Acute UVB Irradiation Research Articles

Macrophage migration inhibitory factor (MIF) and its homolog D-dopachrome tautomerase (D-DT) are significant promotors of UVB- but not chemically induced non-melanoma skin cancer

Non-melanoma skin cancer (NMSC) is the most common cancer in Caucasians worldwide. We investigated the pathophysiological role of MIF and its homolog D-DT in UVB- and chemically induced NMSC using Mif−/−, D-dt−/− and Mif−/−/D-dt−/− mice on a hairless SKH1 background. Knockout of both cytokines showed similar attenuating effects on inflammation after acute UVB irradiation and tumor formation during chronic UVB irradiation, without additive protective effects noted in double knockout mice, indicating that both cytokines activate a similar signaling threshold. In contrast, genetic deletion of Mif and D-dt had no major effects on chemically induced skin tumors. To get insight into the contributing mechanisms, we used an in vitro 3D skin model with incorporated macrophages. Application of recombinant MIF and D-DT led to an accumulation of macrophages within the epidermal part that could be reversed by selective inhibitors of MIF and D-DT pathways. In summary, our data indicate that MIF and D-DT contribute to the development and progression of UVB- but not chemically induced NMSC, a role at least partially accounted by effects of both cytokines on epidermal macrophage accumulation. These data highlight that MIF and D-DT are both potential therapeutic targets for the prevention of photocarcinogenesis but not chemical carcinogenesis.

528 Energy metabolism rewiring following acute UVB irradiation is largely dependent on nuclear DNA damage

Solar ultraviolet B (UVB) radiation is the main risk factor of skin carcinoma via the induction of DNA damage like cyclobutane pyrimidine dimers (CPD). Cells respond to UVB irradiation with a series of signaling cascades called DNA damage response (DDR), which are necessary for maintaining cell integrity. DDR involves sensing the damage and subsequently signaling to downstream effectors. This network includes different cell signaling pathways such as DNA repair systems or apoptosis, however, its crosstalk with energy metabolism is still unclear. To elucidate the link between DDR and energy metabolism, we took advantage of keratinocytes expressing nuclear or mitochondrial CPD photolyases. Applying comprehensive proteomic analysis and metabolomics, we found a biphasic alteration in numerous metabolic pathways (such as glycolysis, oxidative phosphorylation, fatty acid B-oxidation) after acute exposure of keratinocyte to UVB radiation, which was largely dependent on nuclear DNA damage. Using live imaging and bioinformatic analyses, we found that the structure of the mitochondrial network was dynamically modified after UVB irradiation and those modifications were affected by both mitochondrial and nuclear DNA damage. Altogether, our data suggest that energy metabolism could be considered as a sub-program of the DDR network. A better understanding of what role metabolism plays in keratinocyte responses to UVB radiation is crucial for developing adapted metabolic therapies both for prevention and therapy of skin cancers.

Beneficial Effect of Gastrodia elata Blume and Poria cocos Wolf Administration on Acute UVB Irradiation by Alleviating Inflammation through Promoting the Gut-Skin Axis.

Bioactive compounds in some herbs can, directly and indirectly, protect against photoaging. We evaluated the effects of Gastrodia elata Blume (GE) and Poria cocos Wolf (PC) water extracts on ultraviolet (UV) B-induced skin lesions by acute UVB exposure in ICR mice and explored their mechanism of action. After removing the hair on the back of the mice, UVB (280–310 nm) was exposed to the back for 30 min to induce skin damage. Four UVB exposure groups were divided into the following according to the local application (1,3-butanediol extract) on the dorsal skin and oral intake (0.3 g water extract/kg body weight/day): 1,3-butanediol and cellulose(control; UV-Con), retinoic acid (positive-control; UV-Positive), PC extracts (UV-PC), and GE extracts (UV-GE). The fifth group had no UVB exposure with the same treatment as the UV-Con (Normal-control). The erythema, burns, erosion, and wounds of the UV-PC and UV-PC groups were alleviated, and the most significant improvements occurred in the UV-PC group. PC and GE reduced the thickness of the dorsal skin tissue, the penetration of mast cells, and malondialdehyde contents. The mRNA expression of TNF-α, IL-13, and IL-4, inflammatory factors, were also reduced significantly in the dorsal skin of the UV-PC and UV-GE groups. UV-PC, UV-GE, and UV-Positive showed improvements in UV-induced intestinal tissue inflammation. UV-Con deteriorated the intestinal morphology, and PC and GE alleviated it. The α-diversity of the fecal microbiota decreased in the UV-control, and UV-PC and UV-GE prevented the decrease. Fecal metagenome analysis revealed increased propionate biosynthesis in the UV-PC group but decreased lipopolysaccharide biosynthesis in the UV-PC and UV-GE groups compared to UV-Con. In conclusion, the local application and intake of PC and GE had significant therapeutic effects on acute UV-induced skin damage by reducing oxidative stress and proinflammatory cytokines, potentially promoting the gut-microbiota-gut-skin axis.

Thioredoxin Reductase 1 Modulates Pigmentation and Photobiology of Murine Melanocytes in vivo

Pigment-producing melanocytes overcome frequent oxidative stress in their physiological role of protecting the skin against the deleterious effects of solar UV irradiation. This is accomplished by the activity of several endogenous antioxidant systems, including the thioredoxin antioxidant system, in which thioredoxin reductase 1 (TR1) plays an important part. To determine whether TR1 contributes to the redox regulation of melanocyte homeostasis, we have generated a selective melanocytic Txnrd1-knockout mouse model (Txnrd1mel‒/‒), which exhibits a depigmentation phenotype consisting of variable amelanotic ventral spotting and reduced pigmentation on the extremities (tail tip, ears, and paws). The antioxidant role of TR1 was further probed in the presence of acute neonatal UVB irradiation, which stimulates melanocyte activation and introduces a spike in oxidative stress in the skin microenvironment. Interestingly, we observed a significant reduction in overall melanocyte count and proliferation in the absence of TR1. Furthermore, melanocytes exhibited an elevated level of UV-induced DNA damage in the form of 8-oxo-2'-deoxyguanosine after acute UVB treatment. We also saw an engagement of compensatory antioxidant mechanisms through increased nuclear localization of transcription factor NRF2. Altogether, these data indicate that melanocytic TR1 positively regulates melanocyte homeostasis and pigmentation during development and protects against UVB-induced DNA damage and oxidative stress.

Keratinocytes Counteract UVB-Induced Immunosuppression in Mice through HIF-1a Signaling

The transcription factor HIF-1a regulates cellular metabolism under hypoxia but also immune responses and UVB-induced skin reactions. In keratinocytes (KCs), HIF-1a is an environmental sensor orchestrating the adaptation to environmental changes. In this study, we investigated the role of HIF-1a in KCs for skin reactions to acute and chronic UVB exposure in mice. The function of HIF-1a in KCs under UVB exposure was analyzed in KC-specific HIF-1a conditional knockout (cKO) mice. cKO mice were hypersensitive to acute high-dose UVB irradiation compared with wild-type mice, displaying increased cell death and delayed barrier repair. After chronic low-dose UVB treatment, cKO mice also had stronger epidermal damage but reduced infiltration of dermal macrophages and T helper cells compared with wild-type mice. Irradiated cKO mice revealed accumulation of regulatory lymphocytes in dorsal skin-draining lymph nodes compared with wild-type and unirradiated mice. This was reflected by an augmented IL-10 release of lymph node cells and a weaker contact hypersensitivity reaction to DNFB in UVB-exposed cKO mice than in wild-type and unirradiated controls. In summary, we found that KC-specific HIF-1a expression is crucial for adaptation to UVB exposure and inhibits the development of UVB-induced immunosuppression in mice. Therefore, HIF-1a signaling in KCs could ameliorate photoaging-related skin disorders.

Topical Application of Peptide-Chondroitin Sulfate Nanoparticles Allows Efficient Photoprotection in Skin.

