In Vitro Investigations on the Effect of Dermal Fibroblasts on Keratinocyte Responses to Ultraviolet B Radiation

Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280–320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB‐induced damage. To investigate these processes, established two and three‐dimensional culture models were utilized to study the impact of fibroblast–keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase‐3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast‐produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation‐induced damage.     

Prevention of UV radiation-induced premature skin aging in hairless mice by the novel compound Melanocin A

Repetitive exposure of the skin to UV radiation induces various harmful changes, such as thickening, wrinkle formation, inflammation and carcinogenesis. A variety of natural compounds and synthetic compounds have been studied to determine whether they can prevent UV-induced harmful effects. In this study, we investigated the effect of a novel compound, Melanocin A, which was isolated from Eupenicillium shearii F80695, on UV-induced premature skin aging. First, we studied the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT, in vitro. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated the effect of Melanocin A on UV-induced skin changes in hairless mice in vivo. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. Taken together, these results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging.     

Silibinin is a potent sensitizer of UVA radiation-induced oxidative stress and apoptosis in human keratinocyte HaCaT cells

UVA radiation (315-400 nm), which constitutes ca 95% of the UV irradiation in natural sunlight reaching earth surface, is a major environmental risk factor associated with human skin cancer pathogenesis. UVA is an oxidizing agent that causes significant damage to cellular components through the release of reactive oxygen species (ROS) and leads to photoaging and photocarcinogenesis. Here we investigate the effect of silibinin, the flavonolignan from Silybum marianum, on UVA-induced ROS and cell death in human keratinocyte cell line HaCaT. In addition, the effect of silibinin on UVA-induced intracellular ROS-mediated endoplasmic reticulum (ER) stress was also analyzed. UVA irradiation resulted in ROS production and apoptosis in HaCaT cells in a dose-dependent manner, and the ROS levels and apoptotic index were found to be elevated significantly when the cells were treated with 75 μmsilibinin for 2 h before UVA exposure. When the cells were pretreated with 10 mmN-acetyl cysteine, the enhancement of UVA-induced apoptosis by silibinin was compromised. Furthermore, we found that silibinin enhances ER stress-mediated apoptosis in HaCaT cells by increasing the expression of CHOP protein. These results suggest that silibinin may be beneficial in the removal of UVA-damaged cells and the prevention of skin cancer.     

ULTRAVIOLET RADIATION INDUCES A CHANGE IN CELL MEMBRANE POTENTIAL in vitro: A POSSIBLE SIGNAL FOR ULTRAVIOLET RADIATION INDUCED ALTERATION IN CELL ACTIVITY

The regulation of a transmembrane ionic gradient, reflected by the cellular membrane potential, has been shown in several cell systems to be involved in the regulation of cell function. This investigation presents evidence that biologically relevant doses of ultraviolet radiation (UVR) will alter the membrane potential of keratinocytes in vitro. Estimation of the relative change in the steady-state membrane potential of the murine keratinocyte cell line PAM 212, the murine myelomonocytic cell line P388D1, and normal human keratinocytes in culture, were made through the use of the lipophilic cationic membrane potential sensitive probe; triphenylmethylphosphonium. Our observations indicate that UVR composed primarily of UVB (280-320 nm) radiation at doses as low as 100 J/m2 can induce a depolarization in the murine cell lines and a hyperpolarization in human keratinocytes. Evidence suggests that this difference in the direction of the membrane potential response reflects a difference in Na+/K+ ATPase activity following UVR. These results suggest a possible mechanism for modulation of keratinocyte activity induced by UVR.     

Analysis of UVB-modulated Gene Expression in Human Keratinocytes by mRNA Differential Display Polymerase Chain Reaction

Abstract— EUltraviolet (UV) light is the most important environmental insult to skin. Even a single exposure to UVB radiation can result in inflammation and may also lead to DNA damage and apoptosis in the acute response of the cutaneous tissue. To elucidate the complex alterations of gene expression in human keratinocytes underlying these UV responses we took advantage of differential display polymerase chain reaction (DD-PCR) technology's ability to detect qualitative and quantitative changes in gene expression in more than two cell populations simultaneously. We demonstrate that low-dose UVB (100 Jm_-2) leads to both induction and down regulation of different genes during the 24 h after irradiation in a time-dependent manner. In addition to the identification of known genes as possible effectors or targets in the UV response of human keratinocytes, we here identify a new sequence that is negatively regulated by UVB irradiation and was termed HUR 7 (HaCaT UV repressed). In general our results showed that DD-PCR is a useful tool in the analysis of quantitative changes of mRNA levels in human keratinocytes after UV irradiation. The identification of new UVB-repressed genes offers the opportunity to identify unrecognized molecular mechanisms in the UV response of human cells.     

