Human Malignant Melanoma : A Potential Mechanism for High Tumor Aggressiveness

Besides its proinflammatory properties, interleukin-8 (IL-8) has been suggested as an important promoter for melanoma growth. To study the role of IL-8 in melanoma biology, we determined the in vivo expression of IL-8 mRNA by in situ hybridization in primary melanoma lesions and metastases. High levels of melanoma cell-associated IL-8-specific transcripts were exclusively detected in close vicinity of necrotic/hypoxic areas of melanoma metastases, whereas both in primary melanomas and in non-necrotic metastases IL-8 expression was low or absent. To analyze further the up-regulation of IL-8 mRNA expression in necrotic/hypoxic tumor areas, human melanoma cell lines of different aggressiveness exposed to severe hypoxic stress (anoxia) were used as an in vitro model. Anoxia induced IL-8 mRNA and protein expression in the highly aggressive/metastatic cell lines MV3 and BLM but not in the low aggressive cell lines IF6 and 530. As shown by IL-8 promoter-dependent reporter gene analysis and mRNA stability assays, elevated mRNA levels in melanoma cells were due to both enhanced transcriptional activation and enhanced IL-8 mRNA stability. Interestingly, transcriptional activation was abolished by mutations in the AP-1 and the NF-kappaB-like binding motifs, indicating that both sites are critical for IL-8 induction. Concomitantly, anoxia induced an enhanced binding activity of AP-1 and NF-kappaB transcription factors only in the highly aggressive cells. From our in vitro and in vivo data we suggest that anoxia-induced regulation of IL-8 might be a characteristic feature of aggressive tumor cells, thus indicating that IL-8 might play a critical role for tumor progression in human malignant melanoma.

Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-κB control

RelB is an unusual member of the Rel/NF-κB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IκBs, the cytosolic NF-κB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IκBs. However, RelB degradation differs from that of IκBs since it is not induced by TNFα but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelBDM, site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-κB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-κB activity.

Cellular FLICE-inhibitory Protein (cFLIP) Isoforms Block CD95- and TRAIL Death Receptor-induced Gene Induction Irrespective of Processing of Caspase-8 or cFLIP in the Death-inducing Signaling Complex

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory “non-apoptotic” signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIPS, cFLIPL, or mutants of cFLIPL (cFLIPD376N and cFLIPp43). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIPL induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIPS or cFLIPp43 blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.

Gene Transfer of Tissue Inhibitor of Metalloproteinases-3 Reverses the Inhibitory Effects of TNF-α on Fas-Induced Apoptosis in Rheumatoid Arthritis Synovial Fibroblasts

Apart from counteracting matrix metalloproteinases, tissue inhibitor of metalloproteinases-3 (TIMP-3) has proapoptotic properties. These features have been attributed to the inhibition of metalloproteinases involved in the shedding of cell surface receptors such as the TNFR. However, little is known about effects of TIMP-3 in cells that are not susceptible to apoptosis by TNF-α. In this study, we report that gene transfer of TIMP-3 into human rheumatoid arthritis synovial fibroblasts and MRC-5 human fetal lung fibroblasts facilitates apoptosis and completely reverses the apoptosis-inhibiting effects of TNF-α. Although TNF-α inhibits Fas/CD95-induced apoptosis in untransfected and mock-transfected cells, fibroblasts ectopically expressing TIMP-3 are sensitized most strongly to Fas/CD95-mediated cell death by TNF-α. Neither synthetic MMP inhibitors nor glycosylated bioactive TIMP-3 are able to achieve these effects. Gene transfer of TIMP-3 inhibits the TNF-α-induced activation of NF-κB in rheumatoid arthritis synovial fibroblasts and reduces the up-regulation of soluble Fas/CD95 by TNF-α, but has no effects on the cell surface expression of Fas. Collectively, our data demonstrate that intracellularly produced TIMP-3 not only induces apoptosis, but also modulates the apoptosis-inhibiting effects of TNF-α in human rheumatoid arthritis synovial fibroblast-like cells. Thus, our findings may stimulate further studies on the therapeutic potential of gene transfer strategies with TIMP-3.

