Coactivation of AP-1 Activity and TGF-Β1 Gene Expression in the Stress

Oncogene (1997) 15, 981 ± 989  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Coactivation of AP-1 activity and TGF-b1 gene expression in the stress response of normal skin cells to ionizing radiation

M Martin,1 M-C Vozenin,1 N Gault,1 F Crechet,1 CM Pfarr2,3 and J-L Lefaix1

1Laboratoire de Radiobiologie et d'Etude du GeÂnome, CEA, DVS, 91191, Gif sur Yvette, France; 2Unite des Virus OncogeÁnes, CNRS, Institut Pasteur, 75724, Paris, cedex 15, France

Activation of the AP-1 transcription factor and TGF-b1 NFkB families, which encode nuclear transcription growth factor by ionizing radiation was studied both in factors. vivo in pig skin, and in vitro in human ®broblasts and Activator protein-1 (AP-1) is the collective name for keratinocytes. Three and 6 h after irradiation, the Fos a class of transcription factors which recognize and Jun proteins and their binding activity to an AP-1 common regulatory DNA sequences. AP-1 transcrip- consensus sequence were strongly induced by high doses tion factor is a protein complex composed of of g-rays. c-Fos, c-Jun and JunB proteins were found to heterodimers between members of the Fos and Jun be present in gel-shift complexes by probing with speci®c protein families or homodimers of Jun family proteins. antibodies. Both keratinocytes and ®broblasts exhibited Dimerization through the leucine zipper domain is a heightened AP-1 activity following irradiation. As we prerequisite for binding to speci®c DNA sequences, previously found that TGF-b1 is involved in the including the consensus sequence 5'-TGA G/C TCA-3', development of skin lesions induced by radiation, TGF- known as TPA response element or AP-1 binding site. b1 gene expression was also examined. Two and 6 h Jun and Fos can also combine with more distantly after irradiation, the levels of TGF-b1 transcripts were related bZIP proteins such as ATF proteins. AP-1 increased in skin. By immunostaining, TGF-b1 protein factors have been implicated in the regulation of many levels were found to be increased in ®broblasts, cellular processes such as proliferation and transforma- keratinocytes and endothelial cells. As the TGF-b1 tion (Angel and Karin, 1991), di€erentiation and promoter contains AP-1 binding sites, the relation embryonic development (Wang et al., 1992; Hilberg between AP-1 activity and TGF-b1 induction was et al., 1993; Grigoriadis et al., 1995) and programmed addressed. The 7365 TGF-b1 promoter fragment, which cell death (Smeyne et al., 1993; Gillardon et al., 1995). contains a high anity AP-1 site, exhibited increased The transcriptional induction of both fos and jun by binding to Jun and Fos proteins following irradiation. radiation has been widely studied in a variety of These results suggest that stress-inducible TGF-b1 cellular systems (Weichselbaum et al., 1994). The expression is mediated by the activation of AP-1 results substantially support the hypothesis that the transcription factor. Fos and Jun transcription factors are involved in the immediate cellular response to radiation, although they Keywords: AP-1; fos; jun; TGF-b1; skin; g-irradiation have been poorly con®rmed by in vivo studies. Further, the protein level received little attention. We previously demonstrated that in normal skin cells, gamma irradiation induced the jun and fos gene activation in Introduction vivo (Martin et al., 1993a). This activation was found at the mRNA level after high doses of g-rays. The ability of cells to respond to extracellular stress is The ®rst aim of the present work was therefore to essential for their survival. This response involves further characterize fos and jun activation in vivo.In changes in the pattern and rates of gene expression. particular, we wanted to assess variations in AP-1 Ionizing radiation induces such changes within a few proteins, their DNA binding activity and the composi- hours, which can result either in cell-cycle arrest and tion of the active AP-1 dimers. The type of target cell repair or in cell death, depending on the level of in the irradiated skin was also explored. damage. In addition, these changes also trigger a The activation of transcription factors constitutes a cascade of cellular modi®cations over a long period of critical point in the cellular response to radiation. time, which may lead to the late e€ects of ionizing Genes encoding growth factors and cytokines may be radiation, mainly tumour formation and the develop- important downstream targets and e€ectors of this ment of sequelae in normal tissues. activation, since a prominent immediate e€ect of Genes induced by radiation, even in the absence of radiation is to modify the cell cycle in damaged cells. new protein synthesis, are called immediate early Examples of radiation-induced growth factors and response genes (review in Weichselbaum et al., 1994). cytokines include TNFa, IL-1, FGF, PDGF and Examples of such genes include the jun/fos, egr-1, and TGF-b (review in Weichselbaum et al., 1993). We previously demonstrated that the transforming growth factor-b1 (TGF-b1) was involved in the late development of skin lesions induced by ionizing Correspondence: M Martin radiation (Martin et al., 1993b). The second aim of 3 Present address: Department of Cell Biology and Biochemistry, this study was to examine whether or not the TGF-b1 Texas Tech University School of Medicine, 3601 4th Street, Lubbock, Texas 79424, USA growth factor is involved in the immediate early Received 5 June 1997; revised 11 July 1997; accepted 17 July 1997 response of skin cells to ionizing radiation. AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 