In this article, we describe a method of delivery of chondroitin sulfate to skin as nanoparticles and demonstrate its anti-inflammatory and antioxidant role using UV irradiation as a model condition. These nanoparticles, formed through electrostatic interactions of chondroitin sulfate with a skin-penetrating peptide, were found to be homogenous with positive surface charges and stable at physiological and acidic pH under certain conditions. They were able to enter into the human keratinocyte cell line (HaCaT), artificial skin membrane (mimicking the human skin), and mouse skin tissue unlike free chondroitin sulfate. The preapplication of nanoparticles also exhibited reduced levels of oxidative stress, cyclobutane pyrimidine dimer formation, TNF-α, and so on in UV-B-irradiated HaCaT cells. In an acute UV-B irradiation mouse model, their topical application resulted in reduced epidermal thickness and sunburn cells, unlike in the case of free chondroitin sulfate. Thus, a completely noninvasive method was used to deliver a bio-macromolecule into the skin without using injections or abrasive procedures.

Physiological responses of Lepidium meyenii plants to ultraviolet-B radiation challenge

BackgroundUltraviolet-B (UV-B) radiation can affect several aspects ranging from plant growth to metabolic regulation. Maca is a Brassicaceae crop native to the Andes growing in above 3500 m of altitude. Although maca has been the focus mainly due to its nutraceutical properties, it remains unknown how maca plants tolerate to harsh environments, such as strong UV-B. Here, we present the first study that reports the physiological responses of maca plants to counteract and recover to repeated acute UV-B irradiation.ResultsIn detail, plants were daily exposed to acute UV-B irradiation followed by a recovery period under controlled conditions. The results showed that repeated acute UV-B exposures reduced biomass and photosynthetic parameters, with gradual senescence induction in exposed leaves, reduction of young leaves expansion and root growth inhibition. Negative correlation between increased UV-B and recovery was observed, with marked production of new biomass in plants treated one week or more.ConclusionsA differential UV-B response was observed: stress response was mainly controlled by a coordinated source-sink carbon allocation, while acclimation process may require UV-B-specific systemic defense response reflected on the phenotypic plasticity of maca plants. Moreover, these differential UV-B responses were also suggested by multifactorial analysis based on biometric and physiological data.

Linalool prevents oxidative stress activated protein kinases in single UVB-exposed human skin cells.

Ultraviolet-B radiation (285–320 nm) elicits a number of cellular signaling elements. We investigated the preventive effect of linalool, a natural monoterpene, against UVB-induced oxidative imbalance, activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling in HDFa cells. We observed that linalool treatment (30 μM) prevented acute UVB-irradiation (20 mJ/cm2) mediated loss of activities of antioxidant enzymes in HDFa cells. The comet assay results illustrate that linalool significantly prevents UVB-mediated 8-deoxy guanosine formation (oxidative DNA damage) rather than UVB-induced cyclobutane pyrimidine (CPD) formation. This might be due to its ability to prevent UVB-induced ROS formation and to restore the oxidative imbalance of cells. This has been reflected in UVB-induced overexpression of MAPK and NF-κB signaling. We observed that linalool inhibited UVB-induced phosphorylation of ERK1, JNK and p38 proteins of MAPK family. Linalool inhibited UVB-induced activation of NF-κB/p65 by activating IκBa. We further observed that UVB-induced expression of TNF-α, IL6, IL-10, MMP-2 and MMP-9 was modulated by linalool treatment in HDFa cells. Thus, linalool protects the human skin cells from the oxidative damages of UVB radiation and modulates MAPK and NF-κB signaling in HDFa cells. The present findings substantiate that linalool may act as a photoprotective agent against UVB-induced skin damages.

Exopolysaccharides Isolated from Milk Fermented with Lactic Acid Bacteria Prevent Ultraviolet-Induced Skin Damage in Hairless Mice.

Background: We studied the mechanism by which fermented milk ameliorates UV-B-induced skin damage and determined the active components in milk fermented with lactic acid bacteria by evaluating erythema formation, dryness, epidermal proliferation, DNA damage and cytokine mRNA levels in hairless mice exposed to acute UV-B irradiation. Methods: Nine week-old hairless mice were given fermented milk (1.3 g/kg BW/day) or exopolysaccharide (EPS) concentrate (70 mg/kg BW/day) orally for ten days. Seven days after fermented milk or EPS administration began, the dorsal skin of the mice was exposed to a single dose of UV-B (20 mJ/cm2). Results: Ingestion of either fermented milk or EPS significantly attenuated UV-B-induced erythema formation, dryness and epidermal proliferation in mouse skin. Both fermented milk and EPS were associated with a significant decrease in cyclobutane pyrimidine dimers and upregulated mRNA levels of xeroderma pigmentosum complementation group A (XPA), which is involved in DNA repair. Furthermore, administration of either fermented milk or EPS significantly suppressed increases in the ratio of interleukin (IL)-10/IL-12a and IL-10/interferon-gamma mRNA levels. Conclusion: Together, these results indicate that EPS isolated from milk fermented with lactic acid bacteria enhanced DNA repair mechanisms and modulated skin immunity to protect skin against UV damage.

Abstract 806: Modeling human UV-induced skin cancer through genetic manipulation of Src-kinases and Srcasm

Abstract UV-induced skin cancer is the most common form of cancer and its incidence continues to increase. Novel in vivo models that mimic human UV-induced skin cancer are needed to further studies and evaluate new therapies. Keratinocyte growth is regulated through oncogenic signals from EGFR and Src-family tyrosine kinases (SFKs); these tyrosine kinases are primary drivers of keratinocyte growth in UV-induced skin cancer. At the proteomic level, activated EGFR and SFKs are negatively regulated by Srcasm which targets activated tyrosine kinases for lysosomal degradation. Studies have shown that SFK activity is increased and Srcasm levels are decreased in human cutaneous squamous cell carcinomas and related precancerous lesions. We hypothesized that genetic downregulation of Srcasm by gene deletion or lentivirally mediated shRNA knockdown would promote oncogenic signaling and keratinocyte growth thereby promoting UV-induced skin cancer. To test this hypothesis Srcasm null mice and Srcasm lentiviral knockdown in human keratinocytes were generated and studied. Western blotting of epidermal lysates from Srcasm null mice demonstrates that loss of Srcasm promotes activation of EGFR and Src kinases and decreases levels of p53, NICD and p21. Activation of the canonical Erk1/2, STAT-3 and PDK-1 oncogenic pathways also was seen in Srcasm null keratinocytes. The epidermis of Srcasm null mice exhibits a higher Ki-67 index of 8.7 +/- 2.0 compared to 1.5 +/- 1.1 in controls (p<0.001). UVB irradiation (500 mJ/cm2 × 15 doses) produced precancerous lesions in Srcasm null mice resembling human actinic keratoses but not in controls; the precancerous lesions show an increased Ki-67 index and activation of Src kinases, STAT-3 and PDK-1. Acute UVB irradiation (1200 mJ/cm2 × 1 dose) of Srcasm null mice induced prominent erythema, epidermal necrosis and a prominent increase in the TUNEL-positive index (14.0 vs 1.3). Srcasm knockdown in human keratinocytes accelerated keratinocyte growth 130%; it also increased levels of EGFR and accelerated the kinetics of EGFR and MAPK activation in response to EGF stimulation. In human keratinocytes, Srcasm knockdown accelerated cell growth post-UV exposure which could promote neoplasia. These data show that loss of Srcasm is associated with increased oncogenic signaling, impaired p53 function and keratinocyte proliferation. Loss of Srcasm promotes keratinocyte proliferation post-UV exposure leading to the early stages of UVB-induced cutaneous neoplasia. Citation Format: Xiaoping Yang, Sheila Shankar, Hiroshi Maeno, Micheal Gober, Christine Marshall, Tzvete Dentchev, John Seykora. Modeling human UV-induced skin cancer through genetic manipulation of Src-kinases and Srcasm. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2015-806

