Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays

Angela Sesto*, Manuel Navarro*†, Frank Burslem‡, and Jose´L. Jorcano*

*Department of Molecular and Cell Biology, Centro de Investigaciones Energe´ticas, Medioambientales y Tecnolo´gicas (CIEMAT), Avenida Complutense 22, 28040 Madrid, Spain; and ‡Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom

Communicated by A. Garcia-Bellido, Autonomous University of Madrid, Madrid, Spain, December 17, 2001 (received for review October 19, 2001) UV radiation is the most important environmental skin aggressor, To understand the mechanisms of UV response, we sought to causing cancer and other problems. This paper reports the use of obtain the transcriptional profile of primary keratinocytes after oligonucleotide microarray technology to determine changes in UVB irradiation. Cultured human epidermal keratinocytes were expression in human keratinocytes after UVB treatment. treated with three UV doses, and samples were collected at Examination of the effects of different doses at different times different intervals. Oligonucleotide microarrays containing over after irradiation gave a global picture of the keratinocyte response 6,000 (HuGeneFL, Affymetrix, Santa Clara, CA) were to this type of insult. Five hundred thirty-nine regulated transcripts used to quantitatively assess changes in gene expression. The were found and organized into nine different clusters depending subset of regulated transcripts was classified into self-organizing on behavior patterns. Classification of these genes into 23 func- maps according to their different behaviors. Biological functions tional categories revealed that several biological processes are were assigned to these genes, which were then correlated with globally affected by UVB. In addition to confirming a majority expression patterns. These approaches provide new insights into up-regulation of the transcripts related to the UV-specific inflam- the genes and biological processes involved in the UV response matory and stress responses, significant increases were seen in the and demonstrate the complexity of the UVB transcriptional GENETICS expression of genes involved in basal transcription, splicing, and profile in keratinocytes, allowing global characterization of translation as well as in the proteasome-mediated degradation UV-regulated genes and pathways. category. On the other hand, those transcripts belonging to the metabolism and adhesion categories were strongly down- Materials and Methods regulated. These results demonstrate the complexity of the tran- Cell Culture. Primary human keratinocytes from foreskin were scriptional profile of the UVB response, describe several cellular cultured on a fibroblast feeder layer (50-Gy-irradiated 3T3 J2 processes previously not known to be affected by UV irradiation, cells) as described by Rheinwald and Green (12). Cells were and serve as a basis for the global characterization of UV-regulated grown to 80% confluence in 100-cm2 plates in 3:1 DMEM͞ genes and pathways. Ham’s-F12 medium (GIBCO͞BRL Life Technologies) supple- mented with 10% FBS (BioWhittaker)͞5 mg/ml of insulin he is a physiological barrier that protects the (Sigma)͞0.1 nM cholera toxin (Sigma)͞10 ng/ml of epidermal Torganism against pathogens and chemical or physical dam- growth factor (Serono, Geneva)͞0.5 mg/ml of hydrocortisone age. UV radiation is the most important physical carcinogen (Sigma)͞1% antibiotics (GIBCO͞BRL Life Technologies). in the environment, and the skin is its main target. In this tissue, UV induces photochemical changes that may lead to Irradiation. Fresh culture medium was supplied 24 h before acute effects such as erythema or sunburn (1), or chronic treatment. For irradiation, the medium was removed and the effects that include premature skin aging (2, 3) and skin tumors keratinocytes treated with 10, 20, or 40 mJ͞cm2 UVB. The (4, 5). In recent years, the importance of UV radiation has collected medium was then replaced and the cells maintained in increased because of the thinning of the ozone layer (6) and culture for 4 or 24 h. A bank of six filtered UVB Sankyo 615T8E increasing skin cancer rates (7). UV light affects the skin in 15-W lamps, which emit 80% of the energy in the UVB range as different ways depending on its wavelength. UVA (320–400 measured by a UVX radiometer, were used as the radiation nm) effects are primarily oxidative in nature. UVC (200–290 source. nm) hardly ever reaches the surface of the earth. UVB (290–320 nm) is the most important and is considered the RNA Extraction and Preparation for Hybridization. Total RNA was causative agent of many of the effects attributed to UV (8), isolated by using TRIzol (GIBCO͞BRL Life Technologies). giving rise to mutations in DNA and modifying the pattern of RNA was amplified and labeled with biotin as described by gene expression. This transcriptional regulation is part of the Wodicka et al. (13). cellular reaction to UV-induced stress and operates as a defense mechanism. The known UV response comprises, for Array Hybridization and Scanning. Samples for oligonucleotide instance, the rapid activation of genes with reparative, pro- microarray hybridization were prepared as described by Lock- tective, or apoptotic functions and the communication of stress hart et al. (14), except that hybridization was performed in 1ϫ to other cells via secreted signaling molecules (9). Mes buffer (0.1 M Mes, pH 6.7͞1 M NaCl͞0.01% Triton X-100), Although a number of UV-regulated genes and processes have and the chips were washed in 0.1ϫ Mes buffer. The target been described (see refs. 9 and 10 for review), many of the concentration was 15 ␮g of adjusted fragmented cRNA in 300 molecular events involved in the UV response remain unknown. However, many studies on UV lack physiological relevance because they were performed by using UVC and established Abbreviation: cn, cluster n. epithelial cell lines or fibroblasts. Habitually exposed to UV †To whom reprint requests should be addressed. E-mail: [email protected]. radiation, keratinocytes are more resistant to this insult than are The publication costs of this article were defrayed in part by page charge payment. This fibroblasts (11), which probably means they have developed a article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. specialized response. §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.052678999 PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 2965–2970 Downloaded by guest on September 25, 2021 Fig. 1. Analysis of the experimental variability of the method. The data are represented as log͞log scatter plots of the expression values provided by GENECHIP software. For each graph, only those transcripts present in both samples are considered. The upper and lower boundaries represent a 2-fold change in expression. (A) Control 1 vs. Control 1Ј. Technical variability is provided by the comparison of the expression levels of two different chip analyses performed with the same sample of untreated keratinocytes. The vast majority of the points fall within the Ϯ2-fold limit. (B) Control 1 vs. Control 2. Biological variability is provided by the comparison of two different control samples. A slight increase in the number of data points outside the borders indicates changes in the patterns of gene expression attributed to culture-specific characteristics. (C) Control 1 vs. 40 mJ͞cm2 UVB, 24 h. Changes in gene expression because of the UVB response are represented. Up- and down-regulated genes are represented by the points located outside the Ϯ2-fold lines, although most of the transcripts fall within these limits.

