RESUMEN
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-kappaB 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-kappaB 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.
Asunto(s)
Caspasas/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Glicoproteínas de Membrana/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Caspasa 3 , Caspasa 8 , Caspasa 9 , Línea Celular Transformada , Células Cultivadas , Cisteína Endopeptidasas , Humanos , Queratinocitos/citología , Leupeptinas/farmacología , Mitocondrias/metabolismo , Modelos Biológicos , FN-kappa B/metabolismo , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismo , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) exerts a potent cytotoxic activity especially against many tumor cell types such as transformed keratinocytes. The specific role of the different TRAIL receptors in this process, however, is unknown. In this report we examine the role the TRAIL receptors play in both the apoptotic and nonapoptotic responses of HaCaT keratinocytes to leucine zipper TRAIL (LZ-TRAIL). By employing receptor-specific blocking antibodies we demonstrate that TRAIL receptor 1 plays the primary role in mediating caspase activation and apoptosis in HaCaT cells. Furthermore, we show that this receptor mainly mediates nuclear factor kappaB activation and expression of the pro-inflammatory cytokine interleukin-8 and that nuclear factor kappaB activation is critically required for the induction of pro-inflammatory cytokines in response to LZ-TRAIL. Taken together, our data suggest that beside its potent pro-apoptotic role, LZ-TRAIL leads to pro-inflammatory responses that are mainly mediated by TRAIL receptor 1 in HaCaT keratinocytes.
Asunto(s)
Apoptosis/inmunología , Queratinocitos/citología , Glicoproteínas de Membrana/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Reguladoras de la Apoptosis , Línea Celular Transformada , Quimiotaxis/fisiología , Dermatitis/inmunología , Dermatitis/metabolismo , Dermatitis/fisiopatología , Regulación de la Expresión Génica , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-8/genética , Interleucina-8/metabolismo , Queratinocitos/inmunología , Queratinocitos/metabolismo , Leucina Zippers/fisiología , Glicoproteínas de Membrana/genética , FN-kappa B/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Sialoglicoproteínas/genética , Transducción de Señal/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Activación Transcripcional , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Death ligands not only activate a death program but also regulate inflammatory signalling pathways, for example, through NF-kappaB induction. Although tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF both activate NF-kappaB in human keratinocytes, only TRAIL potently induces apoptosis. However, when induction of NF-kappaB was inhibited with a kinase dead IKK2 mutant (IKK2-KD), TNF- but not TRAIL-induced apoptosis was dramatically enhanced. Acquired susceptibility to TNF-induced apoptosis was due to increased caspase-8 activation. To investigate the mechanism of resistance of HaCaT keratinocytes to TNF-induced apoptosis, we analyzed a panel of NF-kappaB-regulated effector molecules. Interestingly, the inhibitor of apoptosis protein (IAP) family member cIAP2, but not cIAP1, X-linked inhibitor of apoptosis, TNF receptor-associated factor (TRAF)-1, or TRAF2, was downregulated in sensitive but not in resistant HaCaT keratinocytes. Surprisingly, however, stable inducible expression of cIAP2 was not sufficient to render IKK2-KD-sensitized keratinocytes resistant to TNF, and reduction of cIAP2 alone did not increase the sensitivity of HaCaT keratinocytes to TNF. In conclusion, we demonstrate that inhibition of NF-kappaB dramatically sensitizes human keratinocytes to TNF- but not to TRAIL-induced apoptosis and that this sensitization for TNF was largely independent of cIAP2. Our data thus clearly exclude the candidates proposed to date to confer TNF apoptosis resistance and suggest the function of an unanticipated effector of NF-kappaB critical for the survival of HaCaT keratinocytes upstream or at the level of caspase-8 activation.
Asunto(s)
Caspasa 8/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Queratinocitos/metabolismo , FN-kappa B/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis/fisiología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular Transformada , Supervivencia Celular/fisiología , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Queratinocitos/citología , Mutagénesis , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/fisiología , Factor 1 Asociado a Receptor de TNF/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Morbus Köhlmeier-Degos is a rare systemic disease characterized by pathognomonic cutaneous lesions with typical histology. We report the case of a 22-year-old woman with a benign course of this disease and discuss therapeutical options. The patient presented with whitish papules on neck, trunk and extremities that slowly developed within seven months. Examination of the skin revealed about 20 papules of 2-5 mm size. The lesions were partly skin-colored, partly with an erythematous rim and showed a central porcelain-like atrophy. Histology showed an interface-dermatitis and a wedge-shaped mucin deposition with sclerosis of the upper reticular dermis. Organ manifestation was absent. The patient was started on a daily therapy of 300 mg acetyl salicylic acid. Over the course of 24 months single new lesions appeared at a reduced frequency. Apart from the malignant form of Morbus Köhlmeier-Degos there exists a benign course which may be successfully controlled with anti-platelet therapy. We speculate that the lack of vessel occlusion in the histology could be a hallmark of a benign form of this rare disease.