Hesperidin methyl chalcone inhibits oxidative stress and inflammation in a mouse model of ultraviolet B irradiation-induced skin damage

Hesperidin methyl chalcone (HMC) is a safe flavonoid used to treat chronic venous diseases, but its effects and mechanisms on UVB irradiation-induced inflammation and oxidative stress have never been described in vivo. Thus, the purpose of this study was to evaluate the effects of systemic administration of HMC in skin oxidative stress and inflammation induced by UVB irradiation. To induce skin damage, hairless mice were exposed to an acute UVB irradiation dose of 4.14J/cm2, and the dorsal skin samples were collected to evaluate oxidative stress and inflammatory response. The intraperitoneal treatment with HMC at the dose of 300mg/kg inhibited UVB irradiation-induced skin edema, neutrophil recruitment, and matrix metalloproteinase-9 activity. HMC also protected the skin from UVB irradiation-induced oxidative stress by maintaining ferric reducing antioxidant power (FRAP), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS) scavenging ability and antioxidant levels (reduced glutathione and catalase). Corroborating, HMC inhibited UVB irradiation-induced superoxide anion generation and gp91phox (NADPH oxidase subunit) mRNA expression. Furthermore, the antioxidant effect of HMC resulted in lower production of inflammatory mediators, including lipid hydroperoxides and a wide range of cytokines. Taken together, these results unveil a novel applicability of HMC in the treatment of UVB irradiation-induced skin inflammation and oxidative stress.

Enhanced biosynthesis of quercetin occurs as A photoprotective measure in Lycopersicon esculentum mill. under Acute UV-B exposure

Lycopersicon esculentum respond to UV-B by enhanced synthesis of flavonoid quercetin, a strong antioxidant that helps the plants to well acclimatize to UV-B stress. Three weeks old plants of L. esculentum were subjected to acute UV-B irradiation for 20, 40 and 60 minutes daily until 28 days and analyzed for the morphological and biochemical changes. UV-B exposure for 40 and 60 minutes considerably affected the growth and biomass of L. esculentum. The leaves were deformed, developed chlorosis and abscised early as compared to the unexposed plants. Biomass declined by 35% and total chlorophyll decreased by 24.7% due to disintegration of chloroplasts. Enhancement was seen in the content of carotenoids, anthocyanins and total flavonoids by 15, 33.3 and 22.8%, respectively, which was attributed to the photoprotective role of these compounds as potential quenchers of excess excitation energy. Quercetin content decreased on UV-B exposure to 20 and 40 min, and thereafter increased significantly by 5.19% on 60 min of exposure. This pattern probably indicated that the over-expression of genes involved in its biosynthesis such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) occurred only after certain threshold exposure (60 min), which could be the strategy for developing tolerance against UV-B stress in L. esculentum.

Effects of various flavonoids isolated from Scutellaria baicalensis roots on skin damage in acute UVB-irradiated hairless mice

Solar ultraviolet (UV) radiation causes skin damage including increasing skin thickness, edema and flush. Scutellaria baicalensis roots have been traditionally used as a remedy for allergic inflammatory diseases in China and Japan. In this study, we examined the effects of four flavonoids isolated from these roots, namely 2',5, 5',7-tetrahydroxy-6',8-dimethoxyflavone (1), skullcapflavone II (2), 2(S)-2',5,6',7-tetrahydroxyflavanone (3) and 2(R), 3(R)-2',3,5,6',7-pentahydroxyflavanone (4), on acute UVB irradiation-induced skin damage in hairless mice. The four flavonoids were orally administered twice daily, at doses of 10 and 50 mg/kg, for 14 consecutive days. The UVB irradiation was performed at a dose of 200 mJ cm(-2) on days 7 and 8 after beginning oral administration of the four flavonoids. Compounds 1 and 4 prevented increases in skin thickness, levels of matrix metalloproteinases (MMP)-2 and MMP-9, and vascular endothelial growth factor (VEGF) induced by UVB irradiation. The other two flavonoids 2 and 3 had no effect. Compounds 1 and 4 isolated from Scutellaria baicalensis roots may be useful for preventing skin inflammation induced by acute UVB irradiation.

