Pigmented contact dermatitis and hair dyes: A retrospective case-control multicentre study in Korea.

BACKGROUND: Pigmented contact dermatitis (PCD), a rare variant of non-eczematous contact dermatitis, is clinically characterized by sudden-onset brown or grey pigmentation on the face and neck. It is hypothesized to be caused by repeated contact with low levels of allergens. OBJECTIVES: This study evaluated the risk of using hair dyes in patients with PCD in Korea. METHODS: A total of 1033 PCD patients and 1366 controls from 31 university hospitals were retrospectively recruited. We collected and analysed the data from the patient group, diagnosed through typical clinical findings of PCD and the control group, which comprised age/sex-matched patients who visited the participating hospitals with pre-existing skin diseases other than current allergic disease or PCD. RESULTS: Melasma and photosensitivity were significantly more common in the control group, and a history of contact dermatitis was more common in the PCD group. There were significantly more Fitzpatrick skin type V participants in the PCD group than in the control group. There was no significant difference in sunscreen use between the groups. Using dermatologic medical history, Fitzpatrick skin type and sunscreen use as covariates, we showed that hair dye use carried a higher PCD risk (odds ratio [OR] before adjustment: 2.06, confidence interval [CI]: 1.60-2.65; OR after adjustment: 2.74, CI: 1.88-4.00). Moreover, henna users had a higher risk of PCD (OR before adjustment: 5.51, CI: 4.07-7.47; OR after adjustment: 7.02, CI: 4.59-10.74), indicating a significant increase in the risk of PCD with henna dye use. Contact dermatitis history was more prevalent in henna users than in those using other hair dyes in the PCD group (17.23% vs. 11.55%). CONCLUSION: Hair dye use is a risk factor for PCD. The risk significantly increased when henna hair dye was used by those with a history of contact dermatitis.

Sorafenib induces pigmentation via the regulation of ß-catenin signalling pathway in melanoma cells.

We conducted large-scale screening test on drugs that were already approved for other diseases to find pigmentation-modulating agents. Among drugs with potential for pigmentation control, we selected sorafenib and further investigated the effect on pigmentation using HM3KO melanoma cells. As a result of treating melanoma cells with sorafenib, pigmentation was promoted in terms of melanin content and tyrosinase activity. Sorafenib increased mRNA and protein levels of pigmentation-related genes such as MITF, tyrosinase and TRP1. To uncover the action mechanism, we investigated the effect of sorafenib on the intracellular signalling pathways. Sorafenib reduced phosphorylation of AKT and ERK, suggesting that sorafenib induces pigmentation through inhibition of the AKT and ERK pathways. In addition, sorafenib significantly increased the level of active ß-catenin, together with activation of ß-catenin signalling. Mechanistic study revealed that sorafenib decreased phosphorylation of serine 9 (S9) of GSK3ß, while it increased phosphorylation of tyrosine 216 (Y216) of GSK3ß. These results suggest that sorafenib activates the ß-catenin signalling through the regulation of GSK3ß phosphorylation, thereby affecting the pigmentation process.

Development of Pigmentation-Regulating Agents by Drug Repositioning.

Skin color is determined by the processes of melanin synthesis and distribution. Problems in various molecules or signaling pathways involved in melanin synthesis contribute to skin pigmentation defects. Several trials have been conducted on the production of pigmentation-regulating agents, and drug repositioning has emerged as a modern technique to identify new uses for existing drugs. Our research team has researched substances or drugs associated with pigmentation control and, as a result, nilotinib, sorafenib, and ICG-001 have been found to promote pigmentation, while 5-iodotubercidin inhibits pigmentation. Therefore, these substances or medications were suggested as potential therapeutics for pigmentation disorders by drug repositioning.

Induction of pigmentation by a small molecule tyrosine kinase inhibitor nilotinib.

Skin color is determined by the melanin pigments that are produced in melanocytes then transferred to surrounding keratinocytes. Despite the growing number of commercial products claiming the pigmentation-regulatory effects, there is still a demand for the development of new materials that are safe and more efficacious. We tried to screen the pigmentation-regulatory materials using a commercially available drugs, and found that nilotinib could induce pigmentation in melanoma cells. When HM3KO melanoma cells were treated with nilotinib, melanin content was increased together with increase of tyrosinase activity. Nilotinib increased the expression of pigmentation-related genes such as MITF, tyrosinase and TRP1. Consistent with these results, the protein level for MITF, tyrosinase, and TRP1 was significantly increased by nilotinib. To delineate the action mechanism of nilotinib, we investigated the effects of nilotinib on intracellular signaling. As a result, nilotinib decreased the phosphorylation of AKT, while increased the phosphorylation of CREB. The pretreatment of PKA inhibitor H89 markedly blocked the nilotinib-induced phosphorylation of CREB. In accordance with, pretreatment of H89 significantly inhibited the nilotinib-induced pigmentation, indicating that nilotinib induces pigmentation via the activation of PKA signaling. Together, our data suggest that nilotinib can be developed for the treatment of hypopigmentary disorder such as vitiligo.

