Inflammatory Mediator TAK1 Regulates Hair Follicle Morphogenesis and Anagen Induction Shown by Using Keratinocyte-Specific TAK1-Deficient Mice

Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the NF-κB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial–mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7fl/flK5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7fl/flK5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7fl/flK14-Cre-ERT2). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance

Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB

Nuclear IL-33 levels are high at the epidermal edges of skin wounds and facilitate wound healing. However, IL-33−mediated regulation of keratinocyte (KC) biology during wound healing remains poorly understood. During skin-wound healing, KC migration and re-epithelialization are mediated predominantly by EGFR signaling activation and depend on the function of signal transducer and activator of transcription 3 (STAT3). We found that migrating KCs at the leading edges of mouse skin wounds exhibited concomitant induction and nuclear colocalization of IL-33 and phosphorylated STAT3. In cultured human KCs, activation of EGFR signaling caused rapid elevation of nuclear IL-33, which directly interacts with phosphorylated STAT3, promoting STAT3 activation. In vitro KC migration and wound-healing assays revealed that high nuclear IL-33 levels were required for KC migration and wound closure. KC mobility associated with a lack of suprabasal epidermal keratins and extracellular matrix degradation mediated by matrix metalloproteinases (MMPs) control cell migration at the intracellular and extracellular levels, respectively. In EGFR-activated KCs, nuclear IL-33 mediated keratin 1 and 10 downregulation and MMP9 upregulation by promoting STAT3 activation and limited MMP1, MMP3, and MMP10 induction by suppressing NF-κB transactivation. Thus, epidermal nuclear IL-33 is involved in KC migration and wound closure by regulating the STAT3 and NF-κB pathways

Eccrine sweat contains IL-1α, IL-1β and IL-31 and activates epidermal keratinocytes as a danger signal.

Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal

A Novel Long Noncoding RNA, Lnc-OAD, Is Required for Bone Morphogenetic Protein 2- (BMP-2-) Induced Osteoblast Differentiation

Long noncoding RNAs (lncRNAs) play very important roles in cell differentiation. Our recent study has demonstrated that a novel lncRNA named lnc-OAD modulated 3T3-L1 adipocyte differentiation. In the present study, we examined the roles of lnc-OAD in bone morphogenetic protein 2- (BMP-2-) induced osteoblast differentiation. Lnc-OAD expression was increased during BMP-2-induced osteoblast differentiation in C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblast cells. Knockdown of lnc-OAD expression by specific siRNA remarkably decreased early osteoblast differentiation. In addition, stable knockdown of lnc-OAD by lentivirus vector also significantly inhibited late osteoblast differentiation and matrix mineralization in vitro. Conversely, stably overexpressed lnc-OAD with lentiviral vector accelerated osteoblast differentiation. Mechanistically, knockdown of lnc-OAD reduced significantly the phosphorylation of AKT and the expression of Osterix induced by BMP-2, while overexpression of lnc-OAD enhanced the phosphorylation of AKT and the expression of Osterix. Taken together, our study suggests that lnc-OAD plays an important role in modulating BMP-2-induced osteoblast differentiation via, at least in part, regulating the AKT-Osterix signaling axis

Vesicular LL-37 contributes to inflammation of the lesional skin of palmoplantar pustulosis.

"Pustulosis palmaris et plantaris", or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin

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