Reactive Oxygen Species In Keratinocytes Research Articles

Benvitimod Inhibits IL-4– and IL-13–Induced Tight Junction Impairment by Activating AHR/ARNT Pathway and Inhibiting STAT6 Phosphorylation in Human Keratinocytes

Tight junctions are involved in skin barrier functions. In this study, the expression of CLDN1, CLDN4, and OCLN was found to decrease in skin lesions of atopic dermatitis by bioinformatics analysis. Immunohistochemistry staining in skin specimens from 12 patients with atopic dermatitis and 12 healthy controls also showed decreased CLDN1, CLDN4, and OCLN expression in atopic dermatitis lesions. Invitro studies showed that IL-4 and IL-13 downregulated CLDN1, CLDN4, and OCLN expression in HaCaT cells as well as CLDN4 and OCLN expression in human primary keratinocytes. This effect, which was mediated through the Jak-signal transducer and activator of transcription 6 signaling pathway, increased paracellular flux of 4-kDa dextran. Benvitimod, a new drug for atopic dermatitis, upregulated CLDN4 and OCLN through the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator pathway. Benvitimod induced nuclear translocation of NRF2 and reduced production of ROS in keratinocytes, thus inhibiting IL-4-/IL-13-induced CLDN1 downregulation and signal transducer and activator of transcription 6 phosphorylation. These results indicate that T helper 2 cytokines are involved in tight junction impairment, and benvitimod can inhibit these effects.

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages.

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.

Sulforaphane controls the release of paracrine factors by keratinocytes and thus mitigates particulate matter-induced premature skin aging by suppressing melanogenesis and maintaining collagen homeostasis

BackgroundSkin aging, potentially caused by exposure to particulate matter (PM)2.5, is characterized by wrinkling, abnormal pigmentation, and skin dryness triggered by several keratinocyte-derived paracrine factors. Sulforaphane (4-methylsulfinylbutyl isothiocyanate, SFN), commonly found in cruciferous vegetables, has diverse biological effects on skin tissue. PurposeIn the present study, we have investigated whether SFN may alleviate PM2.5-induced premature skin aging. MethodsWe used keratinocyte/melanocyte or keratinocyte/fibroblast coculture models of skin cells and measured the parameters of melanogenesis, collagen homeostasis and inflammation. ResultsSFN inhibited the development of reactive oxygen species in keratinocytes exposed to PM2.5. In keratinocyte/melanocyte cocultures, it significantly inhibited the upregulation of melanogenic paracrine mediators (including endothelin-1 and prostaglandin E2) in keratinocytes exposed to PM2.5; the synthesis of melanogenic proteins including microphthalmia-associated transcription factor, tyrosinase-related protein 1, and tyrosinase; and the levels of melanin in melanocytes. SFN treatment of keratinocyte/fibroblast cocultures significantly reduced the PM2.5-induced expression of NF-κB-mediated cytokines including interleukin-1β, interleukin-6, tumor necrosis factor α, and cyclooxygenase-2. In fibroblasts of the keratinocyte/fibroblast coculture system, the expression levels of phospho-NF-κB, cysteine-rich protein 61, and matrix metalloproteinase-1 were significantly decreased whereas procollagen type I synthesis was significantly increased. ConclusionCollectively, our results suggest that SFN mitigates PM2.5-induced premature skin aging by suppressing melanogenesis and maintaining collagen homeostasis. It acts by regulating the release of paracrine factors from keratinocytes.

Miconazole and terbinafine induced reactive oxygen species accumulation and topical toxicity in human keratinocytes

There are an estimated 1 billion cases of superficial fungal infection globally. Fungal pathogens form biofilms within wounds and delay the wound healing process. Miconazole and terbinafine are commonly used to treat fungal infections. They induce the accumulation of reactive oxygen species (ROS) in fungi, resulting in the death of fungal cells. ROS are highly reactive molecules, such as oxygen (O2), superoxide anion (O2•−), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH). Although ROS generation is useful for killing pathogenic fungi, it is cytotoxic to human keratinocytes. To the best of our knowledge, the effect of miconazole and terbinafine on HaCaT cells has not been studied with respect to intracellular ROS stimulation. We hypothesized that miconazole and terbinafine have anti-wound healing effects on skin cells when used in antifungal treatment because they generate ROS in fungal cells. We used sulforhodamine B protein staining to investigate cytotoxicity and 2′,7′-dichlorofluorescein diacetate to determine ROS accumulation at the 50% inhibitory concentrations of miconazole and terbinafine in HaCaT cells. Our preliminary results showed that topical treatment with miconazole and terbinafine induced cytotoxic responses, with miconazole showing higher cytotoxicity than terbinafine. Both the treatments stimulated ROS in keratinocytes, which may induce oxidative stress and cell death. This suggests a negative correlation between intracellular ROS accumulation in keratinocytes treated with miconazole or terbinafine and the healing of fungi-infected skin wounds.

