Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis

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. DOI: http://dx.doi.org/10.7554/eLife.03422.001


Introduction
Tumor necrosis factor receptor 1 (TNFR1)-mediated signaling is regulated by multiple ubiquitination events involving linear (Met1), Lys63 and Lys48 ubiquitination of several target proteins (Wertz and Dixit, 2008;Verhelst et al., 2011;Schmukle and Walczak, 2012). Upon TNF stimulation, a receptor proximal signaling complex (termed complex I) consisting of the TNFR1-associated death domain (TRADD), receptor-interacting protein kinase 1 (RIPK1), TNF receptor-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) is recruited to the intracellular domain of TNFR1 (Haas et al., 2009). Ubiquitination of proteins in complex I, including RIPK1, cIAP1/2, and TRAF2, leads to the recruitment of additional signaling components that facilitate activation of nuclear factor-κB (NF-κB) and mitogen-activated protein (MAP) kinase signaling cascades (Wu et al., 2005;Varfolomeev et al., 2008;Haas et al., 2009). It has been shown that the transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 2 (TAB2) complex is recruited into complex I through the interaction between the TAB2-Npl4 zinc finger (NZF) and Lys63-linked ubiquitin chains (Kanayama et al., 2004;Kulathu et al., 2009;Sato et al., 2009). cIAP-mediated ubiquitination of RIPK1 and cIAPs themselves was shown to result in the recruitment of the E3 ligase complex Linear Results TNFR1 deficiency in keratinocytes prevents skin inflammation in Sharpin cpdm/cpdm mice Previous studies showed that TNF is required for the development of multi-organ inflammation in Sharpin cpdm/cpdm mice (Gerlach et al., 2011). To address whether this function of TNF is mediated by TNFR1, we crossed Sharpin cpdm/cpdm mice with Tnfrsf1a −/− animals. Double deficient Sharpin cpdm/cpdm ;Tnfrsf1a −/− eLife digest In response to an injury or an infection, areas of the body can become inflamed as the immune system attempts to repair the damage and/or destroy any microbes or toxins that have entered the body. At the level of individual cells inflammation can involve cells being programmed to die in one of two ways: apoptosis and necroptosis.
Apoptosis is a highly controlled process during which the contents of the cell are safely destroyed in order to prevent damage to surrounding cells. Necroptosis, on the other hand, is not controlled: the cell bursts and releases its contents into the surroundings.
Inflammation is activated by a protein called TNFR1, which is controlled by a complex that includes a protein called Sharpin. Mice that lack the Sharpin protein develop inflammation on the skin and other organs, even in the absence of injury or infection. However, it is not clear how the Sharpin protein controls TNFR1 to prevent inflammation. Kumari et al. and, independently Rickard et al., have now studied this process in detail. Kumari et al. have found that inflammation in mice lacking Sharpin depends on TNFR1 interacting with another protein called TRADD. The experiments also show that the inflammation is mainly driven by apoptosis, with necroptosis having only a minor role. Further experiments carried out in mammal cells showed that TRADD and another protein (called FADD) work with Sharpin to prevent apoptosis.
At the molecular level, Sharpin is known to induce a special type of protein modification (called linear ubiquitination) with two partner proteins, so the next challenge is to work out exactly how Sharpin uses this process to prevent apoptosis. DOI: 10.7554/eLife.03422.002 mice did not develop skin inflammation, demonstrating that TNF-induced TNFR1 signaling is essential for the pathogenesis of inflammatory skin lesions in Sharpin cpdm/cpdm mice ( Figure 1). We then tried to identify the cellular target of the pathogenic TNFR1 signaling in Sharpin cpdm/cpdm mice. We have recently shown that TNFR1 signaling in NF-κB-deficient epidermal keratinocytes drives psoriasis-like skin inflammation in mice (Kumari et al., 2013), identifying keratinocytes as an important cellular target of pathogenic TNF signaling in skin inflammation. To address whether TNFR1 signaling in epidermal keratinocytes drives the skin inflammation in Sharpin cpdm/cpdm mice, we crossed Sharpin cpdm/cpdm mice with K14Cre-Tnfrsf1a fl/fl (TNFR1 E-KO ) mice that lack TNFR1 specifically in keratinocytes ( Figure 1A). These Sharpin cpdm/cpdm ;TNFR1 E-KO mice did not develop any macroscopic signs of skin inflammation ( Figure 1B). In addition, histological analysis of Sharpin cpdm/cpdm ;TNFR1 E-KO mice skin revealed a normal epidermis without keratinocyte death (cleaved caspase-3 staining in Figure 1C), skin inflammation (F4/80 staining in Figure 1C), or epidermal hyperplasia (H&E, Keratin 6, Keratin 10, and Loricrin staining in Figure 1C and quantification in Figure 1D), similar to Sharpin cpdm/cpdm ;Tnfrsf1a −/− animals ( Figure 1C,D). These results demonstrate that TNFR1 signaling in epidermal keratinocytes is essential for the pathogenesis of skin inflammation in Sharpin cpdm/cpdm mice.
Epidermal FADD-dependent TNFR1-induced death of keratinocytes induces skin inflammation in Sharpin cpdm/cpdm mice Having established keratinocyte-intrinsic TNFR1 signaling as a key spatial event triggering skin inflammation in Sharpin cpdm/cpdm mice, we sought to investigate the cell death mechanisms by which epithelial TNFR1 induces the inflammatory response. We and others have shown that increased numbers of keratinocytes in the epidermis of Sharpin cpdm/cpdm mice undergo apoptosis, as indicated by the presence of cleaved caspase-3 Liang and Sundberg, 2011) (also see Figure 1C). In addition, it was suggested that Sharpin deficiency sensitizes primary keratinocytes to both TNFinduced caspase-dependent apoptosis and RIP-kinase-dependent necroptosis (Gerlach et al., 2011). We therefore used genetic mouse models to address the role of FADD/caspase-8-dependent apoptosis and RIPK3-dependent necroptosis in Sharpin cpdm/cpdm mice. To address the role of RIPK3-dependent necroptosis, we generated mice lacking both Sharpin and RIPK3 by crossing Sharpin cpdm/cpdm with Ripk3 −/− animals ( Figure 2). Double deficient Sharpin cpdm/cpdm ;Ripk3 −/− mice developed skin lesions similar to those of Sharpin cpdm/cpdm mice, demonstrating that RIPK3 deficiency did not prevent the development of skin inflammation (Figure 2A,B). However, the initiation of the skin phenotype was delayed in Sharpin cpdm/cpdm ;Ripk3 −/− animals, which started to show lesions after the age of 10 weeks but showed a large variability in onset and severity with some mice showing only mild lesions even at the age of 19 weeks (Figure 2-figure supplement 1). Sharpin cpdm/cpdm mice also showed variability with lesion onset between 8 and 11 weeks, but all mice showed severe lesions by the age of 12-14 weeks. Quantification of epidermal thickness revealed that RIPK3 deficiency mildly ameliorated the severity of skin lesions ( Figure 2C). These results showed that, although RIPK3-dependent necroptosis contributes to accelerating the onset and exacerbating the severity of the phenotype, it is not essential for the pathogenesis of the inflammatory skin lesions in Sharpin cpdm/cpdm mice.
Next, we sought to address whether FADD/caspase-8-dependent apoptosis of Sharpin-deficient keratinocytes induces TNF-dependent skin inflammation in Sharpin cpdm/cpdm mice. Since deficiency of caspase-8 or FADD alone in epidermal keratinocytes triggers a RIPK3-dependent skin inflammation (Bonnet et al., 2011;Weinlich et al., 2013), we could not directly investigate the role of FADD or caspase-8 in the TNF-induced death of Sharpin-deficient keratinocytes in vivo. However, taking advantage of the fact that concomitant deletion of RIPK3 fully prevents skin lesion formation in mice with keratinocyte-restricted FADD knockout (FADD E-KO ) (Bonnet et al., 2011), we generated Sharpin cpdm/cpdm ;Ripk3 −/− mice that also lacked FADD specifically in keratinocytes ( Figure 2). These Sharpin cpdm/cpdm ;FADD E-KO ;Ripk3 −/− mice did not develop any macroscopic ( Figure 2A) or histological ( Figure 2B) skin lesions up to the age of 4 months, as they showed normal keratinocyte proliferation and differentiation without any signs of inflammation or epidermal hyperplasia ( Figure 2B,C). Moreover, apoptosis of keratinocytes observed in Sharpin cpdm/cpdm mice was completely prevented in Sharpin cpdm/cpdm ;FADD E-KO ;Ripk3 −/− mice, as shown by the absence of cleaved caspase-3 positive cells (compare Figure 2B with Figure 1C). Taken together, these results showed that combined inhibition of FADD/caspase-8-dependent apoptosis and RIPK3-dependent necroptosis prevented keratinocyte death and the development of skin lesions in Sharpin cpdm/cpdm mice, providing in vivo genetic evidence that skin inflammation is triggered by TNFR1-induced death of Sharpin-deficient keratinocytes.