Inhibitory effect of 5-iodotubercidin on pigmentation.

Melanin pigments are the primary contributors for the skin color. They are produced in melanocytes and then transferred to keratinocytes, eventually giving various colors on skin surface. Although many depigmenting and/or skin-lightening agents have been developed, there is still a growing demand on materials for reducing pigmentation. We attempted to find materials for depigmentation and/or skin-lightening using the small molecule compounds commercially available, and found that 5-iodotubercidin had inhibitory potential on pigmentation. When HM3KO melanoma cells were treated with 5-iodotubercidin, pigmentation was dramatically reduced. The 5-iodotubercidin decreased the protein level for pigmentation-related molecules such as MITF, tyrosinase, and TRP1. In addition, 5-iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of AKT and ERK. These data suggest that 5-iodotubercidin inhibits melanogenesis via the regulation of intracellular signaling related with pigmentation. Finally, 5-iodotubercidin markedly inhibited the melanogenesis of zebrafish embryos, an in vivo evaluation model for pigmentation. Together, these data suggest that 5-iodotubercidin can be developed as a depigmenting and/or skin-lightening agent.

Inhibitory effect of cucurbitacin B on imiquimod-induced skin inflammation.

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.

Identification of a possible susceptibility locus for UVB-induced skin tanning phenotype in Korean females using genomewide association study.

A two-stage genomewide association (GWA) analysis was conducted to investigate the genetic factors influencing ultraviolet (UV)-induced skin pigmentation in Korean females after UV exposure. Previously, a GWA study evaluating ~500 000 single nucleotide polymorphisms (SNPs) in 99 Korean females identified eight SNPs that were highly associated with tanning ability. To confirm these associations, we genotyped the SNPs in an independent replication study (112 Korean females). We found that a novel SNP in the intron of the WW domain-containing oxidoreductase (WWOX) gene yielded significant replicated associations with skin tanning ability (P-value = 1.16 × 10(-4) ). To understand the functional consequences of this locus located in the non-coding region, we investigated the role of WWOX in human melanocytes using a recombinant adenovirus expressing a microRNA specific for WWOX. Inhibition of WWOX expression significantly increased the expression and activity of tyrosinase in human melanocytes. Taken together, our results suggest that genetic variants in the intronic region of WWOX could be determinants in the UV-induced tanning ability of Korean females. WWOX represents a new candidate gene to evaluate the molecular basis of the UV-induced tanning ability in individuals.

Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis.

HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DOαP11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ∼6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the α1 and α2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/sDM complex and have implications for the mechanism of DO inhibition of DM.

Serum amyloid A overrides Treg anergy via monocyte-dependent and Treg-intrinsic, SOCS3-associated pathways.

The acute phase protein serum amyloid A (SAA) has been well characterized as an indicator of inflammation. Nevertheless, its functions in pro versus anti-inflammatory processes remain obscure. Here we provide unexpected evidences that SAA induces the proliferation of the tolerogenic subset of regulatory T cells (T(reg)). Intriguingly, SAA reverses T(reg) anergy via its interaction with monocytes to activate distinct mitogenic pathways in T(reg) but not effector T cells. This selective responsiveness of T(reg) correlates with their diminished expression of SOCS3 and is antagonized by T(reg)-specific induction of this regulator of cytokine signaling. Collectively, these evidences suggest a novel anti-inflammatory role of SAA in the induction of a micro-environment that supports T(reg) expansion at sites of infection or tissue injury, likely to curb (auto)-inflammatory responses.

An insertion mutant in DQA1*0501 restores susceptibility to HLA-DM: implications for disease associations.

HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules, and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on Ag presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. In this study, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2-DM interface to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface, and the presentation of α-II-gliadin epitope (residues 62-70) to murine, DQ2-restricted T cell hybridomas. We found that the three α-chain mutations (α+53G, α+53R, or αY22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the α+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that α-II-gliadin 62-70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2.