982 TGF-b1 is a homodimeric protein of 25 kDa, which We found that in irradiated skin cells, the radiation- is ubiquitously produced (for reviews see Roberts and induced expression of the jun and fos genes, AP-1 Sporn, 1990; Massague , 1990). A wide variety of cells binding activity, and TGF-b1 gene expression, all possess TGF-b1 receptors and respond to TGF-b1 occurred with similar kinetics for the same doses of action. TGF-b1 elicits a number of rapid responses in irradiation. These results suggest that stress-inducible cells, including alteration in their proliferation and TGF-b1 expression might be mediated through the apoptosis. activation of AP-1 transcription factor. TGF-b1 can either stimulate or inhibit cell prolifera- tion depending on its concentration and the target cell (Moses et al., 1990). It is a potent negative regulator of Results growth for various cell types, including epithelial, endothelial, and haematopoetic cells. TGF-b1 can Radiation-induced fos and jun gene expression also induce reversible G1 cell cycle arrest through signalling by its receptors (Yingling et al., 1995; Saltis, In a previous publication, we showed that both c-fos 1996). Its downstream targets include kinase inhibitors and c-jun mRNAs were induced in vivo in normal skin of the G1 cyclin-cdk complexes such as p21, which is cells by high doses of gamma rays. Exposure to 16 Gy induced by TGF-b1 (Datto et al., 1995). By contrast, resulted in a signi®cant induction for both pro- TGF-b1 can promote the proliferation of mesenchymal oncogenes. Here therefore, the levels of Fos and Jun cells such as ®broblasts. Thus, treatment of normal proteins were examined after doses of 2 and 16 Gy in WI38 ®broblasts with TGF-b1 stimulates their growth pig skin. Nuclear proteins were isolated from control and represses p21 protein (Raynal and Lawrence, and irradiated skin 6 h after irradiation, in order to 1995). quantify the amounts of c-Fos, c-Jun, and JunD TGF-b1 can also elicit apoptosis in a variety of proteins by Western blot. In control skin, the protein normal and tumoral cells, including hepatocytes contents were low. Signi®cant induction of the three (Oberhammer et al., 1992), endometrial cells (Rotello AP-1 proteins was observed after irradiation with et al., 1991), and prostatic epithelial cells (Hsing et al., 16 Gy (Figure 1 and Table 1). The Jun and Fos 1996). pRb and E2F-1 appear to be involved in the proteins induced by g-rays migrated in SDS-polyacry- pathway of apoptotic death, at least in liver cells (Fan lamide gels with apparent molecular weights of 43 kDa et al., 1996). and 67 kDa respectively in their major bands. The involvement of TGF-b in the cellular response to radiation has been studied in the breast and lung, where TGF-b1 can be rapidly induced. Using indirect immuno¯uorescence, induction of TGF-b1 protein was detected 1 h after irradiation in the mammary gland of c-Fos protein adult BALB/c mice (Barcellos-Ho€, 1993), and its expression remained elevated for 7 days. Changes in the expression of TGF-b mRNAs were found one day — 67 kD after exposure of mouse lungs to gamma rays (Finkelstein et al., 1994; Rubin et al., 1995) and within 6 h in the skin of mice exposed to b-irradiation 0 6 16 (Randall and Goggle, 1995). In our previous study, TGF-b1 mRNA levels were found to be increased in irradiated pig skin on appearance of the lesions, which started 3 weeks after C-Jun protein irradiation by an erythematous phase. Thereafter, sustained expression of the TGF-b1 gene persisted in the irradiated skin, associated with the development of late ®brosis (Martin et al., 1993b). It was recently — 43 kD proposed that in normal tissues, the long-term e€ects of radiation develop as a continuous cascade, which starts immediately after irradiation, during the 0 2 16 immediate early gene response (Rubin et al., 1995). In the present work, we therefore attempted to establish that TGF-b1 was induced during the JunD protein immediate early response of skin cells to radiation, as well as the possible cellular source of this factor within the irradiated skin. — 43 kD Lastly, we explored the possible relationships between TGF-b1 gene induction and AP-1 activation. Little has been reported on the regulation of the TGF- b1 promoter. The human TGF-b1 gene has two 0 2 16 promoters, containing a number of putative binding Figure 1 Fos and Jun Western-blots of skin proteins. Nuclear sites for regulatory proteins, including NF1, SP1, FSE2 proteins were isolated from irradiated and control skin samples of and AP-1 (Kim et al., 1989). Consequently, we studied the same pig 6 h after treatment. 10 mg of proteins was loaded on 10% SDS polyacrylamide gels and reacted with polyclonal rabbit the possible involvement of a high anity AP-1 site c-Fos antibody (1 : 2500), c-Jun antibody (1 : 200), and JunD (Kim et al., 1990) in the stress response to radiation. antibody (1 : 400) AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 983 Table 1 Quanti®cation of Fos and Jun proteins by Western blot AP-1 EMSA, skin c-Fos protein 0 Gy 2 Gy 16 Gy Experiment Standard Dose Gy no.1 no.2 no.3 no.4 no.5 Mean deviation n – AP1 SP1 – AP1 SP1 – AP1 SP1 0 1 1 1 1 1 1 ± 5 2 1.4 1 5 2.5 2.2 2.5 1.4 5 16 1.6 2.6 6 2.7 4.6 3.5 1.6 5 c-Jun protein Experiment Standard Dose Gy no.1 no.2 no.3 Mean deviation n 0 1 1 1 1 3 2 2.2 0.8 1.8 1.6 0.6 3 16 2.2 4.9 5.4 4.2 1.4 3 JunD protein Experiment Dose Gy no.1 no.2 Mean n 0 1 1 1 2 2 2 2 2 2 16 72 11 41 2 Results are expressed as the ratio of induction in irradiated to control skin samples removed 6 h after irradiation. Means were calculated from the results of 2 to 5 independent experiments

AP-1 binding activity to a consensus sequence To study the DNA binding activity of Fos and Jun, nuclear proteins were isolated from irradiated and control skin, and their binding to the AP-1 consensus sequence (5'-TGACTCA-3') was tested. In control skin, constitutive binding activity was very weak. g Figure 2 AP-1 electrophoretic mobility shift assay of skin irradiation induced a signi®cant increase in DNA proteins. Nuclear proteins were isolated from irradiated and binding within 6 h. This increase was maximal for control pig skin 6 h after treatment. The binding activity of these the dose of 16 Gy, as quanti®cation of 3 independent proteins to the AP-1 consensus DNA sequence (5'-TGACTCA-3') 32 experiments yielded a mean induction of 13, s.d.=2.5 was tested. 14 mg of proteins was incubated with 2 ng of P- labelled AP-1 oligonucleotides. Competitors: AP-1 ± 200 ng of (Figure 2 and Table 2). unlabelled AP-1 oligonucleotides; SP1 ± 200 ng of unlabelled AP-1 binding activity was shown to be speci®c by oligonucleotides speci®c to the SP-1 sequence the addition of a molar excess of unlabelled AP-1 oligonucleotide. This competitor abolished the radio- active signal. The speci®city of AP-1 binding was also Table 2 Quanti®cation of skin protein binding to the consensus shown when the addition of unlabelled oligonucleotide AP-1 sequence speci®c to a consensus SP-1 sequence failed to alter Experiment Standard protein binding to the AP-1 sequence. Dose Gy no.1 no.2 no.3 Mean deviation n 0 1 1 1 1 ± 3 2 7.8 2 8 5.9 2.8 3 Cellular source of AP-1 binding activity 16 12 16 10 12.7 2.5 3 The AP-1 response of cultured human primary skin Results are expressed as the ratio of induction in irradiated to control cells to irradiation was also examined. To mimic the skin samples removed 6 h after irradiation. Means were calculated from the results of three independent experiments proliferative status of each cell type in the skin, ®broblasts were irradiated at con¯uency, whereas keratinocytes were irradiated in the exponential phase of growth. g irradiation of cultured cells induced the Jun (Figure 4) and JunB (data not shown) antibodies binding activity to the AP-1 consensus sequence 3 and all displaced the signal, but the JunD antibody did not 6 h after irradiation both in ®broblasts and keratino- (Figure 4). In addition, the c-Fos antibody (Figure 3b) cytes (Figure 3a and b). In keratinocytes, induction by and c-Jun antibody (data not shown) reduced the 20 Gy gamma irradiation was more signi®cant than the binding of nuclear keratinocyte proteins to the AP-1 induction by 100 ng of TPA. A small dose of 0.5 Gy sequence. failed to induce DNA binding. Radiation-induced TGF-b1 gene expression Composition of AP-1 dimers The mRNA levels of the TGF-b1 gene were To ascertain the composition of the dimers which bind determined in irradiated and control skin removed 2, to the AP-1 consensus oligonucleotide, speci®c anti- 6, and 24 h after treatment. In irradiated skin, bodies were added to DNA-protein mixtures. In signi®cant induction of the TGF-b1 gene was found control skin, the AP-1 binding signal was displaced after 2 h for the highest doses (Table 3). For the two by each of the 3 Jun antibodies but not by the c-Fos animals studied at that time, mean induction, antibody. In skin irradiated with 16 Gy, the c-Fos, c- calculated as the ratio of induction in irradiated to AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 984

AP-1 EMSA, skin

a AP-1 EMSA, fibroblasts 0 Gy 16 Gy 0 Gy 16 Gy – cFos cJun JunD JunB – cFos cJun junD 6 H 6 H

– AP1 O* – AP1 O*

Figure 4 Gel supershift detection of Fos and Jun. The composition of the AP-1 dimers was studied by probing the binding reaction with speci®c antibodies. 5 mg of nuclear extracts from skin samples removed 6 h after irradiation was incubated with 2 ml of serum at room temperature for 15 min prior to adding 2 ng of 32P-labelled consensus AP-1 oligonucleotides. Arrowheads indicate the position of speci®c complexes

Table 3 Quanti®cation of TGF-b1 gene expression by Northern blot Time Pig after 0 2 8 16 32 48 64 number treatment Gy Gy Gy Gy Gy Gy Gy 1 2h 1 0.5 1.7 ± 1.4 3.9 7 2 2h 1 1.6 ± ± 2.6 2.5 ± Mean 2h 1 1.1 1.7 ± 2 3.2 7

b AP-1 EMSA, keratinocytes 3 6h 1 1 2.7 ± ± 4 6h 1 1.1 1.3 3.6 3 2.4 2.5 control TPA 0.5 Gy 20 Gy 20 Gy 6 6h 1 ± 1 5.3 1.8 24 11.8 7 6h 1 ± ± ± ± ± 5 – AP1 SP1 – AP1 SP1 – AP1 SP1 – AP1 SP1 – Fos 8 6h 1 ± ± ± ± ± 2.4 Mean 6h 1 1.1 1.1 3.3 2.5 13.3 5.4 s.d. ± 1.8 0.5 ± 3.8 n 2 3 3 2 4 10 24 h 1 0.7 1.2 0.8 0.5 ± ± 11 24 h 1 1.9 0.4 0.9 1 0.9 ± Mean 24 h 1 1.3 0.8 0.9 0.7 0.9 ± Results are expressed as the ratio of induction in irradiated to control skin. Means were calculated from the results of 2 to 4 independent experiments

control skin, was 3.5 (s.d.=1.9, n=5) for doses ranging from 32 to 64 Gy. The maximal TGF-b1 induction was found at 6 h (Table 3). Figure 5 shows that g-rays Figure 3 AP-1 electrophoretic mobility shift assay of proteins induced the 2.5 and 3.5 kb TGF-b1 transcripts in a from cultured skin cells. Cellular sources of the AP-1 binding dose-dependent manner, and that the threshold dose activity were studied by gel mobility shift assays performed with was 16 Gy. For all animals studied at 6 h, the mean nuclear proteins isolated 3 h after irradiation from human primary ®broblasts (a) and human primary keratinocytes (b) induction, for doses ranging from 16 to 64 Gy, was 5.5 cultured in monolayers. (3 Con¯uent ®broblast cultures were (s.d.=6.2, n=12). However, this induction was irradiated with 16 Gy. 1 ng of 32P-labeled AP-1 consensus transient, decreasing to basal values by 24 h post- oligonucleotides was incubated with 5 mg of proteins. Compe- irradiation. titor: AP1 = 200 ng of unlabeled AP-1 oligonucleotides. O* ± The cellular source of the TGF-b1 protein was 1 ng of labeled AP-1 oligonucleotides alone, without proteins. (b) Growing keratinocytes were incubated with 100 ng/ml of TPA as examined in cryostat sections of pig skin by immuno- a positive control or irradiated with 0.5 and 20 Gy. 5 mgof ¯uorescence using a TGF-b1 speci®c antibody (Figure proteins was incubated with 1 ng of 32P-labelled AP-1 consensus 6). In irradiated dermis, ®broblasts and endothelial oligonucleotides. Competitors: AP1 ± 200 ng of unlabelled AP-1 cells were strongly positive. Staining was primarily oligonucleotides; SP1 ± 200 ng of unlabelled oligonucleotides speci®c to the SP-1 sequence; Fos ± nuclear proteins were localized within the cytoplasm of these cells. Further, incubated with 2 ml of serum containing anti-c-Fos antibodies the epidermis was homogeneously stained. Doses of 16 AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 985 and 64 Gy enhanced the TGF-b1 staining in all cell AP-1 proteins. 16 Gy g irradiation signi®cantly types examined. enhanced DNA binding after 1, 3 and 6 h (Figure 7a). This increase was transient, as it was no longer observed after 24 h (Figure 7b). Similar results have AP-1 binding activity to a TGF-b1 promoter sequence been obtained on two human ®broblast cell lines. The Nuclear proteins were isolated from cultured normal mean maximal increase was 2.5 (s.d.=0.5, n=3) for human ®broblasts. Their binding to a DNA sequence HF8 cell line and 3.2 (s.d.=1, n=3) for HF12 cell contained within the human TGF-b1 promoter (5'- line. TGTCTCA-3'), located between 7365 to 7372 (Kim, Binding speci®city was determined by competition 1990), was tested. This sequence has a high anity for experiments using various oligonucleotides. Incubation of nuclear proteins with 200 ng of unlabelled 7365 AP-1 oligonucleotide resulted in loss of binding to the labelled oligonucleotide. Incubation of nuclear proteins β TGF- 1 Northern blot with 200 ng of unlabelled SP-1 oligonucleotide, 200 ng L 0 8 16 32 48 64 Gy of 7365 AP-1 sequence mutated on three bases (Mut 1, 5'-TGTTCGA-3') or 200 ng of 7365 AP-1 sequence — 3.5 kb mutated on four bases (Mut 2, 5'-TAGTACA-3'), did β TGF- 1 — 2.5 kb not change the binding (Figure 7b).

Discussion

L 0 8 16 32 48 64 Radiation-induced expression of fos and jun genes 18S — 1.9 kb Most data in the literature concerning fos and jun Figure 5 TGF-b1 Northern blotting of skin proteins. mRNA activation were obtained at the RNA level and in vitro levels of the TGF-b1 gene were assayed in control and irradiated on cultured cells. We previously demonstrated that g- pig skin removed 6 h after irradiation. For each dose, 5 mgof rays induced fos and jun transcripts in vivo in irradiated mRNA isolated from pig skin was loaded on the gel, except for skin, with a preferential induction for c-fos (Martin et the 48 Gy dose, for which 3 mg was loaded. 10 mg of mRNA from peripheral lymphocytes (L) was used as a positive control for al., 1993a). In this work, we addressed their activation TGF-b1 gene expression. The 18 S probe was used as a control of at the protein level, both in vivo in the skin and in vitro RNA loading in normal skin cells.

ab

Figure 6 Immunocytochemistry of skin: 32 Gy-irradiated skin samples (a) and control skin samples (b) were removed from the same animal 6 h after irradiation. 6 mm cryosat sections were prepared and incubated with a 1 : 50 dilution of the polyclonal rabbit antibody directed against a peptide corresponding to amino acids 328 ± 353 of the carboxy terminal region of human TGF-b1 (Santa Cruz Biotechnology, Inc). Magni®cation: obj.620 for a; obj.640 for b AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 986

a TGF-β1 EMSA b TGF-β1 EMSA 0 Gy 16 Gy 0 Gy 16 Gy 0 Gy 16 Gy 3H 3H 6H 6H 24 H 24 H

– AP1 – AP1 SP1 – AP1 – AP1 – AP1 Mut1 Mut2 SP1 – AP1 Mut1 Mut2 SP1

ëFigure 7 TGF-b1 electrophoretic gel mobility shift assay of proteins from human cultured ®broblasts. (a) Three and 6 h after irradiation, nuclear proteins were isolated from cultured normal human skin ®broblasts (HF8 cells) and their binding activity to the 7365 AP-1 sequence of the human TGF-b1 promoter (5'-TGTCTCA-3') was assayed. 5 mg of proteins was incubated with 1 ng of 32P-labelled AP-1/TGF oligonucleotide. Competitors: AP1 ± 200 ng of unlabelled AP-1/TGF oligonucleotide; SP1 ± 200 ng of unlabelled oligonucleotide speci®c to the SP-1 sequence. (b) Nuclear proteins were isolated 24 h after irradiation from cultured normal human ®broblasts (HF8 cells) and incubated with 1 ng of 32P-labelled 7365 AP-1 sequence of the human TGF-b1 promoter. Competitors: AP1 ± 200 ng of unlabelled 7365 AP-1 oligonucleotides; SP1 ± 200 ng of unlabelled oligonucleotides speci®c to the SP-1 sequence; Mut 1 : 1 ng of 7365 AP-1 sequence mutated on three bases (5'-TGTTCGA-3'); Mut 2 : 1 ng of 7365 AP-1 sequence mutated on four bases (5'-TAGTACA-3'). Arrowhead indicate the position of speci®c complexes

The present results show that activation of fos and anti-JunD antibodies. Our results thus show that the jun gene expression led to a complete response at the composition of the induced AP-1 dimers was distinct protein level, since the AP-1 proteins were translated from the dimers constitutively present in control skin and exhibited increased DNA binding activity. cells. The active dimers induced by radiation exposure Concerning this activity, two processes can account were probably heterodimers composed of c-Fos, c-Jun for the observation. Firstly, under stress conditions, and JunB proteins, whereas the active dimers of the pre-existing AP-1 proteins can exhibit increased AP-1 control skin were mainly Jun homodimers. binding activity through post-translational changes or Similarly, it was found that sarcoma cell proteins changes in interactions with other proteins. Alterna- that bind an AP-1 sequence following irradiation have tively, newly synthesized proteins can participate in epitopes recognized by antiserum to the DNA binding the response. Activation of pre-existing AP-1 proteins domains of Jun and Fos (Hallahan et al., 1993). In this was found by Hallahan in sarcoma RIT-3 cells, as study, the AP-1 DNA binding sequence was found by irradiated cells exhibited a fourfold increase in nuclear reporter gene assay sucient and necessary to confer binding to an AP-1 sequence within 10 ± 20 min, but X-ray mediated gene induction. Consequently, we not after 60 min (Hallahan et al., 1993). This increase propose that, in skin, the AP-1 response is a key was not prevented by incubation of cells with element of radiation-induced signalling. In fact, many cycloheximide. authors propose an important role for AP-1 proteins, The results of our gel shift assays showed that both and more particularly c-Fos, in skin homeostasis ®broblasts and keratinocytes display heightened AP-1 (Basset-Seguin et al., 1994; Welter and Eckert, 1995). activity following irradiation. Moreover, they point AP-1 transcription factors regulate several epidermal once again to an important role for c-Fos activation, as and dermal genes and have been implicated in the skin the largest di€erence in the composition of the AP-1 response to various stress agents like phorbol esters dimers between the control and irradiated cells was the and u.v. irradiation. involvement of the c-Fos protein in the latter. Another Radiation exposure can lead to repair or to cell di€erence was the absence of binding displacement by death, depending on the level of damage. AP-1 AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 987 involvement in death of irradiated cells is probably an Such regulations may not be speci®c to the TGF-b1. important protection against carcinogenesis, as it was Ionizing radiation induce various growth factors and found that stress-inducible fos and jun activation is cytokines, including TNF-a, IL-1, FGF, and PDGFa. lacking in many radioresistant tumour cell lines. This A relation between the basic ®broblast growth factor was demonstrated at the RNA level and DNA binding and AP-1 transcription factor has been reported (Lee level for melanoma cells (Sahijdak et al., 1994; Collart et al., 1995). In fact, ionizing radiation induced bFGF et al., 1995), and at the RNA level for astrocytoma cell expression in human breast carcinoma cells that was lines (Gubitz et al., 1993). mediated through the activation of AP-1. In conclusion, we found that after radiation-induced injury of the skin, the time courses of the expression and Radiation-induced TGF-b1 gene expression activity of the Fos and Jun proteins and of TGF-b1 gene Possible targets of this AP-1 activation can be expression were the same and occurred after the same searched for among the genes controlling the cell doses. We propose that, due to their regulatory roles for cycle and cell proliferation, or genes important for cell genes such as TGF-b1 and collagenase, AP-1 proteins, death pathways. TGF-b1 is an important regulator of and above all c-Fos, play an important role in the all these processes in many cell types, and its role in induction and development of the late e€ects of radiation the control of skin cells has been increasingly in normal tissues. The present results point to the TGF- emphasized. b1 growth factor playing a crucial role in controlling the The present results demonstrate that in normal skin, early response of skin cells to oxidative stress. TGF-b1 growth factor expression is activated by g- rays. Gene transcription is probably induced, as the amount of speci®c transcripts increased in a dose- dependent manner within 6 h following irradiation. At Materials and methods the protein level, immunoreactivity was increased at the same time and the main cellular components of the Radiation exposure skin, namely ®broblasts, keratinocytes and endothelial In vivo studies were performed in pigs. This is the reference cells, all participated in this increase. This appearance species for experimental skin studies, as it allows the best of immunoreactivity may re¯ect either enhanced extrapolation to human skin. Accordingly, 13 Large White synthesis of the TGF-b1 protein or greater epitope pigs were gamma irradiated. They were 5 months old and accessibility. Barcellos-Hoof et al. proposed that the weighed about 70 ± 80 kg. Permission for this animal increased immunoreactivity that they found within 1 h experiment was obtained from the Animal Protection in the irradiated mouse mammary gland was due to the Oce of the French Ministry for Agriculture and Forestry (Permit no. 3255). activation of the latent pre-existing TGF-b1 proteins The in vivo irradiation procedure with the 192iridium source and thus to greater access to the speci®c epitopes has already been described (Lefaix et al., 1993). The (Barcellos-Ho€ et al., 1993). In our model, both collimated source measuring 2 cm in diameter was applied processes can occur. to the surface of the skin on the ¯ank of anaesthetized Next, we examined the relations between AP-1 animals, which were irradiated with 2, 8, 16, 32, 48 and protein activity and TGF-b1 activation. The human 64 Gy. In this model, previous clinical studies showed that no TGF-b1 gene has two promoters which contain several change in the skin was observed after delivery of skin surface AP-1 sequences. One high-anity AP-1 site and two doses below 20 Gy. After doses exceeding 20 Gy, erythema putative, low-anity AP-1 sites have been described in appeared, and doses of over 48 Gy induced necrosis and late the two promoters of the gene, as well as three putative ®brosis (Lefaix et al., 1993). In vitro, normal human skin cells were irradiated at 208C AP-1 binding sites, located about 200 base pairs with a 60cobalt source of medical type, at a dose rate of downstream of this gene (Asiedu et al., 1994). 0.9 Gy min71. The ¯asks were then returned to the incubator Reporter gene assays indicate that the AP-1-like sites and harvested at the indicated times. Control cells were at the 5' end of the human TGF-b1 gene can act as sham-irradiated. TPA response elements (Kim et al., 1989; Kim et al., 1990). These TREs have been implicated in the Tissue removal autoinduction of the TGF-b1 gene mediated by AP-1 (Roberts and Sporn, 1990; Kim et al., 1990). The AP-1 Samples of skin irradiated with the above series of doses sites in 3' region of the gene have also been shown to and the control sample were removed from the same ¯ank stimulate reporter gene expression in response to of each animal. Samples were removed at 2, 6, and 24 h after irradiation. For RNA and protein studies, the skin phorbol ester treatment, through both AP-1 and was cut into pieces and directly frozen in liquid nitrogen. CREB proteins (Asiedu et al., 1994). We found that the high anity AP-1 site within the TGF-b1 promoter bound larger amounts of regulatory Cell culture proteins isolated from g-irradiated human ®broblasts In vitro studies were performed on cultured human skin than from control ®broblasts. Jun and Fos proteins cells. Primary cultures were established from normal skin as might therefore be direct regulators of the TGF-b1 described earlier (Martin et al., 1989; Vozenin et al., 1997). promoter under cellular stress conditions. Again, both The ®broblasts thus obtained were cultured in DMEM pre-existing and newly synthesized AP-1 proteins could medium containing 4.5 g l71 glucose supplemented with 71 71 participate in this DNA binding activity. Reporter gene 10% FCS, 100 U ml penicillin, 50 mgml streptomycin, 1 M HEPES and 200 mM glutamine. Keratinocytes were assays are currently being performed in our skin cell plated at a density of 1 ± 2 104 cells cm72 on dishes coated models to investigate whether the AP-1 DNA binding with a feeder-layer of irradiated normal human skin sequence is sucient to confer g-ray mediated gene ®broblasts. HD culture medium used for the keratinocytes induction. contained 1 g l71 glucose and was supplemented as AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 988 following: 5% FCS, 100 U ml71 penicillin, 50 mgml71 GAGCTGACTCAGATGT-3') contained the consensus streptomycin, 5 mgml71 insulin (Sigma), 200 mM adenine AP-1 sequence 5'-TGACTCA-3'; (2) SP1 oligonucleotide (Sigma), 1077 M cholera toxin (Sigma), 0.4 mgml71 hydro- (5'-GATCTAAACCCCGCCCAGCG-3') was a speci®c cortisone (Sigma), 10 ng ml71 EGF (Sigma), 1 M HEPES SP1 consensus sequence. The two strands of the (Gibco) and 200 mM glutamine (Gibco). corresponding double stranded oligonucleotide were

annealed in 50 mM Tris pH 7.5, 10 mM MgCl2 and 100 mM NaCl, starting at 858C for 5 min and cooling RNA isolation from tissues down slowly to room temperature. 40 mg of the double- Frozen tissues were crushed to powder in liquid nitrogen stranded oligonucleotides was labelled with 10 mCi32 P- (Bioblock Scienti®c crusher, ref B78101), and RNA was dATP (3000 Ci/mmol) by ®lling with the klenow fragment isolated as previously described (Martin et al., 1993a). of DNA Polymerase I. DNA binding reactions were performed in a ®nal volume of 20 ml. 14 mg of proteins from skin samples or 5 mg of proteins from cultured cells Northern-blot analysis was preincubated for 30 min on ice in binding bu€er For Northern blotting, RNA was separated in 1% (17.5 mM HEPES pH 7.6, 0.07 mM EDTA, 80 mM KCl, 71 agarose gels containing 0.66 M formaldehyde and 0.7 mM DTT, 5 mM MgCl2, 7% glycerol, 0.1 mgml poly transferred onto Nytran ®lters (Schleicher and Schuell). dIdC, 0.1 mg ml71 of salmon sperm DNA, and 20 mM Filters were then crosslinked with ultraviolet light and spermidine). One mg of the puri®ed labelled probe was hybridized to the probes at 658C overnight. Probes were then added over 5 min. The DNA protein complexes were labelled with a-32P-dCTP by random priming (Megaprime resolved on 6% polyacrylamide gel in TBE 0.25X and run DNA labeling kit, Amersham) with a speci®c activity of at room temperature for 3 h at 180 V. Gels were dried in 1±26109 c.p.m. mg71. After washing in 0.56SSC and a slab gel drier for 1 h at 808C and the complexes were 0.1% SDS at 608C, ®lms were exposed to blots for identi®ed by autoradiography. Binding speci®city was various times at 7708C using intensifying screens. The determined by competition experiments using either probes for the c-fos,c-jun,TGF-b1 and 18S genes were 200 ng of unlabelled AP-1 oligonucleotide or 200 ng of used as previously described (Martin, 1993a and b). The unlabelled double-stranded SP-1 oligonucleotide. The last probe was used as a control of RNA loading. composition of the DNA-protein complexes was deter- Autoradiograms were analysed using the Biorad imaging mined by competition with speci®c antibodies. Rabbit sera densitometer GS-700. The intensity of hybridization containing polyclonal antibodies against c-Fos, c-Jun, Jun signals were normalized to the signal from the 18S B, or JunD proteins were used. Nuclear extracts were ribosomal probe . incubated with 2 ml of serum at room temperature for 15 min before addition of the radiolabelled probe and loading on the gel. Preparation of nuclear proteins from tissues Nuclear extracts were prepared as described by Deryckere AP-1/TGF-b1 gel retardation assay et al., 1994). One g of frozen minced pig skin was crushed to powder in liquid nitrogen (Bioblock Scienti®c crusher, To examine the AP-1 sequence contained within the TGF- ref B78101). The powder was resuspended in 5 ml cold b1 promoter, methods similar to those described in the bu€er (10 mM HEPES, pH 7.9; 150 mM NaCl; P-40 0.6% previous section were used. Nuclear proteins were isolated Nonidet, 1 mM EDTA and 1 mM PMSF) transferred into a 3, 6 and 24 h after irradiation from normal human 15 ml Dounce tissue homogenizer and homogenized with ®broblasts cultured at passage 7. Experiments were pestles. The supernatant was incubated for 5 min on ice performed three times on two di€erent human cell lines and then centrifuged for 5 min at 5000 r.p.m. at 48C. The (HF8 and HF12). To study the binding activity of proteins pelleted nuclei were resuspended in 500 mlcoldbu€er to the 7365 AP-1 sequence (5'-GATCCCCTGTGTCT- (20 mM HEPES pH 7.9, 420 mM NaCl, 25% glycerol, CATCCCC-3')ofthehumanTGF-b1 promoter (Kim, 1990), 5 mg of protein was incubated with 1 ng of 32P- 1.2 mM MgCl2,0.2mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 2 mM benzamidine, 5 mgpermloftheprotease labelled 7365 AP-1/TGF-b1 oligonucleotide. Competitors inhibitors pepstatin, leupeptin and aprotinin) and incu- were as follows: (1) For AP-1, 200 ng of unlabelled 7365 bated at 48C for 20 min for high salt extraction. The lysed AP-1/TGF-b1 oligonucleotide; (2) For SP1, 200 ng of nuclei were centrifuged for 2 min at 48C and 12 000 r.p.m. unlabelled oligonucleotide speci®c to the SP-1 consensus and the supernatant was frozen at 7808C. The nuclear sequence; (3) For Mut 1, 200 ng of 7365 AP-1/TGF-b1 proteins contents were determined by Bradford assays and sequence mutated on three bases (5'-TGTTCGA-3'); (4) were about 0.4 mg g71 of tissue. andforMut2,200ngof7365 AP-1/TGF-b1 sequence mutated on four bases (5'-TAGTACA-3').

Preparation of nuclear proteins from cultured cells Western blotting Normal human primary keratinocytes were cultured until they had reached 80% con¯uency. Normal human Antibodies were prepared at the Pasteur Institute (Paris, ®broblasts were cultured in monolayers until dense France) in rabbits using glutathione-S-transferase fusion con¯uency. 16106 cells were scraped into 1 ml of Tris- proteins with murine peptides, as previously described bu€ered saline and pelleted by centrifugation at 1500 g for (Pfarr et al., 1994). Anti c-Jun antibodies recognized the N- 5 min. Nuclear extracts were prepared from cultured cells terminal fragment of the protein (residues 1 ± 53), anti c- as described by Schreiber (Schreiber et al., 1989). Protein Fos antibodies recognized the B-ZIP region of the Fos contents were determined by Bradford assays. They were protein, anti JunD antibodies recognized residues 1 ± 114, about 40 mg per million cells for keratinocytes, and 70 mg and anti JunB antibodies recognized the N-terminal part of per million for ®broblasts. the protein (residues 1 ± 53). Nuclear proteins were loaded on 10% SDS polyacrylamide gels and separated according to Laemmli, 1970. Separated proteins were then transferred Consensus AP-1 gel retardation assay to nitrocellulose sheets by wet transfer. Blots were To study the interactions of nuclear proteins with the AP- incubated with antibodies and revealed by chemilumines- 1 consensus sequence, we used the following synthetic cence (ECL Amersham kit). Films were analysed using the oligonucleotides: (1) AP-1 oligonucleotide (5'-CTA- Biorad imaging densitometer GS-700. AP-1 and TGF-b1 coactivation in irradiated skin M Martin et al 989 Immunohistochemistry ranging from 1 : 50 to 1 : 150. The FITC rabbit second antibody was used at a 1 : 100 dilution and incubated for Irradiated and control skin samples were removed from 1 h at room temperature. Sections were observed with an the same animals 6 h after irradiation and immediately Olympus BH2 microscope, with Hg 100 W u.v. epi- frozen in isopentane cooled by liquid nitrogen. 6 mm illumination. cryostat sections were ®xed in 0.5% paraformaldehyde and permeabilized in 0.1% Triton X-100 solution. Sections were saturated in 3% PBS-BSA solution for 1 h and incubated overnight with shaking at 48Cin0.3% Acknowledgements PBS-BSA with a 1 : 50 dilution of the primary antibody The authors wish to thank F Thierry for her helpful advice, (a polyclonal antibody directed against a peptide Jean-Jacques Leplat for technical assistance, and Jean- corresponding to amino acids 328 ± 353 of the carboxy Francois Dossin and Philippe Bacon for animal care. This terminal region of human TGF-b1-SantaCruzBiotech- study was supported by EC grants no. FI3P-CT92-0059 nology, Inc.). This antibody was used at dilutions and F13P-CT92-0059 and F14P-CT95-0029.

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