Study on the roles of β-catenin in hydrogen peroxide-induced senescence in human skin fibroblasts

Oxidative stress is one of the most important causes of the cellular senescence process. Previous studies showed that β-catenin can regulate FoxO3a and this association was enhanced in cells exposed to oxidative stress. It has also been reported that β-catenin can regulate some senescence-related proteins. We propose that β-catenin may play a crucial role in senescence of normal human primary skin fibroblasts (NHSFs). Here, we explored the roles and mechanisms of β-catenin on H(2)O(2)-induced senescence in NHSFs. β-catenin expression was decreased in NHSFs after H(2)O(2) treatment. Overexpression of β-catenin in NHSFs led to a marked delay of many senescent phenotypes induced by H(2)O(2). Furthermore, overexpression of β-catenin in NHSFs can antagonise the alteration of reactive oxygen species accumulation and some senescence-related proteins expression induced by H(2)O(2) treatment. Our data demonstrated that β-catenin can protect NHSFs from H(2)O(2)-induced premature senescence by alleviating oxidative stress and regulating some senescence-related molecules.     

Organ culture of psoriatic skin: effect of TGF-α and TGF-Β on epidermal structure in vitro

Summary Normal skin and uninvolved and involved psoriatic skin specimens were maintained in vitro in organ culture. The 3–4 mm punch-biopsied skin specimens were put freely into the culture medium with or without fetal calf serum, under an atmosphere of 95% O₂ plus 5% CO₂, and rotated at 60 rpm at 37C. In the serum-free culture medium (vitamin A-free) granular layers appeared in the involved psoriatic epidermis in culture. Addition of TGF- caused normal skin and uninvolved and involved psoriatic skin specimens to become acanthotic and to degenerate easily almost to the full thickness of the epidermal layer in proportion to increasing concentrations of TGF- as well as with the duration of the culture, but without disappearance of their granular layers. TGF- caused the normal skin and uninvolved psoriatic skin specimens to become thinned without disappearance of granular layers, but caused the involved psoriatic skin specimens to be thinned without appearance of granular layers in serumcontaining medium or with their disappearance in the serum-free medium. TGF- also antagonized the acanthotic and degenerative effect of TGF-. The results suggest that TGF- and TGF- may partially be related to the induction of psoriatic epidermal lesions.     

Expression of estrogen-related receptor beta (ERRβ) in human skin

Skin is a non classical target tissue for estrogens, whose biological and mechanical properties are affected by the hormonal deprivation occurring at the menopause. Estrogen-related receptors (ERR), closely related to the estrogen receptors (ER), constitute a subfamily of orphan receptors, interfering with ER-mediated signalling pathways. The expression of ERRβ has been detected in only a few adult tissues so far, such as the prostate and the inner ear. Here, we demonstrate the expression of ERRβ in normal human skin. ERRβ was detected in human epidermis, in both keratinocytes and Langerhans cells, by immunohistochemistry. These results were validated on freshly extracted epidermal cells and on monolayer-cultured keratinocytes by RT-PCR and western blotting (WB), suggesting the implication of ERRβ in skin immunity and endocrine effects.     

Specific inhibition of human skin fibroblast chemotaxis to platelet-derived growth factor A-chain homodimer by transforming growth factor-β1

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) have been suggested to play important roles in wound healing. We investigated the effect of TGF-beta1 on the mitogenic and chemotactic activities of PDGF A-chain homodimer (PDGF-AA) and B-chain homodimer (PDGF-BB) in primary cultures of human skin fibroblasts. TGF-beta1 inhibited the growth-promoting activity of both PDGFs. Proliferative responses to basic fibroblast growth factor and epidermal growth factor were also restricted by TGF-beta1. A Boyden chamber chemotaxis assay revealed that the chemotactic migration to PDGF-AA was inhibited by TGF-beta1 pretreatment, but in contrast, the response to PDGF-BB was not affected by the same treatment. Western blot analysis showed that TGF-beta1 downregulated PDGF alpha-receptors, but not beta-receptors, indicating that the isoform-specific inhibition of chemotaxis is related to differential effects of TGF-beta1 on PDGF receptor expression. The present findings suggest that TGF-beta1 may act antagonistically towards PDGFs in humans under certain conditions, and this antagonistic nature of TGF-beta1 must be considered when it is applied to human wounds as a therapeutic agent.     