Pitavastatin Induces Apoptosis of Cutaneous Squamous Cell Carcinoma Cells through Geranylgeranyl Pyrophosphate-Dependent c-Jun N-Terminal Kinase Activation.

BACKGROUND: Pitavastatin is a cholesterol-lowering drug and is widely used clinically. In addition to this effect, pitavastatin has shown the potential to induce apoptosis in cutaneous squamous cell carcinoma (SCC) cells. OBJECTIVE: The purpose of this study is to investigate the effects and possible action mechanisms of pitavastatin. METHODS: SCC cells (SCC12 and SCC13 cells) were treated with pitavastatin, and induction of apoptosis was confirmed by Western blot. To examine whether pitavastatin-induced apoptosis is related to a decrease in the amount of intermediate mediators in the cholesterol synthesis pathway, the changes in pitavastatin-induced apoptosis after supplementation with mevalonate, squalene, geranylgeranyl pyrophosphate (GGPP) and dolichol were investigated. RESULTS: Pitavastatin dose-dependently induced apoptosis of cutaneous SCC cells, but the viability of normal keratinocytes was not affected by pitavastatin at the same concentrations. In supplementation experiments, pitavastatin-induced apoptosis was inhibited by the addition of mevalonate or downstream metabolite GGPP. As a result of examining the effect on intracellular signaling, pitavastatin decreased Yes1 associated transcriptional regulator and Ras homolog family member A and increased Rac family small GTPase 1 and c-Jun N-terminal kinase (JNK) activity. All these effects of pitavastatin on signaling molecules were restored when supplemented with either mevalonate or GGPP. Furthermore, pitavastatin-induced apoptosis of cutaneous SCC cells was inhibited by a JNK inhibitor. CONCLUSION: These results suggest that pitavastatin induces apoptosis of cutaneous SCC cells through GGPP-dependent JNK activation.

High-throughput profiling of peptide-RNA interactions using peptide microarrays.

A rapid and quantitative method to evaluate binding properties of hairpin RNAs to peptides using peptide microarrays has been developed. The microarray technology was shown to be a powerful tool for high-throughput analysis of RNA-peptide interactions by its application to profiling interactions between 111 peptides and six hairpin RNAs. The peptide microarrays were also employed to measure hundreds of dissociation constants (K(d)) of RNA-peptide complexes. Our results reveal that both hydrophobic and hydrophilic faces of amphiphilic peptides are likely involved in interactions with RNAs. Furthermore, these results also show that most of the tested peptides bind hairpin RNAs with submicromolar K(d) values. One of the peptides identified by using this method was found to have good inhibitory activity against TAR-Tat interactions in cells. Because of their great applicability to evaluation of nearly all types of RNA-peptide interactions, peptide microarrays are expected to serve as robust tools for rapid assessment of peptide-RNA interactions and development of peptide ligands against RNA targets.

Adults' Perception of Children's Vowel Production.

The study examined the link between Korean-speaking children's vowel production and its perception by inexperienced adults and also observed whether ongoing vowel changes in mid-back vowels affect adults' perceptions when the vowels are produced by children. This study analyzed vowels in monosyllabic words produced by 20 children, ranging from 2 to 6 years old, with a focus on gender distinction, and used them as perceptual stimuli for word perception by 20 inexperienced adult listeners. Acoustic analyses indicated that F0 was not a reliable cue for distinguishing gender, but the first two formants served as reliable cues for gender distinction. The results confirmed that the spacing of the two low formants is linguistically and para-linguistically important in identifying vowel types and gender. However, a pair of non-low back vowels caused difficulties in correct vowel identification. Proximal distance between the vowels could be interpreted to result in the highest mismatch between children's production and adults' perception of the two non-low back vowels in the Korean language. We attribute the source of the highest mismatch of the two non-low back vowels to the ongoing sound change observed in high and mid-back vowels in adult speech. The ongoing vowel change is also observed in the children's vowel space, which may well be shaped after the caregivers whose non-low back vowels are close to each other.

Inhibitory Effect of Mitoxantrone on Collagen Synthesis in Dermal Fibroblasts.