7,3′,4′-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.

Recent advances in sunlight-induced carcinogenesis using the Xiphophorus melanoma model

Unlike breast and prostate cancers, the nature and sequence of critical genetic and epigenetic events involved in the initiation and progression of melanoma are not well understood. A contributing factor to this dilemma, especially given our current understanding of the importance of UV light in melanoma etiology, is the lack of quality UV-inducible melanoma animal models. In this study we elaborate on the capability of UV light to induce cutaneous malignant melanomas (CMM) in Xiphophorus fishes, which were previously found to develop melanomas after acute neonatal UVB irradiation. In two separate tumorigenesis experiments, we exposed adult Xiphophorus hybrids to either acute UVB irradiations (5 consecutive daily treatments) or chronic solar irradiations (continuous UVA/UVB treatment for 9 months). Acute adult UVB irradiation resulted in the significant induction of melanomas, and moreover, this induction rate is equivalent to that of animals exposed to acute neonatal UVB irradiation. This study represents the first evidence that acute adult UVB irradiation, in the absence of any early life exposures, induces CMM. Similar to the findings conducted on other divergent melanoma models, including HGF/SF transgenic mice and Monodelphis domestica, prolonged chronic solar UV was not a factor in melanomagenesis.

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

The phosphorylated form of histone H2AX, gammaH2AX, is a component of the DNA repair system. Most studies have focused on the role of gammaH2AX 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 gammaH2AX 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 gammaH2AX constitutes a highly sensitive marker relevant for studying subapoptotic doses as well as proapoptotic doses of UVB in human skin.

UVB induces a biphasic response of HIF‐1α in cultured human keratinocytes

Hypoxia in the skin is important in chronic degenerative dermo-epidermal changes, inflammation, photoageing and carcinogenesis. In these processes, vascular endothelial growth factor (VEGF) plays a crucial role and is known to be affected by ultraviolet radiation (UVR). Hypoxia-inducible factor-1 (HIF-1) closely regulates the expression of VEGF in several experimental settings. We set out to study the impact of acute UVB irradiation on the level of HIF-1 as a major regulator of hypoxia-induced genes. Effects of UVB exposure on HIF-1alpha expression were investigated in HaCaT cells after a single irradiation by Western blots. Downstream target gene expression was measured by quantitative real-time polymerace chair reaction (PCR). UVB treatment resulted in an initial decrease of the HIF-1alpha protein level followed by a subsequent prolonged increase. If cells were exposed to additional UVB irradiation, another decrease in HIF-1alpha was provoked, similar to the original effect. The observed changes followed a strict timeline and were dose-dependent. The role of the PI3K/AKT pathway was examined. No change in the total level of AKT after UVB treatment was seen; however, its phosphorylation level was found to be markedly higher. In accordance with these observations, wortmannin, an inhibitor of PI3-kinase effectively blocked the UVB-induced increase in HIF-1alpha. In agreement with previous findings, UVB irradiation increased VEGF and haem oxygenase-1 mRNA levels determined by quantitative real-time PCR. It is concluded that changes in HIF-1alpha expression underlie the alterations in expression of VEGF upon UVB irradiation. Our findings indicate the involvement of PI3K in UVB-mediated HIF-1alpha upregulation.

