Activation of mTORC1 signaling inhibits differentiation.

<p>HaCaT cells (a) or NHK cells (b) were serum starved overnight and treated with increasing doses of MHY1485 or DMSO for 30 min If indicated cells were pre-treated with 100 nM Rapamycin for 30 min. Cells were harvested and protein lysates were analyzed by Western blotting with the indicated antibodies, showing that MHY1485 induces mTORC1 signaling. (c) Increasing numbers of HaCaT cells were seeded and driven into differentiation by post-confluent growth in the presence of the indicated concentrations of MHY1485. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below a quantification of 6–8 similar Western blots is shown. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01). (d-g) MHY1485 or vehicle control was topically applied to the dorsal skin of mice for 12 consecutive days with increasing doses. (d) DSFT (Double Skin Fold Thickness) was measured before the first treatment (day1) and repeated every day. Data shown are mean values from one experiment, with n = 3 mice per treatment. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01, *** p ≤0.001, **** p ≤0.0001). (e) Representative images of H&E-stained sections from dorsal skin of a mouse of control and MHY 1485 treated groups (scale bar, 100 μM). (f) Evaluation of histological features, including number of epidermal layers and epidermal thickness in μM. Data shown are mean values of five measurements per mouse ± SEM. Statistical significance was calculated with Mann-Whitney test (**p ≤0.01). (g) Involucrin staining of vehicle control or MHY1485 treated mice. Overview images and close-ups are shown. MHY1485 induces in vivo a psoriasis like phenotype and interferes with proper differentiation (h) Hypothetical model how mTOR serves as a switch to determine the fate of keratinocytes.</p

mTOR signaling is deactivated during differentiation and only partially contributes to the control of proliferation.

<p>(a) Increasing densities of HaCaT were seeded to promote differentiation and harvested after 72h. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies. (b) NHK were serum-starved and differentiation was induced with 2mM CaCl₂ for the indicated time points. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies was performed. Below a densiometrical quantification of involucrin and P-S6 levels of n = 4–5 similar blots is shown. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (* p ≤0.05, ***p ≤0.001). (c) Keratinocytes stem cells (KSC), transient amplifying (TA) and postmitotic (PM) cells were separated according to their ability to adhere to type IV collagen. Protein lysates were subjected to SDS-PAGE and Western blotting with the indicated antibodies, showing that mTORC1 signaling is mainly present in undifferentiated cells. (d) Normal human skin was stained with P-mTOR S2448 and Ki-67 antibody. Nuclei were stained with DAPI. Single color and overlay images are presented, which show that mTOR is activated in proliferation cells of the basal layer. Bars represent 100 μm. (e) HaCaT cells were reverse-transfected with siRNA targeting Akt, Raptor or control siRNA and seeded in 96 well plates. After 48h proliferation was quantified using the XTT-based assay. Graph presents mean ± SEM (n = 4–8). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (f) To control for the efficiency of knockdown, HaCaT cells were transfected as in (e) and seeded in 6 well plates. Protein lysates were harvested after 48h and a Western blot was performed with the indicated antibodies. To show the consequences of Akt and Raptor knockdown, phosphorylation of the downstream targets GSK-3 and S6 was also captured. (g) HaCaT cells were seeded in 96 well-plates in triplicates and after 24h 50 μM LY294002, 100 nM Rapamycin or 250 nM Torin or solvent (DMSO) were added. After another 48h cell proliferation was measured with a BrdU assay. Graph presents mean ± SEM (n = 6). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (h) To show efficiency of the used inhibitors, HaCatT cells were treated as in (g). Protein lysates were prepared and a Western blot was performed with the indicated antibodies.</p

Inhibition of mTORC1 signaling promotes differentiation.