Ultraviolet A radiation induces rapid apoptosis of human leukemia cells by Fas ligand-independent activation of the Fas death pathways

Endogenous cellular chromophores absorb ultraviolet A radiation (UVA, 290-320 nm), the major UV component of terrestrial solar radiation, leading to the formation of reactive oxidizing species that initiate apoptosis, gene expression and mutagenesis. UVA-induced apoptosis of T helper cells is believed to underlie the UVA phototherapy for atopic dermatitis and other T cell-mediated inflammatory skin diseases. We have evaluated the involvement of the Fas-Fas ligand (FasL) pathway in rapid UVA-induced apoptosis in human leukemia HL-60 cells. UVA-induced apoptosis was not inhibited by pretreatment with a neutralizing anti-Fas antibody, although the same UVA treatment initiated cleavage of caspase-8 and subsequent processing of Bid and caspase-3-like proteases. Inhibition of caspase-8 by Lle-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone completely blocked caspase-3 cleavage and apoptosis in UVA-treated cells, suggesting that apoptosis was initiated by the Fas pathway. This inference was supported by demonstrating that immunoprecipitates obtained from UVA-treated cells using anti-Fas antibody contained caspase-8 and Fas-associating protein with death domain (FADD). In addition, Fas clustering in response to UVA treatment was observed by immunofluorescence microscopy. These data support a mechanism for rapid, UVA-induced apoptosis in HL-60 cells involving initial formation of the Fas-FADD-caspase-8 death complex in an FasL-independent manner.     

Mitochondrial Sirtuins in Skin and Skin Cancers

Mammalian sirtuins (SIRTs 1-7) are a family of NAD+-dependent deacetylases with distinct subcellular localization and biological functions that regulate various important cellular processes. Among these, SIRTs -3, -4 and -5 are located in the mitochondria and have been implicated in caloric restriction, oxidative stress, aging and various human diseases. Emerging evidence has found dysregulation of mitochondrial sirtuins in multiple dermatological conditions, including responses to ultraviolet radiation (UVR), suggesting their importance in maintaining skin health. In this review, we discuss the roles and implications of mitochondrial sirtuins in cutaneous cellular processes, and their emerging potential as a target for the management of skin diseases, including skin cancer. Among mitochondrial sirtuins, SIRT3 is the most studied and linked to multiple skin conditions and diseases (keratinocyte differentiation, wound healing, chronological aging, UVR and ozone response, systemic sclerosis, melanoma, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)). SIRT4 has been connected to keratinocyte differentiation, chronological aging, UVR response, alopecia, BCC and SCC. Further, SIRT5 has been associated with keratinocyte differentiation, melanoma, BCC and SCC. Overall, while there is compelling evidence for the involvement of mitochondrial sirtuins in skin, additional detailed studies are needed to understand their exact roles in skin and skin cancers.     

Effects of intranasal phototherapy on nasal mucosa in patients with allergic rhinitis

RATIONALE: Rhinophototherapy has been shown to be effective in the treatment of allergic rhinitis. Considering that phototherapy with ultraviolet light (UV) induces DNA damage, it is of outstanding importance to evaluate the damage and repair process in human nasal mucosa. METHODS: We have investigated eight patients undergoing intranasal phototherapy using a modified Comet assay technique and by staining nasal cytology samples for cyclobutane pyrimidine dimers (CPDs), which are UV specific photoproducts. RESULTS: Immediately after last treatment Comet assay of nasal cytology samples showed a significant increase in DNA damage compared to baseline. Ten days after the last irradiation a significant decrease in DNA damage was observed compared to data obtained immediately after finishing the treatment protocol. Difference between baseline and 10 days after last treatment was not statistically significant. Two months after ending therapy, DNA damage detected by Comet assay in patients treated with intranasal phototherapy was similar with that of healthy individuals. None of the samples collected before starting intranasal phototherapy stained positive for CPDs. In all samples collected immediately after last treatment strong positive staining for CPDs was detected. The number of positive cells significantly decreased 10 days after last treatment, but residual positive staining was present in all the examined samples. This finding is consistent with data reported in skin samples after UV irradiation. Cytology samples examined two months after ending therapy contained no CPD positive cells. CONCLUSION: Our results suggest that UV damage induced by intranasal phototherapy is efficiently repaired in nasal mucosa.     