TRADD-dependent apoptosis of Sharpin-deficient keratinocytes triggers skin inflammation in Sharpin cpdm/cpdm mice Our findings strongly suggest that FADD-dependent apoptosis of Sharpin-deficient keratinocytes triggers skin inflammation. However, since the role of FADD can only be addressed in a RIPK3-deficient  background, it remains possible that FADD-dependent apoptosis and RIPK3-dependent necroptosis might share a redundant function in inducing the cell death of Sharpin-deficient keratinocytes and triggering skin inflammation. To directly address the role of TNFR1-induced apoptosis in Sharpin cpdm/cpdm mice, we employed mice carrying conditional alleles for TRADD, an adapter molecule that is important for the induction of inflammatory and apoptotic signaling downstream of TNFR1 (Chen et al., 2008;Michallet et al., 2008). It has been shown that TRADD deficiency partially inhibits TNFR1-induced activation of NF-κB and MAP kinase pathways and fully prevents TNFR1-induced apoptosis in mouse embryonic fibroblasts (MEFs) in vitro and in hepatocytes in vivo . To examine the role of TRADD in TNFR1-induced apoptosis and necroptosis, we analyzed the response of wild type and TRADD-deficient primary MEFs to TNF stimulation in the presence of cycloheximide (CHX), caspase inhibitor (Z-VAD-FMK), and RIPK1 inhibitor (Necrostatin-1) ( Figure 3A). As expected, TRADDdeficient MEFs were resistant to apoptosis induced by TNF and CHX. However, in contrast to earlier studies (Pobezinskaya et al., 2008), we found that TRADD-deficient MEFs were sensitive to necroptosis induced by TNF, CHX, and Z-VAD-FMK. These results demonstrated that TRADD deficiency specifically blocks TNFR1-induced apoptosis ( Figure 3A). We therefore generated Sharpin cpdm/cpdm mice lacking TRADD specifically in keratinocytes by crossing Sharpin cpdm/cpdm with K14Cre-Tradd fl/fl mice (Sharpin cpdm/cpdm ; TRADD E-KO ) ( Figure 3B-D). Indeed, keratinocyte-restricted TRADD deficiency prevented skin lesion development in Sharpin cpdm/cpdm mice, as shown by macroscopic and histological analysis ( Figure 3B-D). Collectively, our results show that TNFR1-induced TRADD-and FADD-dependent apoptosis of Sharpindeficient keratinocytes triggers the chronic proliferative dermatitis phenotype in Sharpin cpdm/cpdm mice.

