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Background Antimicrobial peptide derived from insulin-like growth factor binding protein-5 (AMP-IBP5) is a potent antimicrobial agent that possesses various immunomodulatory activities. The parent protein of AMP-IBP5, IGFBP-5, has been shown to exert its effects via an insulin-like growth factor-1 receptor-independent mechanism, including binding to multifunctional low-density lipoprotein receptor-related protein 1 (LRP1), which contributes to several biological processes involved in skin wound healing. Objectives To investigate whether LRP1 is involved in AMP-IBP5-induced migration and proliferation of human epidermal keratinocytes and dermal fibroblasts. Methods The mRNA expression of LRP1 and IGFBP-5 was assessed by quantitative real-time PCR, whereas Western blotting was used to evaluate the protein expression. Production of cytokines was determined by ELISA. Cell migration was measured by the scratch wound assay, whereas cell proliferation was analyzed using the BrdU labeling assay. MAPK activation was determined by Western blotting. Results We found that AMP-IBP5 markedly induced the migration and proliferation of keratinocytes and fibroblasts, and this effect was reversed by specific siRNA and neutralizing antibody targeting the LRP1 receptor. In addition, LRP1 was upregulated by lipopolysaccharide, flagellin and AMP-IBP5 in keratinocytes and fibroblasts. LRP1 knockdown also inhibited MAPK pathway activation, which was required for AMP-IBP5-mediated cell migration and proliferation, as evidenced by the specific inhibitors for extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38. Conclusions Our results suggest that LRP1 expressed in human epidermal keratinocytes and dermal fibroblasts contributes to AMP-IBP5-mediated cell migration and proliferation, supporting its crucial role in cutaneous wound healing process.

Background Topical corticosteroid and calcineurin inhibitor have similar therapeutic benefits in atopic dermatitis (AD), but the differences in therapeutic mechanisms of action of these agents against AD symptoms are not fully understood. Objective This study was performed to examine the different effects of topical betamethasone valerate (BMV), clobetasol propionate (CBP), and tacrolimus (TAC) on itch-related behavior and dermatitis in NC/Nga mice with AD-like symptoms. Methods AD-like dermatitis was induced in the dorsal skin of NC/Nga mice by repeated topical application of Dermatophagoides farinae body (Dfb) ointment twice weekly for three weeks. Mice with dermatitis scores over 5 were divided into five groups with equal dermatitis scores and treated with BMV, CBP, TAC, or Vaseline (Vas) once daily for two consecutive days, or were not treated (NT). Scratching behavior was analyzed using a SCLABA ® -Real system. Transepidermal water loss (TEWL) before and after treatment was measured using a Tewameter ® TM210. Skin collected from each group was analyzed histologically. Results After the second treatment, dermatitis showed significantly greater improvement in the CBP and TAC-treated groups than in the Vas-treated and NT groups. The numbers of scratching bouts were significantly lower in CBP and TAC-treated mice than in Vas-treated mice. TEWL was significantly lower in TAC-, but not in CBP-, treated mice than in Vas-treated mice. Immunohistochemical examination showed that BMV, CBP and TAC did not reduce the increased densities of epidermal protein gene product 9.5- and substance P-immunoreactive fibers. The numbers of dermal CD4-immunoreactive T cells were significantly lower in BMV and CBP-treated mice than in Vas-treated and NT mice. The numbers of dermal eosinophils were significantly lower in BMV, CBP and TAC-treated mice than in Vas-treated and NT mice, with CBP showing the strongest effect. CBP significantly reduced epidermal thickness compared with Vas and NT. There were no significant differences in the numbers of interleukin-31-immunoreactive cells and mast cells, or in expression of epidermal thymic stromal lymphopoietin among all five groups. Conclusion The therapeutic potency of TAC against AD-like symptoms, including pruritus, is equal to that of the corticosteroid CBP. Epidermal innervation of sensory nerves itself might not be related to the therapeutic effects of topical tacrolimus and corticosteroids in its early phase.

Background In addition to their antimicrobial activities, antimicrobial peptides, also known as host defense peptides (HDPs) activate keratinocytes; promote wound healing; and improve the skin barrier. AG-30/5C is a novel angiogenic HDP that activates various functions of fibroblasts and endothelial cells, including cytokine/chemokine production and wound healing. Objectives To investigate whether AG-30/5C activates human keratinocytes and to examine the underlying mechanisms. Methods Production of cytokines/chemokines was assessed by ELISA. Expression of Mas-related G-protein coupled receptors X (MrgXs) in keratinocytes was determined by real-time PCR and Western blot. MAPK and NF-κB activation was analysed by Western blot. Cell migration was assessed by chemotaxis microchamber and in vitro wound closure assay, whereas cell proliferation was analysed using an XTT assay. Results We found that AG-30/5C was more efficient than its parent peptide AG-30 in increasing the production of various cytokines/chemokines and promoting keratinocyte migration and proliferation. Furthermore, MrgX3 and MrgX4 receptors were constitutively expressed in keratinocytes at higher levels than MrgX1 and MrgX2, and were up-regulated upon stimulation with TLR ligands. Because MrgX3 and MrgX4 siRNAs suppressed AG-30/5C-mediated cytokine/chemokine production, keratinocyte migration and proliferation, we propose that AG-30/5C utilizes these MrgXs to stimulate keratinocytes. In addition, AG-30/5C-induced activation of keratinocytes was controlled by MAPK and NF-κB pathways, as evidenced by the inhibitory effects of ERK-, JNK-, p38- and NF-κB-specific inhibitors. Indeed, we confirmed that AG-30/5C enhanced phosphorylation of MAPKs and IκB. Conclusions Our findings provide novel evidence that AG-30/5C may be a useful therapeutic agent for wound healing by activating human keratinocytes.

Background Interleukin (IL)-37, a new member of the IL-1 family, is characterized as a fundamental inhibitor of innate immunity: it dampens the production of proinflammatory cytokines, protects against inflammatory and autoimmune diseases, and plays a potent immunosuppressive role in the pathogenesis of psoriasis. IL-37 is highly expressed in psoriatic skin, in which human β-defensins (hBDs) have been detected. Although hBDs enhance the production of cytokines, including IL-1 cytokines, whether they stimulate the production of IL-37 remains unclear. Objectives To assess the ability of hBDs to stimulate IL-37 expression/production by human keratinocytes and to determine the mechanism involved. Methods Real-time PCR and Western blotting were used to evaluate IL-37 expression. Caspase activities were assessed using colorimetric assay kits. A CCR6 antibody, siRNA, and caspase, Smad3, MAPK and NF-κB inhibitors were used to investigate the signaling mechanism of hBDs. Results Among the four hBDs used, only hBD-3 up-regulated the mRNA and protein expression of IL-37. The combination of TNF-α, EGF and poly (I:C) with hBD-3 synergistically enhanced the mRNA but not the protein expression of IL-37. Furthermore, hBD-3 increased the release of IL-37 into the culture supernatants. Evaluation of the signaling mechanism of hBD-3 suggested that caspases 1 and 4, Smad3, CCR6, MAPKs and NF-κB were required for hBD-3-mediated IL-37 expression. Conclusions The finding that hBD-3 stimulates IL-37 expression, a novel target for the pathogenesis and therapy of cutaneous inflammatory diseases, provides evidence that hBDs contribute to the suppression of inflammatory and innate immune responses through the regulation of IL-37 expression.

Background In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. Objectives To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Methods Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Results Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Conclusions Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes.