Photoprotective properties of Prunella vulgaris and rosmarinic acid on human keratinocytes

UVA radiation provokes the generation of reactive oxygen species (ROS), which induce oxidative stress in the exposed cells leading to extensive cellular damage and cell death either by apoptosis or necrosis. One approach to protecting human skin against the harmful effects of UV radiation is by using herbal compounds as photoprotectants. This study evaluated the protective effects of Prunella vulgaris L. (Labiatae) and its main phenolic acid component, rosmarinic acid (RA), against UVA-induced changes in a human keratinocyte cell line (HaCaT). Human keratinocytes exposed to UVA (10-30 J/cm(2)) were treated with an extract of P. vulgaris (1-75 mg/l) or RA (0.9-18 mg/l) for 4h. P. vulgaris and RA exhibited ability to reduce the UVA-caused decrease in a cell viability monitored by neutral red retention and by LDH release into medium. The P. vulgaris extract and RA significantly suppressed UVA-induced ROS production, which manifests as a decrease in intracellular lipid peroxidation, elevation of ATP and reduced glutathione. Post-treatment with P. vulgaris extract and RA also significantly reduced DNA damage. In addition, UVA-induced activation of caspase-3 was inhibited by treatment with P. vulgaris and RA. The P. vulgaris extract and RA demonstrated a concentration-dependent photoprotection (maximum at 25-50 mg/l and 9 mg/l, respectively). These results suggest that P. vulgaris and RA, used in skin care cosmetics, may offer protection against UVA-induced oxidative stress and may be beneficial as a supplement in photoprotective dermatological preparations.     

Ultraviolet radiation attenuates thrombospondin 1 expression via PI3K-Akt activation in human keratinocytes

Thrombospondin 1 (TSP1) is an extracellular glycoprotein and a recognized inhibitor of angiogenesis. Recent studies have demonstrated that UV radiation induces an angiogenic switch, by which it alters the balance between pro- and anti-angiogenic factors in the skin. Here we describe the effects of acute UV exposure on TSP1 expression in human skin epidermis, primary keratinocytes and the epidermal cell line HaCaT. We found that protein and mRNA expressions of TSP1 are significantly reduced in human skin in vivo and in keratinocytes in vitro by a single UV exposure. In human skin and keratinocytes, UV exposure induced the phosphorylation of Akt, a downstream target of the PI3K pathways. Specific inhibitors of PI3K, wortmannin and LY294002, completely blocked Akt activation and UV-induced TSP1 downregulation in keratinocytes. We showed that a specific Akt phosphorylation inhibitor and small interfering RNA-mediated Akt depletion were also blocked by UV-induced TSP1 downregulation in keratinocytes. In conclusion, our findings demonstrate that acute UV exposure downregulates TSP1 expression via PI3K-Akt activation in human keratinocytes. These novel findings may help us understand the regulatory mechanisms of UV-induced skin angiogenesis.     

Photoprotection by melanin

This paper is an attempt to summarize the current state of information on melanin and epidermal melanin pigmentation (EMP) as photoprotective agents. The chemistry and biochemistry of melanin (the particle) and its interaction, in its various forms, with UV radiation are considered. Methods of attenuation of UV radiation are discussed in terms of structure and chemical constituents. Photoprotection by constitutive and facultative pigmentation is reviewed with minimum erythema dose (MED) as the end point. The issue of acclimatization to UV radiation is discussed in terms of UVB phototherapy for psoriasis. Finally, skin cancer is considered as an end point and the reduction of its incidence with pigment level is discussed. It is concluded that whilst EMP provides protection, its extent depends on the end point chosen for evaluation. MED is a convenient photobiological end point but is rather insensitive, whereas skin cancer is sensitive but impractical for laboratory studies. Our current state of knowledge of melanin lacks information on its absorption and scattering coefficients and its refractive index. Methods for the quantitative measurement of EMP are also urgently required.     

