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Abstract: Epidermal keratinocytes are the main component cells of the epidermis and their function is regulated by various kinds of growth factors, cytokines, and chemokines. Of these, members of the epidermal growth factor and fibroblast growth factor families, as wells as hepatocyte growth factor and insulin-like growth factor, play central roles in keratinocyte proliferation, while transforming growth factor-β, vitamin D3, and interferon-γ are important inhibitors of keratinocyte growth. Keratinocytes are known to produce many of the currently identified growth factors, cytokines and chemokines. Keratinocyte-derived growth factors and cytokines regulate immune and inflammatory responses, and play important roles in pathological skin conditions. This review focuses on the regulation of keratinocytes by growth factors, cytokines, and chemokines. [Copyright &y& Elsevier]

Abstract: Background: Living skin equivalents (LSEs) are being used to treat burn wounds, skin defects, and chronic wounds, and today, several biomaterials are applied as dermal matrices in LSEs. The amnionic membrane (AM) is known to have useful properties as a dermal matrix and can be used to construct a LSE. Objective: To develop a new LSE with human AM as the matrix. Methods: Human AM was de-epithelialized and investigated to determine whether it supported keratinocyte adherence and proliferation, and fibroblast in-growth and proliferation. A new LSE was constructed by seeding keratinocytes on the epithelial side of fibroblast-populated, de-epithelialized AM and was investigated histologically. The LSE was transplanted onto a full-thickness wound on a nude mouse and a histological examination was conducted. Results: De-epithelialized AM supported the adherence and proliferation of keratinocytes and the in-growth of fibroblasts. The new LSE demonstrated good mechanical properties and revealed good morphology, with a well differentiated epidermis and well developed basement membrane. The LSE grafts survived well on nude mice, showing good morphology. Conclusion: A LSE with amnions as a matrix exhibited good morphology, low cost, and good mechanical properties and may be useful as a skin substitute for clinical use. [Copyright &y& Elsevier]

The presence of pre-existing basement membrane (BM) components improves the morphogenesis of epidermis and BM in constructing a human living skin-equivalent (LSE). De-epithelialized amniotic membrane (AM) retains key BM components. We have therefore investigated the usefulness of AM for constructing LSE. De-epithelialized AM was overlaid on type I collagen gel embedded with fibroblasts. Normal human keratinocytes (NHKs) were then seeded onto the epithelial side of the AM to construct an AM-LSE. A conventional LSE was constructed by seeding NHKs on a fibroblast-populated type I collagen gel. When the keratinocytes reached confluence, the LSE was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at various times and investigated morphologically, immunohistochemically, and ultrastructurally. In AM-LSE, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of LSE without AM. A more continuous BM and better-developed hemidesmosomes were found in AM-LSE. The epidermis of AM-LSE outgrew much faster than that of LSE without AM. When transplanted onto nude mice, both LSEs took well; however, the AM-LSE graft showed better morphogenesis of the epidermis, BM, and hemidesmosomes. The better epidermal morphology and better-developed BM in AM-LSE in vitro and in vivo indicates its superiority over LSE without AM for clinical applications. [ABSTRACT FROM AUTHOR]

The role of angiotensin II (Ang II) in the control of systemic blood pressure and volume homeostasis is well known and has been extensively studied. Recently, Ang II was suggested to also have a function in skin wound healing. In the present study, the in vivo function of Ang II in skin wound healing was investigated using Ang II type 1 receptor (AT1R) knock-out mice. Wound healing in these mice was found to be markedly delayed. Keratinocytes and fibroblasts play important roles in wound healing, and thus the effect of Ang II on the migration of these cells was examined. Ang II stimulated keratinocyte and fibroblast migration in a dose-dependent manner. It has been reported that G protein-coupled receptor (GPCR) activation induces epidermal growth factor (EGF) receptor (EGFR) transactivation through the shedding of heparin-binding EGF-like growth factor (HB-EGF). As AT1R is a GPCR, it was hypothesized that Ang II-induced keratinocyte and fibroblast migration is mediated by EGFR transactivation. Ang II induced EGFR phosphorylation, which was inhibited by an AT1R antagonist, HB-EGF neutralizing antibody, and an HB-EGF antagonist in both keratinocytes and in fibroblasts. Moreover, Ang II-induced migration of keratinocytes and fibroblasts was also prevented by these inhibitors. Taken together, these findings clearly demonstrate, for the first time, that Ang II plays an important role in skin wound healing and that it functions by accelerating keratinocyte and fibroblast migration in a process mediated by HB-EGF shedding. [ABSTRACT FROM AUTHOR]

Summary: Background:: Gene transfer to skin is an attractive therapeutic approach because of the accessibility of the skin and the high rate of cure for many cutaneous diseases. However, safety concerns over viral vectors and the low efficiency of most non-viral gene transfer techniques have encumbered their clinical application for gene transfer. By contrast, efficient gene transfers into various cell types using microbubble-enhanced ultrasound has been reported. Objectives:: The purpose of this study was to investigate whether ultrasound with microbubble enhancement allowed effective transfer of foreign genes into living skin equivalents (LSEs). Methods:: Microbubbles and plasmid DNA encoding green fluorescent protein (GFP) were added to the dermal–epidermal junctions of LSEs, which were then exposed to ultrasound. The LSEs were harvested at different time points to investigate transgene expression using confocal laser microscopy. Transfected LSEs were also transplanted onto nude mice, and the in vivo transgene expression was observed. Results:: From days 2 to 7 after transfection, most GFP-positive cells continued to migrate upward from the basal layer, while other GFP-positive cells lagged behind or remained in the basal layer on days 5 and 7. Transfection resulted in 20–30% GFP-positive cells. Multiple transfections further increased the percentage of transfected cells and resulted in multi-layer transgene expression. Grafts from the transfected LSEs survived on nude mice and continued to express GFP up to 2 weeks post-transplantation. Conclusion:: Gene transfer into LSE using ultrasound with microbubble enhancement is an effective alternative to viral and non-viral methods. [Copyright &y& Elsevier]

Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated kerotinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in kerotinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation. [ABSTRACT FROM AUTHOR]

It has been proposed that transforming growth factor-β (TGF-β) is involved in the growth inhibition of normal human epidermal keratinocytes (NHEK) by 1α,25-dihydoxyvitamin D3 (1α,25(OH)2D3), although this is still controversial because of the difficulty in blocking TGF-β activity completely.To determine whether TGF-β is involved in early phase growth inhibition by 1α,25(OH)2D3.TGF-β mRNA was detected by ribonuclease protection assay (RPA), and biological active TGF-β was determined by a luciferase reporter assay. To block intrinsic TGF-β activity completely, we constructed an adenovirus vector expressing a truncated TGF-β type II receptor with a dominant negative effect (AdexTβTR) that blocks TGF-β signal transduction.1α,25(OH)2D3 slightly upregulated TGF-β1 and TGF-β2 after 24h according to an RPA and a luciferase reporter assay, however growth inhibition by 1α,25(OH)2D3 occurred at 6h. The addition of 10-6M of 1α,25(OH)2D3 to NHEK infected with AdexTβTR or AdexLacZ (control vector) reduced DNA synthesis to 59.3 and 62.2% at 6h, respectively. There was no significant difference in cell number after a 3-day incubation with AdexTβTR or AdexLacZ-infected cells treated with 1α,25(OH)2D3.Since 1α,25(OH)2D3 rapidly inhibits NHEK growth regardless of the prevention of TGF-β signal transduction, TGF-β is not involved in early phase growth inhibition by 1α,25(OH)2D3. [Copyright &y& Elsevier]

Vascular endothelial growth factor (VEGF) is a potent, multifunctional, endothelial-cell-specific growth factor. It stimulates proliferation and migration of endothelial cells. Characterization of intracellular signal transduction after VEGF and VEGF receptor (VEGFR) interaction has demonstrated the involvement of the mitogen-activated protein kinase pathway. However, several studies indicated that signal transducers and activators of transcription (STAT) is another important pathway downstream of VEGF/VEGFR interaction. Therefore, we studied the role of STAT3 in the migration and tube formation of the human dermal microvascular endothelial cells (HDMEC). HDMEC expressed phosphorylated forms of STAT1, STAT3, and STAT5, and a marked increase of phosphorylated STAT3 in the nuclear fraction after addition of VEGF was observed by Western blot and immunohistochemical staining. To verify the functional implication of STAT3 phosphorylation in HDMEC migration, we introduced a dominantnegative STAT3 using adenovirus vector system. Dominant-negative STAT3 abolished the VEGF-induced nuclear translocatlon of phosphorylated STAT3 and inhibited HDMEC migration completely. Dominant-negative STAT3 also suppressed VEGF-induced HDMEC tube formation on Matrigel and on collagen gel. These data demonstrate that STAT3 and its phosphorylation are involved in the downstream pathway of VEGF/VEGFR interaction and regulate VEGF-induced HDMEC migration and tube formation. [ABSTRACT FROM AUTHOR]

Background: Cultured epidermal sheet (CES) grafts accelerate wound healing as a result of so-called biological dressing effect, which is thought to be mediated by various growth factors. However, the profile of growth factor expression in CESs is unclear. Objective: To investigate whether CESs produce growth factors along with stratification we investigated the production of growth factors and their regulation in CESs. Methods: CESs conditioned medium was harvested and the concentration of TGF-α, TGF-β1, TGF-β2, and VEGF was measured using ELISA. The mRNA of EGF family, TGF-β family and VEGF was detected by Northern blot or RNase protection assay. Results: The concentration of TGF-α was 100 pg/ml in the monolayer culture, but dramatically increased to 600 pg/ml 2 days after stratification. It decreased to baseline, and then gradually increased to 300 pg/ml in the presence of EGF and remained at that level until day 20. TGF-β1 increased from 50 to 400 pg/ml after stratification, and remained at that level day 20. TGF-β2 was undetectable in the monolayer culture, but dramatically increased to 200 pg/ml 2 days after stratification. Unlike TGF-β1, TGF-β2 gradually increased over time after stratification. VEGF increased with stratification from 500 to 1500 pg/ml. The addition of EGF upregulated EGF family, TGF-β, and VEGF production in CESs, as confirmed by ELISA, Northern blot, and RNase protection assay. Conclusion: These results indicate that so-called biological dressing effect of CESs is mediated by production of the EGF family, TGF-β, and VEGF. Our results also demonstrate the ability of EGF to enhance growth factor production in CESs. [Copyright &y& Elsevier]

Oral mucosal lesions are seen in most cases of pemphigus vulgaris, whereas they are only rarely seen in pemphigus foliaceus; however, both pemphigus vulgaris and pemphigus foliaceus sera show positive immunofluorescence staining on oral mucosa. To explain this apparent paradox, we examined the expression level of desmoglein (Dsg)3, pemphigus vulgaris antigen, and Dsg1, pemphigus foliaceus antigen, in human squamous mucosal epithelia and epidermis by immunofluorescence staining and immunoblotting. For immunofluorescence staining, Dsg isotype-specific antibodies were produced by immunoadsorbing pemphigus vulgaris sera with either recombinant Dsg1 or Dsg3 baculoprotein. In oral mucosa and esophagus both Dsg were immunoreactive on cell surfaces throughout the entire epithelia, but staining intensity was weaker for Dsg1 than for Dsg3. Immunoblotting was performed to compare Dsg1 and Dsg3 expression levels in extracts from epidermis and oral mucosa. The total amount of desmosomal proteins applied was adjusted to give the same degree of staining intensity for desmoplakin, a cytoplasmic plaque protein of desmosomes. In the mucosal extract, the Dsg1 band was much weaker than Dsg3, whereas in the epidermal extract the Dsg1 band was stronger than Dsg3. These data suggest that although Dsg1 and Dsg3 are expressed in a similar distribution throughout squamous mucosal epithelia, Dsg1 is expressed at a much lower level than Dsg3. This finding provides a good explanation for the paradox: even though anti-Dsg1 autoantibodies block the function of Dsg1 in the mucosal epithelia, Dsg3 may be sufficient for cell–cell adhesion, with consequently no apparent oral involvement in pemphigus foliaceus patients. [ABSTRACT FROM AUTHOR]

Presents research which studied the banking of cultured epidermal sheets for the treatment of epidermodysplasia verruciformis (EV). What EV is; Development of multiple cancerous lesions requiring surgery; Increase in the number of lesions; Case of a 50-year-old woman with EV; Eliminating human papilloma virus (HPV) in the sheets; Use of a self-skin banking system.