Wnt/β-catenin pathway forms a negative feedback loop during TGF-β1 induced human normal skin fibroblast-to-myofibroblast transition

Background

Fibroblast-to-myofibroblast transition is a key event during wound healing and hypertrophic scar formation. Previous studies suggested Wnt/β-catenin signaling might be involved in the wound healing. However, its specific role in skin fibroblast-to-myofibroblast transition remains unclear.

Objective

To investigate the specific role of β-catenin during the transforming growth factor-β1 induced normal skin myofibroblasts transition.

Methods

By real-time quantitative polymerase chain reaction, Western-blot and immunocytochemistry, the activation of Wnt/β-catenin pathway in cultured human normal skin fibroblasts during TGF-β1 induced fibroblast-to-myofibroblast transition was investigated. The effects of β-catenin on myofibroblasts transition were also investigated when SB-216763, over-expression and siRNA of β-catenin were utilized. In addition, fibroblasts populated collagen lattices contraction assays were conducted to examine the effects of β-catenin on the contractility of the fibroblasts induced by TGF-β1. Furthermore, the effects of β-catenin on the expression of α-smooth muscle actin and collagen types I and III in hypertrophic scar derived fibroblasts were studied.

Results

The expression of Wnts mRNA and β-catenin protein was up-regulated by TGF-β1 stimulation during the myofibroblasts transition. Both of SB-216763 and β-catenin over-expression was paralleled with decreased expression of α-smooth muscle actin, collagen types I and III, while siRNA targeting β-catenin leads to up-regulation of α-smooth muscle actin, collagen types I and III. The increased contractility and α-smooth muscle actin expression of the fibroblasts in the collagen lattices induced by TGF-β1 was inhibited by SB-216763. In addition, the expression levels of α-smooth muscle actin, collagen types I and III in hypertrophic scar derived fibroblasts were also down-regulated by SB-216763.

Conclusion

Specifically in normal skin fibroblasts, β-catenin might be involved in the myofibroblasts transition and negatively regulate the TGF-β1-induced myofibroblast transition.     

Activity of 3β-hydroxysteroid dehydrogenase Δ4–5 isomerase in the human skin

Summary The activity of 3-hydroxysteroid dehydrogenase ^4–5 isomerase (3-HSD) was assayed in various tissues microdissected from the freeze-dried human skin of 13 subjects. The sebaceous gland possessed the highest activity of 3-HSD in the skin, while the apocrine sweat gland showed only one fourth of that activity. The vellus hair follicle showed nearly one half of the activity of the sebaceous gland, whereas the terminal hair follicle exhibited much lower activity. The activity of the epidermis and of the dermis were negligible. Incubation of fresh whole skin of the forehead with 7-³H-DHA and subsequent isolation of various tissues revealed that the metabolites identified in the sebaceous gland were androstanedione and androstenedione, whereas testosterone and/or dihydrotestosterone were not detected.Supported by a grant from Shiseido Company for basic dermatological research     

TGF-β1 up-regulates transglutaminase two and fibronectin in dermal fibroblasts: a possible mechanism for the stabilization of tissue inflammation

Transglutaminase (TGase) has been reported to stabilize tissue inflammation via the mediation of the polymerization of extracellular matrix proteins. A set of cytokines has been implicated in wound healing processes in the dermis. This study was undertaken in order to evaluate the effects of these cytokines on the expression of TGase 2 in human dermal fibroblasts (hDFs), in that TGase 2 is known to be the principal TGase in the dermis. In Western blot analysis, TGF-β1 (1 ng/ml) treatment was found to steadily up-regulate TGase 2 expression for up to 7 days. However, such increases were not observed when the cells were treated with IL-1β, IL-2, and TNF-α. In the enzyme assay, total TGase activities were closely related to the levels of TGase 2 expression. TGase 2 mRNA expression was up-regulated as the result of TGF-β treatment in competitive RT-PCR. In the denatured SDS-PAGE, TGF-β1 treatment resulted in marked induction of an approximately 220 kDa protein, which was revealed to be a fibronectin (FN) via western immunoblotting with an anti-FN antibody. Next, when the hDFs were treated with TGF-β1 (1 ng/ml), FN expression was induced beginning at the third day after treatment. The immunoprecipitants generated by anti-FN antibody were positive for the anti-TGase 2 antibody, and the immune complexes were identified at molecular weights of 92 kDa. Collectively, TGF-β1 stimulates the polymerization of FN via the action of TGase 2, which is supposed to to be an important mechanism in the stabilization of the inflammatory dermis.     

