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Detection of Differentially Regulated Genes in Keratinocytes by cDNA Array Hybridization: Hsp27 and Other Novel Players in Response to Artificial Ultraviolet Radiation

2001; Elsevier BV; Volume: 116; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2001.01347.x

1523-1747

Bernd Becker, Thomas Vogt, Michael Landthaler, Wilhelm Stolz,

Animal Genetics and Reproduction

cDNA array technology was used to identify novel genes participating in the ultraviolet response of cultured human keratinocytes. cDNA arrays representing more than 50,000 different cDNA clones were hybridized with complex probes generated by SMART-polymerase chain reaction amplification of 150 ng of total RNA extracted 24 h after ultraviolet irradiation. Fifty-one clones with differential hybridization signals were detected, representing 19 different sequences; 10 known genes (seven ultraviolet induced, three ultraviolet suppressed) and further nine expressed sequence tags of unknown genes. In seven of 10 genes the data from cDNA arrays probed with SMART-cDNA could be confirmed by northern blot analysis. The 27 kDa heat shock protein mRNA was induced. Keratins 6 and 17, markers for the hyperproliferative status of keratinocytes, were among the ultraviolet suppressed genes. The change of expression profile of keratins indicates a differentiation towards a phenotype of keratinocytes present in the suprabasal layers of the epidermis. These mechanisms may contribute to the ultraviolet protective function of the epidermis and to the anti-proliferative action of ultraviolet in the therapy of psoriasis. We also detected an induction of adenylyl cyclase associated protein and the suppression of G(s)α (a stimulating subunit of the trimeric membrane bound GTPase). cDNA array technology was used to identify novel genes participating in the ultraviolet response of cultured human keratinocytes. cDNA arrays representing more than 50,000 different cDNA clones were hybridized with complex probes generated by SMART-polymerase chain reaction amplification of 150 ng of total RNA extracted 24 h after ultraviolet irradiation. Fifty-one clones with differential hybridization signals were detected, representing 19 different sequences; 10 known genes (seven ultraviolet induced, three ultraviolet suppressed) and further nine expressed sequence tags of unknown genes. In seven of 10 genes the data from cDNA arrays probed with SMART-cDNA could be confirmed by northern blot analysis. The 27 kDa heat shock protein mRNA was induced. Keratins 6 and 17, markers for the hyperproliferative status of keratinocytes, were among the ultraviolet suppressed genes. The change of expression profile of keratins indicates a differentiation towards a phenotype of keratinocytes present in the suprabasal layers of the epidermis. These mechanisms may contribute to the ultraviolet protective function of the epidermis and to the anti-proliferative action of ultraviolet in the therapy of psoriasis. We also detected an induction of adenylyl cyclase associated protein and the suppression of G(s)α (a stimulating subunit of the trimeric membrane bound GTPase). adenylyl cyclase associated protein expressed sequence tag glycerin aldehyde 3-phosphate dehydrogenase stimulating subunit of the trimeric membrane bound GTPase heat shock protein Integrated Molecular Analysis of Genomes and their Expression keratin macrophage inhibitory cytokine-1 neuroblastoma apoptose-related RNA binding protein ribosomal protein synaptic glycoprotein 2 Ultraviolet (UV) irradiation of keratinocytes induces various cascades of changes of gene expression, including both soluble paracrine molecules, e.g., growth factors and cytokines, as well as nonsecreted molecules, e.g., adhesion molecules, transcription factors, and cell cycle regulators (Inohara et al., 1995Inohara S. Kitano Y. Kitagawa K. Cell cycle regulators in the keratinocyte (cyclin-cdk).Exp Dermatol. 1995; 4: 1-8Crossref PubMed Scopus (10) Google Scholar;Krutmann and Grewe, 1995Krutmann J. Grewe M. Involvement of cytokines, DNA damage, and reactive oxygen intermediates in ultraviolet radiation-induced modulation of intercellular adhesion molecule-1 expression.J Invest Dermatol. 1995; 105: 67S-70SCrossref PubMed Scopus (106) Google Scholar;Kondo et al., 1997Kondo S. Kooshesh F. Sauder D.N. Penetration of keratinocyte-derived cytokines into basement membrane.J Cell Physiol. 1997; 171: 190-195Crossref PubMed Scopus (49) Google Scholar). Detailed knowledge about the regulation of these pleiotropic effects is essential for the understanding of the complex interaction between melanocytes and keratinocytes forming the so-called epidermal melanin unit and thereby the analysis of the carcinogenic effects of UV. The epidermal melanin unit provides the intrinsic sun protection against UVB, which presents the most important environmental hazard to skin. Even a single exposure to UVB radiation may lead to DNA damage, which is the main trigger for carcinogenesis. Until now differential gene expression could be analyzed only in a step by step fashion using techniques such as reverse transcriptase PCR, in situ hybridization, or immunohistochemistry, which are not suitable for a simultaneous analysis of the complex changes of gene expression that characterize the mammalian UV response. With the human genome project, a new era has begun as high-density cDNA arrays became available allowing the analysis of the expression of thousands of genes at a time. In order to study the complex UV response of keratinocytes, we applied a series of high density cDNA arrays each representing 27,648 IMAGE cDNA clones from the human genome project. In this study we demonstrate the value of this technique by showing differential expression of 10 known genes and further nine cDNA clones, which represent genes not yet characterized. A positive correlation of detection of differential expression between cDNA array and northern blot analysis can be found. We show that the gene encoding the heat shock protein 27 kDa (Hsp27) is induced by UV irradiation, suggesting its protective function against UV-induced apoptosis in keratinocytes. In addition, we demonstrate that the new combination of arrays hybridized with probes, which were generated by the amplification of cDNA, synthesized by oligo-dT-primed transcription of 150 ng of total RNA (Clontech SMART system), result in reproducible hybridization patterns. This presents a new dimension of applications in medicine as the array analysis of small tissue biopsies from patients with only minute amounts of RNA available is essential for studying the intricacies of skin cancer evolution. Furthermore, this study confirms previous estimates about the complexity of transcriptional changes upon UVB irradiation (Friedberg et al., 1995Friedberg E. Walker G. Siede W. Regulatory Responses to DNA-Damaging Agents in Eukaryotic Cells. ASM Press, Washington1995Google Scholar;Vogt et al., 1997Vogt T. Mathieu-Daude F. Kullmann F. Welsh J. McClelland M. Arbitrarily Primed PCR of DNA and RNA. John Wiley. & Sons, Inc., New York1997Google Scholar). Primary keratinocytes (CellSystems, St Katharinen, Germany) were grown to 80% confluence with keratinocyte growth medium (keratinocyte growth medium bulletkit, CellSystems) on 150 cm2 culture dishes (Falcon, Heidelberg, Germany). For UVB (312 nm) treatment cells were washed once with phosphate-buffered saline at 37°C and irradiated through a thin layer of phosphate-buffered saline (2 ml) using the Biometra transilluminator (Göttingen, Germany) with 0, 50, 100, and 200 mJ per cm2. Phosphate-buffered saline was subsequently replaced with keratinocyte growth medium and the cells were incubated for 24 h. The spectrum of the UV illuminator as provided by the supplier is very similar to standard FS-40 sunlamps (Brown et al., 2000Brown D.B. Peritz A.E. Mitchell D.L. Chiarello S. Uitto J. Gasparro F.P. Common fluorescent sunlamps are an inappropriate substitute for sunlight.Photochem Photobiol. 2000; 72: 340-344Crossref PubMed Scopus (42) Google Scholar). Cells were washed once with phosphate-buffered saline and lyzed directly in the culture dish containing 18 ml of Trizol (Gibco Lifetechnologies, Karlsruhe, Germany). Total RNA was isolated from the lysate according to manufacturer's instructions. Contaminating DNA was eliminated by DNAase treatment using the RNase-free DNase kit from Qiagen (Hilden, Germany). RNA concentration was determined photometrically and quality was checked by agarose gel electrophoresis. Double-stranded cDNA was synthesized by reverse transcription of 150 ng of total RNA and subsequent amplification of the first strand cDNA using the SMART system (Clontech, Heidelberg, Germany). In order to get a maximum yield as well as to avoid abundance normalization, the optimal number of cycles was determined. Aliquots were taken from the polymerase chain reaction (PCR) reaction after 15, 18, 21, 26, and 32 cycles, and analyzed by agarose gel electrophoresis. In accordance with the manufacturer's instructions an optimal number of cycles was assumed when a moderately strong smear of cDNA ranging from 0.5 to 6 kb with several bright bands was seen, which also corresponds to abundant transcripts. A second reaction was prepared and cycled to the optimal number of cycles. The double-stranded cDNA was purified with the Qiagen PCR purification kit. 100 ng of the double-stranded cDNA was labeled radioactively with α32P-deoxycytidine triphosphate by random primed Klenow fragment synthesis. The cDNA arrays number 10 and 19 from the IMAGE filter collection of the Resource Center Primary Database (RZPD, Berlin, http://www.rzpd.de) were prehybridized in roller bottles at 38°C for 6 h (3 × sodium citrate/chloride buffer, 50 mM Tris–HCl (pH 7.5), 20 mg per ml tRNA, 20 mg per ml boiled single stranded salmon sperm DNA, 1 mM ethylenediamine tetraacetic acid, 1 × Denhardt's solution). Subsequently, the radioactive probe was added to a final concentration of 5 × 106 cpm per ml hybridization solution. Hybridization was performed under the same conditions for 18 h. Nonspecifically bound radioactive probe was washed off with buffer (0.1 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate) at 65°C for 30 min. Filters were exposed for autoradiography for 18 h. Four sets of two different cDNA arrays (nos 10 and 19) were obtained from the RZPD. Filter number 10 represented cDNA clones from the library 188 (Soares melanocyte 2NbHM) and library 189 (Soares fetal placenta 2NbHP8-9W). Filter number 19 represented cDNA clones from the libraries 116, 363, 385, 361, 371, and 382 (Soares ovary tumor NbHOT, Soares subtracted NhHMPu-S1, Soares 8–9 wk fetus Nb2HF8–9w, NCI_CGAP_GCB1 germinal B cell, Soares testis NHT, NCI_CGAP_Pr2 low-grade preneoplastic lesion). Each IMAGE filter contained 27,648 Escherichia coli colonies spotted as duplicates harboring plasmids. E. coli bacteria are spotted from 384 well plates on to the nylon membranes. After growing, the bacteria are lyzed on the membranes and the DNA is cross-linked by UV irradiation on to the membrane. The bacteria contained cDNA fragments representing parts of known transcripts, sequenced expressed sequence tags (EST) or yet unsequenced cDNA fragments. Detailed information about the filters and libraries is available at http://www.rzpd.de Hybridization signals on the autoradiographies were densitometrically analyzed using the PC version of the NIH-Image software, ScionImage (Scion, MD). The co-ordinates of clones exhibiting at least a 3-fold difference in hybridization signal intensity were calculated and entered into the online identification form of the RZPD Berlin (http://www.rzpd.de). The detected clones were obtained from the RZPD as stab cultures. Plasmids were prepared from the clones and sequenced. The sequences were compared with the NCBI databases using the basic local alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST). Total RNA (15 μg) was separated by formaldehyde agarose gel electrophoresis [40 mM 3-[N-morpholino] propane sulfonic acid (pH 7.0), 10 mM sodium acetate, 1 mM ethylenediamine tetraacetic acid, 2.2 M formaldehyde] and transferred with 20 × sodium citrate/chloride buffer (3 M NaCl, 0.3 M sodium citrate, pH 7.0) onto nylon membrane (Magna, Micron Separations, Westborough). The RNA was cross-linked to the membrane by UVC irradiation (125 mJ per cm2) using the Biometra transilluminator. Hybridization conditions were the same as described for the array hybridization. DNA fragments for labeling were generated by restriction of the corresponding IMAGE clone plasmids with EcoRV/NotI. Fragments of the plasmids were separated by agarose gel electrophoresis and the gene-specific fragment was extracted from the agarose gel using the QIAquick Gel extraction kit (Qiagen, Hilden, Germany). The following clones were used: IMAGp998K11559, IMAGp998F191927, IMAGp998G191927, IMAGp998N041937, IMAGp998F101930, IMAGp998E20578, IMAGp998O02594, IMAGp998C09586, IMAGp998I03560, and IMAGp998I121869 representing the genes macrophage inhibitory cytokine-1 (MIC-1), G(S)α, Keratin 6, Keratin 17, ribosomal protein (RP) S12, RPL11, adenylyl cyclase associated protein (CAP), neuroblastoma apoptose-related RNA binding protein (NAPOR), Synaptic glycoprotein 2 (SG-2), Hsp27, respectively. GAPDH (IMAGp998I101860) and β-actin (IMAGp998P211859) served as controls. The probe (50 ng of a purified DNA fragment) was labeled radioactively with α32P-deoxycytidine triphosphate by random primed Klenow fragment synthesis. Nonspecifically bound radioactive probe was washed off with buffer (0.1 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate) at 50°C for 30 min. The membranes were exposed to autoradiography for 18 h. The northern blots were carried out two times on independently cultured and UV-treated keratinocytes. In an attempt to identify genes responsive to varying doses of UV radiation in keratinocytes, cDNA array technology was used allowing us to profile the expression of about 50,000 genes, which were represented as cDNA fragments spotted on one pair of filters (nos 10 and 19). Total RNA was prepared from keratinocytes 24 h after cells had been treated with increasing doses of UV (312 nm) (0, 50, 100, and 200 mJ per cm2). The radioactive probes for hybridizing the cDNA arrays were prepared using 150 ng of total RNA as template. For each UV dose a pair of two cDNA array filters (nos 10 and 19) was hybridized. Hybridization signals showing different intensities were analyzed densitometrically. Only clones displaying at least a 3-fold difference of hybridization signals were taken into consideration. Among the some 27,000 cDNA clones on each filter an average of about 500 signals was observed, which represented about 2% of all EST spotted. Twenty-one clones on filter no. 10 and 30 clones on filter no. 19 displayed a differential hybridization signal (≈ 4% and 6%). Owing to a high degree of redundancy within the libraries spotted on the filters, the 51 clones corresponded to 10 different known genes and nine novel EST sequences. As shown in Table I seven of the identified genes were UV induced, whereas three were UV suppressed. The factor of induction or suppression was calculated from the signal ratio of irradiated (200 mJ) vs nonirradiated cells. Eight of the nine EST without any significant homology to any known gene were UV inducible and one was suppressible (data not shown).Table ITable 1. Detection of differential transcription of genes by array analysis in UVB irradiated keratinocytes Open table in a new tab Northern blot analysis (Figure 1) was used for confirming the differential gene expression as indicated by differing hybridization signals. All northern analysis were carried out two times from independently cultured and UV-treated keratinocytes to demonstrate the reproducibility. The expression levels of the genes analyzed, which were normalized according to the GAPDH signal, are shown in Figure 1(a) for induced genes and in Figure 1(b) for suppressed genes (one experiment of two independent northern blot analysis is shown). Hsp27 expression was concentration-dependently induced on UV irradiation: 2.4-fold at 50, 2.5-fold at 100, and 4.4-fold at 200 mJ per cm2 (Figure 1a). These factors are very similar to those calculated by array analysis (Table II). Similarly, we analyzed the change of expression of CAP and NAPOR in comparison with GAPDH. The expression of both genes showed an increase after UV irradiation, which confirmed the data shown by array analysis, although the factors of induction for CAP and NAPOR assessed by cDNA array hybridization differed from those obtained by northern blots (59 vs 1.3 and 4 vs 1.4, respectively). SG-2 gene expression was relatively constant as rated by northern blot analysis in one experiment, but showed an increase of a factor of two in the second experiment (Table II). Also by array analysis an induction was observed (factor 5.5, Table II). One of the array signals proved to be falsely positive: the gene for the ribosomal protein S12 (RPS12) was UV induced by a factor of 37 as calculated by array analysis. In contrast, both northern analysis revealed a 2-fold suppression of RPS12 (Figure 1a, Table II). MIC-1 as well as the ribosomal protein L11 (RPL11) were not detectable in northern blot. Only the second northern analysis showed an increase of a factor of 1.1 for RPL11 expression. We further analyzed the genes in northern blot, which