It is well known that dermal papilla cells (DPCs) play crucial roles in hair follicle induction. In this study, we examined whether Wnt/β-catenin activation results in maintenance of the hair-inducing ability of human DPCs. Expression of DPC marker genes was maintained under Wnt/β-catenin signalling stimulation by GSK-3β inhibition. Furthermore, human DPCs showed constant hair induction when transplanted with murine epidermal cell fraction. Alu-positive human DPCs were essentially detected adjacent to the reconstructing epidermal structure positive for P-cadherin immunoreactivity. The transplanted human DPCs were abundant in the surrounding dermal sheath portion of the fully regenerated hair follicles. These results support the importance of Wnt/β-catenin signalling in hair follicle induction. This study may provide valuable information to establish a culture method of human DPCs for cell-based therapy. [ABSTRACT FROM AUTHOR]

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13flox/floxK5-Cre). At birth, the skin of the Ubc13flox/floxK5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13flox/floxKS-Cre mouse epidermis. In culture, Ubc13flox/floxK5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13flox/flox keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13flox/flox K5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice. [ABSTRACT FROM AUTHOR]

Summary: Background: 1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3), the active form of vitamin D, suppresses keratinocyte proliferation, promotes keratinocyte differentiation, and induces involucrin expression. Peroxisome proliferation-activated receptors (PPARs) are ligand-activated transcription factors. It has been reported that PPARs stimulate keratinocyte differentiation and regulate the expression of differentiation molecules. Objective: Keratinocytes treated with 1α,25(OH)2D3 induced PPARγ, which was followed by increased involucrin expression. In this study, we investigated whether PPARγ is involved in the 1α,25(OH)2D3-induced involucrin expression in human keratinocytes. Methods: Subconfluent keratinocytes were treated with 10−7 M 1α,25(OH)2D3 for the indicated times, and PPAR and involucrin mRNA expression were determined by real-time RT-PCR. The levels of PPARs, involucrin, p38, and phospho-p38 proteins were assayed by Western blotting, and the DNA binding activities of PPARγ and AP-1 were investigated by electrophoretic mobility shift assays (EMSA). To examine the role of PPARγ in 1α,25(OH)2D3 responses, recombinant adenovirus carrying a dominant-negative form of PPARγ (Axdn-PPARγ) was constructed and transfected into keratinocytes. The p38 inhibitor SB203580 was added to the cultures to evaluate the involvement of p38 in involucrin expression. Results: 1α,25(OH)2D3 induced PPARγ expression and stimulated PPARγ activity. The introduction of dn-PPARγ inhibited the expression of involucrin mRNA and protein induced by 1α,25(OH)2D3, and suppressed AP-1 DNA binding activity. 1α,25(OH)2D3 also triggered the phosphorylation of p38, which contributes to involucrin induction. Moreover, dn-PPARγ prevented the 1α,25(OH)2D3-induced phosphorylation of p38. Conclusions: These results suggest that PPARγ regulates involucrin expression by controlling the AP-1 signal and p38 activation in 1α,25(OH)2D3-induced keratinocyte differentiation. [Copyright &y& Elsevier]

Signal transducers and activators of transcription (STATs) are critical to growth factor-mediated intracellular signal transduction. We observed the rapid expression and activation of STAT5a during keratinocyte differentiation induced by suspension culture. STAT5a expression preceded that of involucrin, an important molecule in the terminal differentiation of keratinocytes. To determine whether STAT5a regulated involucrin expression, we expressed a dominant-negative (dn) STAT5a that blocks the dimerization of STAT5 and inhibits its nuclear translocation. We found that dn-STAT5a inhibited involucrin expression in keratinocytes. Given that STAT5 regulates adipogenesis via activating the peroxisome proliferator-activated receptor (PPAR) γ signal, we hypothesized that STAT5a regulated involucrin expression in the same manner. To test this hypothesis, we examined the expression and transactivation of PPARγ in a suspension culture of keratinocytes. Suspension culture induced PPARγ expression and triggered PPARγ transactivation rapidly and dn-STAT5a downregulated this induction and suppressed PPARγ transactivation. Furthermore, preincubation with the PPARγ/retinoid X-receptor inhibitor HX-531 or the introduction of a dn-PPARγ prevented the activation of involucrin promoter and inhibited its induction. This report provides early evidence of a major role for STAT5a in the differentiation of keratinocytes, where it contributes to involucrin expression by activating the PPARγ signal.Journal of Investigative Dermatology (2007) 127, 1728–1735; doi:10.1038/sj.jid.5700758; published online 1 March 2007 [ABSTRACT FROM AUTHOR]

Summary: Background: Bone morphogenetic protein-2 (BMP-2) and Wnt are involved in the normal development and tumorigenesis of several organs, and in the development of skin and skin appendages as a morphogen. However, the crosstalk between BMP-2 and the Wnt/β-catenin signaling pathway is not clear. Objective: We examined BMP-2-dependent expression of Wnt and its receptor frizzled in normal human keratinocytes. Methods: The mRNA expression of the Wnt and frizzled families was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or ribonuclease protection assay. β-Catenin expression was measured using RT-PCR and Western blotting. T-cell factor/lymphoid enhancing factor activity was analyzed using the luciferase reporter assay. Results: We detected the expression of Wnt-2b/13, -4, -5a, -5b, -7a, -7b, and -10a, frizzled-1, -4, -5, -6, -8, -9, and -10, MFRP, and SFRP-1/SARP-2 in keratinocytes. BMP-2 increased Wnt-2b/13, -5b, and -7b, and frizzled-6, -8, and -10. Conversely, BMP-2 suppressed Wnt-10a and SFRP-1/SARP-2. Although Wnt-4 expression was not affected by BMP-2 in confluent conditioned keratinocytes, BMP-2 suppressed cell density-dependent Wnt-4 induction. The transcriptional activity of TCF/LEF, which is a target of the canonical Wnt pathway, was upregulated by BMP-2 in both time- and dose-dependent manners. However, BMP-2-dependent differentiation of keratinocytes suppressed TCF/LEF transcriptional activity. Conclusion: These results suggest that BMP-2 modulates the expression of molecules involved in Wnt signaling, and activates the canonical Wnt pathway in normal human keratinocytes. Moreover, Wnt signaling may be influenced by the fate of keratinocytes, such as proliferation, migration, and differentiation. [Copyright &y& Elsevier]

