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Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis.

Kumari S, Redouane Y, Lopez-Mosqueda J, Shiraishi R, Romanowska M, Lutzmayer S, Kuiper J, Martinez C, Dikic I, Pasparakis M, Ikeda F - Elife (2014)

Bottom Line: Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation.Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role.Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Center for Molecular Medicine, University of Cologne, Cologne, Germany.

ABSTRACT
Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

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Fas-associated protein with death domain (FADD) plays an important role in the Sharpin-dependent apoptosis signaling.(A) Immunoblot of FADD after stable knocked down of FADD in wild type (+/+) mouse embryonic fibroblasts (MEFs) and Sharpincpdm/cpdm MEFs by shRNA. α-Vinculin antibody was used for the loading control. (B) Fluorescence-activated cell sorting (FACS) analysis of annexin V positive cells after stimulation with tumor necrosis factor (TNF) + cycloheximide (CHX) for 4 hr in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs, and Sharpincpdm/cpdm shFADD MEFs. (C) Caspase-8 activity measurement upon stimulation with TNF with or without CHX for the indicated time in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs and Sharpincpdm/cpdm shFADD MEFs. Results are expressed as mean values ± SD. Statistical significance was determined using ANOVA test (****p ≤ 0.0001).DOI:http://dx.doi.org/10.7554/eLife.03422.017
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fig9s1: Fas-associated protein with death domain (FADD) plays an important role in the Sharpin-dependent apoptosis signaling.(A) Immunoblot of FADD after stable knocked down of FADD in wild type (+/+) mouse embryonic fibroblasts (MEFs) and Sharpincpdm/cpdm MEFs by shRNA. α-Vinculin antibody was used for the loading control. (B) Fluorescence-activated cell sorting (FACS) analysis of annexin V positive cells after stimulation with tumor necrosis factor (TNF) + cycloheximide (CHX) for 4 hr in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs, and Sharpincpdm/cpdm shFADD MEFs. (C) Caspase-8 activity measurement upon stimulation with TNF with or without CHX for the indicated time in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs and Sharpincpdm/cpdm shFADD MEFs. Results are expressed as mean values ± SD. Statistical significance was determined using ANOVA test (****p ≤ 0.0001).DOI:http://dx.doi.org/10.7554/eLife.03422.017

