Conversely, regular differentiation could be restored if mTORC1 signaling was blocked. S3 Fig: mTORC1 does not play a CP 375 major in the proliferative CP 375 control of keratinocytes. HaCaT cells were reverse-transfected with siRNA targeting Raptor or control siRNA and seeded in 96 well plates. After 72h proliferation was quantified using a BrdU assay. Graph presents mean SEM (n = 6).(TIF) pone.0180853.s003.tif (61K) GUID:?26D5793C-E611-40E8-92F8-FB90C48D3E79 S4 Fig: Akt Knockdown blocks differentiation. HaCaT cells were reverse-transfected with siRNA specific for Akt or a siRNA control (si contr) and differentiation was induced by post-confluent growth for 72h. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below each blot a quantification of n 3 comparable blots is usually shown. Statistical significant differences between control and knockdown cells of the same density were calculated with one-way ANOVA and Bonferroni multiple comparison (****p 0.0001).(TIF) pone.0180853.s004.tif (161K) GUID:?4FA35DE3-B3F6-4A56-8DC3-3EAC04198581 S5 Fig: Single cytokines are not able to interfere strongly with differentiation in NHK cells. NHK cells were seeded and 24h later, differentiation was induced by the addition of 2 mM CaCl2 in the presence of 20 ng/ml of CP 375 IL-1, IL-17A, IL-22 or TNF- or a mix of IL-1 , IL-17A and TNF- . After 72h RNA was isolated and quantitative RT-PCR was performed to measure expression of the indicated differentiation markers. Graph present imply SEM (n = 4C8). Statistical significant difference between Ca2+ and the cytokines was calculated with one-way ANOVA and Bonferroni multiple comparison (*p 0.05, **p0.01, ****p 0.0001).(TIF) pone.0180853.s005.tif (91K) GUID:?53172C42-BD53-4B41-8E02-93B72CC769BC S6 Fig: Topical application of MHY does not influence serum cytokine levels. Mice were treated, as explained in Fig 6D. At the end of treatment regimen, serum samples were collected and analyzed for protein expression of 26 cytokines and chemokines using multiplex bead immunoassay. IL-17, IL-23, IL-12, IL-1 , IL-10, IL-6 and GM-CSF levels were not detectable. Data shown are from one experiment, with n = 2C3 mice per treatment group.(TIF) pone.0180853.s006.tif (128K) GUID:?D6EAEFD5-F269-45B0-A34C-776DC9D6FB06 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Psoriasis is usually a frequent and often severe inflammatory skin disease, characterized by altered epidermal homeostasis. Since we found previously that Akt/mTOR signaling is usually hyperactivated in psoriatic skin, we aimed at elucidating the role of aberrant mTORC1 signaling in this disease. We found that under healthy conditions mTOR signaling was shut off when keratinocytes switch from proliferation to terminal differentiation. Inflammatory cytokines (IL-1, IL-17A, TNF-) induced aberrant mTOR activity which led to enhanced proliferation and reduced expression of differentiation markers. Conversely, regular differentiation could be restored if mTORC1 signaling was blocked. In mice, activation of mTOR through the agonist MHY1485 also led to aberrant epidermal business and involucrin distribution. In summary, these results not only identify mTORC1 as an important signal integrator pivotal for the cells fate to either proliferate or differentiate, but emphasize the role of inflammation-dependent mTOR activation as a psoriatic pathomechanism. Introduction To maintain homeostasis of the healthy epidermis keratinocyte stem cells divide asymmetrically, leave the basal layer and successively develop into the spinous, granular and corneal layers, characterized by ordered expression of keratins and other marker such as involucrin, loricrin, filaggrin or transglutaminase . Upon maturation, keratinocytes undergo a form of programmed cell death and are shed as corneocytes . The balance between keratinocyte proliferation and differentiation is tightly CP 375 regulated, but is deregulated in certain skin diseases such as psoriasis. Psoriasis is a chronic inflammatory skin disease presenting with red scaly plaques, mostly on the head, trunk and extensor sites of arms and legs . These lesions are characterized by thickened, irregular stratum corneum with parakeratosis, epidermal thickening with acanthosis and absence of the granular layer. This is caused CP 375 by hyperproliferating keratinocytes that are unable to properly initiate the epidermal differentiation program . The molecular mediators and intracellular signaling pathways of the inflammatory psoriatic process involving Th17/Th22 cells and their effector cytokines acting on keratinocytes are well understood . Goat monoclonal antibody to Goat antiRabbit IgG HRP. However, despite increasing identification of deregulated signal mediators such as STAT1 and 3, kinases of the MAPK family, PKC isoforms as well NF-kB [5C9], a comprehensive concept of the signaling pathways governing epidermal homeostasis and its alterations in diseases such as psoriasis has yet to be established. Previously we found that inflammation dependent dysregulation of the PI3-K/Akt cascade interferes with the equilibrium between.