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Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is involved in many signaling pathways via the ubiquitin-proteasome system. UCHL1 is expressed in the human skin and serves as a neuronal marker; however, its functions in melanogenesis remain unknown. Here, we investigated the role of UCHL1 in melanogenesis and elucidated the underlying mechanism using human melanocytes. UCHL1 downregulation by small interfering RNA resulted in upregulation of microphthalmia-associated transcription factor (MITF), tyrosinase, dopachrome tautomerase, tyrosinase-related protein-1, and melanin. In contrast, overexpression of UCHL1 in melanocytes via adenovirus transfection led to downregulation of tyrosinase, dopachrome tautomerase, and tyrosinase-related protein-1 and decreased melanin contents. Furthermore, UCHL1 reduced the protein, but not mRNA, levels of MITF, the upstream regulator of tyrosinase, dopachrome tautomerase, and tyrosinase-related protein-1. Inhibition of de novo protein synthesis and treatment of normal human primary epidermal melanocytes with proteasome inhibitor MG132 revealed that UCHL1 negatively regulates the stability of MITF by binding to the ubiquitinated protein. Finally, overexpression of MITF via an adenovirus restored the level of melanogenesis reduced by UCHL1. Collectively, our findings indicate a role of UCHL1 in regulating skin pigmentation. Suppression of MITF activity by UCHL1 via protein degradation might aid in the development of new therapeutic approaches for melanoma or dyspigmentation disorders.

The exact physiological function of REIC/Dkk-3 in the development of squamous cell carcinoma(SCC) remains unclear.We aimed to investigate the expression pattern and functional role of REIC/Dkk-3 in the development of SCC.We stained normal skin, actinic keratosis (AK) and SCC tissue with REIC/Dkk-3. The proliferation and migration of SCC 12 over-expressed with REIC/Dkk-3 were observed. For in vivo study, SCC12 cells in PBS, SCC12 cells containing LacZ, and REIC/Dkk-3-transduced SCC 12 cells were injected intra-dermally into the left and right backside flanks of SCID mice respectively, and tumor growth was evaluated.REIC/Dkk-3 staining was detected throughout the full epidermis in normal skin, focally positive in AK. Negative or very low stain of REIC/Dkk-3 was observed in SCC in situ, keratoacanthoma, and SCC. REIC/Dkk-3 mRNA level in SCC was very low compared with that in normal skin tissue. REIC/Dkk-3 significantly decreased the proliferation and migration of SCC12 cells comparing with control (p0.05). Cyclin D1 and CDK4/6 expression was slightly lower and p21 was very higher in REIC/Dkk-3-overexpressed group than in the LacZ group. Fewer ITGA6 cells were found in the REIC/Dkk-3 overexpressed group than in the LacZ control (p0.01). Mean tumor volume was smallest in the REIC/Dkk-3 overexpressed group (p0.01) 21days after the intradermal injection of SCC12 cells.REIC/Dkk-3 could be involved early in SCC development and have inhibitory effect on the development of SCC.

Aims Acne is a common skin disease that originates in the sebaceous gland. The pathogenesis of acne is very complex, involving the increase of sebum production and perifollicular inflammation. In this study, we screened the anti-lipogenic material and demonstrated its effect using cultured human sebocytes. Main methods Normal human sebocytes were cultured by explanting the sebaceous glands. To evaluate the anti-lipogenic effect, sebocytes were treated with test materials and 14C-acetate incorporation assay was performed. Key findings To screen the anti-lipogenic materials, we tested the effect of many herbal plant extracts. We found that Angelica dahurica extract inhibited the insulin-like growth factor-1 (IGF-1)-induced sebum production in terms of squalene synthesis in sebocytes. Furthermore, imperatorin isolated from A. dahurica showed remarkable inhibitory effect on squalene production as well as squalene synthase promoter activity. To investigate the putative action mechanism, we tested the effect of imperatorin on intracellular signaling. The results showed that imperatorin inhibited IGF-1-induced phosphorylation of Akt. In addition, imperatorin significantly down-regulated PPAR-γ and SREBP-1, the important transcription factors for lipid synthesis. Significance These results suggest that imperatorin has a potential for reducing sebum production in sebocytes, and can be applicable for acne treatment.

Psoriasis is a common skin disease, of which pathogenesis involves the increase of inflammatory reaction in epidermal cells. In an attempt to find therapeutics for psoriasis, we found that cucurbitacin B has an inhibitory potential on imiquimod-induced inflammation of keratinocytes. Cucurbitacin B significantly inhibited imiquimod-induced expression of crucial psoriatic cytokines, such as IL-8 and CCL20, via down-regulation of NF-κB and STAT3 signaling pathway in human keratinocytes. In addition, keratinocyte proliferation was markedly inhibited by cucurbitacin B. The potential beneficial effect of cucurbitacin B on psoriasis was further validated in imiquimod-induced psoriasiform dermatitis of experimental animal. Topical application of cucurbitacin B resulted in significant reduction of epidermal hyperplasia and inflammatory cytokines production, and ameliorated the psoriatic symptom. Taken together, these results suggest that cucurbitacin B may be a potential candidate for the treatment of psoriasis.