Mentions: As keratinocyte apoptosis and skin inflammation in Sharpincpdm/cpdm mice were suppressed by epidermal-specific deletion of FADD or TRADD, we sought to investigate how the lack of FADD, TRADD, and RIPK3 proteins impact on TNF-induced cell viability in Sharpin-deficient cells. To address this, primary keratinocytes were isolated from newborn pups, Sharpincpdm/cpdm, Sharpincpdm/cpdm;Ripk3−/−, Sharpincpdm/cpdm;FADDE-KO;Ripk3−/−, and their control littermates, Sharpincpdm/wt or Sharpinwt/wtSharpincpdm/wt;Ripk3−/− and Sharpincpdm/wt;FADDE-KO;Ripk3−/−, respectively. Cells were treated with increasing concentrations of TNF alone or TNF + CHX for 24 hr and their viability was analyzed using the WST-1 assay (Figure 9A). Although TNF + CHX treatment strongly induced the death of Sharpin-deficient keratinocytes, TNF treatment alone had a very small effect in reducing the viability of Sharpin-deficient keratinocytes by about 10% compared with controls. Interestingly, the combined lack of FADD and RIPK3 in Sharpincpdm/cpdm;FADDE-KO;Ripk3−/− keratinocytes fully rescued the increased sensitivity of Sharpin-deficient keratinocytes to TNF + CHX (Figure 9A). However, keratinocytes obtained from Sharpincpdm/cpdm;Faddfl/fl;Ripk3−/− showed a similar response to TNF + CHX as Sharpincpdm/cpdm keratinocytes, demonstrating that RIPK3 deficiency does not prevent the death of Sharpin-deficient keratinocytes. Therefore, Sharpin deficiency primarily sensitizes keratinocytes to FADD-mediated apoptosis and not to RIPK3-mediated necroptosis. To further examine a direct role of FADD in Sharpin-deficient cells without involvement of RIPK3, we generated HaCaT cells in which Sharpin and FADD were both stably knocked down by shRNA (Figure 9B). Upon treatment with TNF alone or TNF + CHX, HaCaT cells lacking both Sharpin and FADD showed reduced caspase-8 activity compared with Sharpin-deficient HaCaT cells (Figure 9C,D). Similar to the keratinocytes, we generated FADD-deficient Sharpincpdm/cpdm MEFs and analyzed the effect of FADD deficiency on apoptosis induced by TNF alone and by TNF + CHX in Sharpincpdm/cpdm MEFs (Figure 9—figure supplement 1A) and observed that FADD deficiency significantly suppressed the annexin V positive cells and caspase-8 activity in Sharpincpdm/cpdm MEFs (Figure 9—figure supplement 1B and C). To address an involvement of TRADD in TNF-induced sensitivity of Sharpin-deficient HaCaT cells, we used HaCaT cells which were knockdown for Sharpin and TRADD expression. In comparison to caspase-8 activity induced by TNF alone or TNF + CHX in Sharpin-deficient HaCaT cells, TRADD deficiency significantly suppressed caspase-8 activation (Figure 9E–G). These data collectively suggest that regulation of Sharpin-dependent anti-apoptosis signaling depends on FADD and TRADD in a cell-intrinsic manner.10.7554/eLife.03422.016Figure 9.Fas-associated protein with death domain (FADD)- and tumor necrosis factor receptor 1-associated death domain (TRADD)-dependent enhanced sensitivity of Sharpin-deficient keratinocytes to tumor necrosis factor (TNF)-induced apoptosis.


Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis.

Kumari S, Redouane Y, Lopez-Mosqueda J, Shiraishi R, Romanowska M, Lutzmayer S, Kuiper J, Martinez C, Dikic I, Pasparakis M, Ikeda F - Elife (2014)