NOX2 Expression Is Increased in Keratinocytes After Burn Injury

Reepithelialization is crucial for effective wound repair in burn wounds. Reactive oxygen species (ROS) have shown to be important in this. Recent studies suggest that NOX proteins produce ROS in keratinocytes. In the present study, we have studied NOX proteins in burn wounds, including the effect of C1-esterase inhibitor (C1inh) hereon, which is the endogenous inhibitor of complement activity whereof we have shown previously that it also increased the rate of reepithelialization in burn wounds. Skin tissue derived from healthy control Wistar rats (n = 6) were compared with burn-injured rats, with (n = 7) or without C1inh treatment (n = 7). After 14 days, rats were terminated. From the burn-injured rats, the entire wound and nonburned skin from the hind leg, that is, internal control was excised. From the control rats, dorsal skin was excised. In these skin samples, NOX2 and NOX4 were analyzed immunohistochemically. In nonburned rats, NOX2 was found in keratinocytes in both the basal layer and suprabasal layer of the epidermis; and the number of NOX2-positive keratinocytes was 367/mm2 (254–378). In burned rats, the number of NOX2-positive keratinocytes was significantly increased in the newly forming epidermis in the burned area to 1019/mm2 (649–1172), especially in the suprabasal layer, but significantly decreased in remote nonburned skin to 22/mm2 (6–89). C1inh treatment counteracted these changes in epidermal NOX2 expression in burned rats, both in the burned area as in remote nonburned skin. No NOX4 expression was found in the epidermis in none of the groups. NOX2 expression was increased in keratinocytes in newly forming epidermis after burn injury. C1inh, a drug that increases the rate of reepithelialization, counteracted this effect. These results suggest a role for NOX2 in the reepithelialization of burn wounds.

330 Allergens or non-allergens can induce Th2-type inflammation via reactive oxygen species in keratinocytes

330 Allergens or non-allergens can induce Th2-type inflammation via reactive oxygen species in keratinocytes

Protocatechuic acid inhibits Toll-like receptor-4-dependent activation of NF-κB by suppressing activation of the Akt, mTOR, JNK and p38-MAPK

Protocatechuic acid has demonstrated to have antioxidant and anti-inflammatory effects. We assessed whether protocatechuic acid may reduce the inflammatory mediator production, which is regulated by the Toll-like receptor-4-dependent Akt, mTOR and NF-κB pathway, and JNK and p38-MAPK in HaCaT cells and primary keratinocytes. Protocatechuic acid, Akt inhibitor, Bay 11-7085 and N-acetylcysteine reduced the lipopolysaccharide-caused production of cytokines and chemokines, expression of cyclooxygenase, increase in the levels and activities of Toll-like receptor-4, p-Akt and mTOR, activation of NF-κB, phosphorylation of the JNK and p38-MAPK, and production of reactive oxygen species in keratinocytes. Inhibitors of the c-JNK (SP600125) and p38-MAPK (SB203580) reduced lipopolysaccharide-caused production of inflammatory mediators, activation of the JNK and p38-MAPK, and production of reactive oxygen species in keratinocytes. These results show that protocatechuic acid may inhibit the lipopolysaccharide-stimulated inflammatory mediator production in keratinocytes by reducing the Toll-like receptor-4-dependent activation of Akt, mTOR and NF-κB pathways, and activation of JNK and p38-MAPK. The suppressive effect of protocatechuic acid appears to be associated with inhibition of the reactive oxygen species production. Protocatechuic acid appears to reduce the microbial product-caused inflammatory skin diseases.

KATP channel block inhibits the Toll-like receptor 2-mediated stimulation of NF-κB by suppressing the activation of Akt, mTOR, JNK and p38-MAPK

Changes in the KATP channel activity have been shown to regulate inflammation and immune responses. Using human keratinocytes, we investigated the effect of KATP channel inhibition on inflammatory mediator production in relation to the Toll like receptor-2-mediated-Akt, mTOR and NF-κB pathways, as well as JNK and p38-MAPK, which regulate the transcription genes involved in immune and inflammatory responses. 5-Hydroxydecanoate (a selective KATP channel blocker), glibenclamide (a cell surface and mitochondrial KATP channel inhibitor), the Akt inhibitor, rapamycin, Bay 11-7085 and N-acetylcysteine reduced the lipoteichoic acid- or peptidoglycan-induced production of cytokines and chemokines, and production of reactive oxygen species and increased the levels and activities of Kir 6.2, NF-κB, phosphorylated-Akt and mTOR, and the activation of JNK and p38-MAPK in keratinocytes. Inhibitors of c-JNK (SP600125) and p38-MAPK (SB203580) attenuated the lipoteichoic acid- or peptidoglycan-induced production of inflammatory mediators, the activation of the JNK and p38-MAPK, and the production of reactive oxygen species in keratinocytes. The results show that KATP channel blockers may reduce the bacterial component-stimulated production of inflammatory mediators in keratinocytes by suppressing the Toll-like receptor-2-mediated activation of the Akt, mTOR and NF-κB pathways, as well as JNK and p38-MAPK. The suppressive effect of KATP channel blockers appears to be achieved by the inhibition of reactive oxygen species production.

Rotundarpene inhibits TNF-α-induced activation of the Akt, mTOR, and NF-κB pathways, and the JNK and p38 associated with production of reactive oxygen species.

Ilex Rotunda Thunb has been shown to have anti-inflammatory and antioxidant effects. In human keratinocytes, we investigated the effect of rotundarpene (4-caffeoyl-3-methyl-but-2-ene-1,4-diol) on the TNF-α-stimulated production of inflammatory mediators in relation to the Akt, mTOR, and NF-κB pathways, and the JNK and p38-MAPK. Rotundarpene, Akt inhibitor, Bay 11-7085, rapamycin, and N-acetylcysteine inhibited the TNF-α-stimulated production of cytokines and chemokines, increase in the levels of p-Akt and mTOR, activation of NF-κB, and production of reactive oxygen species in keratinocytes. TNF-α treatment induced phosphorylation of the JNK and p38-MAPK. Inhibitors of the c-JNK (SP600125) and p38-MAPK (SB203580) reduced the TNF-α-induced production of inflammatory mediators, binding of NF-κB to DNA, and activation of the JNK and p38-MAPK in keratinocytes. The results show that rotundarpene may reduce the TNF-α-stimulated inflammatory mediator production by suppressing the reactive oxygen species-dependent activation of the Akt, mTOR, and NF-κB pathways, and activation of the JNK and p38-MAPK in human keratinocytes. Additionally, rotundarpene appears to attenuate the Akt, mTOR, and NF-κB pathways and the JNK and p38-MAPK-mediated inflammatory skin diseases.

The Effect of Rhus verniciflua Stokes Extracts on Photo-Aged Mouse Skin.

BackgroundRhus verniciflua Stokes (RV) has traditionally been used in Korea as an indigenous food (Rhus chicken soup) and as an herbal medicinal plant. While the anticancer, antimicrobial, and anti-inflammatory properties of RV have been actively studied in the medical field, its antioxidant effects in the skin that resist the reactive oxygen species in keratinocytes and fibroblasts is less understood.ObjectiveWe designed to evaluate the effects of R. verniciflua Stokes extract (RVE) on the photo-aged skin by an in vitro experiment using human fibroblasts and an in vivo experiment using a photo-aged murine model.MethodsFor the in vitro experiments, human fibroblasts irradiated with ultraviolet (UV) B were treated with RVE or vehicle, and the growth levels and the expression level of type 1 procollagen were compared. For the in vivo experiment, photo-aged mice irradiated with UVB and UVA were administered drinking water with or without RVE, and histological changes and the expression level of type 1 procollagen and matrix metalloprotease (MMP)-13 were compared.ResultsIn vitro experiments using fibroblasts irradiated with UVB showed that RVE promoted growth and significantly increased the expression of type 1 procollagen as compared to the control group. In the photo-aged mice, RVE increased collagen content in the dermis and promoted the synthesis of type 1 procollagen without any visible decrease in MMP-13 as compared to control group.ConclusionIn addition to the previously reported antioxidant effects of RVE, oral intake of RVE effectively inhibited photo-aging in hairless mice by enhancing collagen synthesis.

Inhibition of aryl hydrocarbon receptor signaling and induction of NRF2-mediated antioxidant activity by cinnamaldehyde in human keratinocytes

BackgroundDioxins and other environmental pollutants are toxic and remain in biological tissues for a long time leading to various levels of oxidative stress. Although the toxicity of these agents has been linked to activation of the aryl hydrocarbon receptor (AHR), no effective treatment has been developed. ObjectiveTo explore novel phytochemicals that inhibit AHR activation in keratinocytes. MethodsKeratinocytes were used in this study because the skin is one of the organs most affected by dioxin and other environmental pollutants. HaCaT cells, which are a human keratinocyte cell line, and normal human epidermal keratinocytes were stimulated with benzo[a]pyrene to induce AHR activation, and the effects of traditional Japanese Kampo herbal formulae were analyzed. Quantification of mRNA, western blotting, immunofluorescence localization of molecules, siRNA silencing, and visualization of oxidative stress were performed. ResultsCinnamomum cassia extract and its major constituent cinnamaldehyde significantly inhibited the activation of AHR. Cinnamaldehyde also activated the NRF2/HO1 pathway and significantly alleviated the production of reactive oxygen species in keratinocytes. The inhibition of AHR signaling and the activation of antioxidant activity by cinnamaldehyde operated in a mutually independent manner as assessed by siRNA methods In addition, AHR signaling was effectively inhibited by traditional Kampo formulae containing C. cassia. ConclusionCinnamaldehyde has two independent biological activities; namely, an inhibitory action on AHR activation and an antioxidant effect mediated by NRF2/HO1 signaling. Through these dual functions, cinnamaldehyde may be beneficial for the treatment of disorders related to oxidative stress such as dioxin intoxication, acne, and vitiligo.

Apocynin inhibits Toll-like receptor-4-mediated activation of NF-κB by suppressing the Akt and mTOR pathways.

Microbial product lipopolysaccharide has been shown to be involved in the pathogenesis of inflammatory skin diseases. Apocynin has demonstrated to have an anti-inflammatory effect. However, the effect of apocynin on the Toll-like receptor-4-dependent activation of Akt, mammalian target of rapamycin (mTOR), and nuclear factor (NF)-κB pathway, which is involved in productions of inflammatory mediators in keratinocytes, has not been studied. Using human keratinocytes, we investigated the effect of apocynin on the inflammatory mediator production in relation to the Toll-like receptor-4-mediated-Akt/mTOR and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Apocynin, Akt inhibitor SH-5, Bay 11-7085 and N-acetylcysteine each attenuated the lipopolysaccharide-induced production of cytokines, PGE2, and chemokines, changes in the levels of Toll-like receptor-4, p-Akt, mTOR, and NF-κB, and production of reactive oxygen species in keratinocytes. The results show that apocynin appears to attenuate the lipopolysaccharide-stimulated production of inflammatory mediators in keratinocytes by suppressing the Toll-like receptor-4-mediated activation of the Akt, mTOR, and NF-κB pathways. The effect of apocynin appears to be attributed to its inhibitory effect on the production of reactive oxygen species. Apocynin appears to attenuate the microbial product-mediated inflammatory skin diseases.

Flavonoid myricetin inhibits TNF-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR and NF-κB pathways in human keratinocytes

Flavonoid myricetin has been shown to exhibit anti-inflammatory and anti-oxidant effects. Nevertheless, the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we examined the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in relation to the Akt, mTOR and NF-κB pathways, which regulate the transcription genes involved in immune and inflammatory responses. TNF-α stimulated production of the inflammatory mediators and reactive oxygen species in keratinocytes, and activation of the Akt, mTOR and NF-κB pathways in HaCaT cells and primary keratinocytes. Myricetin, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-κB activation), rapamycin (mTOR inhibitor) and N-acetylcysteine attenuated TNF-α-induced activation of Akt, mTOR and NF-κB. Myricetin and N-acetylcysteine attenuated the TNF-α-stimulated production of cytokines and chemokines, and production of reactive oxygen species in keratinocytes. The results show that myricetin may reduce TNF-α-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR and NF-κB pathways. The effect of myricetin appears to be associated with inhibition of the production of reactive oxygen species. Further, myricetin appears to attenuate the proinflammatory mediator-induced inflammatory skin diseases.

Antioxidant Opuntia ficus-indica Extract Activates AHR-NRF2 Signaling and Upregulates Filaggrin and Loricrin Expression in Human Keratinocytes.

quinone oxidoreductase 1 (NQO1), which inhibited the generation of reactive oxygen species in keratinocytes challenged with tumor necrosis factor α or benzo[α]pyrene. The antioxidant capacity of OFIE was canceled in NRF2 knockdown keratinocytes. OFIE exerted this NRF2-NQO1 upregulation through activation of the aryl hydrocarbon receptor (AHR). Moreover, the ligation of AHR by OFIE upregulated the expression of epidermal barrier proteins: filaggrin and loricrin. OFIE also prevented TH2 cytokine-mediated downregulation of filaggrin and loricrin expression in an AHR-dependent manner because it was canceled in AHR knockdown keratinocytes. Antioxidant OFIE is a potent activator of AHR-NRF2-NQO1 signaling and may be beneficial in treating barrier-disrupted skin disorders.

Antioxidant soybean tar Glyteer rescues T-helper-mediated downregulation of filaggrin expression via aryl hydrocarbon receptor.

Soybean tar Glyteer (Gly) has been widely used for the treatment of various inflammatory skin diseases in Japan since 1924 as an alternative to coal tar remedy. Recently, coal tar has been shown to induce barrier repair in atopic dermatitis via aryl hydrocarbon receptor (AhR). In this study, we demonstrated that Gly activated AhR by inducing its cytoplasmic to nuclear translocation in keratinocytes. The AhR ligation by Gly was biologically active, with significant and dose-dependent upregulation of CYP1A1 expression, which is a specific marker for AhR activation. Gly upregulated the expression of filaggrin in an AhR-dependent manner because its enhancing effect was completely abrogated in AhR-knockdown keratinocytes. T-helper (Th)2 cytokines inhibited the expression of filaggrin; however, Gly completely restored the Th2-mediated inhibition of filaggrin expression. Furthermore, Gly coordinately upregulated a series of epidermal differentiation complex genes, including involucrin, loricrin and hornerin. In addition, Gly exhibited potent antioxidant activity through the activation of nuclear factor-erythroid 2-related factor-2 (Nrf2) and downstream antioxidant enzymes such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), which actually inhibited the generation of reactive oxygen species in keratinocytes treated with tumor necrosis factor-α or benzo[α]pyrene. In conclusion, antioxidant Gly rescues the downregulated expression of filaggrin (and plausibly other barrier proteins) in a Th2-skewed milieu via AhR activation, which may partly explain its empirical anti-inflammatory therapeutic effects.

Rotundarpene inhibits Toll-like receptor 2 activation-induced production of inflammatory mediators in keratinocytes by suppressing the Akt and NF-κB pathways

Microbial components have been shown to be involved in the pathogenesis of inflammatory skin diseases. The extract of from the barks of Ilex rotunda Thunb has demonstrated anti-inflammatory and anti-oxidant effects. However, the effect of hemiterpene rotundarpene (4-caffeoyl-3-methyl-but-2-ene-1,4-diol) on the Toll-like receptor (TLR)-2 activation-induced production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we investigated the effect of rotundarpene on the inflammatory mediator production in relation to the TLR-2-mediated-Akt and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Rotundarpene, Akt inhibitor, Bay 11-7085 and N-acetylcysteine each attenuated the lipoteichoic acid- or peptidoglycan-induced production of cytokines and chemokines, expression of TLR-2, activation of NF-κB and Akt, and formation of reactive oxygen species in keratinocytes. Cyclosporine A attenuated the bacterial component-induced production of inflammatory mediators but did not reduce the formation of reactive oxygen species. The results show that rotundarpene may attenuate the bacterial component-stimulated production of inflammatory mediators in keratinocytes by suppressing the TLR-2-mediated activation of the Akt and NF-κB pathways. The effect of rotundarpene may be attributed to its inhibitory effect on the formation of reactive oxygen species. Rotundarpene may exert a preventive effect against the bacterial component-mediated inflammatory skin diseases.

The Protein Deacetylase SIRT3 Prevents Oxidative Stress-induced Keratinocyte Differentiation

Keratinocyte differentiation is a key process in the formation and maintenance of the protective skin barrier. Dysregulation in the balance of reactive oxygen species homeostasis may play a role in keratinocyte differentiation. We have identified the mitochondrial deacetylase SIRT3 as a key regulator of mitochondrial reactive oxygen species in keratinocytes. Our studies demonstrate that SIRT3 expression is down-regulated during keratinocyte differentiation, consistent with an increase in mitochondrial superoxide levels. Importantly, loss of SIRT3 expression in keratinocytes increased superoxide levels and promoted the expression of differentiation markers, whereas overexpression decreased superoxide levels and reduced the expression of differentiation markers. These findings identify a new role for SIRT3 in the suppression of epidermal differentiation via lowering oxidative stress.

Ligand-activated PPARδ inhibits UVB-induced senescence of human keratinocytes via PTEN-mediated inhibition of superoxide production

UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt/Rac1 signalling and on generation of ROS in keratinocytes exposed to UVB. Finally, when HR-1 hairless mice were treated with GW501516 before exposure to UVB, the number of senescent cells in the skin was significantly reduced. Thus ligand-activated PPARδ confers resistance to UVB-induced cellular senescence by up-regulating PTEN and thereby modulating PI3K/Akt/Rac1 signalling to reduce ROS generation in keratinocytes.

Different characteristics of reactive oxygen species production by human keratinocyte cell line cells in response to allergens and irritants

Keratinocytes mount immune responses through the secretion of a variety of inflammatory cytokines, soluble proteins and reactive oxygen species (ROS). However, the role of ROS in keratinocytes in response to allergens and irritants has not yet been elucidated. In this study, we investigated the (i) ROS production; (ii) potential sites of ROS production; (iii) expression of cell surface molecules; (iv) secretion of cytokines; and (v) ROS-dependent protein carbonylation in chemical-treated human keratinocyte cell line (HaCaT) cells. Treatment of HaCaT cells with 2,4-dinitrochlorobenzene (DNCB) and benzalkonium chloride (BKC) increased ROS levels in a time- and dose-dependent manner, as determined with dichlorodihydrofluorescein diacetate (CM-H(2) DCFDA), without reducing cell viability. Potential sources of ROS production were evaluated with pretreatment of diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; rotenone, an inhibitor of the mitochondrial electron transport chain complex or allopurinol, a xanthine oxidase inhibitor. The DNCB-induced ROS was related to both NADPH oxidase and mitochondrial electron transport chain complex. Conversely, BKC-induced ROS was related to NADPH oxidase only. Western blotting using an anti-DNP antibody revealed ROS-dependent protein carbonylation in response to DNCB but not BKC. Both DNCB and BKC increased the secretion of IL-1α from HaCaT cells; however, ROS production as well as other changes, except DNCB-induced secretion of IL-1α, was not inhibited by antioxidants. Although the role of ROS in keratinocytes in response to chemicals was inconclusive, our results suggest that the characteristics of ROS produced by keratinocytes in response to chemicals might differ.

Coenzyme Q10 protects against oxidative stress‐induced cell death and enhances the synthesis of basement membrane components in dermal and epidermal cells

Coenzyme Q10 (CoQ10), which has both energizing and anti-oxidative effects, is also reported to have antiaging action, e.g., reducing the area of facial wrinkles. However, the mechanism of its anti-aging activity is not fully established. Here, we examined the effect of CoQ10 on human dermal and epidermal cells. CoQ10 promoted proliferation of fibroblasts but not keratinocytes. It also accelerated production of basement membrane components, i.e., laminin 332 and type IV and VII collagens, in keratinocytes and fibroblasts, respectively; however, it had no effect on type I collagen production in fibroblasts. CoQ10 also showed protective effects against cell death induced by several reactive oxygen species in keratinocytes, but only when its cellular absorption was enhanced by pretreatment of the cells with highly CoQ10-loaded serum. These results suggest that protection of epidermis against oxidative stress and enhancement of production of epidermal basement membrane components may be involved in the antiaging properties of CoQ10 in skin.