Sharpin regulates TNF-induced apoptosis
Our in vivo genetic experiments showed that TNFR1-induced apoptosis of Sharpin cpdm/cpdm keratinocytes causes skin inflammation in Sharpin cpdm/cpdm mice. We therefore investigated the molecular    mechanisms by which Sharpin prevents TNFR1-induced apoptosis. We first examined the induction of apoptosis in MEFs derived from Sharpin cpdm/cpdm mice stimulated with TNF alone or TNF + CHX using various assays. We observed increased sensitivity to TNF + CHX-induced apoptosis in Sharpin cpdm/cpdm MEFs compared with wild type MEFs, as determined by immunocytochemical detection of cleaved caspase-3 ( Figure 7A), detection of annexin V positive cells by fluorescence-activated cell sorting (FACS) analysis ( Figure 7B), analysis of the cleavage of caspase-3 and Poly (ADP-ribose) polymerase (PARP) by immunoblotting ( Figure 7C) , and luminescent-based caspase-8 activity assay ( Figure 7D). Sharpin cpdm/cpdm MEFs also showed increased cleavage of caspase-3 and PARP ( Figure 7E) and caspase-8 activity ( Figure 7F) in response to stimulation with TNF alone although, in the absence of CHX, the response was considerably weaker. To further analyze the cellular functions of Sharpin in anti-apoptotic signaling, we used a human keratinocyte cell line, HaCaT cells (Boukamp et al., 1988). By using lentiviral-based shRNA knockdown, Sharpin was stably depleted in HaCaT cells ( Figure 8A). Sharpin-deficient HaCaT and control shRNA-introduced HaCaT cells were treated with TNF alone or TNF + CHX, and the induction of apoptosis was assessed by FACS analysis of annexin V positive cells ( Figure 8B) and measurement of caspase-8 activity ( Figure 8C). Similar to Sharpin cpdm/cpdm Figure 6. Epidermal keratinocyte-restricted knockout of tumor necrosis factor receptor 1-associated death domain (TRADD) (TRADD E-KO ) in Sharpin cpdm/cpdm mice has a minor effect on the inflammation of the lung, liver, and splenomegaly. (A and B) H&E staining of liver, lung, and spleen and macroscopic pictures of spleen as well as measurement of spleen weight from mice with the indicated genotypes derived from wild type (+/+), Sharpin cpdm/cpdm , TRADD E-KO or Sharpin cpdm/cpdm ;TRADD E-KO mice at the age of 12 weeks. The mice used here are the littermates. Scale bars in (A) are 100 μm. Results are expressed as mean values ± SD. Statistical significance was determined using ANOVA test (***p ≤ 0.001 and ****p ≤ 0.0001). DOI: 10.7554/eLife.03422.012 MEFs, Sharpin knockdown sensitized HaCaT cells to apoptosis induced by TNF or TNF + CHX. Interestingly, treatment with Necrostatin-1, a RIPK1 inhibitor, suppressed caspase-8 activation in Sharpindeficient HaCaT cells ( Figure 8D), suggesting an involvement of RIPK1 in the induction of TNF-induced death of Sharpin-deficient keratinocytes as suggested recently by Berger et al. (Berger et al., 2014). Our results collectively suggest that Sharpin plays a critical role in protecting against TNF-induced apoptosis. To distinguish between the possibilities that the anti-apoptotic protection afforded by Sharpin is a LUBAC-independent function of Sharpin, we assessed the involvement of the catalytic LUBAC MEFs. Apoptosis in MEFs stimulated with TNF (10 ng/ml) and CHX (1 μg/ml) for 4 hr was examined by immunofluorescent staining using α-cleaved caspase-3 antibody with Alexa488 conjugated secondary antibody (A), by fluorescence-activated cell sorting (FACS) analysis using annexin V staining (B), by immunoblotting using α-cleaved caspase-3 and α-Poly (ADP-ribose) polymerase (PARP) antibodies (C), or by caspase-8 activity assay measured using a luminol-based assay. Scale bars in (A) are 100 μm. (E and F) TNF-induced apoptosis in MEFs analyzed by immunoblotting (E) or by caspase-8 activity assay (F) as in (C and D). Results are expressed as mean values ± SD. Statistical significance was determined using ANOVA test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). DOI: 10.7554/eLife.03422.013 Research article  (Figure 8-figure supplement 1A), and the cells were examined for apoptosis induced by TNF alone or TNF + CHX using FACS analysis (Figure 8-figure supplement 1B) and a caspase-8 activity assay (Figure 8-figure supplement 1C and D). As expected and consistent with a LUBAC-dependent Sharpin function, HOIP deficiency sensitized HaCaT cells to apoptosis induced by TNF alone or by TNF + CHX, akin to that observed in Sharpin cpdm/cpdm MEFs or Sharpin-deficient HaCaT cells. There results provide strong support that a LUBAC-dependent Sharpin function plays a role in the regulation of apoptosis in keratinocytes.