Background: Fibroblasts produce collagen molecules that support the structure of the skin. The decrease and hypersynthesis of collagen causes skin problems such as skin atrophy, wrinkles and scars. Objective: The purpose of this study is to investigate the mechanism of mitoxantrone on collagen synthesis in fibroblasts. Methods: Cultured fibroblasts were treated with mitoxantrone, and then collagen synthesis was confirmed by reverse transcription-polymerase chain reaction and Western blot. Results: Mitoxantrone inhibited the expression of type I collagen in fibroblasts at both the mRNA and protein levels. In the collagen gel contraction assay, mitoxantrone significantly inhibited gel contraction compared to the control group. Mitoxantrone inhibited transforming growth factor (TGF)-ß-induced phosphorylation of SMAD3. Finally, mitoxantrone inhibited the expression of LARP6, an RNA-binding protein that regulates collagen mRNA stability. Conclusion: These results suggest that mitoxantrone reduces collagen synthesis by inhibiting TGF-ß/SMAD signaling and LARP6 expression in fibroblasts, which can be developed as a therapeutic agent for diseases caused by collagen hypersynthesis.

Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes.

Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

Phototoxicity of oriental medicinal plants: measurement and possible applications.

Phototoxicity can be either harmful and induce adverse skin reactions or beneficial and be used therapeutically as in psoralen and UV-A or photodynamic therapy. Hundreds of medicinal plants are widely used in Asia and Western countries in oriental medicine, yet the phototoxicity of oriental medicinal plants is an understudied area. In this contribution, the authors discuss some methods used to measure the phototoxicity of plants and give an overview of the results of their previous and ongoing studies into the phototoxicity of medicinal plants. The authors argue that because they found that more than a quarter of oriental medicinal plants can be phototoxic, such research is helpful for dermatologists and that active phototoxic components extracted from oriental medicinal plants may be used therapeutically.

Palisaded Neutrophilic and Granulomatous Dermatitis in a Patient with Behçet's Disease: A Case Report.

Palisaded neutrophilic and granulomatous dermatitis (PNGD) is an uncommon skin eruption and characterized histopathologically by the presence of granulomatous inflammation with or without leukocytoclastic vasculitis. PNGD is known to be associated with various immune-mediated connective tissue diseases such as rheumatoid arthritis and lupus erythematosus. However, to our knowledge, a case of PNGD in a patient with Behçet's disease is extremely rare and only one case has been reported in foreign literature to date. Herein, we report an unusual case of a 60-year-old female with Behçet's disease who presented multiple erythematous to flesh-colored papules on the extremities, buttocks, and ear lobes and was diagnosed with PNGD. After the treatment of systemic corticosteroids, colchicine and azathioprine, the skin lesions and oral ulcers improved. The patient is under observation without recurrence of skin lesions for 6 months.

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms.

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.

Kinetic and structural characterization of human mortalin.

Human mortalin is an Hsp70 chaperone that has been implicated in cancer, Alzheimer's and Parkinson's disease, and involvement has been suggested in cellular iron-sulfur cluster biosynthesis. However, study of this important human chaperone has been hampered by a lack of active material sufficient for biochemical characterization. Herein, we report the successful purification and characterization of recombinant human mortalin in Escherichia coli. The recombinant protein was expressed in the form of inclusion bodies and purified by Ni-NTA affinity chromatography. The subsequently refolded protein was confirmed to be active by its ATPase activity, a characteristic blue-shift in the fluorescence emission maximum following the addition of ATP, and its ability to bind to a likely physiological substrate. Single turnover kinetic experiments of mortalin were performed and compared with another Hsp70 chaperone, Thermotogamaritima DnaK; with each exhibiting slow ATP turnover rates. Secondary structures for both chaperones were similar by circular dichroism criteria. This work describes an approach to functional expression of human mortalin that provides sufficient material for detailed structure-function studies of this important Hsp70 chaperone.

Complexes of two cohorts of CLIP peptides and HLA-DQ2 of the autoimmune DR3-DQ2 haplotype are poor substrates for HLA-DM.

Atypical invariant chain (Ii) CLIP fragments (CLIP2) have been found in association with HLA-DQ2 (DQ2) purified from cell lysates. We mapped the binding register of CLIP2 (Ii 96-104) to DQ2 and found proline at the P1 position, in contrast to the canonical CLIP1 (Ii 83-101) register with methionine at P1. CLIP1/2 peptides are the predominant peptide species, even for DQ2 from HLA-DM (DM)-expressing cells. We hypothesized that DQ2-CLIP1/2 might be poor substrates for DM. We measured DM-mediated exchange of CLIP and other peptides for high-affinity indicator peptides and found it is inefficient for DQ2. DM-DQ-binding and DM chaperone effects on conformation and levels of DQ are also reduced for DQ2, compared with DQ1. We suggest that the unusual interaction of DQ2 with Ii and DM may provide a basis for the known disease associations of DQ2.