Propionibacterium acne and sebaceous glands are considered to have an important role in the development of acne. Although information regarding the activation of innate immunity by P. acnes in the sebaceous gland is limited, different P. acnes phylotypes and a higher prevalence of follicular P. acnes macrocolonies/biofilms in sebaceous follicles of skin biopsies from acne compared with control skin and occasionally single P. acnes clusters in single sebaceous glands have been detected. In this study, we investigated whether P. acnes activates the inflammasome in human sebaceous glands in vivo and in vitro. We found that IL-1β expression was upregulated in sebaceous glands of acne lesions. After stimulation of human sebocytes with P. acnes, the activation of caspase-1 and secretion of IL-1β were enhanced significantly. Moreover, knocking down the expression of NLRP3 abolished P. acnes-induced IL-1β production in sebocytes. The activation of the NLRP3 inflammasome by P. acnes was dependent on protease activity and reactive oxygen species generation. Finally, we found that NALP3-deficient mice display an impaired inflammatory response to P. acnes. These results suggest that human sebocytes are important immunocompetent cells that induce the NLRP3 inflammasome, and that P. acnes-induced IL-1β activation in sebaceous glands may have a role in combating skin infections and in acne pathogenesis.

Background O-linked β- N -acetylglucosamine (O-GlcNAc) modification is one of the posttranslational modification, emerging as an important regulatory mechanism in various cellular events. Objective We attempted to investigate whether O-GlcNAcylation is involved in keratinocyte differentiation. Methods Immunohistochemistry and Western blot were performed to demonstrate O-GlcNAcylation in keratinocyte differentiation. Results During calcium-induced keratinocyte differentiation, overall O-GlcNAcylation was decreased in a temporal manner. We focused our attention on transcription factor Sp-1, which is implicated in keratinocyte differentiation. Total Sp-1 level did not change during keratinocyte differentiation. However, O-GlcNAcylated Sp-1 was decreased in a keratinocyte differentiation-dependent manner. Interestingly, transcriptional activity of Sp-1, in terms of involucrin and loricrin promoter activities, was markedly increased by overexpression of O-GlcNAcase (OGA). In addition, membrane permeable non-O-GlcNAcylated Sp-1 did show transcriptional activity, while membrane permeable O-GlcNAcylated Sp-1 did not, suggesting O-GlcNAcylated Sp-1 is an inactive form in keratinocyte differentiation. Conclusion Our results reveal that O-GlcNAcylation is a dynamic regulatory mechanism for keratinocyte differentiation.

Background Alopecia areata (AA), a chronic, relapsing hair-loss disorder, is considered to be a T-cell-mediated autoimmune disease. High-mobility group box 1 (HMGB1), released by necrotic cells and in response to various inflammatory stimuli, is currently considered to be a significant target antigen in diverse autoimmune diseases. Objective We sought to investigate the clinical significance of serum HMGB1 levels in AA. Methods We compared levels of HMGB1 in scalp specimens from 7 patients with AA and 8 healthy control subjects and in blood samples from 45 patients with AA and 10 healthy control subjects. Moreover, we evaluated the correlation between HMGB1 level and clinical severity. Results Immunohistochemical staining of scalp tissues from patients with AA revealed higher HMGB1 levels than in healthy control subjects. In addition, serum HMGB1 levels in the AA group were generally higher, and showed concordance with the patients' clinical characteristics, including onset, hair-pull test results, and treatment response. Limitations The number of patients and healthy control subjects evaluated was small. Conclusion These results suggest that HMGB1 plays a significant role in the pathogenesis of AA, and that it is a promising predictor of prognosis and treatment response. Moreover, this study identifies a new potential therapeutic target for the treatment of AA.

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

S100A8 and S100A9 are members of the S100A8 protein family that exist as homodimers and heterodimers in neutrophils, monocytes, and macrophages. Recent studies have shown the pivotal roles of S100A8 and S100A9 in the propagation of inflammation and keratinocyte proliferation in psoriasis. We found significant up-regulation of S100A8 and S100A9 secretion from keratinocytes in psoriatic lesions. To mimic the in vivo secretory conditions of S100A8 and S100A9 from psoriatic epidermal keratinocytes, we used the culture medium (CM) of S100A8 and S100A8/A9 adenovirus-transduced keratinocytes to investigate the functions of S100A8 and S100A9. We detected increased levels of various pro-inflammatory cytokines in the CM, including IL-8 and TNF-α, which are involved in aggravating psoriatic skin lesions, and IL-6 and members of the CXCL family of pro-angiogenic cytokines. The CM increased immune cell migration and increased angiogenesis in human umbilical vein endothelial cells. In conclusion, we found that the upregulated production of S100A8 and S100A9 by psoriatic epidermal keratinocytes activated adjacent keratinocytes to produce several cytokines. Moreover, S100A8 and S100A9 themselves function as pro-angiogenic and chemotactic factors, generating a psoriatic milieu in skin.

