Oncogene (2006) 25, 2601–2614 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE The UVB-induced gene expression profile of human epidermis in vivo is different from that of cultured keratinocytes CD Enk1, J Jacob-Hirsch2, H Gal3, I Verbovetski4, N Amariglio2, D Mevorach4, A Ingber1, D Givol3, G Rechavi2 and M Hochberg1 1Department of Dermatology, The Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 2Department of Pediatric Hemato-Oncology and Functional Genomics, Safra Children’s Hospital, Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University,Tel Aviv, Israel; 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel and 4The Laboratory for Cellular and Molecular Immunology, Department of Medicine, The Hadassah-Hebrew University Medical Center, Jerusalem, Israel In order to obtain a comprehensive picture of the radiation. UVB, with a wavelength range between 290 molecular events regulating cutaneous photodamage of and 320 nm, represents one of the most important intact human epidermis, suction blister roofs obtained environmental hazards affectinghuman skin (Hahn after a single dose of in vivo ultraviolet (UV)B exposure and Weinberg, 2002). To protect itself against the were used for microarray profiling. We found a changed DNA-damaging effects of sunlight, the skin disposes expression of 619 genes. Half of the UVB-regulated genes over highly complicated cellular programs, including had returned to pre-exposure baseline levels at 72 h, cell-cycle arrest, DNA repair and apoptosis (Brash et al., underscoring the transient character of the molecular 1996). Failure in selected elements of these defensive cutaneous UVB response. Of special interest was our strategies may result in propagation of cutaneous finding that several of the central p53 target genes cancers. Handlingof photodamageby the skin constitu- remained unaffected following UVB exposure in spite of tes a crucial survival mechanism, and the regulation and p53 protein accumulation. We next compared the in vivo activity of cutaneous UVB-regulated genes represents an expression profiles of epidermal sheets to that of cultured attractive model for the functioningof central protective human epidermal keratinocytes exposed to UVB in vitro. cellular responses under physiological conditions. We found 1931 genes that differed in their expression The advent of microarray technology allows the profiles between the two groups. The expression profile in generation of global expression profiles and differential intact epidemis was geared mainly towards DNA repair, expression of thousands of genes (Park et al., 2002; Joos whereas cultured keratinocytes responded predominantly et al., 2003; Sellheyer and Belbin, 2004). Several recent by activating genes associated with cell-cycle arrest and studies have employed microarray profilingto study apoptosis. These differences in expression profiles might UVB-regulated gene expression in cultured human reflect differences between mature differentiating kerati- keratinocytes (Becker et al., 2001; Li et al., 2001; nocytes in the suprabasal epidermal layers versus Murakami et al., 2001; Sesto et al., 2002; Takao et al., exponentially proliferating keratinocytes in cell culture. 2002; Dazard et al., 2003; Pisarchik et al., 2004; Lee Our findings show that extreme care should be taken when et al., 2005), revealing that a wide range of genes extrapolating from findings based on keratinocyte cultures regulating transcription factors, cytoskeletal proteins, to changes in intact epidermis. secreted signaling molecules and controlling cellular Oncogene (2006) 25, 2601–2614. doi:10.1038/sj.onc.1209292; functions such as signal transduction, terminal differ- published online 23 January 2006 entiation and apoptosis, are induced by UVB. However, since these studies differ considerably in terms of time Keywords: UVB; epidermis; p53; in vivo; in vitro; kinetics, UVB dose, light sources and hybridization microarrays techniques, it is difficult to generate a comprehensive picture of the complex changes in gene expression taking place in UVB-irradiated keratinocytes. An additional drawback common to all these studies Introduction is the fact that they were performed on cultured keratinocytes under in vitro conditions. Skin tissue The skin constitutes the outer barrier of the human cultures differ substantially from the intact tissues they organism towards external stimuli and ultraviolet (UV) are supposed to imitate: Keratinocytes are usually cultured in the presence of hormones and growth factors Correspondence: Dr CD Enk, Department of Dermatology, Hadassah that might affect cell-cycle regulation, stress response Medical Center, P.O. Box 12000, IL 91010, Israel. and apoptosis (Gibbs et al., 