FADD and TRADD have a major role in Sharpin-regulated TNF-induced apoptosis
As keratinocyte apoptosis and skin inflammation in Sharpin cpdm/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, Sharpin cpdm/cpdm , Sharpin cpdm/cpdm ; Ripk3 −/− , Sharpin cpdm/cpdm ;FADD E-KO ;Ripk3 −/− , and their control littermates, Sharpin cpdm/wt or Sharpin wt/wt Sharpin cpdm/wt ;Ripk3 −/− and Sharpin cpdm/wt ;FADD E-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 Sharpindeficient keratinocytes, TNF treatment alone had a very small effect in reducing the viability of Sharpindeficient keratinocytes by about 10% compared with controls. Interestingly, the combined lack of FADD and RIPK3 in Sharpin cpdm/cpdm ;FADD E-KO ;Ripk3 −/− keratinocytes fully rescued the increased sensitivity of Sharpin-deficient keratinocytes to TNF + CHX ( Figure 9A). However, keratinocytes obtained from Sharpin cpdm/cpdm ;Fadd fl/fl ;Ripk3 −/− showed a similar response to TNF + CHX as Sharpin cpdm/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 Sharpindeficient 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 FADDdeficient Sharpin cpdm/cpdm MEFs and analyzed the effect of FADD deficiency on apoptosis induced by TNF alone and by TNF + CHX in Sharpin cpdm/cpdm MEFs (Figure 9-figure supplement 1A) and observed that FADD deficiency significantly suppressed the annexin V positive cells and caspase-8 activity in Sharpin cpdm/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 Sharpindependent anti-apoptosis signaling depends on FADD and TRADD in a cell-intrinsic manner.

Discussion
Ubiquitination regulates a wide variety of biological functions by generating ubiquitin chains with different linkages on substrates (Ikeda and Dikic, 2008;Komander and Rape, 2012). One of the atypical linkage types is the Met-1/linearly-linked ubiquitin chain which is specifically generated by the LUBAC E3 ligase complex (Kirisako et al., 2006). Sharpin is a critical component of the LUBAC complex (Gerlach et al., 2011;Ikeda et al., 2011;Tokunaga et al., 2011) and its deficiency in mice leads to severe TNF-dependent inflammation in multiple organs including the skin , suggesting that Sharpin has an important role in preventing inflammation. However, the mechanisms We show here that TNFR1 signaling in keratinocytes is essential for the pathogenesis of skin inflammation in Sharpin cpdm/cpdm mice. In addition, we provide genetic evidence that TNFR1-mediated, TRADD-and FADD-dependent apoptosis of Sharpin-deficient keratinocytes induces skin inflammation in these mice. RIPK3 deficiency only mildly delayed and ameliorated the severity of skin lesions in Sharpin cpdm/cpdm mice, showing that RIPK3-dependent necroptosis plays a minor role in driving skin inflammation in this model, which was also shown by Rickard et al. (Rickard et al., 2014). These results demonstrate that Sharpin deficiency triggers skin inflammation by sensitizing keratinocytes to TNFinduced apoptosis. Furthermore, we found that inflammation in other organs including lung and liver as well as splenomegaly and altered splenic structure observed in Sharpin cpdm/cpdm mice also depend on TNFR1 signaling and occur independently of the skin lesions. Together, these findings identified a cellintrinsic function of Sharpin in inhibiting TNFR1-induced apoptosis that is essential for the maintenance of tissue homeostasis and the prevention of multi-organ inflammation.
