Irradiation of light emitting diode at 850nm inhibits T cell-induced cytokine expression

Background

An anti-inflammatory effect of light obtained from light-emitting diodes (LEDs) has been discovered, however, limited ranges of wavelengths have been used and the action mechanism has been rarely demonstrated.

Objective

We sought to analyze the immunomodulatory effect of LED on Jurkat T cells and human T cells.

Methods

Jurkat T cells with/without stimulation were irradiated once or five times using seven ranges of LED wavelengths, from 415 nm to 940 nm. Cytotoxic effects were examined by an MTT assay. Changes in T cell-induced cytokines, including IL-2, IL-4, IL-10, IL-12 and IFN-gamma, and their upstream signaling molecules, ZAP-70 and PKCθ, were examined by real-time PCR, ELISA, and Western blot analysis. The effect of the LED wavelength, whose effect was identified on Jurkat T cells, was also examined in human CD3+ T cells with/without stimulation and in Dermatophagoides farinae-induced atopic dermatitis (AD) NC/Nga mice.

Results

Lower doses of LED irradiation at 850 nm inhibited T cell-derived cytokines without inducing cell death in both Jurkat T cells and human T cells. Repeated exposure resulted in a greater increase of inhibitory effects than that observed with a single exposure, and these effects were identified in the NC/Nga AD model.

Conclusions

Although more remains to be clarified, these results may support the clinical application of LED for immune regulation.     

Optimization of filaggrin expression and processing in cultured rat keratinocytes

Background

In normal mammalian epidermis, cell division occurs primarily in the basal layer where cells are attached to the basement membrane. Upon release from the basement membrane, these basal cells stop dividing and begin to differentiate and stratify producing cornified cells expressing differentiation markers, including the keratin bundling protein filaggrin, and cornified envelope proteins. Little is understood about the regulatory mechanisms of these processes. A rat epidermal keratinocyte cell line synthesizing and processing profilaggrin at confluence in a synchronous manner for 4-5 days provides a useful culture model for epidermal differentiation. Profilaggrin expression in this cell line however decreases with passaging, and its processing involves extensive nonspecific proteolysis.

Objective

Our objective was to identify culture conditions that effect the decrease in profilaggrin expression with passaging and nonspecific proteolysis of profilaggrin in order to study epidermal differentiation more closely.

Method

The large amount of nonspecific proteolysis suggested autophagocytosis. To test this, cells were cultured in the presence of 3-methyladenine (3-MA). Two known gradients in epidermis are decreasing serum components and increasing calcium concentrations in the upper cell layers. To determine whether these gradients effected processing, cells were cultured in serum/DMEM or in serum-free KGM and under varying external calcium concentrations. Cells were also cultured in presence of aminoguanidine in an attempt to maintain profilaggrin expression with passaging.

Results

Profilaggrin expression was enhanced in the presence of 3-MA, with optimum around 6 mM. In the absence of aminoguanidine, profilaggrin expression decreased as a function of increasing passage number; in its presence, profilaggrin expression remained high in some, but not in all of the independently maintained cell lines. Thus, culturing in aminoguanidine was necessary, but not sufficient, for sustained ability to express profilaggrin at confluence. Production of filaggrin from profilaggrin was maximized in a serum-free medium with [Ca²⁺] at 5 mM. Filaggrin associates with phospholipid vesicles in vitro forming aggregates similar to those seen in vivo, suggesting that filaggrin release induces vesicular aggregation and autophagocytosis.

Conclusion

We have used a keratinocyte cell line that synthesizes and processes profilaggrin after confluence as a culture model to study epidermal differentiation. In this system profilaggrin processing must be preceded by inhibition of autophagosome formation and/or modulation of vesicular trafficking, and these processes are regulated by epidermal calcium and serum factor gradients.     

Differentiation-dependent expression of NADP(H):quinone oxidoreductase-1 via NF-E2 related factor-2 activation in human epidermal keratinocytes

Background

NADP(H):quinone oxidoreductase-1 (NQO-1) is known for its protective role in skin carcinogenesis, but the expression of NQO-1 during keratinocyte (KC) differentiation has not been studied.

Objective

The purpose of the current study was to evaluate modulation of NQO-1 and NF-E2-related factor-2 (Nrf2) during KC differentiation.

Methods

Normal human epidermal keratinocytes (NHEKs) were induced to differentiation by prolonged culture after confluency (postconfluence).

Results

NQO-1 was induced at the late stage of differentiation of NHEKs (7th day of postconfluence). The expression of postconfluence-induced NQO-1 was stimulated by 0.1 mM H₂O₂, but attenuated by 5 mM N-acetylcysteine, implying that reactive oxygen species (ROS) are implicated in the expression of NQO-1 in differentiated KCs. Nrf2 was up-regulated at the earlier than NQO-1 induction (3rd day of postconfluence). The Nrf2-dependent expression of NQO-1 was further supported by Nrf2-siRNA experiments. A confocal study confirmed the differentiation-dependent induction and activation of NOQ-1 and Nrf-2 in NHEKs. Immunohistochemistry showed that NQO-1 was accentuated in the upper epidermal layers, supporting the notion that differentiation-dependent NQO-1 expression is functional in human skin in vivo.

Conclusion

These results demonstrate that NQO-1 is modulated during KC differentiation via Nrf2 pathway, suggesting the active role of NQO-1 in the differentiating epidermis.     

Radiation-induced mast cell mediators differentially modulate chemokine release from dermal fibroblasts

Background

Ionizing radiation has been demonstrated to result in degranulation of dermal mast cells. Chemokines are thought to play a crucial role in the early phase of the cutaneous radiation reaction. In human skin, mast cells are located in close proximity to dermal fibroblasts, which thus are a potential target for the action of mast cell mediators.

Objective

In this study, we evaluated the effects of mast cell-derived histamine, serotonin, tumour necrosis factor (TNF)-α and tryptase on chemokine release from dermal fibroblasts.

Methods

Human mast cells (HMC-1) were investigated for histamine release and cytokine production after ionizing radiation using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Receptor expression on human fetal foreskin fibroblasts (HFFF2) and human adult skin fibroblasts (HDFa) was examined by flow cytometry. Chemokine mRNA and protein expression were analyzed by gene array and ELISA, respectively.

Results

Ionizing radiation significantly increased histamine release and cytokine expression by HMC-1 cells. Receptors for histamine, serotonin, TNF-α and tryptase were detected both in HFFF2 and in HDFa cells. Dermal fibroblasts constitutively expressed distinct sets of chemokine mRNA. Mast cell mediators differentially affected the release of chemokines CCL8, CCL13, CXCL4 and CXCL6 by fibroblasts.

Conclusions

Our data suggest that radiation-induced mast cell mediators have a tremendous impact on inflammatory cell recruitment into irradiated skin. We postulate the activation of mast cells to be an initial key event in the cutaneous radiation reaction, which might offer promising targets for treatment of both normal tissue side effects in radiation therapy and radiation injuries.     

Expression of antimicrobial peptides in diabetic foot ulcer

Background

Foot ulcers are one of the main diabetes complications due to its high frequency and difficulty of complete healing. There are several factors that participate in diabetic ulcers development and limited information exists about the role of antimicrobial peptides (AMP) in its pathogenesis.

Objective

The aim of this study was to analyze the expression pattern of the main AMPs: Human Neutrophil Peptide (HNP)-1, Human β-defensin (HBD)-1, HBD-2, HBD-3, HBD-4 and cathelicidin LL-37 in biopsies from diabetic foot ulcers (DFU).

Methods

20 biopsies from DFU grade 3 according to Wagner's classification and 20 biopsies from healthy donors were obtained. Real time PCR, immunohistochemistry and primary cell cultures were performed.

Results

β-Defensins were overexpressed in DFU, whereas LL-37 has low or none expression in comparison with healthy skin. When primary cell culture from these biopsies were performed and infected with Staphylococcus aureus, epidermal cell from diabetic ulcers showed lower LL-37 expression compared with cell cultures from healthy donors skin.

Conclusion

These results suggest that though most AMPs are expressed in DFU, this production is not appropriate to promote wound healing and contain secondary infections.     

Cathepsin K-upregulation in fibroblasts promotes matrigel invasive ability of squamous cell carcinoma cells via tumor-derived IL-1α

Background

Cathepsin K (CTSK), a cysteine protease with strong collagenolytic properties, is involved in extracellular matrix turnover. In the previous studies, CTSK expression was detected in peritumoral fibroblasts (Fbs) around squamous cell carcinoma (SCC), but not in those surrounding benign epidermal tumors. However, the mechanism governing CTSK expression in epidermal tumors remains unclear.

Objective

To study the regulatory mechanisms of fibroblastic CTSK expression in the SCC-stromal interaction.

Methods

We examined dynamic interactions of Fbs with tumorigenic SCC cells (A431 and A253) or normal human keratinocytes.

Results

SCC cells and normal keratinocytes did not synthesize CTSK, while Fbs constitutively expressed CTSK. When cocultured, SCC cells upregulated fibroblastic CTSK expression more potently than did normal keratinocytes, which was mainly attributable to SCC-derived IL-1α. Coculturing Fbs with SCC cells significantly augmented the matrigel invasive ability of SCC cells, which was downregulated when cocultured with CTSK knockdown Fbs or in the presence of neutralizing anti-IL-1α antibody.

Conclusion

The CTSK-upregulated Fbs generated by SCC-derived IL-1α may play a crucial role in the progression and invasion of SCC.     

Dermal carotenoid level and kinetics after topical and systemic administration of antioxidants: Enrichment strategies in a controlled in vivo study

Background

High doses of sun-emitted UV-radiation induce reactive oxygen species (ROS) as major pro-oxidants thus inducing premature skin aging. The best prevention of the destructive action of free radicals in human skin is textile coverings, topical sunscreens and the development of a high antioxidative protective network.

Objective

The effects of topical, systemic and combined application of antioxidants (AO) were investigated on human skin in vivo.

Methods

Topical application of creams and systemic incorporation of tablets both containing AO was investigated in vivo by resonance Raman spectroscopy.

Results

Topical, systemic and combined AO-treatments induced a statistically significant increase of AO levels in human skin while placebo did not show any changes. The highest accumulation was induced by the combination of topical and systemic AO. Carotenoid-tablets combined with placebo-cream induced less carotenoid accumulation than carotenoid-tablets alone. Carotenoid levelling after the end of treatment lasted for around 2 weeks following the topical application of AOs, and up to 5 weeks after systemic administration, depending on the BMI of volunteers.

Conclusion

Topically applied AO are stored in the SC for a short time only due to the rapid AO-depletion by desquamation, textile contact, washing and environmental stress. In contrast to topical application, the systemically applied carotenoids are stored in the body fat tissue and slowly released onto the skin surface with sweat and sebum. The combined topical and systemic application of AO represents an optimal form of protection of the AO-network.     

Imatinib mesylate induction of ROS-dependent apoptosis in melanoma B16F0 cells

Background

Imatinib mesylate (STI571), a protein tyrosine kinase inhibitor, was shown to reduce the viability of several cancer cell lines via apoptosis induction; however, the role of reactive oxygen species (ROS) in STI571-induced melanoma cell apoptosis is still undefined.

Objective

In this study, we investigated the contribution of ROS to STI571-induced apoptosis in melanoma B16F0 cells, and the apoptotic mechanism elicited by STI571 was illustrated.

Methods

Using an in vitro cell culture system, the effects of STI571 on ROS production, cell cycle progression, caspase activation, and mitochondrial functions were examined via Western blotting, a flow cytometric analysis, an enzyme activity assay, and a DNA integrity assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, dipheylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (PD98059, SP600125, and SB203580) were applied to investigate the mechanism.

Results

STI571 reduced the viability of melanoma cells B16F0, but not human skin fibroblasts WS1, via apoptosis induction. Besides, apoptosis induced by STI571 was inhibited by the addition of NAC and DPI, and an increase in the intracellular peroxide level by STI571 was identified in melanoma B16F0 cells. Activation of caspases 3 and 9 enzyme activities accompanied by disrupting the mitochondria membrane potential in according with stimulating JNK and p38 protein phosphorylation was identified in STI571-treated B16F0 cells. STI571-mediated a ROS-dependent apoptosis potentiated by JNK inhibitor SP600125 was first identified in melanoma B16F0 cells.

Conclusion

Our results support the idea that ROS-dependent apoptosis in STI571-treated melanoma cells B16F0. The combination of a JNK inhibitor with STI571 for treating melanomas is suggested for further in vivo studies.     

Semaphorin 7A on keratinocytes induces interleukin-8 production by monocytes

Background

Semaphorin 7A (Sema7A) expressed on activated T cells stimulates cytokine production in monocytes through its receptor, α1β1 integrin.

Objective

To study the significance of Sema7A expressed on keratinocytes in skin inflammation where interaction between keratinocytes and β1-integrin expressing inflammatory cells, such as monocytes, takes place.

Methods

The regulation of Sema7A expression on keratinocytes by various cytokines was studied by flow cytometry and immunoblot. β1-integrin expressing human monocyte cell line, THP-1 cells, were co-cultured with paraformaldehyde-fixed normal human epidermal keratinocytes (NHK) and IL-8 production by THP-1 cells was studied. The significance of β1-integrin or Sema7A within this cell interaction was examined by the experiments using β1-integrin blocking antibody or Sema7A siRNA.

Results

IFN-γ and TNF-α slightly increased Sema7A expression, while IL-4 decreased it. Among cytokines tested, TGF-β1 most strikingly increased the Sema7A expression on NHK. When NHK was stimulated by TGF-β1, paraformaldehyde-fixed, and co-cultured with THP-1 cells, IL-8 production by THP-1 cells was increased compared to THP-1 cells only. When THP-1 cells were pretreated with β1-integrin blocking antibody, this increase in IL-8 production by THP-1 cells was inhibited. Likewise, when NHK were pretreated with Sema7A siRNA before fixation and co-cultured with THP-1 cells, increase in IL-8 production by THP-1 cells was inhibited.

Conclusion

Our results suggest that Sema7A on keratinocytes and β1-integrin on monocytes contribute to monocyte activation by keratinocytes within skin inflammation, such as psoriasis or wound.     

Apigenin attenuates dopamine-induced apoptosis in melanocytes via oxidative stress-related p38, c-Jun NH2-terminal kinase and Akt signaling

Background

Accumulating evidence suggests that the occurrence of oxidative stress leads to melanocyte degeneration in vitiligo. Elevated level of dopamine (DA), an initiator of oxidative stress, reportedly is found in patients with vitiligo and induces melanocyte death in vitro. DA-treated melanocytes have been used as a model to search for antioxidants for treating vitiligo.

Objective

We investigated the protective effects of apigenin against DA-induced apoptosis in melanocytes and the molecular mechanism underlying those effects.

Methods

Melanocytes with or without pretreatment with apigenin were exposed to DA. Then cell viabilities were measured by MTT assay. Cellular reactive oxygen species (ROS) levels and the percentage of apoptotic cells were detected by flow cytometry analysis. Activation of caspase 3, poly(ADP-ribose) polymerase (PARP) and oxidative stress-related signaling, including p38, c-Jun NH2-terminal kinase (JNK) and Akt, were assessed by Western blotting.

Results

Apigenin attenuated DA-induced apoptotic cell death, relieved ROS accumulation and activated caspase 3 and PARP, suggesting the protective effects of apigenin against DA-induced oxidative stress and apoptosis in melanocytes. Moreover, DA induced phosphorylation of p38, JNK and Akt, while inhibitors of p38, JNK and Akt significantly decreased DA-induced apoptosis. However, pretreatment with apigenin significantly inhibited DA-triggered activation of p38, JNK and Akt, suggesting the involvement of p38, JNK and Akt in the protective effects of apigenin against DA-induced cytotoxicity.

Conclusion

These results suggest that apigenin attenuates dopamine-induced apoptosis in melanocytes via oxidative stress-related p38, JNK and Akt signaling and therefore could be a potential agent in treating vitiligo.     

Benzo[c]phenanthridine alkaloids exhibit strong anti-proliferative activity in malignant melanoma cells regardless of their p53 status

Background

Search for new substances with antiproliferative activity towards melanoma cells is important since malignant melanoma is notoriously resistant to conventional chemotherapy. Benzo[c]phenanthridine alkaloids (BAs) are natural products with significant anti-proliferative activities, therefore they are considered as agents promising for cancer therapy.

Objectives

The effects of five BAs (sanguinarine, chelerythrine, chelidonine, sanguilutine, and chelilutine) on human malignant melanoma cell lines were compared. The study focused on BAs effects on DNA, anti-apoptotic and p53 protein levels; and the involvement of p53 in cellular responses to alkaloids treatment.

Methods

Melanoma cell lines, two wild types and two with dysfunctional p53 derived from one of them were used. The mechanism of anti-proliferative and pro-apoptotic effects and the effect on DNA was investigated using MTT assay, flow cytometry, Western blot analysis, fluorescence and electron microscopy.

Results

All tested alkaloids exhibit strong anti-proliferative activity. CHL, CHE and SA induced apoptosis, which was probably mediated by decreasing levels of anti-apoptotic proteins (Bcl-xL, Mcl-1, XIAP) and was accompanied by mitochondrial membrane potential decrease as well as caspase-3 and PARP cleavage. Although all alkaloids caused DNA damage, which was demonstrated by induction of H2AX phosphorylation, none of the tested alkaloids stabilised p53 and their toxicity in cells with non-functional p53 was comparable to wild type cells.

Conclusion

Despite the profound similarity of BAs molecular structures, it is clear that the mechanism of cell death induction is different for each alkaloid. Our results indicate that BAs could be effective in malignant melanoma treatment, including tumours which have lost wild type p53.     

Interactions between myofibroblast differentiation and epidermogenesis in constructing human living skin equivalents

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.     

An association between IL-9 and IL-9 receptor gene polymorphisms and atopic dermatitis in a Korean population

Background

The genes encoding IL-9 and IL-9R have recently been implicated in the genetic basis of asthma and allergy. Although studies performed on transgenic and knockout mice have shown conflicting results, no evidence of skin changes has ever been reported in these animals.

Objective

To find association of the SNPs in IL-9 and IL-9R genes and interaction of these genes in atopic dermatitis.

Method

We genotyped 5 SNPs from the IL-9 and IL-9R genes of 1090 subject samples (631 AD patients and 459 normal controls). A luciferase assay was then performed for the rs31563 (−4091G/A) SNP located in the IL-9 gene promoter region.

Result

The rs31563 (−4091G/A) SNP in the IL-9 gene was significantly associated with the AD phenotype, especially allergic-type AD. In the luciferase assay, the rs31563 G construct was observed to have 1.54 times higher activity than the rs31563 A construct. Although no association was found between SNPs in IL-9R gene and AD, the rs3093467 SNP showed association with non-allergic AD. In the gene-gene interaction analysis, we found that IL-9/IL-9R genotype rs31563 GG/rs3093467 TT conveyed a greater risk for AD phenotype development.

Conclusion

Significant evidence exists to suggest that the rs31563 SNP (−4091G/A) located in the IL-9 gene is associated with an increased susceptibility to AD. Similarly, the rs3093467 SNP in IL-9R gene seems to be associated with an increased risk for developing non-allergic AD. In a subsequent gene-gene interaction analysis, the rs31563 GG/rs3093467 TT genotype combination (IL-9/IL-9R) was found to exert a synergistic effect in the development of the AD phenotype. As the classes of helper T cells are diverse and the function of IL-9 cytokine has not been fully described, the cutaneous function of IL-9 needs to be further explored in future studies.     

Differential expression of matrix metalloproteinases and proteoglycans in Juvenile Hyaline Fibromatosis

Background

Juvenile Hyaline Fibromatosis (JHF) is a rare autosomal recessive disorder, histologically characterized by the production and deposition of an unidentified hyaline material in the skin and other organs. Extracellular matrix molecules are implicated in the development of skin lesion which is debilitating and recurrent and, so far, no treatment is satisfactory.

Objective

To investigate the expression of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and proteoglycans in lesional as compared to site-matched lesion-free skin tissue specimens of a JHF patient, aiming to elucidate the aetiopathological mechanisms involved in the development of JHF skin lesions.

Methods

Gelatinase activity of MMP-2 and MMP-9 was investigated by gelatine zymography. Protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in skin tissue extracts were measured by ELISA. Gene expression of MMPs, TIMPs and proteoglycans was examined by quantitative RT-PCR.

Results

JHF lesions exhibited significantly higher activity as well as elevated protein and gene expression of MMP-2 and MMP-9, as compared to lesion-free skin tissue specimens. Decorin was downregulated and aggrecan was upregulated in lesional skin, as compared to normal skin.

Conclusion

The results presented in this study indicate that MMPs and proteoglycans may be involved in the pathogenesis of JHF and therefore these molecules may offer alternative targets for pharmacological intervention to achieve more radical and effective treatment.     

EC-SOD induces apoptosis through COX-2 and galectin-7 in the epidermis

Background

Extracellular superoxide dismutase (EC-SOD) is an anti-oxidant enzyme found in the extracellular matrix of tissues, and plays an important role in the prevention of many diseases caused by oxidative stress. However, other functions of EC-SOD in epidermis are not well known.

Objective

We investigated the functions of EC-SOD in epidermis using keratinocyte cell line and EC-SOD transgenic mice.

Methods

Expression of galectin-7 in pEC-SOD transfected cells or skin of EC-SOD transgenic mice was detected by western blot analysis. The percentage of apoptotic cells was determined by propidium iodide staining and subsequent FACS analysis. COX-2 siRNA or scrambled siRNA was transfected into HaCaT cells and western blot analysis was performed to detect pro-apoptotic protein levels.

Results

The epidermis of EC-SOD transgenic mice was thinner than wild type mice. In addition, we showed that the thin epidermis of EC-SOD transgenic mice results from the apoptosis of epidermal cells. To elucidate which molecules are involved in EC-SOD-induced apoptosis, we utilized two-dimensional electrophoresis; the results showed that the epidermis of EC-SOD transgenic mice produces more galectin-7, a pro-apoptotic factor, than the wild type. Furthermore, we showed that the transfection of EC-SOD-expressing plasmids induces the production of galectin-7, and pro-apoptotic proteins in keratinocytes. This suggests that EC-SOD induces apoptosis through increased galectin-7 expression. Finally, we demonstrated that EC-SOD-induced galectin-7 results from the production of COX-2.

Conclusion

Our results imply that EC-SOD plays a role not only as a reactive oxygen species scavenger, but also as a pro-apoptotic factor via COX-2/galectin-7 pathways in the epidermis.     

Aging enhances maceration-induced ultrastructural alteration of the epidermis and impairment of skin barrier function

Background

Skin maceration is recognized as a risk factor for the development of certain skin lesions. In health care settings, incontinence-associated skin maceration is highly prevalent in the elderly. However, the effect of senescence on maceration has not been fully elucidated.

Objective

To reveal the enhancement of the maceration-induced ultrastructural alteration and barrier function of the epidermis by aging.

Methods

Skin maceration was reproduced by exposure to agarose gel in human and rat. The ultrastructural alterations in human and rat tissue were observed by transmission electron microscopy. The skin barrier function was evaluated by noninvasive methods in human, and by the transdermal penetration of small- and large-fluorescent molecules in rat. In order to reveal the effect of aging on the skin maceration, we compared these parameters between young and aged rats.

Results

In macerated skin, we observed expansion of the interstices of the stratum corneum, spinosum, and basale of the epidermis; disruption of the intercellular lipid structure in the stratum corneum; a decreased number of cell processes in the stratum spinosum and basale. The transdermal penetration test in the rat using two types of fluorescein indicated that maceration disrupted skin barrier function. Furthermore, senescence-enhanced ultrastructural and functional alterations were revealed in the rodent studies.

Conclusion

This study demonstrates that aging enhances skin maceration. Considering that maceration is a risk factor for the skin damage, the development of technology to promote skin barrier recovery after maceration in the elderly is warranted.     

The hsp27kD heat shock protein and p38-MAPK signaling are required for regular epidermal differentiation

Background

In human epidermal keratinocytes the expression of hsp27 is closely related to differentiation in vitro and in situ.

Objective

We aimed to gain further insight into the role of hsp27 in epidermal differentiation by specific inhibition through siRNA and inhibition of p38-MAPK, the key enzyme of hsp27 phosphorylation.

Methods

Normal human keratinocytes (KC) and organotypic skin cultures (SE-skin equivalents) were used. Expression and phosphorylation of hsp27 was inhibited in these models by siRNA and SB203580, a specific inhibitor of p38-MAPK, respectively. Modification of morphology and expression of hsp27 and other differentiation associated proteins was investigated by immunofluorescence, western blot, and RT-PCR.

Results

Inhibition of p38-MAPK resulted in a downregulation of hsp27 in KC and SE. Additionally, in the presence of SB203580 Ca²⁺ induced expression of pro-filaggrin and loricrin was inhibited at the protein level and expression of filaggrin, keratin 10, and transglutaminase 1 at the mRNA level. Addition of SB203580 to SE, as well as hsp27 knockdown in this model resulted in identical patterns of irregular differentiation, disturbance of epidermal layers, and delayed expression of K10.

Conclusion

These results provide evidence that the expression of hsp27 and its phosphorylation by p38-MAPK are required for keratinocyte differentiation and for the formation of a regularly stratified epidermis.