Cross‐validation of Murine UV Signal Transduction Pathways in Human Skin†

Acute UVB irradiation of mouse skin results in activation of phospatidyinositol-3 (PI-3) kinase and mitogen-activated protein kinase (MAPK) pathways leading to altered protein phosphorylation and downstream transcription of genes. We determined whether activation of these pathways also occurs in human skin exposed to 4x minimal erythemic dose of UVB in 23 volunteers. Biopsies were taken prior to, at 30 min, 1 and 24 h post-UVB. In agreement with mouse studies, the earliest UV-induced changes in epidermis were seen in phospho-CREB (two- and five-fold at 30 min and 1 h) and in phospho-MAPKAPK-2 (three-fold at both 30 min and 1 h). At 1 h, phospho-c-JUN and phospho-p38 were increased five- and two-fold, respectively. Moreover, phospho-c-JUN and phospho-p38 were further increased at 24 h (12- and six-fold, respectively). Phospho-GSK-3beta was similarly increased at all time points. Increases in phospho-p53 (12-fold), COX-2 (four-fold), c-FOS (14-fold) and apoptosis were not seen until 24 h. Our data suggest that UVB acts through MAPK p38 and PI-3 kinase with phosphorylation of MAPKAPK-2, CREB, c-JUN, p38, GSK-3beta and p53 leading to marked increases in c-FOS, COX-2 and apoptosis. Validation of murine models in human skin will aid in development of effective skin cancer chemoprevention and prevention strategies.

The characteristics of aromatase deficient hairless mice indicate important roles of extragonadal estrogen in the skin

The roles of extragonadal estrogen in the skin are poorly understood, due to the lack of proper animal models. We examined the skin phenotypes of aromatase-knockout hairless (ArKO) mice and wild-type hairless (WT) mice, both of which were obtained through crossbreeding of Ar± mice and hairless mice. Differences in the skins of ArKO and WT mice were compared with those of ovariectomized (OVX) and control (Sham) mice. A difference was observed in the skin tone of ArKO mice, which is pale white and differs from the pinkish tone of all other mice. However, both ArKO and OVX mice similarly exhibited deteriorations of skin properties as compared to their respective controls. Furthermore, all the deteriorations were similarly amplified by chronic UVB irradiation in both ArKO and OVX mice as compared to their respective controls. The unique skin phenotype of ArKO mice was observed in sunburn reactions. Specifically, skins of ArKO mice showed no reaction after an acute UVB irradiation at dose intensities caused sunburn in others. However, follow-up observation found delayed reactions associated with brownish skin color and swelling only in ArKO mice, thereby suggesting that the role of extragonadal estrogen may be connected with the protective reactions of skin.

Dietary grape seed proanthocyanidins inhibit UVB-induced oxidative stress and activation of mitogen-activated protein kinases and nuclear factor-κB signaling in in vivo SKH-1 hairless mice

We have shown previously that dietary grape seed proanthocyanidins (GSP) inhibit UVB-induced photocarcinogenesis in mice. As UVB-induced oxidative stress and oxidative stress-mediated signaling has been implicated in photocarcinogenesis, this study was designed to investigate the effect of dietary GSPs on UVB-induced oxidative stress in in vivo SKH-1 hairless mice. Here, we report that provision of dietary GSPs (0.2 and 0.5%, w/w) to mice exposed to either acute UVB irradiation (120 mJ/cm(2)) or chronic irradiation of UVB inhibited depletion of glutathione peroxidase, catalase, and glutathione, and inhibited UVB-induced H(2)O(2), lipid peroxidation, protein oxidation, and nitric oxide in mouse skin. As UV-induced oxidative stress mediates activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways, we determined the effect of dietary GSPs on these pathways. We observed that dietary GSPs inhibited UVB-induced phosphorylation of extracellular signal-regulated kinase 1/2, c-Jun-NH(2)-kinase, and p38 proteins of MAPK family, which seems to be mediated through reactivation of MAPK phosphatases. GSPs inhibited UVB-induced activation of NF-kappaB/p65 through inhibition of degradation of IkappaBalpha and activation of IkappaB kinase alpha (IKKalpha). As NF-kappaB-targeted genes play critical roles in inflammation and cellular proliferation, we assessed the effect of GSPs on proteins encoded by these genes. Dietary GSPs resulted in inhibition of the expression of proliferating cell nuclear antigen, cyclin D1, inducible nitric oxide synthase, and cyclooxygenase-2 in the skin. Collectively, our data show that GSPs have the ability to protect the skin from the adverse effects of UVB radiation via modulation of the MAPK and NF-kappaB signaling pathways and provide a molecular basis for the photoprotective effects of GSPs in an in vivo animal model.