were suppressed after UV irradiation (Figure 1b). As shown in Figure 1(b), the factors of the signal ratios of Krt6/GAPDH gene expression did not change significantly after irradiation with 50 or 100 mJ per cm2 (1.2 and 1.0, respectively) but decreased to less than 0.3 after irradiation with 200 mJ per cm2 (Figure 1b). Similarly, the factors of the expression ratio of coupling protein G(s)α mRNA decreased after 50 mJ per cm2 (2.5-fold), 100 mJ per cm2 (1.4-fold), and 200 mJ per cm2 (1.8-fold) (Figure 1b). The factor of the ratio of the keratin 17 (Krt17) gene expression decreased after UV irradiation 3.9-fold. These results correlate with the data from array analysis as shown in the summary in Table II. When β-actin was used for normalization of the northern blots, the factors of induction or repression were similar indicating the reliability of the comparison with GAPDH (data not shown).Table IIComparison of ratios of gene expression changes as determined by array and northern analysis (factors determined twice)GeneFactor by arrayFactor by northern (experiment 1/2)Hsp274.334.4/2.0CAP591.3/1.2NAPOR41.4/1.2SG-25.51.0/2.0RPS12370.5/0.4RPL113NDaNot detected./1.1MIC-111ND/NDKrt170.290.3/0.2Krt6<0.30.6/0.6G(s)α0.290.6/0.5a Not detected. Open table in a new tab A cDNA array hybridization technique was used for studying the complex effects of UV radiation in keratinocytes. From 27,648 different cDNA clones spotted on two IMAGE cDNA arrays, about 2% hybridized with the complex probes generated from keratinocytes.de Saizieu et al., 1998de Saizieu A. Certa U. Warrington J. Gray C. Keck W. Mous J. Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays [see comments].Nat Biotechnol. 1998; 16: 45-48Crossref PubMed Scopus (195) Google Scholar assumed that a 2-fold change in signal intensity is significant using oligo nucleotide arrays. As the IMAGE cDNA arrays spotted with E. coli colonies could bear slightly more variation in the amount of cDNA spotted, we considered those clones as significantly induced or repressed, which displayed an at least 3-fold difference in signal intensity. When applying this as a threshold 4% and 6% of the clones showed differential hybridization signals on filter no. 10 and no. 19, respectively (Table I). Owing to the redundancy of the cDNA filters these 51 clones represented 10 known genes and nine EST of novel genes. The known genes were selected as candidates for verification in northern blot analysis. Meanwhile filters are also available that are spotted with PCR fragments, thereby ensuring a higher reproducibility; however, the colony filters contain about five times more cDNA, increasing the chance to detect novel players. In order to be able to ensure the reliability of array hybridization and the RNA/cDNA amplification techniques, we confirmed the results from the array analysis for seven of 10 genes by northern blot. One gene turned out to be falsely positive and the remaining two could not be found in northern blot. As shown in Table II the induction (Hsp27, CAP, NAPOR, SG-2) or repression (Krt6, Krt17, G(S)α) detected was quite consistent within the two methods. In the case of CAP, NAPOR, SG-2, Krt6, and G(s)α the array showed a more pronounced difference than northern blot. These differences between the two methods might be explained by the amplification of cDNA for probe preparation, leading to a shift in the profile of the transcribed messages displayed. The expression of MIC-1 and RPL11 is probably too low, as no signal in northern blot could be detected but the amplification for probe preparation might have enhanced the sensitivity of array analysis for these messages; however, there was a clear discrepancy in the results from array and northern blot analysis regarding the RPS12 expression.Bertucci et al., 1999Bertucci F. Bernard K. Loriod B. et al.Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples.Hum Mol Genet. 1999; 8: 1715-1722https://doi.org/10.1093/hmg/8.9.1715Crossref PubMed Scopus (156) Google Scholar showed that the intensity of hybridization signals depends on the amount of target DNA molecules on the array available for hybridization. As the accuracy of spotting E. coli colonies and the amount of plasmid DNA produced by the bacteria determines the amount of target DNA molecules on each spot, some variations in hybridization signals might occur caused by deviations in these factors. Especially small differences between the results of array and northern blot analysis – as demonstrated for NAPOR, SG-2, Krt6, and G(s)α – could be explained by different amounts of target DNA available for hybridization in each spot. Our results indicate that differences in the mRNA expression could be detected despite using a RNA amplification protocol. It appears to be very important that cycling is carefully monitored by agarose gels and overcycling is to be avoided in order to provide probes for hybridization that reliably represent the expression profile of the cells analyzed. Using cDNA array analysis of primary keratinocytes, we detected an induction of Hsp27 upon UV irradiation, which was confirmed by northern blot analysis (Figure 1a). It was demonstrated earlier, that several Hsp (e.g., Hsp72, Hsp90) were inducible by UVB irradiation (Maytin, 1992Maytin E.V. Differential effects of heat shock and UVB light upon stress protein expression in epidermal keratinocytes.J Biol Chem. 1992; 267: 23189-23196PubMed Google Scholar). Hsp27 and Hsp70 are thought to protect cells from apoptosis induced by various stimuli (Jaattela, 1999Jaattela M. Escaping cell death: survival proteins in cancer.Exp Cell Res. 1999; 248: 30-43https://doi.org/10.1006/excr.1999.4455Crossref PubMed Scopus (566) Google Scholar). Hsp27 appears to be also upregulated upon differentiation of keratinocytesTrautinger et al., 1995Trautinger F. Kindas-Mugge I. Dekrout B. Knobler R.M. Metze D. Expression of the 27-kDa heat shock protein in human epidermis and in epidermal neoplasms: an immunohistological study.Br J Dermatol. 1995; 133: 194-202Crossref PubMed Scopus (82) Google Scholar andNozaki et al., 1997Nozaki J. Takehana M. Kobayashi S. UVB irradiation induces changes in cellular localization and phosphorylation of mouse HSP27.Photochem Photobiol. 1997; 65: 843-848Crossref PubMed Scopus (17) Google Scholar found, that murine Hsp27 is induced, phosphorylated and translocated into the nucleus upon UVB stimulation. Similarly, an accumulation of Hsp27 occurring with the differentiation-mediated decrease in embryonic stem cell proliferation (Mehlen et al., 1997Mehlen P. Mehlen A. Godet J. Arrigo A.P. Hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells.J Biol Chem. 1997; 272: 31657-31665Crossref PubMed Scopus (194) Google Scholar) could be observed. According to our data, one could theorize that the increased Hsp27 expression in keratinocytes upon UV trauma prevents premature apoptosis and loss of protective epidermal keratinocytic layers. The prevention of apoptosis upon UVB irradiation by expression of Hsp27 may contribute to the well-documented thickening of UVB-irradiated epidermis to protect the stem cells in the stratum basale. The increase of the epidermal thickness and pigmentation of the skin by increased melanin production are most important for intrinsic UVB protection (Rucker et al., 1991Rucker B.U. Haberle M. Koch H.U. Bocionek P. Schriever K.H. Hornstein O.P. Ultraviolet light hardening in polymorphous light eruption—a controlled study comparing different emission spectra.Photodermatol Photoimmunol Photomed. 1991; 8: 73-78PubMed Google Scholar); however, the functional activity of Hsp27 during differentiation depends on its degree of oligomerization, which in turn depends on its dephosphorylation (Mehlen et al., 1997Mehlen P. Mehlen A. Godet J. Arrigo A.P. Hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells.J Biol Chem. 1997; 272: 31657-31665Crossref PubMed Scopus (194) Google Scholar). Hsp27 could probably mediate both functions, the protection against UV-induced apoptosis as well as the differentiation, depending on the intracellular activation status of the different signaling kinases. Although the detailed mechanisms have to be further elucidated, our findings about Hsp27 significantly show the potential of the array technology to spark subsequent studies of entirely novel mechanisms. Among 10 UV responsive genes detected by cDNA array analysis seven were induced, whereas three were UV suppressed. The ribosomal protein L11, a part of the large 60S ribosomal subunit, can be regarded as a protein generally involved in protein synthesis and may reflect a general activation of the protein metabolism in keratinocytes after UV irradiation. Two signaling proteins were detected to be differentially regulated upon UV irradiation: CAP was induced, whereas G(S)α was suppressed. The trimeric GTPases are signaling subunits of all 7-transmembrane helix receptors (e.g., steroid hormone receptors) (LeVine, 1999LeVine 3rd, H. Structural features of heterotrimeric G-protein-coupled receptors and their modulatory proteins.Mol Neurobiol. 1999; 19: 111-149Crossref PubMed Scopus (24) Google Scholar). In yeast, CAP was shown to be important for the RAS-mediated activation of the adenylyl cyclase and the cytoskeletal rearrangements leading to morphologic changes of the cells (Gerst et al., 1991Gerst J.E. Ferguson K. Vojtek A. Wigler M. Field J. CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex.Mol Cell Biol. 1991; 11: 1248-1257Crossref PubMed Scopus (115) Google Scholar;Zelicof et al., 1996Zelicof A. Protopopov V. David D. Lin X.Y. Lustgarten V. Gerst J.E. Two separate functions are encoded by the carboxyl-terminal domains of the yeast cyclase-associated protein and its mammalian homologs. Dimerization and actin binding.J Biol Chem. 1996; 271: 18243-18252Crossref PubMed Scopus (52) Google Scholar). The reciprocal change of expression of these two molecules in UV-irradiated keratinocytes could reflect a shift of hormone-mediated growth of the cells to other activating stimuli mediated via the RAS pathway. As RAS activation presents a hallmark of the UV response in mammalian cells (Devary et al., 1992Devary Y. Gottlieb R.A. Smeal T. Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases.Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (789) Google Scholar), our findings are consistent with the current concepts of the UV triggered pathways. By array analysis, MIC-1 gene was shown to be induced, which could not be confirmed by northern blot, as no signal could be detected, indicating a very low expression of the MIC-1 gene. MIC-1 was shown to be expressed in macrophages, and it was proposed that it may be an autocrine regulatory factor (Bootcov et al., 1997Bootcov M.R. Bauskin A.R. Valenzuela S.M. et al.MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily.Proc Natl Acad Sci USA. 1997; 94: 11514-11519Crossref PubMed Scopus (773) Google Scholar). In the skin, it could prevent an exaggerated inflammatory response induced by cytokines released by epithelial cells (Kondo et al., 1993Kondo S. Kono T. Sauder D.N. McKenzie R.C. IL-8 gene expression and production in human keratinocytes and their modulation by UVB.J Invest Dermatol. 1993; 101: 690-694Abstract Full Text PDF PubMed Google Scholar;Boxman et al., 1996Boxman I.L. Ruwhof C. Boerman O.C. Lowik C.W. Ponec M. Role of fibroblasts in the regulation of proinflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1.Arch Dermatol Res. 1996; 288: 391-398https://doi.org/10.1007/s004030050068Crossref PubMed Scopus (68) Google Scholar;Gaspari et al., 1996Gaspari A.A. Sempowski G.D. Chess P. Gish J. Phipps R.P. Human epidermal keratinocytes are induced to secrete interleukin-6 and co-stimulate T lymphocyte proliferation by a CD40-dependent mechanism.Eur J Immunol. 1996; 26: 1371-1377Crossref PubMed Scopus (61) Google Scholar). Two other genes coding for the SG-2 and the RNA-binding protein, NAPOR, were also UV induced, which presents evidence of an UV-dependent regulation of these genes. A functional context of this result is still elusive. The keratin 6 and 17 genes were UV suppressed, which was confirmed by northern blot analysis. These keratins form heterodimers and are characteristic for the hyperproliferative state of keratinocytes in culture or under pathologic conditions (Galvin et al., 1989Galvin S. Loomis C. Manabe M. Dhouailly D. Sun T.T. The major pathways of keratinocyte differentiation as defined by keratin expression: an overview.Adv Dermatol. 1989; 4: 277-299PubMed Google Scholar). The expression of keratins 6 and 17 is increased in suprabasal keratinocytes in psoriasis (Leigh et al., 1995Leigh I.M. Navsaria H. Purkis P.E. McKay I.A. Bowden P.E. Riddle P.N. Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro.Br J Dermatol. 1995; 133: 501-511Crossref PubMed Scopus (246) Google Scholar;McKay and Leigh, 1995McKay I.A. Leigh I.M. Altered keratinocyte growth and differentiation in psoriasis.Clin Dermatol. 1995; 13: 105-114https://doi.org/10.1016/0738-081x(95)93817-8Abstract Full Text PDF PubMed Google Scholar) and, recently, keratin 17 was addressed as a major target for autoreactive T lymphocytes in psoriatic lesions (Gudmundsdottir et al., 1999Gudmundsdottir A.S. Sigmundsdottir H. Sigurgeirsson B. Good M.F. Valdimarsson H. Jonsdottir I. Is an epitope on keratin 17 a major target for autoreactive T lymphocytes in psoriasis? [In Process Citation].Clin Exp Immunol. 1999; 117: 580-586https://doi.org/10.1046/j.1365-2249.1999.01013.xCrossref PubMed Scopus (82) Google Scholar).Swensson et al., 1998Swensson O. Langbein L. McMillan J.R. et al.Specialized keratin expression pattern in human ridged skin as an adaptation to high physical stress.Br J Dermatol. 1998; 139: 767-775Crossref PubMed Scopus (92) Google Scholar demonstrated results indicating a specialized role for these keratins in ridge skin regions as an adaptation to physical stress. The loss of expression of these keratins accompanies keratinocyte differentiation towards the suprabasal phenotype of normal skin (Galvin et al., 1989Galvin S. Loomis C. Manabe M. Dhouailly D. Sun T.T. The major pathways of keratinocyte differentiation as defined by keratin expression: an overview.Adv Dermatol. 1989; 4: 277-299PubMed Google Scholar). Thus, downregulation of these cytokeratins may reflect an ongoing differentiation from hyperproliferative keratinocytes towards keratinocytes of the higher epithelial cell layers in vivo triggered by UV light, which may explain the therapeutic effects of UVB irradiation in the treatment of psoriasis. All measurements were performed at 24 h after exposure. As the genes analyzed may have individual time courses of upregulation or downregulation after UV, the single time point may miss important loci and exaggerate the importance of others in the analysis of UV regulation. Taken together, we add several new aspects to the scenario of the UV response in keratinocytes: The induction of Hsp27 as well as the suppression of Krt6 and Krt17 indicate a UV-induced phenotype comparable with keratinocytes of the higher levels of the epidermis, which may explain the value of UV therapy in hyperproliferative skin disorders (e.g., psoriasis). From the downregulation of the G(S)α paralleled by the induction of the CAP gene expression we could conclude that UV-induced differentiation could be mediated by a switch in signal transduction from 7-transmembrane helix receptors via the trimeric G-proteins to the activation of the RAS pathway. The amplification of the cDNA by SMART technology supplied us with a sufficient amount of DNA for probe preparation ensuring signal intensities, which allow the detection of differences between two sources of RNA. The differences detected by array analysis were reliable as we could confirm the differences in gene expression by northern blots for seven of 10 genes. In contrast to protocols used by other groups, we generated probes for array hybridization from as little as 150 ng of total RNA (Bertucci et al., 1999Bertucci F. Bernard K. Loriod B. et al.Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples.Hum Mol Genet. 1999; 8: 1715-1722https://doi.org/10.1093/hmg/8.9.1715Crossref PubMed Scopus (156) Google Scholar;Wang et al., 1999Wang Y. Rea T. Bian J. Gray S. Sun Y. Identification of the genes responsive to etoposide-induced apoptosis: application of DNA chip technology.FEBS Lett. 1999; 445: 269-273https://doi.org/10.1016/s0014-5793(99)00136-2Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). As we were able to reliably probe arrays with SMART PCR products starting with minor amounts of RNA, experiments with ex vivo skin samples are now feasible. This is important for the verification of tissue culture studies and makes array technology applicable for studying skin cancer evolution or other disorders, such as psoriasis, directly in the human system. A better understanding of UV responses may help not only to improve the selection of patients for therapy but also to develop UV-protective pharmacons and to elucidate carcinogenesis further. Our results provide the assurance that array technology in combination with SMART-generated probes for hybridization can be used as a rapid tool with acceptable accuracy in order to examine global changes of gene expression upon a particular stimulus. This study was supported in part by the primary health insurance companies in Bavaria, by the Wilhelm-Sander-Stiftung 97.21.1/2 and by the Deutsche Forschungsgesellschaft Vo 416/3.1.