Caspase-1 Activity Is Required for UVB-Induced Apoptosis of Human Keratinocytes  Gabriel Sollberger, Gerhard E. Strittmatter, Serena Grossi, Martha Garstkiewicz, Ulrich auf dem Keller, Lars E. French, Hans-Dietmar Beer  Journal of Investigative Dermatology  Volume 135, Issue 5, Pages 1395-1404 (May 2015) DOI: 10.1038/jid.2014.551 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 UVB-induced inflammasome activation and apoptosis are temporally separated. Human primary keratinocytes were irradiated with UVB or mock-treated and analyzed at the indicated time points. (a) Western blots of lysate and supernatant for analysis of expression and release of the indicated proteins. The supernatants were concentrated by acetone precipitation. The asterisk indicates the specific band. (b) Percentage of IL-1β release determined by ELISA measurement of the cytokine in the supernatant and the lysate. (c) Percentage of lactate dehydrogenase activity in the supernatant reflecting cell lysis. (d) Percentage of cells with surface Annexin V-binding indicating apoptosis determined by flow cytometry. (e) Percentage of cells positive for cleaved caspase-3 reflecting apoptosis. Keratinocytes were fixed with PFA, stained with an antibody against cleaved caspase-3, followed by a fluorescent secondary antibody, and counted. Nuclei were stained with Hoechst. (b–e) Error bars represent the mean±SD of a representative experiment performed in triplicates. PFA, paraformaldehyde. Journal of Investigative Dermatology 2015 135, 1395-1404DOI: (10.1038/jid.2014.551) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Caspase-1 expression is required for UVB-induced apoptosis. (a–d) Human primary keratinocytes were transfected with siRNA against caspase-1. Scrambled siRNA (ctr.) and a knock down of caspase-5 expression served as controls. After 48 hours, the cells were UVB-irradiated and analyzed 24 hours later (a–c). (a) Cells were fixed and stained with phalloidin (binds to actin) and Hoechst (stains DNA, indicating nuclei). Bar=100μm. (b) Percentage of cells positive for cleaved caspase-3 indicating apoptosis, determined by flow cytometry. Western blots for expression of caspase-1, cleavage of caspase-3, and β-actin, the latter served as loading control. (c and d) Percentage of cells that lost their mitochondrial membrane potential, determined by flow cytometry. (d) Keratinocytes were treated with staurosporine (0.5μM) and analyzed 24 hours later. (e) THP-1 cells with a stable shRNA-mediated knock down of Lamin or caspase-1 expression (Sollberger et al., 2012), which were differentiated with 25 ng ml−1 TPA overnight, were irradiated with UVB. Six hours later, the percentage of cells positive for cleaved caspase-3 was determined by flow cytometry. (b–e) Error bars represent the mean±SD of a representative experiment performed in triplicates. shRNA, short hairpin RNA; siRNA, small interfering RNA. Journal of Investigative Dermatology 2015 135, 1395-1404DOI: (10.1038/jid.2014.551) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Caspase-1 expression is not required for UVB-induced apoptosis in mice. (a) Keratinocytes were isolated from mice lacking expression of caspase-1 and from wild-type controls. After seeding, the cells were grown to 80–90% confluency, irradiated with UVB, and 24 hours later the percentage of apoptotic cells positive for cleaved caspase-3 was determined by flow cytometry. (b) Mice lacking expression of caspase-1/caspase-11 and wild-type controls were shaved, anesthetized, and UVB-irradiated. Twenty-four hours after irradiation, the skin was isolated, quick-frozen, and stained with an antibody against cleaved caspase-3. The number of positive cells per mm epidermis was counted from n=6 mice. (c–f) Human epidermis was incubated in a medium (DMEM) with DMSO (0.1%) (c and d) or with the caspase-1 inhibitor YVAD (100μM, added 1 hour before irradiation and 8 hours after irradiation with a medium change) (e) and irradiated with UVB (d and e) or mock-treated (c). After 24 hours, the skin was quick-frozen and stained with an antibody against cleaved caspase-3, followed by a fluorescent secondary antibody. Nuclei were stained with Hoechst. The dashed line represents the basal membrane separating the epidermis from the dermis. Bar=100μm. (f) Cells positive for cleaved caspase-3 were quantified by counting; strong and intermediate/low fluorescence signals are separately shown. Error bars represent the mean±SD. The P-value in b was 0.132 as calculated by the Mann–Whitney test. YVAD, Tyr-Val-Ala-Asp. Journal of Investigative Dermatology 2015 135, 1395-1404DOI: (10.1038/jid.2014.551) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Caspase-4 but not other inflammasome proteins supports UVB-induced apoptosis. (a–e) Human primary keratinocytes were transfected with siRNAs as indicated (ctr: scrambled siRNA) and 48 hours later irradiated with UVB. Twenty-four hours after irradiation, lysates and supernatants were harvested and analyzed for the expression and activation of the indicated proteins by western blots. Cleavage of caspase-3 represents apoptosis, and β-actin served as loading control. See Supplementary Figure S3a online for additional controls. (f) Human primary keratinocytes were treated with 100μM of the NLRP3 inflammasome inhibitor glybenclamide or with DMSO (0.25% v/v) as control. One hour after treatment, the cells were irradiated with UVB, and after 24 hours the percentage of cells positive for cleaved caspase-3 was determined