Retinoic acid derivatives have been used successfully for the treatment of various dermatoses, such as psoriasis; however, topical application of these compounds often elicits skin irritation. We hypothesized that this irritation was as a result of the local production of interleukin-8 (IL-8). To test this hypothesis, we investigated whether all-trans-retinoic acid (ATRA) induced IL-8 production in normal human keratinocytes. Stimulation with 10−7 M ATRA enhanced IL-8 mRNA expression and induced IL-8 production. We also studied the intracellular signaling mechanisms of ATRA-induced IL-8 production in keratinocytes. ATRA increased the expression of RelA (p65), RelB, nuclear factor (NF)-κB2 (p52), and NF-κB1 (p50), and elevated the DNA-binding activity of p65 and phosphorylation of inhibitorκB (IκB)α. Introduction of a dominant-negative mutant of IκBα completely abolished ATRA-induced IL-8 production, which indicates that this process is NF-κB-dependent. We also studied the role of the p38 mitogen-activated protein kinase (MAPK) pathway in this phenomenon. ATRA phosphorylated the p38 MAPK, and SB202180 inhibited ATRA-induced IL-8 production, which indicates that the p38 MAPK is also involved in ATRA-induced IL-8 production. In summary, ATRA induces IL-8 production in both NF-κB- and p38 MAPK-dependent manners in normal human keratinocytes. [ABSTRACT FROM AUTHOR]

The desmoglein plays an important part in the formation of desmosomes. We constructed recombinant adenoviruses containing desmoglein 1 and desmoglein 3 derivatives partly lacking the extracellular domain (desmoglein 1ΔEC and desmoglein 3ΔEC, respectively), and full-length desmoglein 1 and desmoglein 3 and studied the involvement of desmoglein 1 and desmoglein 3 in desmosome formation. During low-level expression of desmoglein 3ΔEC in transduced HaCaT cells, keratin insertion at cell–cell contact sites was only partially inhibited and desmoplakin was partially stained at cell–cell contact sites. Low-level expression of desmoglein 1ΔEC, however, resulted in complete inhibition of keratin insertion at the cell–cell contact sites, and desmoplakin was stained in perinuclear dots. These results indicate the dominant-negative effect of desmoglein 1ΔEC on desmosome formation was stronger than that of desmoglein 3ΔEC. Desmoglein 1ΔEC coprecipitated plakoglobin to approximately the same extent as desmoglein 3ΔEC. Therefore, we conclude that the dominant-negative effect of desmoglein 1ΔEC is not simply due to plakoglobin sequestration. On the other hand, during low-level expression of full-length desmoglein 3 and desmoglein 1, they both colocalized with desmoplakin. During high-level expression, however, keratin insertion at cell–cell contact sites was inhibited in desmoglein 1 but not in desmoglein 3, and desmoplakin was stained at cell–cell contact sites in desmoglein 3 but not in desmoglein 1. These data suggest desmoglein 1 and desmoglein 3 expressed at low level were incorporated into desmosome but at high-level expression, desmoglein 1 disrupted desmosomes but desmoglein 3 did not. Our findings provide biologic evidence that desmoglein 1 and desmoglein 3 play a different functional role in cell–cell adhesion of keratinocytes. [ABSTRACT FROM AUTHOR]

Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that regulates autologous complement activation, by preventing assembly of alternative or classical C3/C5 convertases, and has been shown to have a wide tissue distribution. In this study, DAF antigen has been demonstrated at the intercellular spaces of normal human epidermis with monoclonal antibody against DAF using the peroxidase- anti-peroxidase method. The amount of DAF was greater at the granular layer than the basal cell layer as judged by intensity of the staining. Western blot analysis of DAF of the epidermis showed a 55-kD band, whereas that of buffy coat cells was approximately 67 kD. When DAF of the epidermis was treated with ncuraminidase, the molecular weight was reduced to 53 kD, whereas that of buffy coat cells was 56 kD. These results indicated that the content of sialic acid of DAF in the epidermis was different from that of buffy coat cells. In phosphatidylinositol-specific phospholipase C (PIPLC) - treated normal human skin, DAF was not demonstrated in the epidermis, whereas DAF remained unchanged on the elastic fibers. After the treatment of the epidermis by PIPLC, DAF was released into the buffer shown by Western blot analysis. These results suggested that DAF on the epidermis was anchored to keratinocyte via phosphatidylinositol (P1), whereas the anchoring mechanism of DAF on the elastic fibers was not through P1. [ABSTRACT FROM AUTHOR]

Membrane co-factor protein (MCP; CD46) is an integral membrane protein with molecular weight (MW) of the two species of 63 kD and 55 kD, and regulates autologous complement activation, with the activity of factor i cofactor. The quantity of each species is genetically regulated, and two codominantly inherited allelic variants account for the three phenotypic patterns. By immunohistochemical study, MCP was found both in the intercellular spaces of the epidermis and on the endothelial cells in the dermis of normal human skin in vivo. The intensity of the staining pattern was higher in the basal layer than in the granular layer. By Western blot analysis with use of a monoclonal antibody, MCP in the epidermis appeared as several bands ranged from 60-50 kD, with a major band of 56 kD, which was different from those in either polymorphonuclear cells, platelets, and cultured keratinocytes. No other variants were found in the epidermis obtained from skin may be regulated at several steps, including DAF and HRF20, thereby protecting cells from autologous complement attack. [ABSTRACT FROM AUTHOR]

Background Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs). Objective To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF. Methods Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay. Results 24 h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor. Conclusion These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF. [ABSTRACT FROM AUTHOR]

Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal–epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal–epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin–eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two–three layers after five days and five–seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-β1, TGF-β2, and TGF-β3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-β family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal–epithelial cell interactions, and the results suggest that stromal cells and epithelial cells may act in concert in the cornea. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Findings from large epidemiologic studies indicate that there is a link between smoking and extrinsic skin ageing. We previously reported that matrix metalloproteinases ( MMPs) mediate connective tissue damage in skin exposed to tobacco smoke extracts. Tobacco smoke contains more than 3800 constituents, including numerous water-insoluble polycyclic aromatic hydrocarbons ( PAHs) that trigger aryl hydrocarbon receptor ( Ah R) signalling pathways. To analyse the molecular mechanisms involved in tobacco smoke-induced skin ageing, we exposed primary human fibroblasts and keratinocytes to tobacco smoke extracts. Hexane- and water-soluble tobacco smoke extracts significantly induced MMP-1 m RNA in both human cultured fibroblasts and keratinocytes in a dose-dependent manner. To clarify the involvement of the Ah R pathway, we used a stable Ah R-knockdown Ha Ca T cell line. Ah R knockdown abolished the increased transcription of the Ah R-dependent genes CYP1 A1/ CYP1 B1 and MMP-1 induced by either of the tobacco smoke extracts. Furthermore, the tobacco smoke extracts induced 7-ethoxyresorufin-O-deethylase activity, which was almost completely abolished by Ah R knockdown. Likewise, treating fibroblasts with Ah R pathway inhibitors, that is, the flavonoids 3-methoxy-4-nitroflavone and α-naphthoflavone, blocked the expression of CYP1 B1 and MMP-1. These findings suggest that the tobacco smoke extracts induce MMP-1 expression in human fibroblasts and keratinocytes via activation of the Ah R pathway. Thus, the Ah R pathway may be pathogenetically involved in extrinsic skin ageing. [ABSTRACT FROM AUTHOR]

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease associated with lymphoproliferative neoplasms and characterized by antibodies against plakins and desmoglein 3 (Dsg3). Anti-Dsg3 antibodies have a primary role in blister formation in PNP. In this study, we used phage display to clone monoclonal anti-Dsg3 antibodies from a PNP patient to further characterize their pathogenicity. We isolated 20 unique Dsg3-reactive mAbs, which we classified into four groups according to the heavy-chain complementarity-determining region 3 (CDR3) region. Genetic analyses demonstrated that three antibody groups used the VH1-46 gene (18 clones) and one group used the VH1-02 gene (2 clones). The results of an in vitro keratinocyte dissociation assay and a human skin organ culture injection assay showed that three antibodies displayed pathogenic activity in blister formation with different potencies. Epitope mapping using domain-swapped Dsg3/Dsg2 showed that these pathogenic mAbs bound Ca2+-dependent conformational epitopes in the middle portion of the extracellular region of Dsg3 (EC2 and EC3 domains), in contrast to most previously characterized pathogenic pemphigus vulgaris antibodies, which bound to the EC1 domain of Dsg3. These mAbs reflect the unique polyclonal nature of anti-Dsg3 antibodies in PNP and represent an important tool for detailing the pathophysiological mechanisms of blister formation in PNP. [ABSTRACT FROM AUTHOR]

Abstract: Background: During skin wounding and healing, skin homeostasis is interrupted. How the altered epithelial–mesenchymal interactions influence scar formation and epidermogenesis should be investigated using three-dimensional models that are similar to in vivo structures. Objective: In this study, we assessed the effects of epithelial–mesenchymal interactions on myofibroblast differentiation and how myofibroblasts influence epidermogenesis using a human living skin equivalent (LSE) model. Methods: We constructed a fibroblast-populated type I collagen gel upon which LSEs were formed by seeding with normal human keratinocytes. Samples of the collagen gel and LSEs were collected at different time points. Myofibroblast differentiation, epidermal differentiation, and proliferation status were investigated immunohistochemically. Several measures were taken to suppress α-smooth muscle actin (α-SMA) expression to determine the effects of myofibroblasts on epidermogenesis, including the addition of basic fibroblast growth factor or a transformation growth factor-β (TGF-β) kinase inhibitor to the culture medium and the inclusion of an amniotic membrane (AM) in the dermal matrix. Results: The myofibroblast/fibroblast ratio in the fibroblast-populated collagen gel kept rising during culture. In the LSEs, most fibroblasts were α-SMA-negative, except for those along the dermal–epidermal junction. The suppression of α-SMA expression enhanced epidermal differentiation and decreased TGF-β1 expression in the epidermis. The inhibition of TGF-β kinase completely suppressed α-SMA expression in the dermal matrix. Conclusions: Epidermogenesis suppressed α-SMA expression in the fibroblast-rich dermal matrix, except near the dermal–epidermal junction. The α-SMA-positive cells at the dermal–epidermal junction contributed to the hyperproliferative phenotype of the epidermis. In contrast, the hyperproliferative epidermis expressed more TGF-β1, which is responsible for myofibroblast differentiation. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions. [Copyright &y& Elsevier]

Abstract: Background: Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain. Objective: The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA. Methods: We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera. Results: When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity. Conclusion: We conclude that the NC1+NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients. [Copyright &y& Elsevier]