␮m of hybridization solution. Samples were hybridized to Af- Results and Discussion fymetrix GeneChip HuGeneFL arrays. Images were scanned at Experimental System. Primary human epidermal keratinocytes ␮ 3- m resolution by using a GeneArray Scanner made for Af- were grown to subconfluence using culture conditions that fymetrix by Hewlett–Packard. maintain many of the characteristics these cells show in vivo (12). The UVB irradiation doses applied (10, 20, and 40 mJ͞cm2) Analysis. All samples were prepared and hybridized in duplicate. represent low, middle, and high exposures to UV considering GENECHIP ANALYSIS SUITE software (Affymetrix) was used to that, in our experimental system, few changes in expression are analyze image data. Values representing genes not expressed at seen at 10 mJ͞cm2, and nondesired high apoptotic rates are a given experimental point are unreliable and are referred to as observed only at doses over 40 mJ͞cm2 (data not shown). ‘‘absent call’’ entries. To gain a more complete picture, those Irradiation with 40 mJ͞cm2 caused undetectable apoptosis at transcripts that showed at least one ‘‘present call’’ in one 24 h and less than 4% of apoptotic cells at 48 h, as estimated by experimental point in both duplicates were included. When cell viability assays and analysis of DNA content by flow required, the expression levels provided by the ‘‘absent call’’ cytometry (data not shown). These conditions approach the entries were modified to up- or down-regulation values no higher situation in skin during moderate to intense exposure to the sun. than 4-fold. The results of the whole experiment are published Samples were collected at 4 and 24 h after irradiation, time as supporting information on the PNAS web site (www. points for early- and late-regulated genes. The final analysis pnas.org). To classify the temporal profiles of gene expression, therefore comprises the effects of both low and high doses in the cluster analysis was performed by using the GENE CLUSTER 1.1 short and long term. program [http:͞͞www.genome.wi.mit.edu͞MPR͞GeneCluster Microarray experiment results were represented in log͞log (15)]. Genes that, after irradiation, showed differences in their scatter plots to check the consistency of the data. Fig. 1 shows the expression levels of at least a factor of 2.0 in at least one expression pattern of untreated keratinocytes, on the horizontal experimental point in both duplicates were selected for cluster- axis, against its duplicate (Fig. 1A), a control sample from a ing. Biological functions were assigned by using the GENECARDS different keratinocyte culture (Fig. 1B), and a sample of kera- database [http:͞͞bioinfo.weizmann.ac.il͞cards (16)]. tinocytes collected 24 h after irradiation with 40 mJ͞cm2 (Fig. 1C), on the vertical axis. The borderline for discriminating Cloning of Probes for Northern Blots. Primers were designed differential expression was set at Ϯ2-fold. The comparison of the by using GCG’s Primer tool and NetPrimer (http:͞͞www. two duplicate samples (Fig. 1A), with an almost perfectly premierbiosoft.com͞netprimer). Primer sequences can be pro- matched scatter plot, demonstrates the reproducibility of the vided on request. Total RNA was prepared from cultured experimental approach. The representation of untreated vs. keratinocytes by using TRIzol (GIBCO͞BRL Life Technolo- irradiated cells (Fig. 1C) displays a remarkable number of gies). Single-stranded cDNA was synthesized by using Super- regulated genes that fall outside the borderlines. The comparison ScriptII RNase H Reverse Transcriptase (GIBCO͞BRL Life of untreated samples from different cultures (Fig. 1B) shows a Technologies). Probes were amplified by standard PCR by using reduced number of data points outside the Ϯ2-fold lines, which Taq polymerase (Promega). PCR fragments were cloned into the probably indicates the existence of variability associated with the pGEM-T Easy Vector (Promega) and the identity of the PCR culture. These analyses illustrate the reproducibility of the fragments verified by sequencing. method and demonstrate the suitability of this approach for studying the UVB keratinocyte response. Northern Blotting. Twenty micrograms of each RNA sample were To identify UVB-regulated genes, the expression levels of electrophoresed through 1.1% formaldehyde-agarose gels and each irradiated sample were compared with those of control transferred to Hybond-N nylon membranes (Amersham Phar- keratinocytes. Transcripts that showed at least a 2-fold change macia Biotech). Hybridization was performed in 50% form- and a ‘‘present call’’ in at least one experimental point in both amide͞1% SDS͞5 ϫ SSC͞10 mM Tris, pH 7.5͞10% dextran duplicates were selected. On the basis of these criteria, 539 sulfate͞100 mg/ml of salmon sperm. Probes were 32P-dCTP- regulated transcripts were identified (see supporting informa- labeled with Rediprime II (Amersham Pharmacia Biotech) and tion, which is published on the PNAS web site), accounting for Northern blots analyzed and quantified by phosphorimaging by approximately 10% of the genes represented in the HuGeneFL using QUANTITY ONE 4.1.1 software (Bio-Rad). Array.

2966 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052678999 Sesto et al. Downloaded by guest on September 25, 2021 GENETICS

Fig. 2. Cluster analysis of gene expression after UV irradiation. Five hundred thirty-nine genes that changed their expression in at least one experimental point after UV irradiation were grouped into nine clusters with different kinetics. The number of genes in each cluster is indicated. Each cluster is represented by the centroid (average pattern) for genes in the cluster. For each graph, the x axis represents time after irradiation (0, 4, and 24 h) and the y axis the normalized factor of induction or repression. Doses: {,10mJ͞cm2; ᮀ,20mJ͞cm2; ⌬,40mJ͞cm2.

Transcriptional Profiling of the UV Response in Keratinocytes. The are repressed at 24 h in a dose-dependent manner (e.g., PEPCK). GENECLUSTER 1.1 (http:͞͞www.genome.wi.mit.edu͞MPR͞ Cluster 8 (77 transcripts) represents genes whose expression is GeneCluster) program, based on self-organizing map algo- decreased at 4 h but, in contrast to cluster 5, continue to be rithms, was used to organize the regulated genes into different down-regulated at 24 h in a dose-dependent way. Throm- expression patterns. Nine clusters were obtained, representing bospondin 2 is a good example of this group. specific patterns of regulation (Fig. 2). Roughly, clusters 0–4 Of the set of 539 regulated genes, only 249 transcripts changed correspond to five different kinetics of gene activation, whereas their expression4hafterUVirradiation: 183 were repressed and clusters 5–8 show kinetics of repression. Cluster 0 (117 tran- 66 induced. However, the 539 were regulated at 24 h, and the scripts) includes genes with a specific late response to high doses; number of induced and repressed genes was similar at this time they are induced only at 40 mJ͞cm2 and 24 h. An example of this (297 and 242, respectively). These data suggest that the early UV group is Bag-1, a potent inhibitor of apoptosis and Fas-induced response (4 h) leads mainly to transcriptional arrest with the cell death (17). Cluster 1 (79 transcripts) comprises genes that induction of a small number of genes (those included in cluster respond to UV radiation at 24 h in a dose-dependent manner; 3), whereas the late response (24 h) is more complex, involving a good example is SprpII, a constituent of the cornified envelope a higher number of genes up- and down-regulated in similar that appears during epidermal differentiation, previously shown proportions. to be induced by UV (18). Cluster 2 (57 transcripts) includes Northern blots were performed to confirm microarray results. genes slightly repressed at 4 h and induced at 24 h in a Some examples are shown in Fig. 3, which presents genes from dose-independent manner, for example, cyclin G1, a DNA six of the nine clusters. The results for Bag-1, cyclin G1, IL-8, damage-induced transcript (19). Cluster 3 (24 transcripts) is the PEPCK, thrombospondin 2, and sprp2 followed the patterns only one that shows a generalized pattern of induction main- obtained with the microarrays, whereas APR showed slight tained from 4 to 24 h in a dose-dependent manner. Some deviations at 24 h. Northern blots confirm the experimental well-known UV-responding genes, such as IL-8, IEX1, and approach but also indicate that the degree of change in gene COX2, belong to this cluster. In cluster 4, 20 interesting tran- expression calculated by the GENECHIP software is clearly un- scripts are present that do not change at 4 h but are specifically derestimated, in agreement with other reports (20). induced at 24 h with a dose of 20 mJ͞cm2. Clusters 5–8 represent kinetics of repression by UV. Cluster Groups of Genes Regulated by UVB According to Their Biological Role. 5 (49 transcripts) includes genes (e.g., integrin ␤4) down- The different behaviors revealed by clustering analysis reflect regulated at4hinadose-independent manner, which return to the complexity of the UV response and should help elucidate the basal levels at 24 h, except for 40 mJ͞cm2. In cluster 6 (42 intricate processes that take place in UV-irradiated cells. To this transcripts), genes suffer up-regulation at 4 h and down- end, the regulated transcripts were classified according to bio- regulation at 24 h when 40 mJ͞cm2 are provided; examples of this logical function. Because of the relatively small size of the group are junD and gadd45. Genes in cluster 7 (74 transcripts) sample, it was not convenient to use a very detailed classification

Sesto et al. PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 2967 Downloaded by guest on September 25, 2021 Some genes may have several functions, so we have chosen the function for which each gene is best known. In some cases, transcripts in the same category may have opposite roles (for instance, activators and inhibitors). Correlations were sought between the genes in functional groups and their expression patterns (activation͞repression). In many categories, correlations were not evident, perhaps in part because of the complexity of the categories, as shown by the transcription and intracellular signaling groups. However, there were eight functional categories (marked in bold type in Table 1) in which more than two-thirds of the components behaved in a similar manner, indicating the existence of biological processes globally induced or repressed by UV. A generalized down- regulation, shared by at least 75% of the components, was observed in the cell-adhesion and metabolism categories. On the other hand, global induction was seen in the stress response, structural , inflammatory response, mRNA splicing, proteasome-related proteins, and translation categories.

Genes Involved in Stress Response, Inflammation, and Apoptosis. Genotoxic stress, such as that caused by UV irradiation, is known to increase the transcription of certain genes. Among our data, transcripts involved in the oxidative stress response, such as manganese superoxide dismutase [cluster 1 (c1)] and some heat-shock proteins (c0 and c1), are up-regulated after UVB Fig. 3. Confirmation of microarray results by Northern blot analysis. RNAs treatment, as described by others (reviewed in ref. 21). Up- were prepared from keratinocytes treated with the indicated doses either 4 or regulation of genes related to DNA damage, such as IEX-1 (c3), 24 h after exposure to UVB. Representative Northern blots for genes with altered expression after UVB irradiation are shown. 7S RNA was used as a XPC-complementing proteins (c0 and c4), and Gadd45 (c6) was loading control. The molecular weights of the hybridizing bands agreed with also apparent. It is also well known that UV light produces an the reported sizes of the corresponding transcripts. inflammatory reaction in the skin in which inflammation-related genes are induced (reviewed in ref. 22). In agreement, our data show that 83% of the altered genes in the inflammatory response of gene function [such as that at reported for yeast by the MIPS category are up-regulated. These include cytokines such as IL-8 (http:͞͞mips.gsf.de͞proj͞yeast͞catalogues͞funcat͞)]. There- (c3), melanoma growth stimulatory activity (c0), and the met- fore, we established a list of 23 different categories (or functional abolic enzyme COX2 (c3), previously shown to be induced by groups) to sort the 539 transcripts according to their biological UV (23, 24). The pathway of IL-1 is particularly activated, with roles as defined by the GENECARDS database (16) (Table 1). increases in the transcription of several forms of the cytokine and