Background: Melanocyte dendrites serve as the principal conduit for melanosome transfer. The dendrite formation requires actin polymerization mediated by Rho family GTPases including RhoA, Rac1 and Cdc42. Objective: The aim of this study is to investigate and explore the involvement of p38 MAPK in melanocyte dendrite formation. Methods: We transduced melanoma cells with adenovirus harboring the expression cassette for constitutive active form of MKK6, an upstream MAPKK for p38 MAPK. Results: We investigated the effect of melanogenic inducers on melanocyte dendricity, using SK-mel-24 melanoma cells because that this cell line is refractory to several melanogenic inducers in terms of melanogenesis. TPA-induced the phosphorylation of p38 MAPK and the elongation of dendrite length, suggesting that MKK6 may be involved in this process. Overexpression of the constitutive active form of MKK6 resulted in significant elongation of dendrites in the melanoma cell line SK-mel-24. Moreover, overexpression of MKK6 ultimately led to the upregulation of Cdc42 and Rac1, suggesting that MKK6 acts as a crucial upstream signaling molecule for Rho family GTPases. When overexpressed in normal human epidermal melanocytes, MKK6 led also the increase of dendrite length. Conclusion: These results suggest that MKK6 is an authentic regulator for melanocytes dendricity, through the modulation of Rho family GTPases.

We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription–PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. We previously investigated the gene expression profile and found that many of differentiation-related genes expressed in a temporally regulated manner. In this study, we attempted to find the hub-molecules and their intracellular signaling networks during keratinocyte differentiation using in silico analysis of data obtained from previous studies. We used protein-protein interaction prediction software called PSIMAP, and drew a hypothetical signaling network. We chose one candidate hub-molecule SHC1 that were predicted to link EGFR and MAPK signal, and then evaluated the protein-protein interactions experimentally. As predicted, SHC1 bound to the MEK1 in an EGF-regulated manner. Furthermore, SHC1 bound to the MEK1 and p38 MAPK in a keratinocyte differentiation dependent manner. These results demonstrate that in silico protein-protein interaction prediction system can be used to efficiently and cost-effectively select the experimental candidates.

Alopecia areata (AA) is a common disease of patchy hair loss on the scalp that can progress to cover the entire scalp and eventually the entire body. Intralesional injection of corticosteroids is the first-line therapy for adult patients, however some patients do not respond to glucocorticoid treatment effectively. To delineate the molecular mechanism underlying glucocorticoid insensitivity, we examined the expression of glucocorticoid receptor (GR) and thioredoxin reductase 1 (TrxR1). In some case of glucocorticoid-resistant AA patients, the expression of TrxR1 was decreased in outer root sheath (ORS). We then investigated the effect of TrxR1 on GR activity using recombinant adenoviruses. Overexpression of TrxR1 markedly increased GR activity in ORS cells cultured in vitro. In addition, TrxR1 protected GR activity against H2O2. Finally, TrxR1-enhanced GR activity was significantly inhibited by the overexpression of dominant negative form of Trx (TrxC32S/C35S). These results suggest that decreased TrxR1 may be one putative cause for glucocorticoid resistance in AA, through the impact on intracellular redox system.

Summary Background Sphingosylphosphorylcholine (SPC) has been reported as a novel lipid mediator that exerts various actions on wound healing process. Objective The aim of this study is to evaluate the involvement of interleukin-6 (IL-6) in SPC-induced wound healing acceleration. Methods We performed immunohistochemical analysis to demonstrate the IL-6 induction by SPC. To analyze the signaling events, skin fibroblasts were treated with SPC, and then RT-PCR, ELISA and Western blot analyses were carried out. Results SPC markedly induced interleukin-6 (IL-6) expression in rabbit ear wound. SPC also induced IL-6 expression at both the mRNA and protein levels in human dermal fibroblasts cultured in vitro. SPC rapidly phosphorylated p42/44 extracellular signal-regulated kinase (ERK). Pretreatment with PD 98059, a specific MAPK kinase 1/2 inhibitor, markedly suppressed SPC-induced IL-6 expression in a dose-dependent manner. Protein kinase C (PKC) activation by phorbol myristate acetate (PMA) potentiated IL-6 mRNA expression, whereas PKC inhibition by bisindolylmaleimide blocked SPC-induced p42/44 ERK phosphorylation and IL-6 expression. Over-expression of PKCα markedly induced the IL-6 expression and p42/44 ERK activation. Conclusion These results suggest that SPC-induced IL-6 production is mediated by PKC-dependent p42/44 ERK activation in human dermal fibroblasts cultured in vitro.

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation. Citation: Shi G, Sohn K-C, Li Z, Choi D-K, Park YM, et al. (2013) Expression and Functional Role of Sox9 in Human Epidermal Keratinocytes. PLoS ONE 8(1): e54355. doi:10.1371/journal.pone.0054355 Editor: Andrzej T. Slominski, University of Tennessee, United States of America Received June 20, 2012; Accepted December 11, 2012; Published January 18, 2013 Copyright: 2013 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a grant of the National Research Foundation of Korea (KRF-2008-314-E00152), and a grant of the Korea Health 21 R&D Project from the Korea Ministry of Health, Welfare and Family Affairs (01-PJ3-PG6-01GN12-0001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jhoon@cnu.ac.kr