Abstract: Background: Production of antimicrobial peptides (AMPs) is the primary mechanism by which skin innate immunity protects against infection. Hormonally active vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D3) is a vital regulator of skin innate immunity, and has been shown to increase the expression and function of AMPs. Objective: PPARγ is a ligand-activated nuclear receptor and plays a role in keratinocyte differentiation and cutaneous homeostasis. In this study, we investigate whether 1,25D3-activated PPARγ signaling regulates AMP expression in keratinocytes. Methods: Subconfluent keratinocytes were treated with 1,25D3 for the indicated times. The mRNA and protein levels of AMPs were detected by RT-PCR and Western blot, and the DNA binding activation of PPARγ, VDRE and AP-1 was investigated by EMSA. To examine the role of PPARγ, the recombinant adenovirus carrying a dominant-negative form of PPARγ (dn-PPARγ) was constructed and transfected into keratinocytes. Results: We show here that 1,25D3 significantly enhances hBD-3 and cathelicidin expression in keratinocytes. Expression of dn-PPARγ did not affect binding to the vitamin D-responsive element (VDRE), which is crucial for cathelicidin induction by VD3; however, it did decrease 1,25D3 induction of both hBD-3 and cathelicidin. Inhibition of the p38, ERK, and JNK signaling pathways blocked hBD-3 expression, whereas only p38 inhibition suppressed cathelicidin induction. dn-PPARγ had no effect on ERK and JNK activity, but inhibited p38 phosphorylation and suppressed 1,25D3-induced AP-1 activation via effects on Fra1 and c-Fos proteins. Conclusions: In conclusion, PPARγ regulates the 1,25D3-induced hBD-3 and cathelicidin expression in keratinocytes through the regulation of AP-1 and p38 activity. [ABSTRACT FROM AUTHOR]

Transforming growth factor-b-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 keratinocytespecific 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. [ABSTRACT FROM AUTHOR]

The immunological significance of IL-27 has been reported and discussed in various Th1/Th17-mediated inflammatory diseases. However, its importance in psoriasis is unknown. We investigated pathophysiological roles of IL-27 in psoriasis in this study. Serum IL-27 levels in psoriatic patients were significantly higher than those in healthy controls, and correlated with disease severity and serum IFN-γ levels. An immunohistochemical analysis revealed the infiltration of IL-27-secreting cells in the papillary dermis of psoriatic skin lesions but not in skin lesions with atopic dermatitis or normal skin. Furthermore, IL-27 alone greatly induced in vitro CXCL9, CXCL10, and CXCL11 production and tyrosine phosphorylation of signal transducer and activator of transcription 1 in normal human keratinocytes, while it suppressed the tumor necrosis factor-α-induced production of IL-1α and CCL20. These results indicate that IL-27 may promote the onset of psoriasis, while it may simultaneously attenuate the expanded inflammation in this disease. Our results implicate potential therapeutic effects of IL-27 for psoriasis. [ABSTRACT FROM AUTHOR]

Purpose: To present a detailed clarification of the symptoms at disease onset of Stevens-Johnson syndrome (SJS) and its more severe variant, toxic epidermal necrolysis (TEN), with ocular complications and to clarify the relationship between topical steroid use and visual prognosis. Design: Cross-sectional study. Participants: Ninety-four patients with SJS and TEN with ocular complications. Methods: A structured interview, examination of the patient medical records, or both addressing clinical manifestations at disease onset were conducted for 94 patients seen at Kyoto Prefectural University of Medicine. Any topical steroid use during the first week at the acute stage also was investigated. Main Outcome Measures: The incidence and the details of prodromal symptoms and the mucosal involvements and the relationship between topical steroid use and visual outcomes. Results: Common cold-like symptoms (general malaise, fever, sore throat, etc.) preceded skin eruptions in 75 cases, and extremely high fever accompanied disease onset in 86 cases. Acute conjunctivitis and oral and nail involvements were reported in all patients who remembered the details. Acute conjunctivitis occurred before the skin eruptions in 42 patients and simultaneously in 21 patients, whereas only 1 patient reported posteruption conjunctivitis. Visual outcomes were significantly better in the group receiving topical steroids compared with those of the no-treatment group (P<0.00001). Conclusions: Acute conjunctivitis occurring before or simultaneously with skin eruptions accompanied by extremely high fever and oral and nail involvement indicate the initiation of SJS or TEN. Topical steroid treatment from disease onset seems to be important for the improvement of visual prognosis. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. [Copyright &y& Elsevier]

The epidermis is the primary boundary between the body and the environment, and it serves as the first line of defense against microbial pathogens. Production of chemokines and cytokines is an important step in the initiation of innate immune responses to viral infections. Epidermal keratinocytes produce IFN-α, -β and macrophage inflammatory protein (MIP)-1α in response to double-stranded RNA (dsRNA) or viral infections. We showed that human keratinocytes produced cytokines [tumor necrosis factor (TNF)-α, IL-1β and IL-15] and chemokines [MIP-1β, RANTES and liver and activation-regulated chemokine (LARC)] in response to dsRNA, with activation of the nuclear factor κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription 1 (STAT1) pathways. To study the roles of these pathways in their production, we transfected keratinocytes with adenoviral vectors (Ax) carrying a dominant-negative form of inhibitor κB α (IκBα) (IκBαM), a dominant-negative mutant form of STAT1 (STAT1F) or suppressors of cytokine signaling 1 (SOCS1). Transfection with AxIκBαM or addition of a p38 inhibitor (SB203580) significantly decreased the dsRNA-mediated production of TNF-α, IL-1β and MIP-1α, but not of IFN-β, IL-15, MIP-1β, RANTES or LARC. Transfection with AxSTAT1F or AxSOCS1 inhibited the dsRNA-mediated production of TNF-α, IL-15, MIP-1α, MIP-1β, RANTES and LARC, but not IFN-β or IL-1β. In conclusion, the NF-κB, p38 MAPK and STAT1 pathways differentially regulate dsRNA-mediated innate immune responses in epidermal keratinocytes. [ABSTRACT FROM PUBLISHER]

Activation of growth factor receptors by ligand binding leads to an increased expression of c-Myc, a transcriptional regulator for cell proliferation. The activation of transcriptional factors via the activated receptors is thought to be the main role of c-Myc gene expression. We demonstrate here that epidermal growth factor receptor (EGFR)- and fibroblast growth factor receptor (FGFR)-mediatedc-Myc induction and cell cycle progression in primary cultured mouse embryonic fibroblasts (MEFs) are abrogated by knockout of the heparin-binding EGF-like growth factor (Hb-egf) gene, or by a metalloproteinase inhibitor, although molecules downstream of the receptors are activated. Induction of c-Myc expression by EGF or basic FGF is recovered in Hb-egf-depleted MEFs by overexpression of wild-type proHB-EGF, but no recovery was observed with an uncleavable mutant of proHB-EGF. The uncleavable mutant also inhibited EGF-induced acetylation of histone H3 at the mouse c-Myc first intron region, which could negatively affect transcriptional activation. We conclude that signal transduction initiated by generation of the carboxyl-terminal fragment of proHB-EGF (HB-EGF-CTF) in the shedding event plays an important intermediary role between growth factor receptor activation and c-Myc gene induction. J. Cell. Physiol. 214: 465–473, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Hair follicle regeneration involves epithelial–mesenchymal interactions (EMIs) of follicular epithelial and dermal papilla (DP) cells. Co-grafting of those cellular components from mice allows complete hair reconstitution. However, regeneration of human hair in a similar manner has not been reported. Here, we investigated the possibility of cell-based hair generation from human cells. We found that DP-enriched cells (DPE) are more critical than epidermal cells in murine hair reconstitution on a cell number basis, and that murine DPE are also competent for hair regeneration with rat epidermal cells. Co-grafting of human keratinocytes derived from neonatal foreskins with murine DPE produced hair follicle-like structures consisting of multiple epidermal cell layers with a well-keratinized innermost region. Those structures expressed hair follicle-specific markers including hair keratin, and markers expressed during developmental stages. However, the lack of regular hair structures indicates abnormal folliculogenesis. Similar hair follicle-like structures were also generated with cultured human keratinocytes after the first passage, or with keratinocytes derived from adult foreskins, demonstrating that epidermal cells even at a mature stage can differentiate in response to inductive signals from DP cells. This study emphasizes the importance of EMI in follicular generation and the differentiation potential of epidermal keratinocytes.Journal of Investigative Dermatology (2007) 127, 2106–2115; doi:10.1038/sj.jid.5700823; published online 12 April 2007 [ABSTRACT FROM AUTHOR]

Transforming growth factor-α-activated kinase 1 (TAK1) is a member of the mitogen-activated protein (MAP) kinase family and is an upstream signaling molecule of nuclear factor-κB (NF-κB). Given that NF-κB regulates keratinocyte differentiation and apoptosis, TAK1 may be essential for epidermal functions. To test this, we generated keratinocyte-specific TAK1-deficient mice from Map3k7flox/flox mice and K5-Cre mice. The keratinocyte-specific TAK1-deficient mice were macroscopically indistinguishable from their littermates until postnatal day 2 or 3, when the skin started to roughen and wrinkle. This phenotype progressed, and the mice died by postnatal day 7. Histological analysis showed thickening of the epidermis with foci of keratinocyte apoptosis and intra-epidermal micro-abscesses. Immunohistochemical analysis showed that the suprabasal keratinocytes of the TAK1-deficient epidermis expressed keratin 5 and keratin 14, which are normally confined to the basal layer. The expression of keratin 1, keratin 10, and loricrin, which are markers for the suprabasal and late phase differentiation of the epidermis, was absent from the TAK1-deficient epidermis. Furthermore, the TAK1-deficient epidermis expressed keratin 16 and had an increased number of Ki67-positive cells. These data indicate that TAK1 deficiency in keratinocytes results in abnormal differentiation, increased proliferation, and apoptosis in the epidermis. However, the keratinocytes from the TAK1-deficient epidermis induced keratin 1 in suspension culture, indicating that the TAK1-deficient keratinocytes retain the ability to differentiate. Moreover, the removal of TAK1 from cultured keratinocytes of Map3k7flox/flox mice resulted in apoptosis, indicating that TAK1 is essential for preventing apoptosis. In conclusion, TAK1 is essential in the regulation of keratinocyte growth, differentiation, and apoptosis. [ABSTRACT FROM AUTHOR]

Toll-like receptor (TLR)3 is a receptor for virus-associated double-stranded RNA, and triggers antiviral immune responses during viral infection. Epidermal keratinocytes express TLR3 and provide an innate immune defense against viral infection. Since the intracellular regulatory mechanism is unknown, we hypothesized that the signal transducers and activators of transcription (STAT)–suppressors of cytokine signaling (SOCS) system regulates the innate immune response of keratinocytes. Treatment with polyinosinic–polycytidylic acid (poly(I:C)) resulted in the rapid translocation of IFN regulatory factor (IRF)-3 into the nucleus, followed by phosphorylation of STAT1 and STAT3. The activation of STATs by poly(I:C) probably occurs in an indirect fashion, through poly(I:C)-induced IFN. We infected cells with the dominant-negative forms of STAT1 (STAT1F), STAT3 (STAT3F), and SOCS1 using adenovirus vectors. Infection with STAT1F suppressed the induction of macrophage inflammatory protein (MIP)-1α by poly(I:C), whereas STAT3F had a minimal effect, which indicates that STAT1 mediates MIP-1α induction. SOCS1, which is a negative feedback regulator of STAT1 signaling, was induced by treatment with poly(I:C). SOCS1 infection inhibited the phosphorylation of STAT1 and significantly reduced poly(I:C)-induced MIP-1α production. Furthermore, STAT1–SOCS1 regulated poly(I:C)-induced TLR3 and IRF-7 expression. However, SOCS1 did not affect NF-κB signaling. Thus, the STAT1–SOCS1 pathway regulates the innate immune response via TLR3 signaling in epidermal keratinocytes.Journal of Investigative Dermatology (2006) 126, 1574–1581. doi:10.1038/sj.jid.5700294; published online 20 April 2006 [ABSTRACT FROM AUTHOR]

Abstract: MIP-1α, a CC chemokine, recruits monocytes, natural killer cells, lymphocytes, and neutrophils, and plays a critical role in viral infection. Since, the lesional epidermis of herpes zoster expressed MIP-1α, we hypothesized that keratinocytes produce MIP-1α in response to virus-associated dsRNA via TLR3. To investigate this, we examined cultured human keratinocytes for MIP-1α production induced by poly(I:C), a TLR3 ligand. Poly(I:C) treatment induced MIP-1α production, interestingly, poly(I:C)-induced IFN-α and -β production preceded MIP-1α production. A neutralizing antibody for IFN-β significantly inhibited the poly(I:C)-induced MIP-1α production indicating that MIP-1α production is via IFN-β. IFN-α priming enhanced TLR3 expression and MIP-1α production in poly(I:C)-treated keratinocytes. This suggests that IFN-α enhanced the TLR3 expression and reinforced the response of keratinocytes to poly(I:C), which resulted in an increase in MIP-1α production. In conclusion, normal human keratinocytes produce MIP-1α in response to dsRNA via TLR3, and this production is regulated by IFN-α/β. [Copyright &y& Elsevier]

Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed anin vitroquantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4±21.4) than controls (n=11, 16.0±9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies. [ABSTRACT FROM AUTHOR]

Abstract: Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes. Because HGF has strong effects on the motility of keratinocytes and is produced by fibroblasts, HGF is thought to regulate keratinocyte migration during wound healing. However, the intracellular signaling mechanism of HGF-induced keratinocyte migration is poorly understood. In this report, we clarify the roles of STAT3 and SOCS/CIS family in HGF-induced keratinocyte migration. HGF activated STAT3 and strongly induced keratinocyte migration. Transfection with the dominant-negative mutant of STAT3 almost completely abolished HGF-induced keratinocyte migration and STAT3 phosphorylation. Next, we studied the mechanisms that regulate STAT3 phosphorylation. HGF enhanced the expression of SOCS3/CIS3 by sixfold within 1h, but had minimum effect on SOCS1/JAB expression. Transfection with SOCS3/CIS3 almost completely abolished HGF-induced STAT3 phosphorylation and keratinocyte migration, indicating that SOCS3/CIS3 acts as a negative regulator of HGF-induced keratinocyte migration. In conclusion, SOCS3/CIS3 regulates HGF-induced keratinocyte migration by inhibiting STAT3 phosphorylation. [Copyright &y& Elsevier]

1,25-dihydroxyvitamin D3 (1,25[OH]2VD3) has an antiproliferative effect on keratinocyte growth, and its derivatives are used for the treatment of psoriasis. It was reported previously that 1,25[OH]2VD3 induced cell cycle arrest not only at the G0/G1 phase but also at the G2/M phase. However, the mechanism of 1,25[OH]2VD3-induced G2/M phase arrest in keratinocytes has not been fully understood. The addition of 10−8 to 10−6 M 1,25[OH]2VD3 to cultured normal human keratinocytes enhanced the expression of Myt1 mRNA preceding Wee1 mRNA; 10−6 M 1,25[OH]2VD3 unregulated Myt1 mRNA from 6 h to 24 h and Wee1 mRNA from 12 to 48 h. Interestingly, the levels of phosphorylated Cdc2 were increased between 6 h and 48 h after 1,25[OH]2VD3 treatment, although the expression levels of Cdc2 mRNA and its protein production were reduced. 1,25[OH]2VD3 also decreased the expression of cyclin B1, which forms a complex with Cdc2. These data indicated that the increase of Myt1 and Wee1 induced the phosphorylation of Cdc2 leading to G2/M arrest. In conclusion, the induction of Cdc2 phosphorylation due to the increase of Wee1 and Myt1 as well as the reduction of Cdc2 and cyclin B1 are involved in 1,25[OH]2VD3-induced G2/M arrest of keratinocytes. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor β is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor β contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor β1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor β1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor β1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor β-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor β type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor β acts in an autocrine manner in keratinocytes.... [ABSTRACT FROM AUTHOR]

Investigates the mechanism regulating signal transducers and activators of transcription and the suppressor of cytokine signaling/cytokine-inducible SH2 containing protein family in keratinocytes. Impact of interleukin-6 on cytokine signaling; Transfection of the adenovirus vector expressing suppressor of cytokine; Relationship between signal transducers and suppressor of cytokine signaling.

Background: thymus and activation-regulated chemokine (TARC)/CCL17 is a CC chemokine that selectively attracts Th2-type lymphocytes. Immunohistochemical analyses have revealed that TARC is expressed in the epidermal keratinocytes of atopic dermatitis (AD), suggesting TARC involvement in the pathogenesis of the disease. However, keratinocyte TARC production has been described only in the transformed keratinocyte cell line HaCaT. Objective: to examine TARC production in normal human epidermal keratinocytes (NHEK) in vitro. Methods: the expression of TARC mRNA and protein were examined in NHEK and HaCaT cells stimulated with various cytokines. Results: stimulation with inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-10, interferon (IFN)-α, IFN-β, IFN-γ, and tumor necrosis factor (TNF)-α failed to induce TARC mRNA expression in NHEK. However, stimulation with IFN-γ and TNF-α together enhanced expression slightly. ELISA analysis failed to detect TARC protein in NHEK culture supernatant, even following stimulation with IFN-γ and TNF-α. In contrast, HaCaT cells produced TARC protein even without stimulation of cytokines. Conclusion: these results indicate that production of TARC by HaCaT cells is a phenomenon specific to the cell line and the observation on TARC in HaCaT cells can not be generalized. NHEK do not produce TARC protein in vitro. [Copyright &y& Elsevier]

Bullous pemphigoid (BP) is an acquired autoimmune subepidermal blistering disease against hemidesmosomal cytoplasmic BP230 and transmembrane BP180 proteins. Epitope mapping studies have shown that the membrane-proximal noncollagenous (NC) 16a domain of BP180 harbors clusters of antigenic sites recognized by the vast majority of BP sera. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) using bacterial recombinant NC16a protein and evaluated its clinical benefit for diagnosis and monitoring disease activity. Fifty four (84.4%) of 64 sera from BP patients were positive, while only one (1.1%) of 91 sera from collagen disease patients and five (1.5%) of 336 sera from normal control barely exceeded the cut-off value. None of 69 pemphigus vulgaris sera and none of 42 pemphigus foliaceus sera exceeded the cut-off value. Thus, the sensitivity and specificity of NC16a ELISA were 84.4 and 98.9%, respectively. The correlation between ELISA scores and disease activity along the time course was examined using seven BP patients. NC16a ELISA scores tended to fluctuate in parallel with the disease activity along the time course and reflected the disease activity much better than indirect immunofluorescence. These findings indicate that NC16a ELISA will be a valuable tool not only for the diagnosis of patients with BP but also for the monitoring of the disease activity. [Copyright &y& Elsevier]