<p>(a) Increasing cell numbers of HaCaT cells were seeded and after 24h 100 nM Rapamycin, 250 nM Torin or solvent control (DMSO) were added and differentiation was allowed to proceed for 72h. Protein lysates were subjected to Western blotting and proteins were detected with the indicated antibodies. Quantification of 3–6 similar blots is depicted below. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison, comparing different treatments with the corresponding cell density in the control group (*p ≤0.05, ***p ≤0,001, ns: non-significant). (b) In NHK cells differentiation was induced with 2mM CaCl₂ in the presence of 100nM Rapamycin, 250 nM Torin or solvent control for 48h. Protein lysates were subjected to Western blotting and proteins were detected with the indicated antibodies. Below a quantification of six similar blots is shown. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (*p ≤0.05). (c) NHK cells were treated with 2 mM CaCl₂ and 100nM Rapamycin or DMSO control. RNA was isolated and quantitative RT-PCR was performed to measure expression of the indicated differentiation markers. Graph presents mean ± SEM (n = 5–8). Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison. (*p ≤0.05, ****p ≤0.0001, ns: non-significant). (d+e) HaCaT (d) or NHK (e) cells were transfected with siRNA specific for Raptor (Rap), mTOR or an siRNA control (si contr) and differentiation was induced either by post-confluent growth (d) or with 2mM CaCl₂ (e) for 48h. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below each blot a quantification of IVL and P-S6 band relative to actin bands of 4–6 similar blots is shown. Statistical significance was calculated with 2-way ANOVA and Bonferroni multiple comparison, comparing Raptor or mTOR knockdown with the corresponding differentiation state in the control group (*p ≤0.05, ** p ≤0,01, ****p ≤0,0001).</p

mTORC1 and its downstream mediator are strongly activated in psoriatic lesions.

<p>Punch biopsies from lesional psoriatic skin (a,c,e,g) of 20 patients and five healthy donors (b,d,f,h) were stained with antibodies for specific for Rheb, Raptor, P-PRAS40 and P-4E-BP. Nuclei were stained with DAPI. Representative overlay images from one patient and one healthy donor are shown. Bars represent 100 μm.</p

Psoriatic cytokines induce mTOR signaling.

<p>Starved HaCaT (a) or NHK cells (b) were treated for 30 min with 20 ng/ml of the indicated cytokines. Serum starved HaCaT (c) or NHK cells (d) were treated for 30 min with the indicated inhibitors (1 μM Wortmannin, 50 μM LY294002, 50 μM U0126 or 100 nM Rapamycin), followed by a 30 min stimulation with IL-1 β or TNF- α (20ng/ml). Cell lysates were analyzed by Western blotting with antibodies for the indicated proteins. (e) HaCaT cells were seeded in triplicates in 96 well plates, starved overnight and treated with 50 μM LY 294002, 100 nM Rapamycin or 250 nM Torin or solvent (DMSO) as well as 20 ng/ml IL-1 β or TNF- α. After 48h cell proliferation was measured with a BrdU assay. Graph presents mean ± SEM (n = 6). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, ****p ≤0.0001). This shows that mTORC1 does not play a major role in controlling keratinocyte proliferation.</p

Cytokine mediated activation of mTOR interferes with differentiation.

<p>(a,c) HaCaT cells were seeded at proliferating (P; 0,6 *10⁵ cells/12 well) or differentiating (Diff; 6* 10⁵ cells/12 well) conditions and treated with 20 ng/ml of the indicated cytokines or a mix of IL-1 β, Il-17A and TNF- α. (b,d) NHK cells were treated with 2mM CaCl₂ to induce differentiation and 20 ng/ml of the indicated cytokines or the mix of these cytokines. After 72h protein and RNA were isolated. (a,b) Protein samples were analyzed by Western blotting with the indicated antibodies. Below each blot quantification of n≥ 3 similar Western blots is depicted. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01). (c,d) Quantitative RT-PCR was performed to measure expression of the indicated differentiation markers. Graphs present mean ± SEM (n<i>≥</i>5). Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison. (****p ≤0.0001). (e) HaCaT cells were reverse-transfected with siRNA specific for Raptor, mTOR or control siRNA and seeded at 0,6 *10⁵ (P) or 6*10⁵ (Diff) cells per 12 well. After 24h cells were treated with 20 ng of IL-1 β, IL-17A and TNF- α (Mix) and harvested after another 72h. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below a quantification of 7–9 similar blots is shown. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison. For P-S6 statistical significant difference is shown for proliferating cells of knockdown cells compared to proliferating control cells (*p ≤0.05, **p ≤0.01, **** p ≤0.0001). (f) NHK cells were transfected with siRNA specific for Raptor or control siRNA. After 24h the cytokine mix and 2 mM CaCl₂ were added. Differentiation was allowed to proceed for 48h, cells were harvested and protein lysates were analyzed by Western blotting with the indicated antibodies. Below a quantification of seven similar blots is shown. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01). Raptor knockdown rescues the cytokine induced repression of keratinocyte differentiation.</p