Corticosteroids induce proliferation but do not influence TNF- or IL-1Β-induced ICAM-1 expression of human dermal microvascular endothelial cells in vitro

The effects of hydrocortisone, dexamethasone, betamethasone 17-valerate and clobetasol propionate at concentrations of 10^–5–10^–12 M on the proliferation of human dermal microvascular endothelial cells (HDMEC) were studied in vitro. In addition, confluent HDMEC were treated with TNF (1000 U/ml) or IL-1 (1000 U/ml) alone or in combination with the corticosteroids (10^–5 M, 10^–6 M) for 24 h, and cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1) was assessed by immunocytochemistry. Controls were treated either with growth medium or with the corticosteroids alone. All tested corticosteroids stimulated HDMEC growth after 4 and 6 days of treatment in a dose-dependent manner, as assessed by ³H-thymidine incorporation and the 4-methyl-umbelliferyl heptanoate (MUH) assay. The minimal effective concentration was 10^–9 M for hydrocortisone, 10^–10 M for dexamethasone and betamethasone, and 10^–12 M for clobetasol. In untreated and in corticosteroid-treated cultures, less than 5% of the cells expressed ICAM-1. TNF and IL-1 were strong inducers of ICAM-1 expression on 74% and 82% of the cells, respectively. None of the tested corticosteroids significantly influenced cytokine-induced ICAM-1 expression, suggesting that the anti-inflammatory effects of corticosteroids are not related to ICAM-1 modulation on HDMEC.     

Human hair follicle dermal cells and skin fibroblasts show differential activation of NF-κB in response to pro-inflammatory challenge

The underlying mechanism of immune privilege in hair follicle cell dermal papilla (DP) and sheath (DS) populations is not well understood, and the responsiveness of hair follicle dermal cells to pro-inflammatory challenge presently remains unknown. In this work, we describe acute NF-κB activation in human DS, DP and dermal fibroblast (DF) cells challenged with TNF-alpha and IL1-beta. In contrast, the DS and DP cells revealed an unexpected tolerance to bacterial LPS challenge relative to DF cells. Understanding follicle cell responses to typical pro-inflammatory stimuli is critical for diseases where collapse of hair follicle immune privilege is observed, and to further applications in autologous stem cell/wound healing therapeutics.     

Upregulation of transforming growth factor-β1 and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity

Keloids are tumor-like lesions that result from excessive scar formation during healing of wounds. Histologically, keloids show an increased blood vessel density compared with normal dermis or normal scars. However, the angiogenic activity of keloid fibroblasts remains unknown. In this study, we investigated angiogenic activity of keloid fibroblasts. Transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-β1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-β1 in the production of VEGF was also investigated with addition of TGF-β1 and a neutralizing anti-TGF-β1 antibody. A modified Boyden chamber assay was performed to assess the chemotactic activity of vascular endothelial cells. Angiogenic activity in vivo was evaluated by neovascularization of nodules formed by implantation of fibroblasts into severe combined immunodeficiency (SCID) mice. EIA showed that the concentrations of TGF-β1 and VEGF in conditioned medium were increased 2.5- and 6-fold, respectively, after the culture of keloid fibroblasts compared with normal fibroblasts. Northern blot analysis revealed that the expression of TGF-β1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-β1 to keloid fibroblast cultures increased VEGF production by 3.5-fold, while there was a 6-fold in culture of normal fibroblasts. A neutralizing anti-TGF-β1 antibody reduced VEGF secretion to control levels, suggesting that TGF-β1 mediated the upregulation of VEGF expression. A modified Boyden chamber assay demonstrated that the chemotactic activity of vascular endothelial cells was more strongly (sevenfold) induced by keloid fibroblast-conditioned medium than by normal fibroblast-conditioned medium. Anti-VEGF antibody inhibited chemotaxis to basal levels. When SCID mice underwent implantation of fibroblasts into the back, the nodules formed by keloid fibroblasts were three times larger than those formed by normal fibroblasts. Although abundant neovascularization was observed in keloid fibroblast nodules, neovascularization was scarce in normal fibroblast nodules. Both in vitro and in vivo studies confirmed the significantly higher angiogenic activity of keloid fibroblasts compared with normal fibroblasts, and TGF-β1 and VEGF were clearly shown to be involved. These results suggest that angiogenesis in keloids is promoted by endogenous TGF-β1 and VEGF.     

Suppression of TGF-β1/SMAD pathway and extracellular matrix production in primary keloid fibroblasts by curcuminoids: its potential therapeutic use in the chemoprevention of keloid

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix (ECM) in the dermis. It is a late spreading skin overgrowth and may be considered a plastic surgeon’s nightmare. In nature, curcuminoid is composed of curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (bDMC). Curcuminoids have been found to inhibit fibrosis. However, their role in the synthesis of ECM in the keloid fibroblasts (KFs) has remained unclear. In this series of studies, a total of seven primary KFs cultures were used as the KFs model for investigating the inhibitory effect of curcuminoids on the expression of ECM and TGF-β1. A sensitive and reproducible HPLC method was developed to provide a quantitative analysis on the cellular uptake of curcuminoids onto the KF cells. The level of ECM in the primary KFs was elevated. The elevation of ECM and TGF-β1/p-SMAD-2 level was substantially blocked by the cellular uptake of curcumin in a dose-dependent manner in all the seven primary KFs. The results have led to the conclusion that the excessive production of ECM in the KF cells could be blocked and/or rapidly decreased by curcumin.     

ER-α agonist induces conversion of fibroblasts into myofibroblasts, while ER-β agonist increases ECM production and wound tensile strength of healing skin wounds in ovariectomised rats

Oestrogen deprivation is one of the major factors responsible for many age-related processes, including poor wound healing in women. Previously, it has been shown that oestrogens have a modulatory effect in different wound-healing models. Therefore, in this study, the effect of selective oestrogen receptor (ER) agonists (PPT - ER-α agonist, DPN - ER-β agonist) on excisional and incisional wound-healing models was compared in ovariectomised rats in vivo as well as on human dermal fibroblasts (HDF) and human umbilical endothelial cells (HUVEC) in vitro. In the in vivo study, 4 months after either ovariectomy or sham ovariectomy, Sprague-Dawley rats were randomly divided into four groups and subjected to two incisional and excisional wounds: (i) control - sham operated, vehicle-treated; (ii) ovariectomised, vehicle-treated; (iii) ovariectomised, PPT treated; (iv) ovariectomised, DPN treated. In the in vitro study, HDFs and HUVECs were used. After treatment with ER agonists, cells were processed for immunocytochemistry and gelatin zymography. Our study shows that stimulation of ER-α leads to the differentiation of fibroblasts into myofibroblasts both in vivo and in vitro. On the other hand, the formation of extracellular matrix was more prominent, and wound tensile strength (TS) was increased when ER-β was stimulated. In contrast, stimulation of ER-α led to a more prominent increase in the expression of MMP-2 and decrease in wound TS. New information is presented in this investigation concerning oestrogen replacement therapy (ERT) in different wound-healing models. This study demonstrates that the ERT should be both wound and receptor-type specific.     

Palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB,p38, and ERK in human dermal fibroblasts

It has been suggested that free fatty acids (FFA) such as palmitate, which are secreted from enlarged adipocytes in the subcutaneous fat of obese subjects, serve as a link between obesity and altered skin functions. Cyclooxygenease-2 (COX-2) and prostanoids participate in the induction of impaired dermal function. In the current study, we investigated the issue of whether palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB or mitogen-activated protein kinase (MAPK) pathways in human dermal fibroblasts. Palmitate treatment significantly induced COX-2 expression and prostaglandin E₂ (PGE₂) release in human dermal fibroblasts. In addition, pre-treatment with triacsin C, an inhibitor of acyl-CoA synthetase in de novo ceramide synthesis, was found to reduce palmitate-induced COX-2 expression and PGE₂ release in human dermal fibroblast. The findings also show that palmitate-induced COX-2 expression and PGE₂ release are mediated by the NF-κB, p38, and extracellular signal-regulated kinase (ERK) MAPK pathways. These findings point to a new mechanism for explaining the link between increased FFAs in obesity and impaired dermal function.