2005 ANNUAL SCIENTIFIC MEETING 187 SIRTl, THE HUMAN HOMOLOGUE TO SIR2, IS EXPRESS:ED :IN HUMAN SKIN AND IN CULTURED KERATINOCYTES FIBROBLASTS AND HaCaT CELLS AND ITS LEVEL IS CLOSELY RELATED TO STRESS AND AGING Claude Dal Farra, Ph.D. and N. Domloge Vincience Research Center, Sophia Antipolis, France Introduction The SIR2 (silent infonnation regulator 2) gene family was first studied in yeast, and it is a family of protein deacetylases (Sirtuins) that are NAD(+)-dependent enzymes. Evidence progressively showed that SIR2, a NAD-dependent histone deacetylase, is the founding member of the family of sirtuins. Studies in yeast have shown that the SIR2 family has diverse biological functions including gene silencing, DNA repair, cell cycle progression, development, metabolism, apoptosis, heterochromatin fonnation, and aging.1-6 The discovery that SIR2 requires NAD for its activity immediately suggested a link between SIR2 activity and caloric restriction. This link was strengthened by the observation that life span extension by caloric restriction requires the SIR2 protein.7-IO The discovery that overexpression of SIR2 is sufficient to extend life span in yeast elevated SIR2 to the central position of aging research in this organism. In mammalian cells, studies have identified SIRTl as the homologue of the Saccharomyces cerevisiae chromatin silencing factor SIR2. In the studies that followed, 4 types of human SIRT were identified. SIRTl has been found to associate with the tumor suppressor protein p53, and the deacetylation ofp53 by SIRTl has been shown to negatively regulate p53-mediated transcription. Therefore, the role ofSIRTl in preventing cellular senescence and apoptosis induced by DNA damage and stress has been strongly suggested. Hence, it is becoming increasingly apparent that SIRTl is a key regulator of cell defense and cell survival in response to stress. Results Immunostaining studies of cultured human keratinocytes, HaCaT cells and fibroblasts demonstrated that SIRTl exhibits a clear nuclear staining in these cells. Irnmunofluorescence studies showed that, with low doses ofUVB stress, SIRTl expression increases in a dose-dependent manner, while p53 expression shows very little variation. In contrast, at higher UV doses (above 40-50m.J/cm2) SIRTl expression decreases while p53 expression increases significantly. SIRTl expression in human fibroblasts exposed to different low UVB doses pSJ expression In human fibroblasts exposed to different low UVB doses Control cdls (A), cells irradiated with 15 mJICJ111 (B), 50 ,,,J/cm1 (CJ, and 75 mJlcm2 (D) This result corroborates SIRT's role in the protection of cells by suppressing p53 after a moderate injury of cells by UVB after stronger, damaging UV doses, p53 takes over, leading the cell to cell cycle arrest or apoptosis. mRNA studies confumed this finding and showed that SIRTl mRNA increased 3 h after low doses of UVB and lasted for 24 h. In response to caloric restriction, SIRTl expression increased, in a dose-dependent manner, to reach maximum expression with total glucose deprivation (for 24 and 48 h), while p53 levels varied very little under these conditions.

188 JOURNAL OF COSMETIC SCIENCE SIRTl ei:pression in human fibroblasts e:iposed to caloric restriction Control cells (A), cells culllued in medlll ctJnulining ollly 5tr-' of tlu glMc:ou of tlle control, for 14 II (a), � a,tu,red in IIUIIJa whll""t glMcou,for 14 II (CJ Studies of SIR T expression in different human and animal tissues (but not the skin) have shown that SIRTI expression is nuclear. Our studies on ex vivo skin involved comparative studies between frozen and fresh skin samples, and between skin samples at different ages. These studies showed that in fresh ex vivo skin samples, SIRTI exhibited a predominant nuclear staining throughout the epidennis. Some cytoplasmic staining was also seen. SIRTI expression in fresh human a vivo skin Comparative studies of skin samples from donors of different ages (30 to 55) did not reveal a significant age-related difference in SIRTI expression under a stress-free condition. SIRTI expression in ski donor irradia 1 Interestingly, when skin samples were irradiated with UVB (50-200 mJ/cm2), fresh skin samples exhibited a clear dose-dependent increase in SIR Tl nuclear expression, up to 100 mJ/cm2, which correlated with low tissue damage and low p53 expression, whereas high UVB doses yielded strong p53 expression. Amazingly, these results appeared more evident in young skin. No UVB (A) JOO mJ/cm (B) Conclusion These studies demonstrate that SIRTI is expressed in human skin and in cultured human keratinoctes, and fibroblasts. These studies also confirm the close relationship ofSIRTI with p53, stress, and cell aging they show that SIRTI expression correlates with its ability to protect human skin and is related to UV stress dosage. I Shore D, Squire M, Nasmyth KA, EMBOJ., 3(12), 2817-23, (1984) 2 Rine J, Strathem JN, Hicks JB, Herskowitz I, Genetics., 93(4), 877-901, (1979) 3 Gotta M, Strahl-Bolsinger S, Renauld H, Laroche T, Kennedy BK, Grunstein M, Gasser SM, EMBO J., 16(11), 3243-55, (1997) 4 Tissenbaum HA, Guarente L, Nature., 410(6815), 227-30, (2001) 5 Brachmann CB, Sherman JM, Devine SE, Cameron EE, Pillus L, Boeke ID, Genes Dev., 9(13), 2888- 902, (1995) 6 Dryden SC, Nahhas FA, Nowak JE, Goustin AS, Tainsky MA, Mo/ Cell Biol., 23(9), 3173-85, (2003) 7 Kaeberlein M, Andalis AA, Fink GR, Guarente L, Mo/ Cell Biol., 21(22) , 8056-66, (2002) 8 Masoro EJ, Mech Ageing Dev., 125(9), 591-4, (2004) 9 Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, Kessler B, Howitz KT, Gorospe M, de Cabo R, Science., 305(5682), 390-2, (2004) IO Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclaire D, Nature., 430(7000), 686-9, (2004)