1998), thereby modifying E-mail: [email protected] Received 11 July 2005; revised 6 October 2005; accepted 28 October the response to UVB. Furthermore, confluence 2005; published online 23 January 2006 in keratinocyte cultures affects the regulation of UVB-induced gene expression profile of human epidermis CD Enk et al 2602 programmed cell death, and a significant difference in sensitivity to UVB-induced apoptosis between subcon- fluent cultures and exponentially growing cells have been reported (Gniadecki et al., 1997). Finally, keratino- cyte cultures constitute a homogeneous population of cells lackingthe additional cellular constituents of intact epidermis such as Langerhans cells and intraepidermal inflammatory cells that together with the keratinocytes play important roles in the response of human epidermis to UV exposure (Baadsgaard, 1991). Usinga unique model system based on UV-exposed human epidermis followed by isolation of epidermal cells from suction blister roofs, we have recently studied the global expression profile in intact human epidermis usingoligonucleotide microarrays (Enk et al., 2004). With this in vivo setup that overcomes several of the limitations outlined above, we identified more than 800 UVB-regulated genes, some of which not previously known to be UVB sensitive. In the present study, we expand this investigation by monitoring the differen- Figure 1 Number up- and downregulated genes at different time- tially expressed genes over a 72 h period following UVB points followingUVB irradiation of intact human epidermis. exposure. We found that the majority of the in vivo UVB-regulated genes changed their expression at the 24 h time-point, but had returned to background levels within 72 h. We next compared the expression profile of sion profiles (Kannan et al., 2001). Applyingthis the in vivo exposed epidermal cells to that of cultured method to genes that were at least twofold changed in UVB-exposed normal human epidermal keratinocytes all volunteers at least at one time-point, triggered (NHEK), thus providingthe first direct comparison distinct, well-ordered gene expression and partitioned between the UV response of intact epidermis and the samples accordingto interval after UV exposure keratinocyte cultures. We here report that the expression rather than accordingto individuals (Figure 2). This profile in intact epidermis was geared mainly towards profilingpattern testifies to the robustness of the data set DNA repair, whereas cultured keratinocytes responded and indicates that our results reflect an overall UVB predominantly by activatinggenesassociated with cell- response. The algorithm showed that the most closely cycle arrest and apoptosis. related expression profiles were those of the 0, 2 and 72 time-points, whereas the 24 h expression profile dis- played greatest dissimilarity from the other time-points. Results and Discussion This suggests that the majority of UVB-induced changes in epidermal gene expression is transient and has UVB-induced gene expression reversed to background expression within 72 h. The To study the global gene expression profile in intact findingthat the early UVB response involves a relatively human epidermis following a single physiological UVB small number of genes, whereas the 24 h response is exposure, we exposed the inner forearms of three more complex and expresses a larger number of genes volunteers to 4 MED of UVB in vivo. Suction blisters corresponds well with the recent findings of Lee et al. were raised at 2, 24 and 72 h followingexposure, and (2005) who examined the differentially expressed genes mRNA extracted from the blister roofs underwent gene of UVB-irradiated immortalized HaCat keratinocytes at profilingusingAffymetrix Human Focus oligonucleotide 0.5, 6 and 24 h. arrays as described in Methods. Applyingan arbitrary The dendogram revealed eight major, stable clusters filter level of twofold changes in the ratios of gene containingdifferent expression kinetics of up- and expression in all volunteers at least at one time-point, we downregulated genes at 2, 24 and 72 h after UV found 619 genes whose expression was modified by UVB exposure (Figure 2). Interestingly, in five of the eight (Figure 1), of which 246 genes were upregulated and 373 clusters (cluster 1, 2, 5, 7 and 8), representing316 of the were downregulated, representing approximately 12% of 619 UVB-regulated genes (51%), the expression pattern the ‘valid’ genes (1.5–2% of the human genome). In our had returned to pre-exposure baseline levels a 72 h, list, the majority, 53% of the differentially expressed again underscoring the transient character of the genes, were found at the 24 h time-point, whereas 21 and genomic changes taking place in human skin