To investigate environmental factors that contribute to ultraviolet A (UVA)-induced oxidative stress, which accelerates the senescence and toxicity of skin cells, we irradiated human fibroblasts cultured in commonly used essential media with UVA and evaluated their viability and production of reactive oxygen species. The viability of fibroblasts exposed to a single dose of 3.6 J/cm2 UVA was not reduced when cultured in Hanks balanced salt solution, but it was significantly decreased when cultured in Dulbecco’s modified Eagle’s medium (DMEM), which contains various amino acids and vitamins. Furthermore, cell viability was not reduced when fibroblasts were cultured in DMEM and treated with a hydrogen peroxide (H2O2) scavenger such as glutathione or catalase added after UVA irradiation. In addition, we confirmed that the production of H2O2 was dramatically increased by UVA photosensitization when riboflavin (R) coexisted with amino acids such as tryptophan (T), and found that R with folic acid (F) produced high levels of H2O2 after UVA irradiation. Furthermore, we noticed that R and F or R and T have different photosensitization mechanisms since NaN3, which is a singlet oxygen quencher, suppressed only R and T photosensitization. Lastly, we examined the effects of antioxidants (L-ascorbic acid, trolox, L-cysteine, and L-histidine), which are singlet oxygen or superoxide or H2O2 scavengers, on R and F or on R and T photosensitization, and found that 1 mM ascorbic acid, Trolox, and L-histidine were strongly photosensitized with R, and produced significant levels of H2O2 during UVA exposure. However, 1 mM L-cysteine dramatically suppressed H2O2 production by UVA photosensitization. These data suggest that a low concentration of R-derived photosensitization is elicited by different mechanisms depending on the coexisting vitamins and amino acids, and regulates cellular oxidative stress by producing H2O2 during UVA exposure.

The incidence of melanoma has increased dramatically in recent years. Over-the-counter sunscreen use in childhood has been shown to lower the lifetime risk of melanoma in Australia, but previous studies in the U.S. have failed to demonstrate benefit. We hypothesize that short follow-up and a failure to take into account inappropriate sunscreen application practices may contribute to the lack of positive findings. Here, we created a predictive model for the impact of different regimens of sunscreen use on melanoma incidence in the pediatric New York State Medicaid population. A Markov model was developed to analyze ideal use conditions (almost daily), some use (50% of ideal), and none. Total sunscreen use for children was estimated at about 15-20 ml per application, with the number of applications depending on time of year. The model population was based on SEER data and published literature. The model incorporated various salient population characteristics including gender, ethnicity (white, non-white), and sunbed use. The model time frame was 79 years, the average life expectancy in the U.S. Our model found that compared to no sunscreen use, ideal sunscreen application is associated with a significant 43% risk reduction of lifetime melanoma development, while some sunscreen application is associated with a 17.5% risk reduction. These findings suggest that in order to demonstrate reduction in melanoma incidence, it is important to ensure appropriate sunscreen application conditions and also to look at long-term followup.

Differentiated cells of the epidermis have an essential role in generating the epidermal barrier, but their communication with and regulation of their stem cell progenitors is poorly understood. Here we use novel mouse transgenic lines to define roles for differentiated suprabasal cells in epidermal morphogenesis. Two mouse models demonstrate that increasing contractility of differentiated cells results in profound changes to basal cell proliferation and fate decisions. Increased proliferation resulted in hyperthickening of the epidermis and premature barrier formation. In addition, hair cell fates were inhibited. Pharmacologic inhibition of the contractility pathway rescued both basal cell proliferation and hair follicle morphogenesis. These data suggest that intra-tissue tension regulates stem cell proliferation and fate decisions, similar to the known roles of extracellular matrix rigidity. Importantly, this demonstrates that differentiated cells are part of the stem cell niche that regulates development and homeostasis of the skin. 884LRRK1 regulates hair follicle (HF) growth via coordinating Wnt and SHH signaling The hair follicle growth cycle consists of growth (anagen), remodeling (catagen), and relative "quiescence" (telogen). At the telogen to anagen transition, HF stem cells (HFSCs) receive cues to initiate proliferation, which results in the downgrowth and formation of the mature follicular epithelium. How these early signals are regulated is not completely understood. Leucine Rich Repeat Kinase 1 (LRRK1) is an understudied multi-domain kinase. Here, we show that LRRK1 is preferentially expressed in: (i) subsets of epidermal basal cells; (ii) the epithelial cells of the HF bulge; and (iii) limbal epithelial basal cells; sites of epidermal, follicular and corneal epithelial stem cells, respectively. Importantly, loss of LRRK1 results in an aberrant induction of anagen during the mouse hair growth cycle, suggesting that LRRK1 is a novel regulator that is essential for proper follicular growth. To elucidate the LRRK1 downstream signaling pathways in stratified epithelium, RNA sequencing revealed that loss of LRRK1 in mouse skin downregulated sonic hedgehog (SHH) and Wnt signaling, two key pathways critical for the induction of the HF growth cycle. Such downregulation was also validated in: (1) human epidermal keratinocytes with reduced LRRK1 by quantitative PCR; and (2) LRRK1 null mouse HFs in vivo by immunostaining. These observations demonstrate that LRRK1 plays an essential role in regulating SHH and wnt pathways. To elucidate further the underlying molecular mechanisms of such regulation, we show that inhibition of LRRK1 increased: (i) Ca2 + -calmodulin-dependent protein kinase II, which inhibits Wnt signaling via phosphorylating b-catenin; and (ii) Glypican 3, which blocks SHH signaling via sequestering SHH proteins. Collectively, these findings indicate that LRRK1 controls a novel regulatory network, which is essential for the induction of early anagen. Furthermore, this LRRK1/Wnt/ SHH signaling axi...

The nucleotide excision repair (NER) system removes pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] and cyclobutane pyrimidine dimers (CPDs) from DNA in the form of small (w30-nt-long), excised, damage-containing DNA oligonucleotides (sedDNAs). However, the detection of UV photoproduct-containing sedDNAs as a biochemical read-out for NER activity has thus far been limited to defined in vitro cell-free systems and to cultured cells. Using small punch biopsies of human skin, we show here that the sedDNA products of NER can be isolated, detected, and quantified in skin epidermis within minutes of UVB exposure and following low, sub-erythemal doses of UVB. Interestingly, a partially degraded form of the sedDNAs can be detected in human skin epidermis for several hours following UVB irradiation, which indicates that sedDNA degradation occurs slowly following their release from epidermal genomic DNA. Treatment of skin explants with spironolactone, which depletes the epidermis of the essential NER protein XPB (xeroderma pigmentosum group B), inhibits sedDNA generation following UVB exposure. Our results indicate that the detection of sedDNAs offers a sensitive and quantitative assay for nucleotide excision repair activity in human skin samples and may therefore be useful for determining how genetic, environmental, and pharmacological factors contribute to inter-individual differences in NER activity in human skin.

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