Table 1. Functional classification of UV-regulated genes Function c0 c1 c2 c3 c4 c5 c6 c7 c8 Total

Adhesion 2 3 3 1 2 6 7 13 37 Angiogenesis 2 3 5 Apoptosis and survival 1 4 1 2 1 1 3 13 Cell cycle 1 2 3 1 1 3 2 13 Cell growth 2 1 2 2 7 Development 3 1 2 1 3 10 Homeostasis 4 3 1 8 Inflammatory response 6 3 1 5 1 2 18 Intracellular signaling 22 10 9 5 3 13 6 8 11 87 Lysosomal enzymes 2 1 1 1 2 1 8 Metabolism 4 2 4 0 2 10 19 10 51 Proteases 2 2 1 2 1 4 12 Protease inhibitors 2 2 1 2 7 Proteasome 6 8 4 1 1 1 21 Replication 1 2 1 1 2 2 9 Respiratory chain 2 1 1 1 5 Splicing 3 2 3 3 1 1 13 Stress response 7 5 2 2 1 2 4 2 25 Structural proteins 5 12 5 3 1 245542 Transcription 21 3 8 2 1 13 4 4 4 60 Translation 6 4 1 1 1 1 2 1 17 Transport 8 7 2 2 414533 Others 9 5 6 1 1 523638

Columns c0–c8 indicate the number of genes of each cluster in the functional group. Functions in bold type are globally up- or down-regulated.