ULTRAVIOLET ACTION SPECTRA FOR PHOTOBIOLOGICAL EFFECTS IN CULTURED HUMAN LENS EPITHELIAL CELLS

Abstract— The action spectrum for cell killing by UV radiation in human lens epithelial (HLE) cells is not known. Here we report the action spectrum in the 297–365 nm region in cultured HLE cells with an extended lifespan (HLE B-3 cells) and define their usefulness as a model system for photobiological studies. Cells were irradiated with monochromatic radiation at 297, 302, 313, 325, 334 and 365 nm. Cell survival was determined using a clonogenic assay. Analysis of survival curves showed that radiation at 297 nm was six times more effective in cell killing than 302 nm radiation; 297 nm radiation was more than 260, 590, 1400 and 3000 times as effective in cell killing as 313, 325, 334 and 365 nm radiation, respectively. The action spectrum was similar in shape to that for other human epithelial cell lines and rabbit lens epithelial cells. The effect of UV radiation on crystallin synthesis was also determined at different wavelengths. To determine whether exposure to UV radiation affects the synthesis of β-crystallin, cells were exposed to sublethal fluences of UV radiation at 302 and 313 nm, labeled with [³⁵S]methionine and the newly synthesized βY-crystallin was analyzed by immunoprecipitation and western blotting using an antibody to β-crystallin. The results show a decrease in crystallin synthesis in HLE cells irradiated at 302 and 313 nm at fluences causing low cytotoxicity. The effect of radiation on membrane perturbation was determined by measuring enhancement of synthesis of prostaglandin E₂ (PGE₂). Synthesis of PGE₂ occurs at all UV wavelengths tested in the 297–365 nm region. The slope of the PGE₂ response curves was higher than that of cell killing curves in cultured HLE cells. These data show that cultured HLE cells with extended lifespan are a suitable system for investigating photobiological responses of cells to UV radiation.     

Influence of UVB,UVA and UVA1 irradiation on histamine release from human basophils and mast cells in vitro in the presence and absence of antioxidants

UV irradiation is widely used for the treatment of atopic eczema. In recent years, UVA1 phototherapy has gained increasing attention. This study analyzed the influence of different UV wavelengths--especially UVA1--on histamine release from human basophils and mast cells. The modulation of this parameter might be responsible for some of the therapeutic effects of UV irradiation. Enriched human basophils and human mast cells (HMC1 cell line) were irradiated with increasing doses of UVB, UVA and UVA1 in vitro. After irradiation, different stimulants were added to induce histamine release. In additional experiments, basophils were preincubated with superoxide dismutase, ascorbate or trolox to study the role of antioxidants in the modulation of histamine release after UV irradiation. UVA and UVA1 significantly inhibited histamine release from basophils and mast cells. UVB only had an inhibitory effect on mast cells. Preincubation with superoxide dismutase and ascorbate did not influence the inhibitory effect of UVA1 on basophil histamine release, whereas trolox decreased significantly the histamine release from nonirradiated basophils.     

The Xanthophyll Carotenoid Astaxanthin has Distinct Biological Effects to Prevent the Photoaging of the Skin Even by its Postirradiation Treatment

Exposure of human skin to ultraviolet (UV) radiation causes significant damage to that tissue. The effects of UV on the skin mainly include acute inflammation (erythema/edema) and abnormal keratinization wherein prostaglandin E₂ (produced by cyclooxygenase‐2), interleukin‐8 and transglutaminase 1 (a major regulatory factor of keratinization) play pivotal roles. Later phases of UV‐induced skin reactions include hyperpigmentation, wrinkle formation and carcinogenesis, the former two being associated with the UVB‐induced production and/or secretion of endothelin‐1, stem cell factor and granulocyte‐macrophage colony‐stimulating factor by keratinocytes in the epidermis. Those paracrine factors then stimulate expression of the critical melanogenic enzyme tyrosinase by melanocytes in the epidermis and increase expression of neprilysin, an enzyme that degrades elastin, by fibroblasts in the dermis. This review summarizes the biological effects of the xanthophyll carotenoid astaxanthin, which prevents UV‐induced cutaneous inflammation, abnormal keratinization and wrinkling as well as pigmentation of the skin even by its postirradiation treatment.