Background Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by skin barrier dysfunction, allergic inflammation and intractable pruritus resistant to conventional antipruritic treatments, including H 1 -antihistamines. Granzymes (Gzms) are a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer cells that have been shown to modulate inflammation. However, the relationship between Gzms and pathology in AD remains unclear. Objective This study assessed the correlation between plasma GzmB levels and severity of pruritus and dermatitis, in AD patients. Methods Plasma was collected from 46 patients with AD, 24 patients with psoriasis, and 30 healthy controls. AD severity was assessed with the scoring atopic dermatitis (SCORAD) index, psoriasis severity with the psoriasis area and severity index (PASI), and degree of pruritus by visual analogue scale (VAS) score. GzmA, GzmB and gastrin releasing peptide (GRP) levels were measured by enzyme-linked immunosorbent assays. Results Plasma GzmB concentrations were significantly higher in patients with AD and psoriasis than in healthy controls. Correlation analyses showed that plasma GzmB concentrations positively correlated with SCORAD and serum levels of severity markers such as thymus and activation-regulated chemokine, and lactate dehydrogenase in AD patients. Moreover, plasma levels of GRP, an itch-related peptide, were higher in patients with AD, positively correlating with VAS score and plasma GzmB level. In addition, plasma GzmB concentration was significantly lower in the treatment group than the untreated group with AD. Meanwhile, there were no correlations among GzmB levels, VAS score and PASI score in patients with psoriasis. In contrast to the results of plasma GzmB, plasma GzmA levels were unchanged among AD, psoriasis and healthy groups, and showed no correlations with VAS score and SCORAD index in patients with AD. Conclusion Plasma GzmB levels may reflect the degree of pruritus and dermatitis in patients with AD.

Background Thymic stromal lymphopoietin (TSLP), highly expressed in keratinocytes in atopic dermatitis patients and bronchial epithelial cells in asthma patients, plays a key role in allergic diseases. Two forms of TSLP mRNA (long and short) have been reported. Objective We compared the expression of the long-form and total TSLP transcripts in primary human keratinocytes. Methods Primary human keratinocytes were stimulated with Toll-like receptor (TLR) ligands, cytokines, and vitamin D receptor agonists. Gene expression was analyzed by quantitative real-time PCR. The amount of TSLP released was measured by ELISA. Results PolyI:C (TLR3 ligand), FSL-1 (TLR2–TLR6 ligand) and flagellin (TLR5 ligand) upregulated long-form TSLP expression, which predominantly contributed to upregulation of total TSLP expression. A glucocorticoid or an endosomal acidification inhibitor inhibited the polyI:C-dependent upregulation of total TSLP and the decrease of the total TSLP was due to the decrease of the long-form. An atopic cytokine milieu (TNF-α+IL-4+IL-13) or TNF-α alone also upregulated the long-form. These stimuli also induced the release of TSLP. In contrast, a high concentration of calcitriol (1,25-dihydroxyvitamin D 3 , the active form of vitamin D 3 ) or its analog MC903 upregulated total TSLP significantly but not the long-form, and did not induce the release of TSLP. Conclusion TLR ligands or cytokines predominantly upregulate the gene expression of the long TSLP form, which contributes to the TSLP protein production, in primary human keratinocytes. Specific measurement of the long-form rather than total TSLP should be useful for accurate detection of functional human TSLP gene expression.

Background Increasing evidence suggests that stress can trigger and exacerbate atopic dermatitis (AD). Psychotherapy is becoming more important in the treatment of AD patients. Yokukansan (YKS, Yi-Gan San in Chinese), a traditional Japanese medicine, has been widely utilized in the treatment of neurosis, insomnia and anxiety especially in Asian countries. Furthermore, it was reported that YKS inhibited skin lesions in socially isolated mice but not in group-housed mice. Therefore, in the present study it was investigated whether or not YKS was effective in the treatment of AD using socially isolated NC/Nga mice. Objective The present study was designed to assess the effect of YKS on the development of AD-like lesions in socially isolated NC/Nga mice to obtain information about its usefulness in the treatment of AD. Methods Ten-week-old male NC/Nga mice were socially isolated under conventional conditions. YKS was administered orally to mice at the dose of 0.5% or 1.0% together with diet. The efficacy of YKS was evaluated by assessing skin lesion severity, scratching behaviors, skin hydration, and infiltration of inflammatory cells in the skin. Grooming behaviors evoked by social isolation stress and serum corticosterone levels were also measured. Results Oral administration of YKS to socially isolated NC/Nga mice resulted in the inhibition of exacerbation of AD-like skin lesions. It seemed that the inhibition of exacerbation of AD-like skin lesions observed in NC/Nga mice might be due to suppression of the scratching and grooming behaviors, inhibition of the infiltration of mast cells and eosinophils, and retention of humidity in the skin. Serum corticosterone levels were also significantly inhibited in the 1%-YKS-treated mice as compared with those of the control mice. There were no significant differences in the levels of serum total IgE and nerve growth factor (NGF) between the YKS-treated mice and the non-treated control mice. Conclusion YKS inhibited the development of AD-like skin lesions in socially isolated NC/Nga mice by suppressing scratching and infiltration of inflammatory cells in the skin. These results indicate that YKS possesses an anti-itching property, and its anti-itching may be partly through attenuation on social isolation stress. It is expected that YKS might provide an effective alternative therapy for AD in human patients.