2968 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052678999 Sesto et al. Downloaded by guest on September 25, 2021 receptor, as well as TRAF6 (c0), a signal transducer in the NF␬B the cornified envelope was found. These up-regulated genes pathway, which activates I␬B kinase in response to the proin- include keratinocyte transglutaminase (c1), its substrates sprpII flammatory cytokines IL-1 (25) and the IL-1 adapter (c1) and (c4), as well as genes from other categories MyD88 (c3) (26). Other molecules, such as major histocompat- also involved in this process, such as PAI-2 (c1), annexin (c2) ibility complex class I E (c0), are also up-regulated. (34), and cystatin M (c1) (35). Only the up-regulation of sprpII Depending on the dose, UVB radiation can drive cells to has been previously reported (17). Although the genes described apoptosis. As previously mentioned, these experiments were above code for proteins characteristic of suprabasal, differenti- performed by using physiological doses of between 10 and 40 ated skin keratinocytes and were also expressed in control cells ͞ 2 mJ cm —and even at the latter, apoptotic levels were not (in agreement with the observation of sporadic foci of differ- significant. When genes involved in apoptosis and survival are entiation), the cultures showed a strong prevalence of keratin K5 analyzed, a complex pattern appears. In control keratinocytes, ͞ and K14 mRNAs (the most abundant transcripts according to apoptosis survival transcripts in the array are called absent or the microarray data and characteristic of proliferative keratin- are expressed only at very low levels. After UV irradiation, the ocytes) over keratins K1 and K10 (typical of differentiated cells). expression of some of the most important genes related to ␣ Other components of the late steps of differentiation (for keratinocyte apoptosis [tumor necrosis factor (TNF) , FasL, and instance, filagrin and loricrin) were not detected (data not bak, for instance] remained unaltered (note that genes like Bax, shown). UV did not affect other keratins or keratinocyte-specific bad, and bid are not represented in the chip). However, several cytoskeletal genes except for the induction of K18 (c1), present other transcripts, some previously undescribed in keratinocytes, at very low levels in nonirradiated cultures (not shown). underwent changes in expression. At the short interval (4 h), two proapoptotic genes were induced in a dose-dependent manner: Proteasome Degradation. IPL (c3), also known as TSSC3, whose murine homologue Genes involved in the proteasome- TDAG51 is implicated in FasL-mediated apoptosis (27), and ubiquitin degradation pathway form another important group. APR (c3), the ortholog of the murine p53-dependent proapo- Nineteen of the 21 transcripts in this category were up-regulated ptotic gene Noxa (28). In addition, three of the four genes only at 24 h. This generalized behavior indicates that irradiated repressed at 4 h are antiapoptotic [semaphorin E (c8), the cells increase the turnover of many proteins, which could be due FAS-associated protein tyrosine phosphatase 1 (c8), and MCL1 either to the replacement of damaged molecules or to the need to significantly change the proteome of the cell. Only two genes (c5)], whereas only one [TRAIL (c8)] is proapoptotic. At 24 h GENETICS after irradiation, however, there was an induction of both were down-regulated in this category, one of which [cyclin- proapoptotic [BNIP3 (c1), TIAR (c2), and TNFR (c4)] and selective ubiquitin carrier protein (c7)] is involved in the de- antiapoptotic [PEA15 (c1), NAIP (c1), TNFAIP3 (c1), and struction of mitotic cyclins in the exit from mitosis into G1. This BAG-1 (c0)] transcripts. Although the functions of many of these transcript was down-regulated at 24 h, and its repression may be genes are still insufficiently clear to develop a model, it seems required for cell cycle arrest, because a dominant negative form that after UV irradiation, there could be an initial shift toward of this protein prevents entry into the cell cycle (36). apoptosis (five of the six gene changes at 4 h favor apoptosis), which stabilizes at 24 h when the expression of proapoptotic and Transcription, mRNA Splicing, and Translation. Transcription is a antiapoptotic genes is balanced. Because no significant apoptosis complex category that includes many genes and has no evident was observed in the cultures, the ‘‘proapoptotic’’ response at 4 h bias toward activation or repression. However, there was a could represent preparation of the cells to undergo apoptosis in generalized up-regulation of basal transcriptional machinery, the case of severe cell damage. which included different subunits of RNA Pol II such as TFIIA (c0), TFIIB (c0), TAFII32 (c0), and TAFII55 (c0), as well as Structural Proteins. Forty-two genes coding for structural proteins up-regulation of genes involved in mRNA elongation. Ten of the changed their expression in response to UV, and most of them 11 transcripts of this type were induced, most belonging to cluster were induced. Transcripts in this category can be subdivided into 0 (induction at 24 h, 40 mJ͞cm2). In addition, many transcrip- three groups: cytoskeletal, nuclear-related, and keratinocyte- tional activators [e.g., HIF-1␣ (c2), AP-2 (c2), p65 (c0)] and specific genes. The group of transcripts coding for cytoskeletal repressors [e.g., YY1 (c2), SAP18 (c0), ATF3 (c3)] were in- proteins showed no common behavior. However, 9 of the 13 creased with different kinetics. Some of these transcriptional genes coding for nuclear structural proteins were up-regulated, regulators are highly relevant in keratinocyte biology, and their including several histones [H2A (c3), H2B (c3), and H3 (c0) regulation by UVB may affect different cellular functions, e.g., isoforms]. Interestingly, the induction of genes involved in AP-2 (c2) a transcription factor proposed to be involved in transcriptional repression by histone deacetylation was also epidermal specificity (37). AP-2-binding sites are often found in observed, as were those of histone deacetylase HDAC1͞RPD3 epidermal promoters, particularly in cytoskeletal genes (ref. 37 (c1) and its associated proteins SAP18 (c0), involved in tran- and refs. therein; refs. 38, 39). However, AP-2-responsive genes scriptional repression via the tethering of the sin3 complex to core histone proteins (29), and the GLI-Kruppel-related protein are not up-regulated in our experiments, in agreement with the YY1 (c2) (these last two factors are included in the ‘‘transcrip- described requirement for cooperation of AP-2 with other tion’’ category). It has been proposed that the complex HDAC1͞ transcription factors (37). sin3 is recruited to the promoters of specific genes by factors like mRNA splicing-related transcripts underwent generalized in- YY1, where it deacetylates histones in a targeted way, leading to duction (11 of 13), as did those involved in protein translation (13 local changes in chromatin structure and specific inactivation of of 17). Nine of these latter genes are related to the initiation gene expression (30, 31). SMRT (c5), a corepressor involved in phase of translation, and eight were up-regulated. Moreover, the this mechanism of inhibition for retinoid and thyroid-hormone only down-regulated gene in this group was the INF-inducible receptors genes (32, 33), is repressed at 4 h and recovered at 24 h. RNA-dependent protein kinase PKR (c5), an inhibitor of pro- Although currently it is not known which genes are silenced by tein synthesis. In addition, four genes related to posttranslational these factors (except in the case of SMRT), it is conceivable that modifications were also up-regulated. The generalized increase this mechanism could be responsible for at least part of the of transcripts involved in translation further confirms that the transcriptional inhibition caused by UV. experimental conditions do not induce significant levels of There are also keratinocyte-specific proteins in this group. A apoptosis, because this process rapidly inhibits the rate of protein generalized induction of transcripts involved in the formation of synthesis at the level of polypeptide chain initiation (40).

Sesto et al. PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 2969 Downloaded by guest on September 25, 2021 Metabolism. More than 75% of the genes in this category were and dose-independent transcriptional arrest during the early UV down-regulated, especially those related to glucidic, lipidic, and response (4 h), plus the induction of a concrete set of genes (c3) amino acidic metabolism. This was the case with key enzymes involved in the activation of well-known UV-response pathways such as hexokinase 1 (c8), phosphoenolpyruvate carboxykinase (inflammation, stress response, etc.) At 24 h, the transcriptional (c7), and isocitrate and succinate dehydrogenases (c7 and c8, arrest is overcome, and more complex transcriptional patterns respectively), which are involved in glucose metabolism; fatty are found. Besides confirming the induction of genes related to acid and long chain acyl-CoA synthetases (c8 and c7, respec- the UV-specific inflammatory and stress responses, the regula- tively), which are involved in fatty acid metabolism; and several tion of several processes mainly affected at 24 h postirradiation amino acid synthetases. However, some important transcripts in was revealed. Analysis of functional categories suggests that this group were up-regulated, such as glycogen synthase kinase keratinocytes undergo an activation of cornified envelope syn- 3␣ (c0) and glycogenin (c1), which take part in glycogen thesis (induction of keratinocyte-specific structural proteins), metabolism. This complex pattern of gene regulation might changes in their energetic requirements (repression of glycolysis reflect the alteration of energetic requirements and metabolic and other catabolic pathways and induction of gluconeogenesis), pathways that cells undergo to cope with the effects of UVB and a generalized decrease in their adhesive properties. At 40 irradiation, in particular an inhibition of glycolysis and an mJ͞cm2, the data reflect a targeted elimination of proteins increase in gluconeogenesis. because of the need to either remove UV-damaged proteins or change the proteome of the cells, as well as an increase in general Adhesion. The adhesion category was also clearly affected by mechanisms of transcription and translation, probably to obtain UVB radiation. Remarkably, 75% of changes were down- new molecules involved in the UV response or in the replace- regulations. Analysis of the altered genes strongly suggests a ment of UV-damaged proteins. These results unveil the com- decrease in the adhesive properties of irradiated keratinocytes. plexity of the UVB response in keratinocytes and serve as a basis For instance, hemidesmosomal [e.g., integrins ␣6 (c7) and ␤4 for undertaking a global characterization of UV-regulated genes (c5), BPAG1 (c8) and BPAG2 (c8)], desmosomal [e.g., desmo- and pathways. plakin I (c8) and desmocollin I (c8)], gap junctional [e.g., connexin 26 (c8)], nonhemidesmosomal integrins [e.g., ␣3 (c8), Note Added in Proof. Recently, Li et al. (41) also reported the transcrip- ␤1 (c7) and ␤5 (6)], and other cell-to-matrix attachment-related tional program of human epidermal keratinocytes in response to UV. components were down-regulated. To our knowledge, this is the We thank Dr. C. Johnson (Pfizer) for help with the microarray exper- first study reporting global repression of these molecules after iments and Drs. M. Del Rı´o, P. Perez, and F. Larcher (CIEMAT) for UVB irradiation. assisting with keratinocyte cultures. The technical assistance provided by In summary, these results describe and help explain the P. Hernandez is acknowledged. This project was supported by Grant processes that occur during the UV response. The expression SAF98–0047 from the Spanish Comision Interministerial de Ciencia y patterns obtained in the cluster analysis are suggestive of a wide Tecnologia (Ministry for Science and Technology).

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