Fas-associated protein with death domain (FADD) plays an important role in the Sharpin-dependent apoptosis signaling.(A) Immunoblot of FADD after stable knocked down of FADD in wild type (+/+) mouse embryonic fibroblasts (MEFs) and Sharpincpdm/cpdm MEFs by shRNA. α-Vinculin antibody was used for the loading control. (B) Fluorescence-activated cell sorting (FACS) analysis of annexin V positive cells after stimulation with tumor necrosis factor (TNF) + cycloheximide (CHX) for 4 hr in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs, and Sharpincpdm/cpdm shFADD MEFs. (C) Caspase-8 activity measurement upon stimulation with TNF with or without CHX for the indicated time in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs and Sharpincpdm/cpdm shFADD MEFs. Results are expressed as mean values ± SD. Statistical significance was determined using ANOVA test (****p ≤ 0.0001).DOI:http://dx.doi.org/10.7554/eLife.03422.017
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fig9s1: Fas-associated protein with death domain (FADD) plays an important role in the Sharpin-dependent apoptosis signaling.(A) Immunoblot of FADD after stable knocked down of FADD in wild type (+/+) mouse embryonic fibroblasts (MEFs) and Sharpincpdm/cpdm MEFs by shRNA. α-Vinculin antibody was used for the loading control. (B) Fluorescence-activated cell sorting (FACS) analysis of annexin V positive cells after stimulation with tumor necrosis factor (TNF) + cycloheximide (CHX) for 4 hr in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs, and Sharpincpdm/cpdm shFADD MEFs. (C) Caspase-8 activity measurement upon stimulation with TNF with or without CHX for the indicated time in +/+ shCtr MEFs, +/+ shFADD MEFs, Sharpincpdm/cpdm shCtr MEFs and Sharpincpdm/cpdm shFADD MEFs. Results are expressed as mean values ± SD. Statistical significance was determined using ANOVA test (****p ≤ 0.0001).DOI:http://dx.doi.org/10.7554/eLife.03422.017
Mentions: As keratinocyte apoptosis and skin inflammation in Sharpincpdm/cpdm mice were suppressed by epidermal-specific deletion of FADD or TRADD, we sought to investigate how the lack of FADD, TRADD, and RIPK3 proteins impact on TNF-induced cell viability in Sharpin-deficient cells. To address this, primary keratinocytes were isolated from newborn pups, Sharpincpdm/cpdm, Sharpincpdm/cpdm;Ripk3−/−, Sharpincpdm/cpdm;FADDE-KO;Ripk3−/−, and their control littermates, Sharpincpdm/wt or Sharpinwt/wtSharpincpdm/wt;Ripk3−/− and Sharpincpdm/wt;FADDE-KO;Ripk3−/−, respectively. Cells were treated with increasing concentrations of TNF alone or TNF + CHX for 24 hr and their viability was analyzed using the WST-1 assay (Figure 9A). Although TNF + CHX treatment strongly induced the death of Sharpin-deficient keratinocytes, TNF treatment alone had a very small effect in reducing the viability of Sharpin-deficient keratinocytes by about 10% compared with controls. Interestingly, the combined lack of FADD and RIPK3 in Sharpincpdm/cpdm;FADDE-KO;Ripk3−/− keratinocytes fully rescued the increased sensitivity of Sharpin-deficient keratinocytes to TNF + CHX (Figure 9A). However, keratinocytes obtained from Sharpincpdm/cpdm;Faddfl/fl;Ripk3−/− showed a similar response to TNF + CHX as Sharpincpdm/cpdm keratinocytes, demonstrating that RIPK3 deficiency does not prevent the death of Sharpin-deficient keratinocytes. Therefore, Sharpin deficiency primarily sensitizes keratinocytes to FADD-mediated apoptosis and not to RIPK3-mediated necroptosis. To further examine a direct role of FADD in Sharpin-deficient cells without involvement of RIPK3, we generated HaCaT cells in which Sharpin and FADD were both stably knocked down by shRNA (Figure 9B). Upon treatment with TNF alone or TNF + CHX, HaCaT cells lacking both Sharpin and FADD showed reduced caspase-8 activity compared with Sharpin-deficient HaCaT cells (Figure 9C,D). Similar to the keratinocytes, we generated FADD-deficient Sharpincpdm/cpdm MEFs and analyzed the effect of FADD deficiency on apoptosis induced by TNF alone and by TNF + CHX in Sharpincpdm/cpdm MEFs (Figure 9—figure supplement 1A) and observed that FADD deficiency significantly suppressed the annexin V positive cells and caspase-8 activity in Sharpincpdm/cpdm MEFs (Figure 9—figure supplement 1B and C). To address an involvement of TRADD in TNF-induced sensitivity of Sharpin-deficient HaCaT cells, we used HaCaT cells which were knockdown for Sharpin and TRADD expression. In comparison to caspase-8 activity induced by TNF alone or TNF + CHX in Sharpin-deficient HaCaT cells, TRADD deficiency significantly suppressed caspase-8 activation (Figure 9E–G). These data collectively suggest that regulation of Sharpin-dependent anti-apoptosis signaling depends on FADD and TRADD in a cell-intrinsic manner.10.7554/eLife.03422.016Figure 9.Fas-associated protein with death domain (FADD)- and tumor necrosis factor receptor 1-associated death domain (TRADD)-dependent enhanced sensitivity of Sharpin-deficient keratinocytes to tumor necrosis factor (TNF)-induced apoptosis.

Bottom Line: Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation.Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role.Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Center for Molecular Medicine, University of Cologne, Cologne, Germany.

ABSTRACT
Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

Show MeSH
Related in: MedlinePlus