J. Cosmet. Sci., 66, 57–63 (January/February 2015) 57 Temporal variations in sirtuin expression under normal and ultraviolet B-induced conditions and their correlation to energy levels in normal human epidermal keratinocytes EDWARD PELLE, KELLY DONG, and NADINE PERNODET, Estee Lauder Research Laboratories, Melville, NY 11747 (E.P., K.D., N.P.), and Environmental Medicine, New York University School of Medicine, New York, NY 10987 (E.P.). Accepted for publication January 12, 2015. Synopsis Sirtuins are post-translational modifi ers that affect transcriptional signaling, metabolism, and DNA repair. Although originally identifi ed as gene silencers capable of extending cell lifespan, the involvement of sirtuins in many different areas of cell biology has now become widespread. Our approach has been to study the tem- poral variation and also the effect of environmental stressors, such as ultraviolet B (UVB) and ozone, on sir- tuin expression in human epidermal keratinocytes. In this report, we measured the variation in expression of several sirtuins over time and also show how a low dose of UVB can affect this pattern of expression. More- over, we correlated these changes to variations in hydrogen peroxide (H2O2) and ATP levels. Our data show signifi cant variations in normal sirtuin expression, which may indicate a generalized response by sirtuins to cell cycle kinetics. These results also demonstrate that sirtuins as a family of molecules are sensitive to UVB- induced disruption and may suggest a new paradigm for determining environmental stress on aging and provide direction for the development of new cosmetic products. INTRODUCTION Post-translational modifi cations exert control across a diverse array of cellular functions (1). One area that has generated much recent interest has been the enzymatic activity of sirtuins on various proteins affecting aging (2), metabolism (3), and response to environ- mental trauma (4,5). Although sirtuins have several different modes of action, they are generally classifi ed as Class III nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases that remove an acetyl group from a lysine side chain of its protein substrate (6). In mammalian cells, seven sirtuin (SIRT1-7) homologs have been characterized that are distributed in several sub-cellular compartments (7). Human skin is constantly exposed to environmental hazards, such as ultraviolet B (UVB) radiation, leading to photo aging and skin cancer (8). Although it is well established that Address all correspondence to Edward Pelle at epelle@estee.com.

JOURNAL OF COSMETIC SCIENCE 58 sirtuins can contribute to the longevity of many organisms by silencing genes during times of nutritional deprivation (9), they may also increase cell survival after UV-induced stress using the same mechanisms. In a previous report (4), we described the effects of low levels of UVB radiation on sirt3 expression and its inverse relationship to sirt4 expression in normal human epidermal keratinocytes ( NHEK). We further demonstrated how UVB can disrupt the normal activity and cycle of expression of sirtuins, both of which appear necessary for maintaining cutaneous health. In this report, we expand these fi ndings to include sirt1 and sirt6 and correlate these results with the effects of UVB on H2O2 and energy metabolism. Our objective was to determine the relative levels of expression of sirt1, sirt3, and sirt6 over time in NHEK synchronized by starvation. Additionally, we determined sirtuin expression after UVB irradiation. If the patterns of sirtuin expression were similar, this might imply a generalized response by sirtuins to environmental challenge that might also be related to ATP levels and reactive oxygen species (ROS). MATERIALS AND METHODS CELL CULTURE NHEK were obtained from a commercial supplier (Cascade Biologics, Portland, OR) and cultured according to the manufacturer’s recommendations at 37°C in a 5% CO2 hu- midifi ed incubator. Cells were incubated in EpiLife medium (Cascade) without any h u- man keratinocyte growth supplement (HKGS Cascade) during starvation. The supplement included bovine pituitary extract, human epidermal growth factor, hydrocor- tizone, insulin, and transferrin. Cells were released from starvation by incubating cells in full EpiLife medium with HKGS. CELL VIABILITY Cell toxicity was determined with an Alamar Blue solution (Invitrogen, Carlsbad, CA), which was prepared at 10% in cell media and added to cells for 2 hours. After incubation, cellular fl uorescence (Ex535nm/Em612nm) was measured in a SpectraMax Gemini fl uores- cence plate reader (Molecular Devices, Sunnyvale, CA). A decrease in fl uorescence is indicative of cytotoxicity. UV RADIATION Cells were irradiated before being released from starvation. Cells were washed with Dulbecco’s modifi ed phosphate-buffered saline, pH 7.4 (D-PBS), covered with a thin layer of D-PBS (3 ml in a 100 mm plate) and then irradiated with FS40 UVB bulbs (Philips Fisher Scientifi c, Pittsburgh, PA) through a Kodacel fi lter (Kodak, Rochester, NY). UVB fl uences were measured with an IL1400A radiometer (International Light, Newburyport, MA).