The PAK1 autoregulatory domain is required for interaction with NIK in Helicobacter pylori-induced NF-κB activation

Abstract Helicobacter pylori, the etiological agent of various human gastric diseases, induces the transcription factor nuclear factor κB (NF-κB) and proinflammatory cytokines/chemokines. We have characterised the direct interaction between p21-activated kinase 1 (PAK1) and NF-κB-inducing kinase (NIK) in H. pylori-infected epithelial cells. The dimerisation (DI) motif, which is part of the NH2-terminal autoregulatory domain of PAK1, is critical for this interaction, whereas NIK forms complexes with PAK1 through its carboxy-terminal IκB kinase α (IKKα) binding site. Since the identified interaction sites are also crucial for the binding of activator (Rac/Cdc42 in the case of PAK1) or effector molecules (IKKα in the case of NIK), sequential stepwise signalling is suggested. Furthermore, we show that mitogen-activated protein kinase kinase kinases (MAP3K), like TPL2 (tumour progression locus 2) and transforming growth factor β-activated kinase 1 (TAK1), have no impact on H. pylori-induced activation of NF-κB. These results identify the roles of PAK1 and NIK in a unique pathway involved in H. pylori-induced NF-κB activation, which is crucial for the induction of the innate immune response.

Synthesis of acetal-αḡlucosides. A stereoselective entry into a new class of compounds

Abstract Acetal-glycosides are a new class of compounds, which became of special interest as enzyme inhibitors and cytostatics for the treatment of cancer. In a highly stereoselective glucosidation, acetyl-protected acetal-α-glucosides such as methoxymethyl 2,3,4,6-tetra- O -acetal-α- d -glucopyranoside ( 5a ) were obtained in 60–80% yield by treatment of 2,3,4,6-tetra- O -acetyl-1- O -trimethylsilyl-α- d -glucopyranose ( 1 ) with acetals, e.g. dimethoxymethane ( 3a ) in the presence of a catalytic amount of trimethylsilyl trifluoromethanesulfonate ( 4 ) at −70°> The glucosidation involves a new principle, where the reaction does not take place at the anomeric carbon, but with retention at the oxygen of the trimethylsilyloxy group on C-1. The trimethylsilyl derivative 1 may be used as a pure anomer or prepared by in situ anomerisation from a mixture of 1 and its β anomer 26 . The yields in the glucosidation can be increased by the addition of acetone or the aldehydes that correspond to the acetals (except formaldehyde). In a one pot synthesis, the reaction of phenylacetaldehyde ( 23e ), trimethylsilyl methyl ether ( 14a ), and 1 at −70° in the presence of 4 led directly to 1-methoxy-2-phenylethyl 2,3,4,6-tetra- O -acetyl-α- d -glucopyranoside ( 5e ). O -Deacetylation of the obtained acetyl-protected acetal-α-glucosides gave the free acetal-α-glucosides in excellent yield. Thus 5e afforded 1-methoxy-2-phenylethyl α- d -glucopyranoside ( 6e ). Since the acetal-α-glucosides can be cleaved by enzymic hydrolysis to the corresponding aldehydes under mild conditions, glucose may be used as a protective group for the CHO function. Similarly, 2,3,4,6,-tetra- O -benzyl-1- O -trimethylsilyl-α- d -glucopyranose ( 2 ) was used to afford benzyl-protected acetal-α-glucosides.

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor–inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)–induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L–CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L–CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2–cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L–CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.

TNF Receptor-Associated Factor 1 is a Major Target of Soluble TWEAK.

Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB pathway using the IKK2 inhibitor, TPCA1. In contrast, in some cell lines, TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924, however, which inhibits classical and alternative NFκB signaling, blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NFκB signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction, but potentiated the ability of soluble TWEAK to trigger production of the classical NFκB-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NFκB pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets, which can be equally well induced by soluble and membrane TWEAK, remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies.