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Regulation of Expression by Ultraviolet Radiation: Differential and Speci®c Effects of Ultraviolet B and Ultraviolet A Exposure

FrancËoise Bernerd, Sandra Del Bino, and Daniel Asselineau L'OreÂal, Life Sciences Research, Clichy, France

Skin, the most super®cial tissue of our body, is the scription of and to a lesser extent the ®rst target of environmental stimuli, among which is keratin 6, , and . The DNA solar ultraviolet radiation. Very little is known about sequence responsible for keratin 19 induction was the regulation of keratin gene expression by ultravio- localized between ±130 and +1. In contrast to ultra- let radiation, however, although (i) it is well estab- violet B, ultraviolet A irradiation induced only an lished that ultraviolet exposure is involved in skin increase in , showing a differential gene cancers and photoaging and (ii) represent regulation between these two ultraviolet ranges. The the major epidermal . The aim of this study induction of keratin 19 was con®rmed by studying was to analyze the regulation of human keratin gene the endogenous in keratinocytes in classical expression under ultraviolet B (290±320 nm) or ultra- cultures as well as in skin reconstructed in vitro and violet A (320±400 nm) irradiation using a panel of normal human skin. These data show for the ®rst constructs comprising different human keratin pro- time that keratin gene expression is regulated by moters cloned upstream of a chloramphenicol acetyl ultraviolet radiation at the transcriptional level with a transferase reporter gene and transfected into normal speci®city regarding the ultraviolet domain of solar epidermal keratinocytes. By this approach, we dem- light. Key words: gene expression/keratins/UVB, UVA onstrated that ultraviolet B upregulated the tran- radiations. J Invest Dermatol 117:1421±1429, 2001

pidermis is the most external tissue of our body and et al, 1984; Stoler et al, 1988; Bernerd et al, 1992; Paladini et al, plays an important protective role. The epidermal 1996). Keratin 17 could be found during cutaneous in¯ammatory keratinocyte illustrates this physiologic need by follow- reactions (Jiang et al, 1994), in psoriatic epidermis (Leigh et al, ing a speci®c program of differentiation, ensuring the 1995), and in basal cell carcinomas (Markey et al, 1992). In skin Econstant renewing of epidermis and leading to the tumors and carcinoma-derived cell lines, decreased expression of formation of functional horny layers (Fuchs, 1990; Eckert et al, differentiation-speci®c keratins 1 and 10 is observed (Kartasova et 1997a). During this differentiation process, cells follow an ordered al, 1992; Markey et al, 1992), and induction of embryonic keratins program of gene expression, including the keratin genes. Normal 8 and 18 (Moll et al, 1982; Quinlan et al, 1985; Markey et al, 1991) interfollicular epidermal cells express ®ve keratins (Fuchs, 1993; and in some cases keratin 19 (Moll et al, 1982; Markey et al, 1992; Fuchs and Byrne, 1994). Basal keratinocytes express the pair of MoleÁs et al, 1994) could be observed. keratins 5 and 14 (Nelson and Sun, 1983) whereas when they enter Mechanisms that regulate the expression of keratins occur at the the suprabasal compartment they stop synthesizing keratins 5 and transcriptional level. Promoter regions of keratin genes contain 14 and express keratins 1 and 10 (Fuchs and Green, 1980; several AP1, AP2, or SP1 binding sequences (Eckert et al, 1997b), Schweizer et al, 1984; Bernerd et al, 1992; Byrne et al, 1994). In the and for some keratins responsive elements for growth factors, granular layer, keratinocytes express a novel keratin, keratin 2e cytokines, hormones, or vitamins (Jiang et al, 1993; 1994; Tomic- (Collin et al, 1992). Canic et al, 1996a). The pro®le of keratin expression varies with the type of Skin is exposed to various environmental stimuli, such as solar (simple, strati®ed, squamous) as well as the stage of ultraviolet (UV) radiation, responsible for short-term effects, i.e., differentiation (Moll et al, 1982). It is tightly regulated and is sunburn and suntan, but also severe long-term consequences such generally modi®ed under pathologic situations, or perturbations of as UV-induced cancers and photoaging (Gilchrest, 1989; Elmets, the steady state level of the tissue. For example, the expression of 1992). Although the effects of UV radiation with regard to keratins 6 and 16 in epidermis is observed in hyperproliferative modi®cations of skin physiology are widely studied, very little is conditions such as wound healing, psoriasis, or carcinomas (Weiss known about the effects of UV radiation on regulation of keratin gene expression. Horio et al (1993) have shown that UV irradiation Manuscript received January 27, 2001; revised June 18, 2001; accepted of guinea pig skin led to an increase in keratins 5 and 14. Another for publication August 14, 2001. study performed on human skin in vivo revealed that UVA induced Reprint requests to: Dr. FrancËoise Bernerd, L'OreÂal, Life Sciences a decrease in keratins 1 and 10, UVC an increase in keratins 5 and Research, C. Zviak Center, 90 rue du GeÂneÂral Roguet, 92583 Clichy, France. Email: [email protected] 14, and UVB an increase in keratins 5, 14, 1, and 10 (Smith and Abbreviation: RA, all-trans retinoic acid. Rees, 1994).

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 1421 1422 BERNERD ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

This study reports the effects of both UVB and UVA radiation (Clonetics). Reconstructed skin in vitro was prepared as previously on keratin gene expression, at the transcriptional level and described (Asselineau et al, 1985; 1989). regarding some endogenous keratins. Normal epidermal keratino- Irradiation sources UVB irradiation was applied using a Philips cytes were transfected using a panel of DNA constructs comprising TL20W/12 ¯uorescent tube ®ltered with a Kodacel ®lter. A UVASUN portions of different human keratin promoters cloned upstream of a 3000 lamp (Mutzhas, Germany) allowed us to obtain a UVA1 spectrum chloramphenicol acetyl transferase (CAT) reporter gene and (340±400 nm) and a 1000 W xenon lamp (Arcane, France) ®ltered by exposed to UVB (280±320 nm) or UVA (320±400 nm). By this UG11 (1 mm) and WG335 (3 mm) Schott ®lters displayed a UVA1 + 2 approach, a differential regulation depending on the UV range was spectrum (320±400 nm). UV wavelengths and irradiance were checked found. Keratin 19 displayed the highest UVB induction, although with a Macam SR3010 PC spectroradiometer (Fig 1). Cell cultures were irradiated in phosphate-buffered saline (Seromed, France). Normal keratins 5, 14, and 6 were slightly induced. No effect was observed human skin and skin reconstructed in vitro were irradiated as described on keratins 17, 16, and 10. In contrast, UVA irradiation previously (Bernerd and Asselineau, 1997). upregulated only keratin 17 and had no effect on the others tested. The induction of keratin 19 by UVB was con®rmed at the level of DNA constructs DNA constructs corresponding to portions of the endogenous protein, in classical keratinocyte cultures as well as in promoter region of human keratin genes cloned upstream of the CAT reporter gene were generously given by Dr. M. Blumenberg and are skin reconstructed in vitro or human skin ex vivo. Deleted or schematically represented in Fig 2. 5¢ deletions of the K19±CAT mutated DNA plasmids from the K19±CAT construct allowed us construct and mutated ±130K19±CAT constructs were obtained by to localize the responsive element between ±130 and +1, but polymerase chain reaction (Ohtsuki et al, 1992) using the K19±CAT excluded the role of two AP1-derived sequences located within this construct as template. Primers are listed in Table I. Internal mutation region. corresponding to the ±37±30 sequence of ±387K19±CAT construct was obtained by replacing the original sequence TGAGACCA by an 8 bp Not1 sequence GCGGCCGC (Tomic-Canic et al, 1996b). Primers used MATERIALS AND METHODS for this procedure correspond to m-40Not1 primers (Table I). Transfection experiments Normal human keratinocytes were Skin samples and cell cultures Normal human keratinocytes were transiently transfected at 70% con¯uence with 10 mg of keratin±CAT obtained and cultured from mammary skin as described previously construct and 5 mg of pRSVZ using the polybrene-mediated transfection (Rheinwald and Green, 1975). For transfection experiments cells were method (Jiang et al, 1991a). The cells were irradiated with UVB or UVA used at the second passage and cultured in a semide®ned medium KGM 24 h later and placed back in culture for 48 h. Cellular extracts were obtained as described previously (Jiang et al, 1991a). b-galactosidase activity was determined on 50 ml of cellular extract supernatant (Jiang et al, 1991a) and protein content was measured using the Bio-Rad Protein Assay (Bio-Rad). CAT levels were evaluated using the CAT-ELISA assay (Boehringer Mannheim, Germany). Retinoic acid treatment Subcon¯uent keratinocytes were treated with medium supplemented with 10±6 M all-trans retinoic acid (RA) (Sigma).

Figure 2. Keratin constructs. Bars represent the length of the keratin Figure 1. Emission spectra obtained from UVB and UVA gene promoter, indicated in base pairs. The arrows show the direction of sources. Spectra have been multiplied by a scaling factor, which allows transcription and the link to the CAT reporter gene. the data to be represented on the same chart.

Table I. Oligonucleotidesa

±320 PF TTT CTG CAG AAG GCC AGA ACA GGG TCT GCA ±221 PF TTT CTG CAG TCT CTG GGA GGG GAG GGA ATT ±130 PF TTT CTG CAG CTG ACA CCA TTC CTC CCT TC ±121 PF TTT CTG CAG TTC CTC CCT TCC CCC CTC CAC ±107 PF TTT CTG CAG CCT CCA CCG GCC GCG GGC ATA A ±95 PF TTT CTG CAG GCG GGC ATA AAA GGC GCC AG ±1 HR TTT AAG CTT GGC GAG GCG GAG CAC GGA GC ±130m1 PF TTT CTG CAG CGG ACA CCA TTC CTC CCT TC ±130m2 PF TTT CTG CAG CTT TCA CCA TTC CTC CCT TC ±130m3 PF TTT CTG CAG CTG TCA CTA TTC CTC CCT TC ±40Not1 NF AGC TTT GCG GCC GCG GGT TGC TCC GTC CGTGCT CCG CCT CGC CAA GCT TAA A ±1Not1 NR TTT AAG CTT GGC GAG GCG GAG CAC GGA CGG AGC AAC CCG CGG CCG CGA AGC T ±29 NR TTT GCG GCC GCG AAG CTG CGA TTC GCG GG

aAll the oligonucleotides are indicated in the 5 ¢to 3 ¢orientation. P, PstI restriction site CTG CAG; H, HindIII restriction site AAG CTT; N, Not1 restriction site GCGGCCGC; F, primer forward; R, primer reverse. Mutations are underlined. VOL. 117, NO. 6 DECEMBER 2001 REGULATION OF KERATIN GENES BY UV RADIATION 1423

12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment Keratinocytes were transfected and treated with TPA (Sigma) at 10±20 ng per ml. Immunolabeling Mouse monoclonal antibodies (MoAb) were against human keratin 19 (clone K4.62, Sigma, 1/100 for Western blot, 1/75 for immuno¯uorescence; or clone Ks19.1, ICN Biomedicals, 1/ 1000 for Western blot, 1/10 for immuno¯uorescence) and human keratin 17 (MoAb 1677, Chemicon, 1/1000 for Western blot, 1/100 for immuno¯uorescence). Fluorescein isothiocyanate conjugated rabbit antimouse Ig (Dako, Denmark, 1/100) for immuno¯uorescence and peroxidase-conjugated sheep antimouse Ig (Amersham, France, 1/1000) for Western blot were used as second antibodies. Keratinocytes cultured on glass coverslips were treated with UVB, UVA, or RA and immunolabeled 48 h after. Immunostaining of human skin ex vivo or reconstructed skin in vitro was performed as described previously (Bernerd and Asselineau, 1997). Western blot analysis Keratinocytes were cultured in 100 mm culture dishes and treated as described previously. Forty-eight hours after treatment, cell extraction was performed in 200 ml of 8 M urea, 50 mM Tris-HCl pH 7.6, 0.1 M b-mercaptoethanol, 1 mM dithiothreitol, and 100 mg per ml phenylmethylsulfonyl ¯uoride followed by disruption using a Pellet Pestle Motor (Kontes). Western blot analysis was performed on 4±6 mg of proteins using a standard method (Sambrook et al, 1989) except that the stacking gel was at 4% acrylamide and a semiliquid transfer method was used (Semi-Phor, HSI). Revelation and stripping were performed according to the ECL kit procedure (Amersham, France). RNA preparation, probes, and northern blotting Total RNA (15±20 mg) and cDNA probes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and keratin 17 were obtained and used for northern blotting as described previously (Bernerd and Asselineau, 1997). Autoradiographies were analyzed by densitometry using a Vilbert Lourmat image analyzer equipped with the Bio 1D program (BioPro®l, France).

RESULTS UV doses The range of noncytotoxic UV doses on cultured human normal keratinocytes was found as follows: 0±50 mJ per cm2 for UVB, 0±50 J per cm2 for long UVA (340±400 nm), and 0± 40 J per cm2 for total UVA (320±400 nm). Regulation of keratin promoter activity by UVB irradiation Normal human epidermal keratinocytes were transfected with DNA±CAT constructs containing portions of the keratin promoters and exposed to UVB. CAT assays were performed 48 h after UVB treatment. Figure 3(A) summarizes the results obtained, after 0, 25, or 42 mJ per cm2 UVB exposure, on the level of CAT protein. Keratins 10 and 16 but also keratin 17 promoter activity was not signi®cantly modi®ed after UVB irradiation. Transcription of keratins 5, 14, and 6 was increased more than 2-fold by UVB exposure at 42 mJ per cm2. The most striking induction after UVB exposure was obtained using the keratin 19 construct, the transcription of which was increased 3- fold at a dose of 42 mJ per cm2. A precise UVB dose±response curve shows a maximum of keratin 19 promoter induction at 34 mJ per cm2 with a 3.5-fold increase (Fig 3B). Study of the keratin 19 promoter sequence One of the mechanisms previously described in UVB response involves the AP1 transcription factor (Stein et al, 1989; Fisher et al, 1996). To Figure 3. UVB effects on keratin gene promoter activity. assess an AP1 response element in keratin 19 promoter, normal Keratinocytes were cotransfected with the keratin±CAT construct and human keratinocytes were transfected with K19±CAT construct pRSVZ plasmid. UVB treatment was performed 24 h after the and treated with phorbol ester TPA at 20 ng per ml, as TPA acts transfection and cellular extracts were collected 48 h after irradiation. through AP1 transcription factor (Angel et al, 1987). TPA Each value represents the mean of three samples and each experiment treatment was indeed able to upregulate the level of transcription was done at least twice. The CAT values were normalized using of keratin 19 promoter (Fig 4). The analysis of the 387 bp DNA b-galactosidase activity, and are represented as fold induction above the sequence of the keratin 19 promoter region in this construct did basal level (= 1). (A) Analysis of the different keratin constructs using 0, 2 not reveal any AP1 consensus sequence TGAG/CTCA (Angel et 25, or 42 mJ per cm UVB. Note that some keratins, such as K10, K16, al, 1987). In order to attempt to localize an AP1-like site, however, and K17, are not affected by UVB treatment. Keratins K5, K14, and K6 promoter activities are moderately upregulated. Keratin 19 promoter is which could be responsible for UVB or TPA induction, three the most upregulated by UVB. (B) UVB dose±response curve on keratin strategies were conducted in parallel. First, the K19±CAT construct 19 promoter activity. Note that K19 promoter is activated in a dose- was cotransfected with a c-jun expression vector (a generous gift dependent manner, with a higher induction at 34 mJ per cm2. 1424 BERNERD ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

from Dr. Binetruy). This approach gave positive results in HeLa cells using a control plasmid (pColl-S-CAT construct, corresponding to the ±73+63 portion of the promoter region of collagenase 1 gene, containing the consensus AP1 site; Angel et al, 1987). Using K19±CAT construct in normal human keratinocytes, however, the over-expression of c-jun did not allow us to show induction of promoter activity of the K19±CAT construct (data not shown). In a second approach to further characterize the promoter sequence of keratin 19, several deletions of K19±CAT construct were performed. Their ability to respond to TPA or UVB treatments was studied using transfection experiments. Results indicated that the constructs ±320/K19±CAT, ±221/K19±CAT, and ±130/K19±CAT were induced by both UVB and TPA (Fig 4A, B). Unfortunately, the basal level of CAT using the shorter constructs (± 121/K19±CAT, ±107/K19±CAT, ±95/K19± CAT) was not suf®cient to consider the values. The identi®cation of UVB and TPA responsive elements within the 130 bp upstream of ATG, however, led us to re-examine this particular sequence and to ®nd two AP1-derived sequences, localized, respectively, between ±129/±122 (TGACACCA) and ±37/±30 (TGAGACCA) (Fig 5A). Evaluation of the mutated constructs corresponding to these two AP1-like sequences did not reveal a role of these DNA sequences in the UVB or TPA response (Fig 5B, C). Analysis of endogenous keratin 19 expression after UVB irradiation The behavior of endogenous keratin 19 was analyzed after UVB irradiation on normal cultured human keratinocytes. A positive control for keratin 19 induction was obtained using a 10±6 M RA treatment (Crowe et al, 1991; Crowe, 1993; Asselineau and Darmon, 1995; Gao and Mackenzie, 1996). The two different antibodies directed against human keratin 19 (clone K6.62 and clone Ks19.1) gave similar results. Nontreated conditions revealed only very rare keratin 19 positive cells (Fig 6). Both treatments induced a signi®cant increase in labeling intensity and revealed numerous keratin 19 positive keratinocytes (Fig 6). The highest ¯uorescence intensity was obtained 48 h after UVB or RA treatment. In contrast, UVA irradiation failed to upregulate keratin 19 expression (data not shown). Protein extracts analyzed by Western blots revealed that a tiny band of low intensity was observed in the absence of treatment. The intensity of this band was increased after 10±6 M RA or 25 mJ per cm2 UVB treatment (Fig 7A). The molecular weight of the protein was determined to be around 40 kDa, which is the expected size for keratin 19 (Wu and Rheinwald, 1981; Savtchenko et al, 1988). As a control for normalization of the quantity of proteins loaded on the gel, a second immunolabeling using an antikeratin 17 antibody was performed on the same membrane after removing the ®rst antibody. The band obtained corresponds to a size of approximately 46 kDa, which is the molecular weight of keratin 17 (Troyanovsky et al, 1992). The intensity of the band was not modi®ed after RA or UVB treatment (Fig 7B). Normal human skin ex vivo and skin reconstructed in vitro were exposed to UVB as described previously (Bernerd and Asselineau, 1997). Doses of 50±100 mJ per cm2 were shown to induce typical UVB damage, such as sunburn cell formation 24 h after irradiation. In these experimental conditions, immunolabeling allowed us to visualize keratin 19 positive cells in the suprabasal compartment of the epidermis in the UVB-exposed samples compared to controls (Fig 8). Effect of UVA irradiation on keratin gene expression Using the long UVA source (340±400 nm), none of the keratin promoter constructs showed modi®cation of CAT level (data not shown). Using total UVA (320±400 nm) at 20 J per cm2, however, keratin 17 promoter activity was upregulated by a 4-fold increase (Fig 9A). Figure 4. Effects of UVB and TPA treatments on K19±CAT This construct was the only one modi®ed by UVA irradiation. A constructs. Deletions of K19±CAT constructs were obtained as complete dose±response curve (0±40 J per cm2) showed that described in Materials and Methods. Deleted constructs were analyzed for upregulation of keratin 17 promoter activity began at 20 J per cm2 their ability to respond to TPA (A) and UVB (B) treatments after UVA (Fig 9B). The study of endogenous keratin 17 by transfection into normal human keratinocytes. Note that the ±298/K19± immuno¯uorescence was impeded by its high constitutive CAT, ±221/K19±CAT, and ±130/K19±CAT constructs respond to both expression in keratinocytes cultured in vitro. Western blot analysis TPA (10 ng per ml) and UVB (25 mJ per cm2) treatments. VOL. 117, NO. 6 DECEMBER 2001 REGULATION OF KERATIN GENES BY UV RADIATION 1425

Figure 5. Analysis of ±130/±1 DNA sequence of the keratin 19 promoter. (A)DNA sequence corresponding to the 387 bp of the keratin 19 promoter region in the K19±CAT construct. Two AP1- derived sequences are underlined, located at ±129/±122 and ±37/±30. Accession number: GenBank J03607. (B)Analysis of the ±129/±122 sequence. Three different mutated constructs (m1, m2, m3130/K19± CAT) were obtained as described in Materials and Methods using mutated forward oligonucleotides. Normal human keratinocytes were transfected using the wildtype ±130/K19±CAT construct or the three mutated ± 130/K19±CAT constructs, and treated with TPA (20 ng per ml). Mutations are as follows: wildtype, TGACACCA; m1, GGACACCA; m2, TTTCACCA; m3, TGTCACTA. Note that none of the mutations in the ±129/±122 DNA sequence was able to abolish the induction by TPA. (C)Analysis of the ±37/±30 sequence. The modi®cation of the internal AP1-like site (TGAGACCA) to the Not1 sequence (GCGGCCGC) has been described in Materials and Methods. Transfections and treatments (TPA 10 ng per ml, or UVB 25 and 34 mJ per cm2) were performed as previously. Note that the replacement of the AP1-like site by the Not1 sequence did not abolish the induction of promoter activity by TPA or by UVB exposure. performed on protein extracts obtained 48 h after UVA treatment Bernerd and Asselineau, 1997). Actually, these keratins have been revealed a slight increase in keratin 17 protein (Fig 10A). On the shown to be upregulated in hyperproliferative situations such as same extracts, a Western blot using an antikeratin 19 antibody was psoriasis, carcinogenesis, or wound healing (Weiss et al, 1984; performed and did not reveal modi®cations of keratin 19 after UVA Roop et al, 1988; Bernerd et al, 1992). The participation of irradiation (Fig 10B). Modi®cations of keratin 17 mRNA after regulatory sequences that could trigger keratin 6 induction, i.e., an UVA irradiation were analyzed by northern blot, which also EGF-RE (Jiang et al, 1993) and an AP1 site (Bernerd et al, 1993; showed a slight increase in keratin 17 mRNA level after UVA Navarro et al, 1995), has been examined. Absence of induction of exposure (Fig 11). K16±CAT construct by UVB, however, seems to exclude the role of EGF-RE, also present in the K16 promoter (Magnaldo et al, 1993a). The strongest promoter induction by UVB was found DISCUSSION using the keratin 19 construct, however. This result was con®rmed This study shows that the transcription of keratins 5, 14, 6, and 19 at the level of endogenous keratin using normal human was induced after UVB irradiation. Among these keratins, two keratinocytes in culture but also in more physiologic systems such (keratins 5 and 14) are constitutively expressed in normal epidermis as normal human skin ex vivo and reconstructed skin in vitro. As the (Byrne et al, 1994), and our results regarding their upregulation K19±CAT construct was also inducible by TPA treatment, we support those obtained after UVB irradiation of human skin in vivo assessed the possibility of the involvement of an AP1-derived (Smith and Rees, 1994). Together, the induction of keratins 5, 14, sequence in the response to UVB. Actually the AP1 binding site and 6 may be related to the transient hyperproliferation of and the corresponding transcription factor, comprising members of epidermal keratinocytes following UVB irradiation (Olsen, 1988; fos and jun families, participate in the UV response. Fos and jun are 1426 BERNERD ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 6. Immunolabeling of keratin 19 after UVB exposure on normal human keratinocytes in culture. Cells were cultured on coverslips until 70% con¯uence, sham irradiated (A, B), exposed to UVB (C), or treated with 10±6 M RA (D). Immunolabeling using antikeratin 19 antibody (clone Ks19.1) was performed 48 h after treatment. (A, B) Nontreated keratinocytes; (B) the same ®eld as (A) using an adequate ®lter to visualize the nuclei (red) stained with propidium iodide; (C) UVB (25 mJ per cm2) treated keratinocytes; (D) 10±6 M RA-treated keratinocytes. Note that UVB as well as retinoic acid, used as a positive control, induced numerous positive keratin 19 keratinocytes (green cells). Scale bar: 25 mm.

Figure 7. Western blot analysis of keratin 19 in normal human keratinocytes. Cells were cultured in 100 mm cell culture dishes until 70% con¯uence, and treated with 10±6 M RA or 25 mJ per cm2 UVB. Cellular extracts were collected 48 h after treatment. (A) Western blot analysis performed using the antikeratin 19 antibody (Ks19.1). Note that RA as well as UVB induced an increase in the 40 kDa band, which corresponded to keratin 19 protein. (B) Western blot analysis performed using the antikeratin 17 antibody (MoAb 1677), after stripping of the membrane used above.

Figure 8. Immunolabeling of keratin 19 on normal human skin ex vivo or skin reconstructed in vitro exposed to UVB. Samples of fresh normal human breast skin (A, B) or skin reconstructed in vitro (C, D) were sham irradiated (A, C) or exposed to 75 mJ per cm2 UVB (B, D). Immunostaining using antikeratin 19 antibody was performed using either peroxidase (A, B) or ¯uorescent (B, D) conjugated second antibody. Note the presence of keratin 19 positive keratinocytes located within the suprabasal compartment of the epidermis in UVB-exposed samples only. Scale bar: 25 mm.

upregulated by UV as well as AP1-containing genes such as Rossi et al, 1998) of two AP1-derived sequences located within the collagenase I, stromelysin I (Stein et al, 1989; Garmyn et al, 1991; 130 bp upstream of ATG did not demonstrate the involvement of Devary et al, 1993; Hibi et al, 1993; Rozek and Pfeifer, 1993; Fisher AP1 in the keratin 19 induction. This could be due to the relative et al, 1996). The results obtained after the over-expression of c-jun, importance of the respective members of the AP1-complex family however, as well as those concerning the mutations (according to and the fact that no consensus AP1 sequence has been identi®ed in VOL. 117, NO. 6 DECEMBER 2001 REGULATION OF KERATIN GENES BY UV RADIATION 1427

Figure 10. Western blot analysis of keratin 17. Cell cultures were performed as described in Fig 8, and UVA irradiated (20, 25 J per cm2) using the total UVA source (320±400 nm). Forty-eight hours after UVA irradiation, cellular extracts were collected as mentioned in Materials and Methods.(A) Western blot analysis using antikeratin 17 antibody. Note that the apparent size of the bands corresponds to 46 kDa. An increase in the band intensity could be detected after UVA treatment. (B) Western blot analysis of the same extracts using antikeratin 19 antibody. The apparent size of the band was determined as 40 kDa. No modi®cation of the band intensity was detected after UVA irradiation.

nonkeratinizing strati®ed epithelia (Ouhayoun et al, 1985; Ermich et al, 1988; Lindberg and Rheinwald, 1989) could be detected in epidermis in particular situations such as precancerous epidermal dermatoses (Ichikawa et al, 1995) or cutaneous tumors (basal cell carcinomas and squamous cell carcinomas) (Markey et al, 1992; Perkins et al, 1992; MoleÁs et al, 1994; Krekels et al, 1997). As suggested by others (Stasiak et al, 1989; Ichikawa et al, 1995), the expression of keratin 19 could re¯ect a malignant transformation. We have previously shown that UVB treatment repressed the expression of several keratinocyte differentiation markers (Bernerd and Asselineau, 1997). The expression of keratin 19 could be associated with an undifferentiated phenotype of the keratinocyte as suggested by its expression in regions containing skin stem cells (Michel et al, 1996), in tumor cells (Wu and Rheinwald, 1981), or in fetal epidermis (Dale et al, 1985; Oliver, 1990). Although the function of keratin 19 remains unclear, it has been evoked as a ``switch'' keratin expressed in a ``labile state of differentiation'', with a role in maintaining tissue homeostasis (Stasiak et al, 1989). In contrast to UVB, UVA irradiation speci®cally activated keratin 17 promoter and had no effect on the other keratin constructs. This differential regulation has to be considered, however, with regard to the length of the promoter region examined, which is relatively short. The use of longer 5¢-upstream sequences may reveal additional UVA-responsive sites in other keratin genes. This result re¯ects the differences between UVB and UVA wavelengths regarding their physical properties as well as their mode of action. A major UVA molecular pathway is related to the generation of reactive oxygen species (Basu-Modak and Tyrell, 1993). Two identi®ed molecular mechanisms of UVA involved activation of (i) NF-kB (Vile et al, 1995), or (ii) AP2 transcription factor as shown for intercellular adhesion molecule 1 (ICAM-1) gene (Grether-Beck et al, 1996). NF-kB recognition sequence is not present in keratin 17 promoter. Further investigations concerning the involvement of AP2 in UVA-induced keratin 17 are currently under way as AP2 sites have been identi®ed in several keratin promoters, such as those of keratins 5, 14, 16, and 17 (Leask et al, 1991; Ohtsuki et al, 1992; Magnaldo et al, 1993b; Milisavljevic et al, 1996). The high variability of AP2 sites from the consensus Figure 9. Effects of UVA irradiation on promoter activity of sequence (Imagawa et al, 1987) may contribute to the induction of human keratin genes. Transfection experiments were performed as for some keratins rather than others. The upregulation of ICAM-1 Fig 3 but using a UVA source (320±400 nm). (A) Analysis of the panel gene by UVA has been linked to the role of UVA radiation in the of keratin constructs after a dose of 20 J per cm2 UVA. Note that only keratin 17 promoter activity is increased by UVA. (B) UVA dose± induction of photosensitive skin reactions characterized by release response experiment using K17±CAT construct. of pro-in¯ammatory cytokines (Norris, 1993). In the same way, the expression of keratin 17 has also been associated with in¯ammatory skin diseases (Jiang et al, 1994; Komine et al, 1996). In conclusion, these data show for the ®rst time that UV these 387 bp. The biologic signi®cance of the induction of keratin radiation is able to alter the transcription of epidermal keratin genes, 19 by UVB has been examined, considering the particular situations in a wavelength-related manner, which re¯ects different UVB and when this keratin is expressed. Keratin 19, a marker of basal cells of UVA cellular targets. Some of the tumor-associated keratins such as 1428 BERNERD ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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