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

Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10 7 /ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.

BackgroundPsoriasis is one of the most frequent skin diseases world-wide. The disease impacts enormously on affected patients and poses a huge financial burden on health care providers. Several lines of evidence suggest that the nuclear hormone receptor peroxisome proliferator activator (PPAR) β/δ, known to regulate epithelial differentiation and wound healing, contributes to psoriasis pathogenesis. It is unclear, however, whether activation of PPARβ/δ is sufficient to trigger psoriasis-like changes in vivo.Methodology/Principal FindingsUsing immunohistochemistry, we define the distribution of PPARβ/δ in the skin lesions of psoriasis. By expression profiling, we confirm that PPARβ/δ is overexpressed in the vast majority of psoriasis patients. We further establish a transgenic model allowing inducible activation of PPARβ/δ in murine epidermis mimicking its distribution in psoriasis lesions. Upon activation of PPARβ/δ, transgenic mice sustain an inflammatory skin disease strikingly similar to psoriasis, featuring hyperproliferation of keratinocytes, dendritic cell accumulation, and endothelial activation. Development of this phenotype requires the activation of the Th17 subset of T cells, shown previously to be central to psoriasis. Moreover, gene dysregulation in the transgenic mice is highly similar to that in psoriasis. Key transcriptional programs activated in psoriasis, including IL1-related signalling and cholesterol biosynthesis, are replicated in the mouse model, suggesting that PPARβ/δ regulates these transcriptional changes in psoriasis. Finally, we identify phosphorylation of STAT3 as a novel pathway activated by PPARβ/δ and show that inhibition of STAT3 phosphorylation blocks disease development.ConclusionsActivation of PPARβ/δ in the epidermis is sufficient to trigger inflammatory changes, immune activation, and signalling, and gene dysregulation characteristic of psoriasis.

Psoriasis is a common skin disease involving keratinocyte proliferation and altered differentiation, as well as T-cell activation. Here, we show that altered gene transcription in psoriatic skin lesions is highly reproducible between independent data sets. Analysis of gene expression confirmed dysregulation in all expected functional categories, such as IFN signaling and keratinocyte differentiation, and allowed molecular fingerprinting of a previously characterized dendritic cell subset associated with psoriasis tumor necrosis factor alpha (TNF-alpha)- and inducible nitric oxide synthase (iNOS)-producing CD11b(INT) DC (Tip-DC). Unexpectedly, a large group of dysregulated transcripts was related to fatty acid signaling and adipocyte differentiation, exhibiting a pattern consistent with the activation of peroxisome proliferator-activated receptor delta (PPARdelta). PPARdelta itself was strongly induced in psoriasis in vivo. In primary keratinocytes, PPARdelta was induced by the transcription factor activator protein 1, in particular by junB, but not by canonical WNT signaling, in contrast to its regulation in colon carcinoma cells. Activation of PPARdelta enhanced proliferation of keratinocytes, while this was inhibited by knockdown of PPARdelta. Finally, heparin-binding EGF-like growth factor (HB-EGF), known to induce epidermal hyperplasia and itself overexpressed in psoriasis, was identified as a direct target gene of PPARdelta. The present data suggest that activation of PPARdelta is a major event in psoriasis, contributing to the hyperproliferative phenotype by induction of HB-EGF.

BackgroundWnt5a is a member of the wingless-type patterning regulators important in pre-natal development. The expression and distribution of Wnt5a and its receptors frizzled (fzd) 3 and fzd 5 in adult human skin have not been comprehensively studied to date.Methodology/Principal FindingsWe here show that Wnt5a, fzd3, fzd5, as well as fzd6 are restricted to specific layers in normal epidermis, analogous to their zonal distribution in hair follicles, suggesting a role in adult skin differentiation. In line, Wnt5a and fzd5 are both overexpressed and re-distributed in the epidermis of psoriasis which involves disturbed keratinocyte differentiation. Functionally, Wnt5a lowers the concentration of IFN required to induce target genes, and increases the magnitude of IFN target gene induction, suggesting a molecular mechanism underlying IFN hypersensitivity in psoriasis. Finally, we identify nedd8 and the amyloid precursor APP, previously shown to be upregulated in psoriasis, as targets of synergistic IFNα/Wnt5a induction.Conclusions/SignificanceThe present data (i) suggest that Wnt5a regulates epidermal differentiation even in adult skin and (ii) identify synergistic induction of type 1 IFN target genes as a novel mode of Wnt5a action. Targeting Wnt5a in the skin may reduce IFN hypersensitivity and be of therapeutical value.

