Tina Wachter

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.

cFLIPL Inhibits Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated NF-κB Activation at the Death-inducing Signaling Complex in Human Keratinocytes

Human keratinocytes undergo apoptosis following treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via surface-expressed TRAIL receptors 1 and 2. In addition, TRAIL triggers nonapoptotic signaling pathways including activation of the transcription factor NF-κB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIPL interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIPL in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIPL. Functional analysis revealed that relative cFLIPL levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIPL specifically blocked TRAIL-induced NF-κB activation and TRAIL-dependent induction of the proinflammatory target gene interleukin-8. Biochemical characterization of the signaling pathways involved showed that apoptosis signaling was inhibited at the DISC in cFLIPL-overexpressing keratinocytes, although cFLIPL did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIPL-overexpressing cells. Taken together, our data demonstrate that cFLIPL is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-κB activation and subsequent proinflammatory target gene expression. Hence, cFLIPL modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation.

cFLIPL inhibits TNF-related apoptosis-inducing ligand-mediated NF-κB activation at the death inducing signalling complex (DISC) in human keratinocytes

Human keratinocytes undergo apoptosis following treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via surface-expressed TRAIL receptors 1 and 2. In addition, TRAIL triggers nonapoptotic signaling pathways including activation of the transcription factor NF-κB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIPL interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIPL in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIPL. Functional analysis revealed that relative cFLIPL levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIPL specifically blocked TRAIL-induced NF-κB activation and TRAIL-dependent induction of the proinflammatory target gene interleukin-8. Biochemical characterization of the signaling pathways involved showed that apoptosis signaling was inhibited at the DISC in cFLIPL-overexpressing keratinocytes, although cFLIPL did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIPL-overexpressing cells. Taken together, our data demonstrate that cFLIPL is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-κB activation and subsequent proinflammatory target gene expression. Hence, cFLIPL modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation.

[What's your diagnosis? Lichenification of flexures, tetraspasm and mental retardation].

DOI 10.1007/s00105-005-1060-0 Online publiziert: 19. Januar 2006

Lichenifikation der großen Beugen, Tetraspastik und mentale Retardierung@@@Lichenification of flexures, tetraspasm and mental retardation

DOI 10.1007/s00105-005-1060-0 Online publiziert: 19. Januar 2006

Lichenifikation der großen Beugen, Tetraspastik und mentale Retardierung

DOI 10.1007/s00105-005-1060-0 Online publiziert: 19. Januar 2006