In this study we have shown that Sharpin and HOIP, components of LUBAC, negatively regulate apoptotic pathways in keratinocytes. Our data suggest that Sharpin and HOIP act in an active ligase complex and raise the intriguing possibility that linear ubiquitination of an unknown target(s) inhibits TNF-induced apoptosis in keratinocytes. Ubiquitin signals have been implicated in the regulation of the death-inducing signaling complex (DISC). For example, cullin 3-based ubiquitination of caspase-8 by both Lys48-and Lys63-linked polyubiquitin chains brings caspase-8 into p62-containing aggregates leading to its activation and to commitment to apoptotic cell death (Jin et al., 2009). Furthermore, it was shown that FADD ubiquitination induced by Makorin Ring Finger Protein 1 (MKRN1) E3 ligase leads to the proteasome-dependent degradation of FADD, and MKRN1 depletion in breast cancer cells accelerates TNF-related apoptosis-inducing ligand (TRAIL)-induced DISC formation and apoptosis (Lee et al., 2012). Since we did not detect significant changes in total FADD protein levels in Sharpin cpdm/cpdm MEFs in comparison to the wild type cells (Figure 9-figure supplement 1A), it suggests that protein stability is not controlled by Sharpin. It is also important to elucidate spatial and temporal regulation of DISC and TNFR complex II component ubiquitination and to identify the E3 ligases that mediate these ubiquitinations for the better understanding of biological functions of TNF signaling. Lastly, our study sheds new light on the specific functions of Sharpin as an integral component of the LUBAC E3 ligase complex. For example, both HOIL-1L and Sharpin are expressed in keratinocytes and have redundant roles in activating the NF-κB signaling pathway in MEFs (Haas et al., 2009;Gerlach et al., 2011). Yet, Sharpin-deficient mice develop inflammation in the skin whereas HOIL-1L knockout mice showed no obvious dermatitis phenotypes . Interestingly, previous studies have shown that the analysis of complex formation of Sharpin, HOIL-1L, and HOIP suggested that some populations of Sharpin and HOIL-1L may exist in different molecular complexes from HOIP (Gerlach et al., 2011;Tokunaga et al., 2011). This raises the question whether Sharpin and HOIL-1L could individually regulate biological functions depending on the cell types, tissues, and pathogenic conditions. Precise examination of the effects of tissue-specific depletion of the LUBAC components in mice is needed to better understand the functional roles of each of the LUBAC components in vivo. Here we report the specific function of Sharpin in the regulation of apoptosis and skin inflammation, which are mediated through FADD and TRADD. Together, further studies of the regulatory mechanisms controlling inflammation in Sharpin cpdm/cpdm mice will be important for the better understanding of the unique functions of Sharpin in vivo.
WST-1 assay. Bars represent mean values ± SEM. Statistical significance was determined using the Student's t test (**p ≤ 0.01, *p ≤ 0.05). (B-G) Sharpin with FADD (B) or TRADD (E) was stably knocked down in HaCaT cells. Knockdown efficiency was analyzed by immunoblotting using α-Sharpin, α-FADD, and α-TRADD antibodies. Caspase-8 activity measurement in Sharpin knockdown, FADD knockdown, and double knockdown of Sharpin and FADD HaCaT cells (C and D) as well as Sharpin knockdown, TRADD knockdown, and double knockdown of Sharpin and TRADD HaCaT cells (F and G) upon treatment with TNF alone (C and F) or TNF + CHX (D and G) for the indicated time. Results are expressed as mean values ± SD. Statistical significance was determined using ANOVA test (****p ≤ 0.0001). DOI: 10.7554/eLife.03422.016 The following figure supplement is available for figure 9: and seeded 20,000 cells/well from the indicated genotypes in 96-well plates and stimulated with TNF (20, 50 and 100 ng/ml) in the presence or absence of CHX (1 μg/ml) for 24 hr followed by incubation with WST-1 reagent (Roche) and measurement as per the manufacturer's instructions. For the caspase-8 activation assay, 50,000 cells/well in a 96-well plate were plated and treated with mTNF, CHX, or Necrostatin-1 for the indicated time. Lysates were used for the determination of caspase-8 activity in a luminescent signal-dependent manner following the manufacturer's protocol (Promega, caspase-Glo 8 Assay Systems).

Lentiviral production
Lentiviral production and infection was performed according to the Addgene's pLKO.1 protocol with a minor modification. Briefly, pLKO.1 vector with a packaging and envelope plasmids were transfected into HEK293T cells using Gene Juice (Novagen). After 36 hr of transfection, released lentivirus particles were filtered and used for infection of target MEFs or HaCaT cells using polybrene (4 μg/ml). After 48 hr of infection, cells were selected with puromycin (2 μg/ml). Expression of infected protein was monitored by western blotting.

Funder
Grant reference number Author

Deutsche Forschungsgemeinschaft SFB829 Manolis Pasparakis
Deutsche Forschungsgemeinschaft SPP1656 Manolis Pasparakis The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.