SummaryPreviously we showed that spatial and developmental modulation of ARNT (HIF1b) expression in mouse epidermis is essential for maintenance of keratinocyte differentiation, proper formation of the barrier and normal desquamation. Here, using lentiviral suppression or induction of ARNT in TERT-immortalized (N-TERT) and HaCaT cells we assessed the nature and mechanisms of ARNT involvement in control of differentiation in human epidermal keratinocytes. ARNT depletion did not affect the levels of basal keratins K5 and K14, but significantly induced expression of several key differentiation markers (an effect abolished by EGF supplementation). Furthermore, ARNT deficiency resulted in the downregulation of amphiregulin (AREG) -the most highly expressed EGFR ligand in human keratinocytes -whereas upregulation of ARNT showed the opposite. In ARNT-deficient monolayer cultures and 3D epidermal equivalents, the downregulation of AREG was concurrent with a decline of EGFR and ERK1/2 phosphorylation. TSA, a potent suppressor of HDAC activity, abolished the effects of ARNT deficiency, implying a role for HDACs in ARNT-dependent modulation of the AREG-EGFR pathway and downstream epidermal genes. Total HDAC activity was significantly increased in ARNT-depleted cells and decreased with ARNT overexpression. ARNT-dependent shifts in HDAC activity were specifically attributed to significant changes in the levels of HDAC1, HDAC2 and HDAC3 proteins (but not mRNA) in both monolayer and 3D cultures. Collectively, our results suggest that ARNT controls AREG expression and the downstream EGFR-ERK pathway in keratinocytes, at least in part, by modulating HDAC activity. This novel regulatory pathway targeting advanced stages of epidermal differentiation might have important implications for skin pathology such as psoriasis, atopic dermatitis and cancer.

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of nonmelanoma skin cancer (NMSC), and its incidence is increasing rapidly. Metastatic cSCC accounts for the majority of deaths associated with NMSC, but the genetic basis for cSCC progression remains poorly understood. A previous study identified small deletions (typically <1 Mb) in the protein tyrosine phosphatase receptor Type D (PTPRD) gene that segregated with more aggressive cSCC. To investigate the apparent association between deletion within PTPRD and cSCC metastasis, a series of 74 formalin-fixed paraffin-embedded tumors from 31 patients was analyzed using a custom Illumina 384 SNP microarray. Deletions were found in 37% of patients with metastatic cSCC and were strongly associated with metastatic tumors when compared to those that had not metastasized (p 5 0.007). Subsequent mutation analysis revealed a higher mutation rate for PTPRD than has been reported in any other cancer type, with 37% of tumors harboring a somatic mutation. Conversely, bisulfite sequencing showed that methylation was not a mechanism of PTPRD disruption in cSCC. This is the first report to observe an association between deletion within PTPRD and metastatic disease and highlights the potential use of these deletions as a diagnostic biomarker for tumor progression. Combined with the high mutation rate observed in our study, PTPRD is one of the most commonly altered genes in cSCC and warrants further investigation to determine its significance for metastasis in other tumor types.Nonmelanoma skin cancer (NMSC) has a predicted prevalence equal to that of all other cancers combined and its incidence is increasing.1 There are more than 81,500 new cases of NMSC diagnosed annually in the United Kingdom alone, placing a heavy burden on both patients and healthcare resources.2 Despite accounting for only 20% of total NMSC cases, cutaneous squamous cell carcinoma (cSCC) is responsible for the majority of NMSC deaths, largely as a result of metastatic disease. A subset of immunosuppressed individuals, such as organ transplant recipients on long-term immunosuppressive drugs, develop particularly aggressive tumors with an increased risk of metastasis.3 Risk factors for metastasis include poor differentiation of the tumor cells, large tumor size, tumor depth >5 mm, immunosuppression and localization on the ear and lip. [4][5][6][7] Metastatic cSCC is currently treated by surgical intervention and/or chemotherapy or radiotherapy and is associated with a poor outcome. 8Despite the well-established role of ultraviolet radiation (UVR) in the etiology of cSCC, the molecular events underlying its development remain largely undefined. Inactivation of the tumor suppressor genes TP53 and p16INK4A is a common and early event in cSCC pathogenesis and is characteristic of all histological grades of tumor. 9,10 Recently, genotyping of 60 cSCC identified high rates of loss of heterozygosity (LOH)

Cel pracyZbadano związek stylu radzenia sobie ze stresem i motywacji do spożycia alkoholu w kontekście ryzyka uzależnienia od alkoholu u studentów kierunków medycznych.MetodaBadaniem objęto 268 studentów kierunków medycznych W badaniach wykorzystano Test AUDIT (Alcohol Use Disorders Identyfication Test), Kwestionariusz Radzenia Sobie w Sytuacjach Stresowych (CISS), oraz autorską ankietę badającą poza danymi demograficznymi także motywację do picia alkoholu i sytuacje, w jakich badani sięgają po alkohol.WynikiW badanej grupie 94% osób spożywało alkohol w okresie ostatnich dwunastu miesięcy. W badanej grupie 16% studentów kierunku medycznego i 20% studentów stomatologii pije w sposób ryzykowny lub szkodliwy. W 2% przypadków zaobserwowano duże ryzyko uzależnienia od alkoholu Ponad połowa respondentów sięga po alkohol w celu radzenia sobie ze stresem. Dane dotyczące stylu radzenia sobie ze stresem nie wykazują różnic istotnych statystycznie w zakresie głównego stylu radzenia sobie ze stresem: skoncentrowanego na zadaniu, skoncentrowanego na emocjach, skoncentrowanego na unikaniu. Studenci kierunku lekarskiego istotnie częściej niż studenci stomatologii radzili sobie ze stresem w sposób unikowy (poszukiwanie kontaktów towarzyskich). Nie wykazano istotnego związku między stylem radzenia sobie ze stresem, motywacją do picia i ryzykiem uzależniania od alkoholu.WnioskiBrak związków między badanymi zmiennymi może wskazywać na stosowanie przez badanych innych czynności niż sięganie po alkohol w radzeniu sobie ze stresem. Ważna jest profilaktyka i stworzenie warunków sprzyjających rozwojowi strategii radzenia sobie ze stresem w grupie studentów kierunków medycznych.