The activation of IL-31 signal in keratinocytes.

<p>(A) Keratinocytes were treated with IL-31 (0∼10 ng/ml) for 8 h, the mRNA expression of CCL2, IL-8 and IL-1β were examined by real time PCR. Relative mRNA levels are expressed as means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of control. (B) Keratinocytes were treated with IL-31 (10 ng/ml) for the indicated times, the levels of phospho-ERK, ERK, phospho-STAT3 and STAT3 were decided by western blotting. (The bands for phospho-STAT1 and phospho-AKT were not detected) (C) Keratinocytes were primed with IL-1Ra for 1h before stimulated with IL-31 (10 ng/ml) or IL-1α (10 ng/ml) for 8 h, the mRNA expression of CCL2, IL-1β and IL-8 was detected by real time PCR of total RNA and their relative mRNA levels were expressed as means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that without IL-1Ra (D) Keratinocytes were treated with IFN-γ (100 mg/ml), IL-4 (10 ng/ml), or IL-13 (50 ng/ml) for 6 h, the mRNA expression of IL-31RA and OSMR was examined by RT-PCR. (E) Keratinocytes were pretreated with IFN-γ for 24 h, then stimulated with or without IL-31 for 8 h, the mRNA expression of CCL2 were examined by real time PCR. Relative mRNA levels are expressed as means SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of IFN-γ stimulation only.</p

The bioactivity of sweat IL-1 is required for sweat-mediated keratinocyte activation.

<p>(A) To block the activation of IL-1 signaling, different concentration of IL-1Ra was applied into cultures for 1 h before stimulation with IL-1β for 24 h, the secretion of IL-8 was detected by ELISA of supernatants. Concentrations represent means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of IL-1β stimulation only. (B) 100 ng/ml IL-1Ra was applied into cultures for 1 h before the addition of concentrated sweat obtained from volunteer 3, keratinocytes were collected after 10 min of stimulation and the phosphorylation of IκBα, ERK and JNK was investigated by Western blotting. Keratinocytes were primed with IL-1Ra for 1 h then stimulated with concentrated sweat obtained from volunteers for 6 h, the mRNA expression of IL-8, IL-1β, RIG-I, NOD2 (C), and CCL2 (E) was detected by real time PCR. Relative mRNA levels were expressed as means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of sweat stimulation without IL-1Ra. (D) Keratinocytes were primed with IL-1Ra for 1h then stimulated with concentrated sweat obtained from volunteers for 24 h, IL-8 secretion from cultures was detected by ELISA of supernatants. Concentrations represent means±SD (<i>n</i> = 3). *<i>p</i><0.05: compared to that of sweat stimulation without IL-1Ra.</p

IL-1α, IL-1β and IL-31 were quantified in the sweats obtained from volunteers.

<p>(A) Sweat samples obtained from 11 healthy volunteers were subjected to ELISA, and the concentrations of IL-1α, IL-1β and IL-31 were detected and quantified against the levels of total sweat protein, respectively. Concentrations (expressed as ng/mg sweat protein) represent means±SD (<i>n</i> = 11). (B) Immunohistochemistry showed the specific expression of IL-31 protein in eccrinne gland in normal skin. (a) The specific staining of IL-31 protein in the eccrine gland apparatus (original magnification: 40X) (b) IL-31 staining in eccrine gland and duct (original magnification: 200X) (c) IL-31 staining in eccrine gland and duct (original magnification: 400X) (d) the marked IL-31 staining in eccrine straight duct (original magnification: 400X) (e) the marked IL-31 staining in the coiled eccrine ducts (original magnification: 200X) (f) negative staining with mouse IgG and then nuclear staining with haematoxylin (original magnification: 40X) (small arrows indicate eccrine glands, and big arrows indicate eccrine ducts).</p

Keratinocyte-specific TAK1 deletion delays hair cycle progression in adolescent mice.

<p>(A) Schedule of tamoxifen application. The hair cycle was synchronized to anagen phase by wax depilation, and tamoxifen was topically applied to the dorsal skin of <i>Map3k7</i>^fl/flK14-Cre-ER^T2mice to delete TAK1. As controls, <i>Map3k7</i>^fl/fl mice or <i>Map3k7</i>^fl/flK14-Cre-ER^T2 mice were treated with tamoxifen or ethanol, respectively. (B) Clinical appearance at the indicated time point after the depilation. At 2 weeks, hair shaft formation was noted in the control mice, while hair shaft growth was not seen in the tamoxifen-treated <i>Map3k7</i>^fl/flK14-Cre-ER^T2 mice. (C) Histological analysis at the indicated time point after the depilation. Anagen progression was severely delayed in TAK1-deleted mice at 1–3 weeks. Scale bar, 100 µm.</p

Keratinocyte-specific TAK1 deletion causes a transition from anagen to dystrophic catagen in adolescent mice.

<p>(A) Schedule of tamoxifen application. The hair cycle was synchronized to anagen phase by wax depilation. At 7 days after depilation, tamoxifen was applied for 5 days. (B) Clinical appearance of the mice 2 weeks after depilation. (C) Histological analysis of the mice 2 weeks after depilation. (D) Higher magnification of the tamoxifen-treated <i>Map3k7</i>^fl/flK14-Cre-ER^T2 mice in (C). Scale bar, 100 µm. (E) Dystrophic catagen was defined according to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011275#pone.0011275-Hendrix1" target="_blank">[28]</a>. Then, the rate (%) of a certain hair follicle stage per total hair follicles was calculated. Quantitative analyses confirmed that most of the hair follicles in tamoxifen-treated <i>Map3k7</i>^fl/flK14-Cre-ER^T2 mice were in late dystrophic catagen, while those of controls were in anagen.</p

Keratinocyte-specific TAK1 deletion results in hair loss in adolescent mice.

<p>Tamoxifen was topically applied to the dorsal skin of the <i>Map3k7</i>^fl/flK14-Cre-ER^T2 mice for 5 consecutive days to delete TAK1. The clinical appearance of the mice 4 weeks after the application is shown. As controls, <i>Map3k7</i>^fl/fl mice or <i>Map3k7</i>^fl/flK14-Cre-ER^T2 mice were treated with tamoxifen or ethanol, respectively.</p

Model for the signaling pathways involved in hair follicle development.

<p>In the development of primary hair placodes, TAK1 is involved in the Edar/NF-κB pathway. In secondary hair placodes, NF-κB (TAK1)-independent pathways regulate hair placode development.</p

Southern blot analysis.

<p>Genomic DNA prepared from the ear skin of mice treated with tamoxifen solution (15 µL/ear) or ethanol for 5 consecutive days was digested with <i>Xba</i>I and <i>Eco</i>RI. Southern blot analysis for the deletion of the floxed <i>Tak1</i> allele was performed as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011275#pone.0011275-Sato1" target="_blank">[1]</a>. Cre expression resulted in excision of the floxed allele (<i>flox</i>) and generated the deleted allele (Δ) of <i>Map3k7</i>.</p