Background Epidermal nerve densities are increased in patients with atopic dermatitis (AD), suggesting that it is partly responsible for the intense itching in the skin. Epidermal hyperinnervation in AD patients is decreased by ultraviolet (UV) phototherapy, although the underlying mechanisms are poorly understood. Interestingly, abnormal expression of axonal guidance molecules, such as nerve growth factor (NGF) and semaphorin 3A (Sema3A), is found in the epidermis of AD patients. Therefore, UV phototherapy may alter levels of axonal guidance molecule expression in atopic skin. Objective This study was performed to investigate whether epidermal Sema3A and NGF levels in AD are influenced by psoralen-UVA (PUVA) therapy. Methods Skin biopsies obtained from chronic AD patients before and after PUVA therapy were used. Both Sema3A and NGF in the skin were examined at mRNA and protein levels by quantitative RT-PCR and immunohistochemistry, respectively. Nerve fibers in the skin were stained with anti-PGP9.5 antibody, and the number of epidermal nerve fibers was counted. Results PUVA therapy decreased epidermal nerve densities in AD patients, concomitant with decreases in both visual analog scale (VAS) scores for pruritus and clinical severity scores. Increased fluorescence intensity of Sema3A and decreased fluorescence intensity of NGF were observed in the epidermis of PUVA-treated group. Moreover, Sema3A mRNA levels were upregulated in the PUVA-treated skins compared with untreated controls, while NGF mRNA levels in the skin were downregulated by the treatment. Conclusion PUVA therapy may reduce epidermal hyperinnervation of AD by normalization of abnormal Sema3A and NGF expression in the epidermis.

Summary Background Skin dryness is apparent in several pruritic skin diseases, such as xerosis and atopic dermatitis. Our previous study has demonstrated an increase of nerve fibers in the epidermis of patients with xerosis, suggesting the contribution of nerve fibers to itching. Objective This study was conducted to reveal a direct linkage between dry skin and intraepidermal nerve growth. Methods ICR mice treated with acetone were used as a dry skin model. Time-dependent measurement of transepidermal water loss (TEWL) and stratum corneum (SC) hydration was performed on the treated areas. Moreover, both the distribution of intraepidermal nerve fibers and the expression of epidermal nerve growth factor (NGF) and amphiregulin (AR) were examined sequentially with immunohistochemistry and quantitative RT-PCR. The same experiments were carried out in control mice treated with sterile water. Results Enhanced TEWL and decreased SC hydration were observed in the acetone-treated skins during the first hour after the treatment. These parameters gradually returned to the normal range within 48 h. In the acetone-treated mice, we found that there were many nerve fibers in the epidermis between 16 and 48 h after the treatment. No changes of the parameters for barrier disruption and intraepidermal nerve growth were observed in the control skins. Moreover, the expression of epidermal NGF and AR at the protein and mRNA levels was increased before the penetration of nerve fibers into the epidermis. Conclusions These results suggest that increases of epidermal NGF and AR levels are associated with intraepidermal nerve growth in acetone-treated mice.

Summary Background Pruritus is a symptom in atopic dermatitis (AD). Previous studies have reported that increased intraepidermal neurites are observed in AD, suggesting that the neuritogenesis is related to itching in the skin. Objective This study was conducted to reveal the mechanism of intraepidermal neurite formation in AD. Methods In this study, we used conventional (Conv) NC/Nga mice with AD. NC/Nga mice maintained in specific pathogen-free (SPF) condition were used as a control with no AD. Distribution of intraepidermal neurites and expression patterns of growth factors (NGF and amphiregulin (AR)) and cell–cell junctional molecules (E-cadherin, zona occludens 1 (ZO-1) and desmoglein 3 (Dsg3)) were examined in the skins by immunohistochemistry or quantitative RT-PCR. Furthermore, detection of gelatinase activity was performed with in situ zymography. The same experiments were conducted in ICR mice for comparison with NC/Nga mice. Results Neurite density and expression levels of growth factors and gelatinase were remarkably increased in the epidermis of Conv-NC/Nga mice compared with those of SPF-NC/Nga mice. Decreased expression of E-cadherin and ZO-1 and misexpression of Dsg3 were also observed in the atopic skins. In comparison with ICR mice, increases of neurite density and gelatinase activity were found in the skins of SPF-NC/Nga mice but expression levels of growth factors and cell–cell junctional molecules were unchanged. Conclusions Increases of growth factors and gelatinase activity may be related to neurite outgrowth in the epidermis of atopic NC/Nga mice. Additionally, abnormal expressions of cell–cell junctional molecules in the epidermis may provide intercellular spaces for the neurite formation.

Beside its physical barrier against invading microorganisms, the skin has the ability to produce a number of antimicrobial peptides and proteins, including human beta-defensins, cathelicidin LL-37 and lysozyme that participate in the innate host defense. These antimicrobial agents are strongly active against a wide spectrum of various pathogens such as bacteria, viruses and fungi. Thus, antimicrobial agents are proposed to be promising candidates for innovative anti-infective drugs, and some antimicrobial peptides are currently used in clinical trials for treatment of various skin infections. In addition to their direct antimicrobial functions against invading pathogenic microorganisms, antimicrobial agents have also multiple roles as mediators of inflammation with the effects on epithelial and inflammatory cells, influencing cell proliferation, wound healing, cytokine/chemokine production and chemotaxis. This review describes the biology of these antimicrobial molecules and discusses their structure, expression and functions. Understanding the actions of antimicrobial agents in skin will provide further insight into the mechanism of innate cutaneous disease control, and yield novel therapeutic approaches to the treatment of skin disorders.

Summary Background: The umbilical cord (UC) is composed of connective tissue called Wharton Jelly, covered by a simple epithelium believed to derive from amniotic membrane epithelium. In previous studies, we observed that the umbilical cord epithelium (UCE) in situ displayed stratified epithelial structures, in some areas that expressed cytokeratins and differentiation markers as characteristic of keratinocytes under airlifted condition in vitro, UCE cells grown on collagen gel displayed more keratinocytes characteristics. Objective: To study the ability of UCE cells to undergo terminal differentiation when grown in the most proper environment. Methods: UCE cells were seeded onto the surface of a fibroblast-populated collagen gel then grafted onto the back of nude mice and examined using immunohistochemical techniques and by transmission electron microscope (TEM). Results: Post-grafted UCE cells formed a stratified epithelial structure similar to that formed by keratinocytes. Although immunohistochemical staining of UCE cells in skin grafts showed a similar pattern to that seen with the keratinocyte controls, UCE cells maintained many of their own intrinsic charactersitics, such as stronger expression of mucous membrane cytokeratins and expression of simple epithelial cytokeratin. Notably, with longer transplant periods, expression of keratinocyte characteristics in UCE cells increased while expression of simple epithelial properties decreased. We observed formation of a complete basement membrane, which had not been achieved using an in vitro model. Conclusions: Grafted UCE cells in an animal model maintain their own intrinsic characteristics, but display the stratified morphogenesis, terminal differentiation and ultrastructures similar to those seen in keratinocytes.

Summary Background: Atopic dermatitis (AD) and psoriasis are common inflammatory skin diseases. Although many reports implicate Th2 cytokines in the pathophysiology of AD and Th1 cytokines in psoriasis, the precise etiology of these diseases remains elusive. Objective: We investigated novel AD- or psoriasis-related genes to further understand the pathogenesis of these diseases. Methods: We performed a comprehensive analysis of mRNA expression in skin biopsies from AD or psoriasis patients using DNA microarrays. Quantitative PCR was then used to monitor the expression of novel disease-related genes in human keratinocytes or pinnae from NC/Nga mice. Results: Levels of mRNA for IDO (indoleamine 2,3-dioxygenase) and kynureninase, enzymes constituting the tryptophan degradation pathway, were found to be upregulated in the skin lesions as compared to the uninvolved skin of patients with AD or psoriasis. Expression of these two genes was induced in human epidermal keratinocytes stimulated with IFN-γ in vitro. Moreover, in NC/Nga mice, the expression of kynureninase mRNA in the ear skin was induced following development of AD-like skin lesions. Conclusion: The tryptophan degradation pathway may play an important role in the pathophysiology of AD and psoriasis.

Background: A Th2 chemokine, thymus and activation regulated chemokine (TARC/CCL17), produced by keratinocytes, is implicated in the development of atopic dermatitis by recruiting CLA+CCR4+ lymphocytes into lesional skin and its expression was induced by proinflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). However, it remains unknown how TARC expression is negatively regulated in keratinocytes. Objective: We sought to determine whether transforming growth factor-β1 (TGF-β1) regulated TARC expression in keratinocytes. Methods: The effect of TGF-β1 on mRNA and protein expression of IFN-γ and TNF-α-induced TARC in a human keratinocyte cell line, HaCaT cells, was evaluated by using RT-PCR and ELISA. Adenovector-mediated gene transfer was used to determine the effect of Smad proteins on TARC expression in HaCaT cells. Results: TGF-β1 inhibited mRNA and protein expression of IFN-γ and TNF-α-induced TARC in HaCaT cells. The inhibitory effect of TGF-β1 on the TARC expression was suppressed by overexpression of Smad7, a major inhibitory regulator of Smad pathway for transforming growth factor-β (TGF-β) signaling, but not by PD98059, an inhibitor for ERK/mitogen-activated protein kinase (MAPK) pathway. In addition, overexpression of Smad2 or Smad3, major signal transducing Smads, was sufficient to inhibite the IFN-γ and TNF-α-induced TARC production in HaCaT cells. Conclusion: TGF-β1 inhibited IFN-γ and TNF-α-induced TARC production in HaCaT cells via Smad2/3, suggesting that modulation of TGF-β/Smad signaling pathway may be beneficial for the treatment of atopic dermatitis.

Various detergents are used as skin cleansing products. In some cases, skin cleanser removes not only dirt but also valuable skin lipids. Therefore, detergents may disrupt epidermal barrier function despite that using of detergents are required for good skin hygiene. Lipid supplements can reverse detergent-induced dysfunction of the skin barrier. Elevated transepidermal water loss (TEWL) and riboflavin penetration in 5% SLS-treated rat and human skin were reversed by supplementation of monoglyceride (MG), squalene (SQ), cholesterol ester (CE) and pseudo-ceramide (Cer2). MG and Cer2 achieved the best results. MG appears to inhibit elution of intercellular ceramides, since more ceramides remained when the detergent was supplemented with MG. Topical application of Cer2 is not effective for recovery from artificially induced barrier disruption, but supplemented Cer2 into skin cleanser has a beneficial effect for prevention of detergent-induced barrier disruption. In conclusion, the prevention of barrier disruption is most important matter for maintaining skin health and barrier function. Therefore, we think that Cer2-supplemented skin cleanser is useful for conservation of skin barrier function.

Skin sulfhydryl oxidase (SOx) is an enzyme that catalyzes disulfide (S–S) cross-linking through the oxidation of sulfhydryl compounds in the skin. In this study, using the enzyme purified from rat seminal vesicle, we obtained peptide sequences for SOx by mass spectrometry. We then searched for SOx nucleotides corresponding highly to the rat peptide sequences by assembling murine-expressed sequence tags (ESTs) from the GeneBank database. The assembled mouse SOx cDNA has an open reading frame of 1704-bp nucleotides, translating into a size of 568 amino acids. The calculated molecular mass of the mouse SOx protein is 65 kDa. This mouse sequence can be amplified from total RNAs of various mouse tissue samples by reverse transcription polymerase chain reaction, especially highly amplified from those of the seminal vesicles and epidermis. The cDNA fragment was subsequently cloned into the mammalian expression vector (pTARGET-MSSOx), allowing us to express mouse recombinant SOx protein in cultured cells. When pTARGET-MSSOx was transfected, Western blot analysis using anti-SOx antiserum could detect a 65 kDa-band of recombinant SOx in both samples from the whole cell extract and the medium after the harvest of the HEK cells. In immunohistochemical analysis, the Pt-K2 cells, following the introduction of pTARGET-MSSOx, seemed to generate a SOx protein reactive to anti-SOx antiserum in the cells. Moreover, the indirect staining of the S–S bonds using N -(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM), following the addition of N -ethylmaleimide and dithiothreitol, showed that the formation of S–S bridges almost matched the localization of SOx expression in the Pt-K2 cells after the transfection. In essence, we cloned skin SOx cDNA and characterized it as one of the S–S cross-linking enzymes. The SOx clone from mouse epidermis seems to be useful for investigating the potential function of the enzyme in the epidermis, especially for understanding the physiological role of SOx in the differentiation of keratinocytes.

In order to investigate the role of cyclin-dependent kinase (CDK) inhibitors in hair growth, we analyzed the expressions of p21(waf1/cip1) and p27(kip1) during the synchronized hair cycle of rat coat. The mRNAs of both p21(waf1/cip1) and p27(kip1) were detected in anagen hair follicles by reverse transcription and polymerase chain reaction and their localization was clearly demonstrated in the upper half portion of the hair bulb and the cortex by in situ hybridization. The dermal tissue containing hair follicles was then excised from the anterior dorsal skin of the 5-12-week-old rats at 0.5 week intervals and the expressions of p21(waf1/cip1) and p27(kip1) were analyzed by northern blot hybridization. The mRNA of both CDK inhibitors was expressed at relatively high levels during anagen than during telogen, a fact which correlated with the mRNA expression levels of hair differentiation markers, type I hair keratin (Ha3) and high sulfur protein B2. These results imply that CDK inhibitors, p21(waf1/cip1) and p27(kip1), are involved in the differentiation of follicular epithelial cells.

a Department of Dermatology, Matsue City Hospital, 101 Nada-machi, Matsue 690-8509, Japan b Department of Dermatology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan c Department of Dermatology, Juntendo Uni6ersity School of Medicine, 3-1-3 Hongo, Bunkyoku 113-8431, Tokyo, Japan d Department of Dermatology, Faculty of Medicine, Uni6ersity of Tokyo, 7-3-1 Hongo, Bunkyoku 113-8655, Tokyo, Japan www.elsevier.com/locate/jdermsci

Epimorphin was originally identified as a mesenchymal cell surface-associated protein that modulates epithelial morphogenesis in embryonic skin and lung epithelia. A previous report which utilized embryonic mouse skin, showed that epimorphin was localized non-homogeneously in a region adjacent to the epidermis and in a mesenchymal cell condensation located in front of growing hair follicles. We report herein a further detailed localization of this protein in adult mouse skin using immunoelectron microscopy. Epimorphin was found to be localized on the undersurface of basal cells, in the cytoplasm of cell processes of fibroblasts, as well as on the plasma membrane of fibroblasts, endothelial cells, pericytes, perineurium and endomysium. Our present finding indicated that epimorphin is one of the factors involved in multiple biological functions in a variety of structures derived from various origins and that it is not a specific epithelial morphogenetic factor.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) on human hair follicle growth was examined using a serum-free organ culture system. The DNA synthesis in human hair follicles and elongation of the hair shaft were measured subsequent to the follicle isolation and culture at 31 degrees C in 95% O2-5% CO2 for 72 h. Results showed that HGF/SF significantly increased 3H-thymidine (P0.001) incorporation and hair follicle length (P0.05). The effect of HGF/SF was dose-dependent with a maximal stimulation at 10 ng/ml.