Asunto(s)
Papulosis Atrófica Maligna/clasificación , Papulosis Atrófica Maligna/diagnóstico , Diagnóstico Diferencial , Femenino , Humanos , Pronóstico , Enfermedades Raras/clasificación , Enfermedades Raras/diagnóstico , Adulto JovenRESUMEN
Beta1-integrin protects keratinocyte stem cells (KSC) from cell-detachment apoptosis ('anoikis'). Here we show that caspase-8 active protein is detected in both young transit amplifying (TA) cells and TA cells, but not in KSC. On suspension, caspases are activated earlier in young TA than in KSC, whereas anti-beta1-integrin neutralizing antibody accelerates caspase activation in both KSC and young TA. Caspases 8 and 10 are the first caspases to be activated whereas caspase-8 inhibitor zIETD-fmk delays the activation of Bid, caspase-9 and caspase-3. However, the caspase-9 inhibitor zLEDH-fmk does not block the activation of caspase-8, Bid, caspase-10 and caspase-3. Moreover, caspase-8, but not caspase-9 inhibitor partially prevents keratinocyte anoikis. As FLIP inhibits caspase-8 processing, we retrovirally infected HaCaT keratinocytes with c-FLIP(L). Anti-beta1-integrin fails to activate caspase-8, Bid, caspase-9 and to induce the release of cytochrome c in c-FLIP(L) overexpressing keratinocytes. Finally, overexpression of c-FLIP(L) partially prevents anoikis in both suspended and anti-beta1 integrin-treated cells. Taken together, these results indicate that the extrinsic apoptotic pathway triggered by caspase-8 predominates in keratinocyte anoikis. However, the release of cytochrome c and the later activation of caspase-9 seem to suggest that the intrinsic mitochondrial pathway may intervene as a positive feedback loop of caspase activation.
Asunto(s)
Caspasas/metabolismo , Integrina beta1/metabolismo , Queratinocitos/citología , Anoicis , Proteínas Reguladoras de la Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/metabolismo , Caspasa 10 , Caspasa 3 , Caspasa 8 , Caspasa 9 , Muerte Celular , Línea Celular , Células Cultivadas , Citosol/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Queratinocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocondrias/metabolismo , Retroviridae/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Low dose UVB irradiation of dendritic cells (DC) dose-dependently decreases their allostimulatory capacity and inhibits alloreactive T cell proliferation. The reduction of the stimulatory capacity is not associated with a perturbation of CD28 costimulation. To examine the underlying mechanism, cell cycle analysis of T cells from cocultures with UVB-irradiated DC (UVB-DC) was performed, revealing no cell cycle arrest, but an increased number of apoptotic T cells in sub-G(0) phase. We confirmed T cells to undergo apoptosis after coincubation with UVB-DC by TUNEL staining and DNA laddering. To analyze whether T cell apoptosis requires the Fas/Fas ligand (FasL) pathway, MLRs were performed with Fas-, FasL-deficient, and wild-type DC and T cells. No differences were found on comparison of wild-type DC with Fas-/FasL-deficient DC or T cells. Likewise, addition of a neutralizing anti-TNF-alpha mAb to cocultures could not overcome inhibition of T cell proliferation by UVB-DC, excluding involvement of the TNF-alpha/TNF-alphaR pathway. FACS analysis of CD69 and CD25 revealed no up-regulation on T cells cocultured with UVB-DC, suggesting a perturbation of early T cell activation. Analysis of UVB-DC by confocal microscopy demonstrated impaired filamentous actin bundling, a process critical for T cell stimulation. To investigate the functional relevance of these observations, time lapse video microscopy was performed. Indeed, calcium signaling in CD4(+) T cells was significantly diminished after interaction with UVB-DC. In conclusion, UVBR of DC impairs their cytoskeletal rearrangement and induces apoptosis in CD4(+) T cells by disruption of early DC-T cell interaction, resulting in a reduced Ca(2+) influx in T cells.
Asunto(s)
Apoptosis/inmunología , Apoptosis/efectos de la radiación , Linfocitos T CD4-Positivos/citología , Citoesqueleto/metabolismo , Citoesqueleto/efectos de la radiación , Células Dendríticas/inmunología , Células Dendríticas/efectos de la radiación , Rayos Ultravioleta , Animales , Antígeno B7-1/farmacología , Antígenos CD28/farmacología , Linfocitos T CD4-Positivos/inmunología , Comunicación Celular/inmunología , Comunicación Celular/efectos de la radiación , División Celular/inmunología , División Celular/efectos de la radiación , Células Cultivadas , Células Dendríticas/metabolismo , Relación Dosis-Respuesta en la Radiación , Proteína Ligando Fas , Ligandos , Activación de Linfocitos/efectos de la radiación , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Transgénicos , Transducción de Señal/inmunología , Transducción de Señal/efectos de la radiación , Receptor fas/fisiologíaRESUMEN
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-kappaB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIP(L) interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIP(L) in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIP(L). Functional analysis revealed that relative cFLIP(L) levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIP(L) specifically blocked TRAIL-induced NF-kappaB 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 cFLIP(L)-overexpressing keratinocytes, although cFLIP(L) did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIP(L)-overexpressing cells. Taken together, our data demonstrate that cFLIP(L) is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-kappaB activation and subsequent proinflammatory target gene expression. Hence, cFLIP(L) modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation.