γH2AX,an Accurate Marker That Analyzes UV Genotoxic Effects on Human Keratinocytes and on Human Skin

The phosphorylated form of histone H2AX, γH2AX, is a component of the DNA repair system. Most studies have focused on the role of γH2AX during cell transformation and human cancer, but little is known about its role in keratinocytes and the skin during UV irradiation. We analyzed the response to UV irradiation focusing on the phosphorylation of histone H2AX both in vitro, in keratinocyte cultures and in artificial epidermis, and then in vivo, in human skin. Acute UVB irradiation of human keratinocytes increased the phosphorylation of H2AX in a dose-dependent manner; two types of γH2AX response were observed either in vitro or in vivo. After a low nonapoptotic UVB irradiation, cells contained phosphorylated H2AX and arrested their cell cycle to repair the DNA damages. For a stronger and proapoptotic UVB irradiation, keratinocytes dramatically increased the phosphorylation of H2AX and committed apoptosis. Our results indicate that γH2AX constitutes a highly sensitive marker relevant for studying subapoptotic doses as well as proapoptotic doses of UVB in human skin.     

Hairless and NFκB Form a Positive Feedback Loop After UVB and TNFα Stimulation(†)

Hairless (HR) is a nuclear protein with corepressor activity whose exact function in the skin remains to be determined. Mutations in both human and mouse Hairless lead to hair loss accompanied by the appearance of papules, a disorder called atrichia with papular lesions. Furthermore, mice with mutations in HR are known to have a higher susceptibility to ultraviolet radiation-induced tumorigenesis, suggesting that HR plays a crucial role in the epidermal UVB response. Using normal human keratinocytes (NHKs) and keratinocytes containing a mutation in HR, we found that HR is an early UVB response gene that negatively regulates NFκB mRNA expression. HR mutant keratinocytes have a dysregulated UVB response that includes increased proliferation and the aberrant activation of NFκB effector genes. Additionally, we show that another UVB response gene, TNFα, negatively regulates HR mRNA expression. TNFα-induced negative regulation of HR occurs through a direct interaction of the p65 subunit with a single NFκB-binding domain located in the HR promoter region. Therefore, we show for the first time that HR and NFκB participate in a positive feedback loop that can be initiated either by UVB or TNFα.     

胶内差异双向电泳-电喷雾串联质谱筛选UVB损伤皮肤细胞潜在的蛋白质靶标

联用胶内差异双向电泳(2D-DIGE)和高效液相色谱-电喷雾串联质谱(HPLC-nESI MS/MS)鉴定人角质形成细胞HaCaT应答中波紫外线(UVB)损伤的差异表达蛋白,筛选UVB影响皮肤细胞正常生理功能潜在的靶标蛋白.结果表明:UVB辐射明显影响HaCaT细胞的蛋白质表达谱,DeCyder软件在每块DIGE凝胶上...     

Cis-UROCANIC ACID SYNERGIZES WITH HISTAMINE FOR INCREASED PGE2 PRODUCTION BY HUMAN KERATINOCYTES: LINK TO INDOMETHACIN-INHIBITABLE UVB-INDUCED IMMUNOSUPPRESSION

Abstract— There is considerable evidence that suppression of the immune system by UVB (280–320 nm UV) irradiation is initiated by UVB-dependent isomerization of a specific skin photoreceptor, urocanic acid (UCA), from the trans to the cis form. Previous studies have confirmed that cis -UCA administration to mice 3–5 days prior to hapten sensitization at a distant site, suppresses the contact hypersensitivity (CHS) response upon challenge. This study demonstrates in mice that cis -UCA, like UVB, suppresses CHS to trinitrochlorobenzene by a mechanism partly dependent on prostanoid production. In vitro experimentation showed that human keratinocytes, isolated from neonatal foreskin, increased prostaglandin E₂ (PGE₂) production in response to histamine but not UCA alone. However, cis -UCA synergized with histamine for increased PGE₂ production by keratinocytes. cis -urocanic acid also increased the sensitivity of keratinocytes for PGE₂ production in response to histamine. Prostaglandin E₂ from keratinocytes exposed to cis -UCA and histamine may contribute directly, or indirectly, to the regulation of CHS responses by UVB irradiation.     

Effects of a Single Exposure to UVB Radiation on the Activities and Protein Levels of Copper-Zinc and Manganese Superoxide Dismutase in Cultured Human Keratinocytes

Abstract— Ultraviolet B irradiation has been believed to decrease or impair the activity of reactive oxygen species (ROS) scavenging enzymes such as superoxide dismutase (SOD) in the skin. It has been recently reported that two isozymes of SOD, namely copper-zinc SOD (Cu-Zn SOD) and manganese SOD (Mn SOD), exist in mammalian cells and that the two enzymes play different roles in living systems. The aim of this study was to investigate changes in SOD activities and protein levels in cultured human keratinocytes after acute UVB irradiation. In addition, the protein levels of Cu-Zn SOD and Mn SOD were quantified separately. A single exposure to UVB irradiation produced an increase in SOD activity and protein level that peaked immediately after UVB irradiation, after which a decline was observed, with subsequent recovery to baseline levels 24 h after irradiation. In individual assays of Mn SOD and Cu-Zn SOD, the amount of Mn SOD protein decreased and then gradually recovered 24 h after irradiation. In contrast, the amount of Cu-Zn SOD protein increased immediately after UVB irradiation, and then gradually declined. To evaluate the mechanisms of these changes, we examined the effects of the cytokines, interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), which can be secreted from keratinocytes after UVB irradiation, on the SOD activity and protein levels in keratinocytes. Interleukin-la and TNF-α enhanced both the SOD activity and protein level of Mn SOD, while these cytokines had no effect on Cu-Zn SOD protein levels in cultured human keratinocytes after incubation for 24 h. Furthermore, when neutralizing antibodies against IL-1α and TNF-α were added separately or together to the culture medium before UVB irradiation, the recovery of total SOD activity and Mn SOD protein level were markedly inhibited 24 h after irradiation. Our results suggest that significant increases in SOD activity and protein level occur as a cutaneous antioxidant defense mechanism that protects against the cytotoxicity as a result of UVB irradiation, and that this increase in SOD is attributed to Cu-Zn SOD. The Cu-Zn SOD and Mn SOD protein levels changed in a different manner after UVB irradiation. The former may participate in an early phase and the latter in a late phase defense mechanism directed against oxidant cytotoxicity through UVB irradiation. In addition, the recovery of Mn SOD to baseline levels 24 h after UVB irradiation seems to be mediated through cytokines such as IL-1α and TNF-α, which are secreted from keratinocytes.     

Effect of N-acetylcysteine on UVB-induced Apoptosis and DNA Repair in Human and Mouse Keratinocytes

The incidence of skin cancer is increasing rapidly, particularly in the Caucasian population. Epidemiological and experimental studies demonstrated that ultraviolet radiation (UVR) is the primary cause for the increasing incidence of skin cancer. It is well known that UV irradiation induces DNA damage. If the damage is not repaired or removed in time, it can lead to mutations and skin carcinogenesis. N-acetylcysteine (NAC) has been shown to be an effective protector against UVB-induced immunosuppression and to modulate the expression of some oncogenes and tumor suppressor genes. To test further the protective effect of NAC against UVR, we used both in vitro and in vivo models to investigate the effect of NAC on UVB-induced apoptosis and repair of DNA damage in human and mouse keratinocytes. Our data indicate that the intracellular glutathione level was increased after treatment with NAC at 10-20 mM but decreased with 40 mM NAC treatment due to the toxicity. At concentrations up to 20 mM NAC did not have a significant effect on UVB-induced apoptosis of cultured human keratinocytes. In addition, in an in vivo mouse model, topical application of NAC (3 mumol cm-2) that has been shown to inhibit UVB-induced immunosuppression did not have any effect on UVB-induced apoptosis and did not reduce the formation or enhance the repair of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. Our results indicate that NAC is ineffective in preserving the genomic stability of keratinocytes against UVB irradiation.     

Narrow‐band UVB‐induced Externalization of Selected Nuclear Antigens in Keratinocytes: Implications for Lupus Erythematosus Pathogenesis†

The aim of this study was to analyze whether sera obtained from patients with lupus erythematosus (LE) react with membrane structures found on keratinocytes irradiated with narrow‐band ultraviolet B (NB‐UVB). We applied atomic force microscopy (AFM) to visualize cell surface structures expressing nuclear antigens upon apoptosis following NB‐UVB irradiation. Immortalized human keratinocytes (HaCaT) were cultured under standard conditions, irradiated with 800 mJ cm^?2 NB‐UVB light and imaged by AFM mounted on an inverted optical microscope. It was observed that NB‐UVB irradiation provoked significant alterations of the keratinocyte morphology and led to the membrane expression of antigens recognized by anti‐La and anti‐Ro 60 kDa sera but not by antidouble‐strand DNA sera. The presence of La and Ro 60 kDa antigens on keratinocyte surfaces after NB‐UVB irradiation was limited mainly to the small bleb‐like protrusions found on the keratinocytes by AFM. A closer investigation by AFM also revealed that some structures positively stained with anti‐Ro 60 kDa serum were also located submembranously. We hypothesize that the externalization of some nuclear antigens because of NB‐UVB exposure might be responsible for exacerbation of skin symptoms in patients suffering from LE.     

Photoprotective Effect of Black Tea Extracts Against UVB-induced Phototoxicity in Skin

In previous studies, we showed that green tea and black tea extracts and their major polyphenolic constituents protect against UVB light-induced carcinogenesis in murine skin. All of these studies required chronic administration of tea extracts or specific constituents either topically or orally. However, it is not known whether acute or subchronic administration of black tea extracts or constituents can ameliorate UVB-induced early effects in skin. In the present study, cultured keratinocytes and mouse and human skin were employed to assess the effect of both oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic subfractions namely BTF1 and BTF2 against UVB-induced photodamage. In SKH-1 hairless mice, topical application of SBTE (0.2 mg/cm2) prior to UVB exposure (180 mJ/cm2) resulted in 40% reduced incidence and 64% reduced severity of erythema and 50% reduction in skinfold thickness by day 6 when compared to nontreated UVB-exposed animals. The SBTE was also effective in protecting against UVB-induced erythema in human volunteers. Administration of SBTE 5 min after UVB irradiation was similarly effective in reducing UVB-induced inflammation in both murine and human skin. The major polyphenolic subfractions, BTF1 and BTF2, were also effective in protecting in mouse skin. The SBTE subfractions inhibited UVB-induced tyrosine phosphorylation of epidermal growth factor receptor (EGFR). The UVB irradiation of human epidermoid carcinoma cells resulted in 3.3-fold induction of tyrosine phosphorylation of EGFR. Pretreatment with BTF1 and BTF2 reduced tyrosine phosphorylation of EGFR by 53% and 31%, respectively. The UVB-mediated enhanced expression of the early response genes, c-fos and c-jun in human epidermal keratinocytes was reduced in a dose-dependent manner by SBTE. Topical application of SBTE was also effective in reducing accumulation of c-fos and p53 proteins by 82% and 78%, respectively, in UVB-exposed mouse skin. These data provide evidence that constituents of black tea can abrogate UVB-induced erythema and associated early events in murine and human skin.     

Cell Cycle Kinetics Following UVA Irradiation in Comparison to UVB and UVC Irradiation

Abstract— There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G₁, S and G₂/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.     

UVA and UVB decrease the expression of CD44 and hyaluronate in mouse epidermis, which is counteracted by topical retinoids

The transmembrane glycoprotein CD44 is currently thought to be the main cell surface receptor for the glycosaminoglycan hyaluronate. We previously showed that (1) CD44 regulate keratinocyte proliferation; (2) topical retinoids dramatically increase the expression of CD44, hyaluronate and hyaluronate synthase (HAS)s in mouse epidermis; (3) topical retinaldehyde restores the epidermal thickness and CD44 expression which are correlated with clinical improvement in lichen sclerosus et atrophicus lesions; and (4) retinaldehyde-induced proliferative response of keratinocytes is a CD44-dependent phenomenon and requires the presence of HB-EGF, erbB1 and matrix metalloproteinases. In this study, we analyzed the effect of UV irradiation on the levels of epidermal hyaluronate and CD44 in mice, as well as its potential prevention by topical retinoids. UVA (10 J/cm(2)) or UVB (1 J/cm(2)) irradiation significantly decreased the expression of CD44 and hyaluronate in the epidermis of hairless mice after 2 h. Expression of both epidermal CD44 and hyaluronate was reconstituted within 24 h. Topical application of retinaldehyde for 3 days prior to UVA or UVB irradiation prevented the decrease of CD44 and hyaluronate expression. Topical retinol and retinoic acid also increased the basal levels of epidermal CD44 and hyaluronate, although their preventive effect on UV-induced decrease of these molecules was less pronounced as compared to topical retinaldehyde. These data confirm the relationships between retinoid and CD44 pathways, although the primary target(s) of UV leading to CD44 and hyaluronate degradation remain to be elucidated.     

UVA and UVB Radiation Differentially Regulate Vascular Endothelial Growth Factor Expression in Keratinocyte-derived Cell Lines and in Human Keratinocytes

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC-derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2 and 20 J/cm2) and UVB (8 mJ/cm2 and 16 mJ/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC-derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2 led to a marked decrease in soluble VEGF, whereas doses as high as 32 mJ/cm2 UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC-derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.     

In vitro and in vivo transfer of bcl-2 gene into keratinocytes suppresses UVB-induced apoptosis.

Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2-expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 x 10(6)) were transfected at 1 x 10(8) plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 x 10(9) PFU in 200 microL. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.