Skin Involvement in Psoriatic Arthritis Worsens Overall Disease Activity, Patient-Reported Outcomes, and Increases Healthcare Resource Utilization: An Observational, Cross-Sectional Study

<p></p><p><b>Article full text</b></p> <p><br></p> <p>The full text of this article can be found here<b>. </b><a href="https://link.springer.com/article/10.1007/s40744-018-0120-8">https://link.springer.com/article/10.1007/s40744-018-0120-8</a></p><p></p><p></p><p> </p><p><br></p> <p><b>Provide enhanced content for this article</b></p> <p><br></p> <p>If you are an author of this publication and would like to provide additional enhanced content for your article then please contact <a href="http://www.medengine.com/Redeem/”mailto:[email protected]”"><b>[email protected]</b></a>.</p> <p><br></p> <p>The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.</p> <p><br></p> <p>Other enhanced features include, but are not limited to:</p> <p><br></p> <p>• Slide decks</p> <p>• Videos and animations</p> <p>• Audio abstracts</p> <p>• Audio slides</p><br><p></p

Downregulation of PAX2 decreases the proliferation, migration and invasion of melanoma cells.

<p>Anchorage-dependent (<b>A</b>) and anchorage-independent (<b>B</b>) cell growth was investigated by using a MTT proliferation assay. Twenty-four hours after siRNA transfection, SkMel5 cells treated with transfection reagents alone (mock) or transfected with scrambled siRNA (sc-siRNA) or PAX2-specific siRNAs were seeded into uncoated anchorage dependent cell growth) or polyHEME coated (anchorage independent cell growth) 96 well plates and cell growth was measured 24, 48 and 72 hours later using a MTT-assay. 3 independent experiments have been performed and statistical analysis has been performed using Anova post-hoc analysis. ***P<0.001 considered statistically significant compared to control transfected cells (Mock). <b>###</b>P<0.001 considered statistically significant compared to scrambled-siRNA transfected cells, *P<0.01 considered statistically significant compared to scrambled-siRNA transfected cells. (<b>C</b>) Migration assay of SkMel5 cells was performed 48 h after the transfection with control siRNA (sc-siRNA) or PAX2 specific siRNA (PAX2-siRNA). ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA). (<b>D</b>) The invasive capacity of SkMel5 cells was analyzed 48 h after the transfection of contol (sc-siRNA) or PAX2 siRNA (PAX2-siRNA) in an invasion assay as described under material and methods ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA).</p

Immunohistochemical analysis of PAX2 expression in tissue sections of benign nevi and malignant melanoma.

<p>(<b>A</b>) In normal sweat glands, PAX2 is expressed in gland epithelial cells (black arrows) while intermingled stromal cells only show very weak or absent nuclear PAX2 expression (green arrow) Bar represent 100 µm. (<b>B, C</b>) Normal appearing epidermal cell layers adjacent to (<b>B,</b> bar represent 100 µm) nevi or (<b>C,</b> bar represent 200 µm) malignant melanoma show a differentially PAX2 expression with strongest PAX2 levels in germinal basal cell layers (black arrows) decreasing in higher differentiated keratinocytes and finally being absent in corneocytes (green arrows). (<b>D</b>) Malignant melanoma cells constantly exhibit a heterogeneous nuclear PAX2 expression. Strongest expression is observed in large atypical nuclei with prominent nucleoli (black arrows). Bar represent 50 µm. (<b>E, F</b>) PAX2 expression in intradermal nevi was heterogeneous and did not correlate with histological features (Original magnification: A–C: 20×; D–F: 40×). Bars represent 50 µm.</p

PAX2 is expressed in melanocytes of benign nevi and melanoma cells of patients with malignant melanoma.

<p>Tissue sections of benign nevi (<b>A</b>) and malignant melanoma (<b>B</b>) were investigated by double immunofluorescence analysis with S100 (melanocyte marker) and PAX2 specific antibodies. S100 expression was visualized by Cy3 coupled secondary antibodies (red) and PAX2 expression was detected with Alexa488 coupled goat anti-rabbit secondary antibodies (green). White arrows in the higher magnified insets indicate PAX2 expression in nucleoli of melanocytes of benign nevi (<b>A</b>), yellow arrows in the higher magnified insets specify PAX2 expression in nucleoli of melanoma cells (<b>B</b>).</p