by flow cytometry. See Supplementary Figure S3g online for additional controls. Human primary keratinocytes were treated with the antioxidant pyrrolidinedithiocarbamate (PDTC, 500μM) (g) or with the membrane permeable Ca2+ chelator BAPTA-AM (12.5μM) (h), irradiated with UVB, and 24 hours later analyzed for the expression or activation of the indicated proteins by western blot or for the fraction of cells without mitochondrial membrane potential by flow cytometry. See Supplementary Figure S3h–j online for additional controls. Error bars represent the mean±SD of a representative experiment performed in triplicates. siRNA, small interfering RNA; YVAD, Tyr-Val-Ala-Asp. Journal of Investigative Dermatology 2015 135, 1395-1404DOI: (10.1038/jid.2014.551) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Caspase-1 activity is required for UVB-induced apoptosis. (a) Human primary keratinocytes were treated with 20μM of the pan-caspase inhibitor VAD (pan-caspase inhibitor), 50μM YVAD (caspase-1 inhibitor) 1 hour before and 8 hours after irradiation, the latter with a medium change, or with the solvent DMSO (0.1%) (ctr.) and irradiated with UVB. After 24 hours, cells were fixed with PFA, stained with an antibody against cleaved caspase-3, followed by a fluorescent secondary antibody, and counted. (b) Inhibitor treatment as in a followed by staurosporine (0.5μM). After 8 hours, the percentage of cells positive for cleaved caspase-3 was determined by flow cytometry. (c) Keratinocytes were transfected with siRNAs as indicated (ctr: scrambled siRNA) and 48 hours later irradiated with UVB. After 8 hours, YVAD (50μM) or DMSO (0.1%) was added, and 16 hours later cells were collected and analyzed for apoptosis by the determination of the percentage of cells positive for cleaved caspase-3 by immunofluorescence as described. Western blots for analysis of expression of caspase-1, secretion/maturation of proIL-1β and mature IL-1β, indicating inflammasome activation, and expression of β-actin (loading control). (d) Keratinocytes were transfected with siRNAs as indicated (ctr: scrambled siRNA) and after 48 hours irradiated with UVB or mock-treated. Twenty-three hours after irradiation, cells were incubated with 50μM biotinylated YVAD for 1 hour, which binds to active caspase-1. After fixation (2% PFA) and incubation with FITC-coupled streptavidin, cells were analyzed by flow cytometry. (e) Keratinocytes were transfected with scrambled (ctr.) or caspase-1 siRNA targeting its 3′-UTR. Thirty hours after transfection, cells were transduced with a lentiviral vector coding for either wild-type (wt) or enzymatically inactive caspase-1 with a FLAG tag or eGFP as control. After incubation with the virus overnight, the medium was changed, and after 6–7 hours, siRNA was retransfected. The next day, expression was induced by the addition of doxycycline (5μg ml−1) for 30 hours. Keratinocytes were irradiated with UVB and harvested after 16 hours; the supernatants were concentrated by acetone precipitation. Western blots for expression of caspase-1, cleavage of caspase-3 (readout for apoptosis), secretion of mature IL-1β (readout for inflammasome activation), and expression of β-actin (loading control). (a–c) Error bars represent the mean±SD of triplicate measurements of a representative experiment. PFA, paraformaldehyde; siRNA, small interfering RNA; UTR, untranslated region; YVAD, Tyr-Val-Ala-Asp. Journal of Investigative Dermatology 2015 135, 1395-1404DOI: (10.1038/jid.2014.551) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Caspase-1-dependent cleavage of Bap31 is required for UVB-induced apoptosis. (a) Human primary keratinocytes were irradiated with UVB and analyzed at the indicated time points for expression, processing, and secretion of Bap31 by western blot. Supernatants were concentrated by acetone precipitation. The asterisks indicate the band for Bap31 p20. Same samples as in Figure 1a. (b) Human primary keratinocytes were transfected with siRNAs as indicated (ctr: scrambled siRNA) and 48 hours later irradiated with UVB. After 24 hours, lysates were harvested and analyzed for expression and processing of Bap31 by western blot. (c) COS-1 cells were transfected with expression plasmids encoding either wild-type Bap31 (left panel, Bap31 wt) or cleavage-resistant Bap31 (Asp to Val mutations in the caspase recognition sequences, right panel, Bap31 cr) and wild-type (wt) or enzymatically inactive (A) mutants of caspase-1 and caspase-4 (Cys to Ala mutations in the active site) as indicated. Expression and cleavage of Bap31 were analyzed by western blots. (d) Human primary keratinocytes were transfected with siRNAs as indicated (ctr.: scrambled siRNA) and after 48 hours mock-treated or irradiated with UVB and harvested after 12 or 24 hours. Western blots indicating expression or processing of the indicated proteins. (e) Human primary keratinocytes were transduced with lentivirus encoding eGFP or FLAG-tagged Bap31. After overnight infection and subsequent incubation for 30 hours, expression of the indicated proteins was induced by the addition of doxycycline (5μg ml−1) overnight. Keratinocytes were irradiated with UVB or mock-treated and 24 hours later analyzed by flow cytometry for the fraction of apoptotic cells (cleaved caspase-3 positive) expressing GFP or FLAG-Bap31. siRNA, small interfering RNA. Journal of Investigative Dermatology 2015 135, 1395-1404DOI: (10.1038/jid.2014.551) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions