Ultraviolet B-induced activated protein-1 activation does not require epidermal growth factor receptor but is blocked by a dominant negative PKClambda/iota.

The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors such as activated protein-1 (AP-1) and NFκB. It is postulated that epidermal growth factor (EGF) receptor, but not protein kinase C (PKC), is the major membrane mediator in UV-induced signal transduction. Since UVB is responsible for most of the carcinogenic effects of sun exposure, we investigated the role of EGF receptors and PKC in UVB-induced AP-1 activation. Our results indicated that while the down-regulation of novel PKC (nPKC) and conventional PKC (cPKC) by pretreatment of cells with 12-O-tetradecanoyl phorbol-13-actetate cannot block UVB-induced AP-1 activity, it can block 12-O-tetradecanoyl phorbol-13-acetate-induced AP-1 activity. Further, the dominant negative mutant PKCλ/ι blocked UVB-induced AP-1 activity in all doses and time courses studied. In contrast, UVB-induced AP-1 activity from cells devoid of EGF receptor (B82) was not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721) or those with a wild-type EGF receptor (B82L) at all UVB irradiation doses and time courses studied. All of this evidence indicated that aPKC, but not EGF receptor, is involved in UVB-induced AP-1 activation.

UV irradiation plays a major role in the development of human skin cancers (1). Experimentally, UV irradiation acts as a tumor initiator and as a tumor promoter in animal models (2,3). The exposure of mammalian cells to UV irradiation, including short (UVC, 200 -280 nM) and long (UVA, 320 -400 nM) wavelengths as well as mid-wavelengths (UVB, 280 -320 nM), leads to a large number of changes in cells, such as activation of transcription factors AP-1 1 and NFB (4 -6). In light of AP-1 activation by tumor promoters and the important role of induced AP-1 activity in tumor promoter-induced transformation, the UV-induced AP-1 response may have an important role in tumor promotion (7,8). Reports from a number of laboratories have established that UVC irradiation activates signal transduction pathways that extend from the cell mem-brane to the nucleus (9,10). The current concept is that the EGF receptor, but not protein kinase C (PKC), plays a major role in UV-induced responses (9,10). This point of view is based on the results from Sachsenmaier et al. (9), which demonstrate that UVC responses were inhibited by pretreatment with EGF, by suramin, or by expression of a dominant-negative EGF receptor mutant and were not inhibited by down-regulation of PKC with TPA pretreatment (9,10). A similar study with UVB has not been reported. Since UVC light does not penetrate the atmosphere (11), UVB irradiation is believed to be responsible for most of the carcinogenic effects of sun exposure (12). Therefore, the present study was aimed at determining whether UVB-induced signals are initiated in a similar way as UVCinduced signals.
Transient Transfection of B82 and B82L-Murine fibroblasts in 10% FBS DMEM were seeded 5 ϫ 10 4 to each well of a 6-well plate. After being cultured at 37°C for 12-14 h, the cells of each well were transiently transfected with 3 g of AP-1 luciferase reporter plasmid together with 0.3 g of ␤-galactosidase plasmid. After 12 h, the medium was replaced with 10% FBS DMEM and cultured for an additional 10 -12 h. The cells were starved in serum-free medium for 24 h and then were or were not exposed to either UVB (1 kJ/m 2 ) or EGF (20 ng/ml). The cells were extracted with lysis buffer 24 h later, and the luciferase activity was measured according to the manufacturer's instructions (Promega). The luciferase activity was corrected with ␤-galactosidase activity, and the results were shown with relative AP-1 activity as described previously (7).
Transient Transfection of JB6 Cells-JB6 P ϩ cells, Cl 41, were cultured in a 6-well plate until they reached 85-90% confluence. We used 2 g of AP-1 luciferase reporter plasmid and 0.3 g of ␤-galactase plasmid either with or without 6 g of dominant negative mutant of Xenopus PKC/ plasmid (CMV-Zn / PKC mutant) and 15 l of Lipo-fectAMINE reagent to transfect each well in the absence of serum. After 10 -12 h, the medium was replaced with 5% FBS MEM. Approximately 30 -36 h after the beginning of the transfection, the cells were starved with 0.1% FBS MEM for 12 h, and cells were then either exposed or not exposed to UVB (1.5 kJ/m 2 ) irradiation. 12 h later, the cells were extracted with lysis buffer, and the luciferase activity was measured as described above (7).
Generation of Stable Cotransfectants with AP-1 Reporter and Dominant Negative PKC/ Mutant-JB6 P ϩ cells, Cl 41, were cultured in 6-well plates until they reached 85-90% confluence. We used 2 g of AP-1 luciferase reporter plasmid, 0.3 g of CMV-neu marker vector either with or without 6 g of dominant negative mutant of Xenopus CMV-Zn, PKC/ mutant, and 15 l of LipofectAMINE reagent to transfect each well in the absence of serum. After 10 -12 h, the medium was replaced with 5% FBS MEM. Approximately 30 -36 h after the beginning of the transfection, the cells were digested with 0.033% trypsin, and the cell suspensions were transferred to 75-ml culture flasks and cultured for 24 -28 days with G418 selection (300 g/ml). Stable transfectants were screened by assay of the luciferase activity and Western blotting with rabbit polyclonal IgG against PKC/. Stable transfected cells, Xenopus PKC/ mutant mass 1 or AP-1 mass 1 , were cultured in G418-free MEM for at least two passages before each experiment.
Generation of Stable Transfectants with AP-1 Reporter in B82L, B82, or B82M721 Cells-B82, B82L, or B82M721 cells were cultured in a 6-well plate until they reached 85-90% confluence. We used 2 g of AP-1 luciferase reporter plasmid and 0.3 g of CMV-neu marker vector with 15 l of LipofectAMINE reagent to transfect each well in the absence of serum. After 10 -12 h, the medium was replaced with 10% FBS DMEM. Approximately 30 -36 h after the beginning of the transfection, the cells were digested with 0.033% trypsin, and cell suspensions were transferred to 75-ml culture flasks and were cultured for 24 -28 days with G418 selection (300 g/ml). Stable transfectants were screened by assay of the luciferase activity. The cell clones were ringisolated and transfected to 12-well tissue culture dishes with 10% FBS DMEM containing 500 g/ml G418. Stable transfected cells were cultured in G418-free MEM for at least two passages before each experiment.
Assay for AP-1 Activity-Confluent monolayers of AP-1 reporter stable transfectants were trypsinized, and 5 ϫ 10 3 viable cells suspended in 100 l of 5% FBS MEM were added into each well of a 96-well plate. Plates were incubated at 37°C in a humidified atmosphere of 5% CO 2 and 95% gas air. 12-24 h later, cells were starved for 12-40 h prior to exposure to either UVB or EGF. The cells were exposed to either UVB or EGF at time and dose as indicated (Figs. [1][2][3][4][5][6]8). The cells were extracted with lysis buffer and luciferase activity was measured as described previously (7). The results were expressed as either relative AP-1 activity or relative luciferase units. Relative AP-1 activity was presented as the fraction of the luciferase activity in medium control cells.
Western Blot Analysis-2 ϫ 10 5 of JB6 Cl 41 transfectants AP-1 mass 1 , Xenopus PKC/ mutant mass 1 , or mass 2 were cultured in each well of 6-well plates to 95% of confluency with 5% FCS MEM. Cells were washed once with ice-cold phosphate-buffered saline and extracted with SDS-sample buffer. The cell extracts were separated on a 8% polyacrylamide-SDS gel, and then transferred and probed with the rabbit polyclonal IgG against PKC/ using ECL detection system.
Statistical Analysis-Data were analyzed by Student's t test.

RESULTS AND DISCUSSION
As we studied the role of EGFR in UVB-induced AP-1 activation, we observed that cells pretreated for 30 min with Lavendustin C methyl ester (a specific EGF receptor-tyrosine kinase inhibitor (17)) did not show significant inhibition of UVBinduced AP-1 activation at noncytotoxic concentrations (doses ranging from 1 to 2 M) in P ϩ 1-1, which is an AP-1 luciferase reporter (Col-Luc) stable transfectant of JB6 cells (p Ͼ 0.05) (Fig. 1). Further, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline, a potent selective inhibitor of EGF receptor tyrosine kinase (18) used in our study, did not show any inhibitory effects on UVBinduced AP-1 activity(p Ͼ 0.05) as it blocked EGF-induced AP-1 activity(p Ͻ 0.05) (Fig. 1). These results suggested that the EGF receptor is not involved in UVB-induced AP-1 activation. To examine more directly whether or not EGFR or EGFR tyrosine kinase is involved in UVB-induced AP-1 activation, we transiently transfected an AP-1 reporter plasmid (Col-Luc) together with ␤-galactosidase plasmid into the well characterized min and sequentially exposed to 1 kJ/m 2 of UVB irradiation. After a 12-h culture, the AP-1 activity was measured by luciferase activity assay as described previously (9). The results were presented as relative AP-1 activity. Each bar indicates the mean and standard deviation of nine assay wells from three independent experiments.
FIG. 2. Full response of B82 and B82M721 cells to UVB in AP-1 induction. 5 ϫ 10 4 of murine fibroblasts in 10% FBS DMEM were seeded into each well of a 6-well plate. After being cultured at 37°C for 12-14 h, the cells of each well were transiently transfected with 3 g of AP-1 luciferase reporter plasmid together with 0.3 g of ␤-galactosidase plasmid. After 12 h, the medium was replaced with 10% FBS DMEM. The cells were cultured at 37°C overnight and starved in serum-free medium for 24 h. The cells were then exposed to UVB (1 kJ/m 2 ) (solid bar) or EGF (20 ng/ml) (hatched bar) as indicated. The cells were extracted with lysis buffer 24 h later, and the luciferase activity was measured according to the manufacturer's instructions (Promega). The luciferase activity was corrected with ␤-galactosidase activity, and the results were shown with relative AP-1 activity as described previously (9). Each bar indicates the mean and standard deviation of four assay wells from two independent experiments. murine fibroblast variants (B82, B82L, and B82M721 cells). B82 cells are murine fibroblasts devoid of endogenous EGFR (19,20). The variant cell lines B82L and B82M721 stably express either wild-type human EGFR (B82L) or a kinasedeficient human EGFR (B82M721) (20). The results show that high levels of AP-1 activity were induced by UVB in B82L, B82M721, and B82 cells (p Ͼ 0.05). EGF induced high levels of AP-1 activity in B82L cells and low levels in B82M721 cells, but no apparent induction occurred in parental B82 cells (p Ͻ 0.05) (Fig. 2). These results suggested that there is no role for EGFR in UVB-induced AP-1 activity. To further confirm our data, we established 10 single cloned, stable AP-1 reporter (Col-Luc) transfectants from B82, B82L, and B82M721 cells, respectively. To avoid any bias from the pressure of cell selection, we also generated Col-Luc "mass stable clones." After transfecting cells with Col-Luc in a 100-mm dish and selecting with G418, more than 30 colonies per dish were grown. Instead of ring cloning these cells, all colonies in the dish were grown as a "mass stable culture." The results observed from these stable transfectants are consistent with those from transient transfectants. The UVB-induced AP-1 activity in the B82 or B82M721 stable transfectants showed similar fold induction of AP-1 activity with B82L stable transfectants (p Ͼ 0.05) (Fig. 3). These data further support the conclusion that EGFR is not involved in UVB-induced AP-1 activation.
The UV-induced AP-1 activity is mediated both by induction of c-Jun and c-Fos expression and by post-translational modification of c-Jun (21-24). Shah et al. (25) reported that UVB induced an immediate early response of c-fos that is downregulated within 2 h and a strong second phase of fos expression with a maximum at 8 h that only returned to control levels after 24 h. Similar prolonged dynamic changes were observed in UVB-induced AP-1 activity in JB6 cells. 2 To be sure that the EGFR-independent manner of UVB-induced AP-1 activity was not due to our study of one time point and one UVB dose, we performed a time course and dose response of UVB-induced AP-1 activation in AP-1 reporter stable transfectants. The UVB-induced AP-1 activity in B82 cells was not significantly different from that of B82L cells at different time points (p Ͼ 0.05) (Fig. 4A). The fold induction of AP-1 activity by different doses of UVB was similar among B82, B82L, and B82M721 cells (p Ͼ 0.05) (Fig. 4B). All of these results argue strongly that UVB-induced AP-1 activity is not dependent on either EGFR or tyrosine kinase of EGFR.
Down-regulation of PKC by pretreatment of cells with TPA blocks TPA-induced signal transduction but not UVC-induced signal transduction (9). This has been interpreted as key evidence supporting the concept that PKC is not involved in the UV-induced signal transduction pathway; however, this point of view requires further testing. Isozymes of the PKC family can be grouped into at least three subfamilies: conventional PKC (cPKC ϭ ␣, ␤, ␤I, ␤II, and ␥), novel PKC (nPKC ϭ ␦, ⑀, , and ), and atypical PKC (aPKC ϭ /,) (26,27). cPKCs exhibit a strict requirement for phospholipids and Ca 2ϩ for their activities, while nPKCs do not need Ca 2ϩ for their activities. Diacylglycerol or phorbol esters bind with cPKC and nPKC and cause activation, while aPKC does not bind with diacylglycerol or phorbol ester. Phorbol ester can only down-regulate cPKC or nPKC and not aPKC (26,27). Pretreatment of JB6 cells with 20 ng/ml of TPA for 24 -36 h did not block UVB-induced AP-1 activity (p Ͼ 0.05) while it completely blocked TPA-induced AP-1 activity (p Ͻ 0.05) (Fig. 5). This result agrees with an-2 C. Huang and Z. Dong, unpublished data.

FIG. 3. UVB-induced AP-1 activity in AP-1 reporter stable transfectants among B82L, B82M721, or B82 cells. 5 ϫ 10 3 cells from AP-1
reporter stable transfectants as indicated were seeded into each well of a 96-well plate. After overnight culture at 37°C, the cells were starved for 36 h with serum-free DMEM. The cells were then exposed to UVB (1 kJ/m 2 ) or EGF (20 ng/ml) and incubated for 24 h before assaying for AP-1 activity. The results were presented as described in the legend to Fig. 2. Each bar indicates the mean and standard deviation of nine assay wells from three independent experiments. other report that UVC-induced signals cannot be blocked by pretreatment of TPA (9). Also, previous results indicate that the ability of UV to induce membrane association of PKC is weak when compared with TPA, and that c-fos induction by UV and TPA is additive (28). The data suggested that inductions of AP-1 activity by TPA and UV are through different mediators. Taken together, all of these experiments can rule out the involvement of cPKC and nPKC in UVB-induced AP-1 activation but cannot rule out the role of aPKC or other unknown phorbol ester-insensitive PKCs in UVB-induced AP-1 activation.
The aPKC family is composed of PKC/ and PKC (29 -31). The PKC/ and PKC show a high degree of homology in both the catalytic and regulatory domains in different species (32). Dominguez et al. (34) had cloned cDNA of an atypical PKC isotope from Xenopus laevis by using the regulatory domain of rat PKC as a probe. This cDNA encoding a protein was shown to be highly homologous to rat PKC; therefore, this Xenopus cDNA was renamed Xenopus PKC in previous reports (13)(14)(15)(16). A very recent report from the same group indicates that the sequence originally named as Xenopus PKC shows the highest degree of homology with PKC/, with an overall 90% identity at the amino acid level; thus, this cloned cDNA is actually Xenopus PKC/ (33). Due to the near identity of catalytic doming of the PKC/ and PKC, these two isotypes of aPKC showed a similar stimulation effect on the B-dependent promoter activity. Interestingly, Diaz-Meco et al. (33) demonstrated that using either the dominant negative mutant of PKC/ or the dominant negative mutant of PKC could block the tumor necrosis factor-␣-induced B-dependent promoter activity. Microinjection of a peptide with the sequence of aPKC isotypes but not of PKC␣ or PKC⑀ dramatically inhibited maturation (34) and NFB activation (35) in X. laevis oocytes. FIG. 4. The time course and dose-response of UVB-induced AP-1 activity. 5 ϫ 10 3 cells from AP-1 reporter stable transfectants (B82-mass 2 , B82L-mass 2 , or B82M721-mass 2 ) were seeded into each well of a 96-well plate. After overnight culture at 37°C, the cells were starved for 36 h by replacing medium with serum-free DMEM. A, for the time course studies, the cells were or were not exposed to UVB (1 kJ/m 2 ), and the AP-1 activity was determined in time as indicated. B, for the dose response study, cells were treated with the indicated doses of UVB and incubated for 24 h before assaying for AP-1 activity. The results were presented as described in the legend to Fig. 2. Each bar indicates the mean and standard deviation of eight assay wells from two independent experiments.
Previous studies have shown that PKC/ is critically involved in many cellular functions such as cell proliferation and Xenopus maturation (13,34). Platelet-derived growth factor can induce the interaction and association of PKC/ (previously reported as PKC) with Ras in mouse fibroblasts (16). Expression of an active mutant of PKC/ activated mitogen-activated protein kinase, and the activation of mitogen-activated protein kinase by tumor necrosis factor-␣ can be blocked by a kinasedefective dominant negative PKC/ (36). Akimoto et al. (29) reported that EGF-or platelet-derived growth factor-induced AP-1 activation is through PKC (35). To test the role of aPKC in the UVB-induced AP-1 activation, we transiently transfected a well characterized dominant negative mutant construct of Xenopus PKC/ (13-16, 38) into JB6 cells. This dominant negative mutant contains the lysine 275 to tryptophan mutation. As shown in Fig. 6, transient transfection of Xenopus dominant negative mutant PKC/ blocks UVB-induced AP-1 activation completely (p Ͻ 0.05) while inhibiting only 13.8% of serum-induced AP-1 activity. This result suggested that aPKC is involved in UVB-induced AP-1 activity. To further confirm this finding, we established mass stable cultures of Xenopus PKC/ dominant negative mutant cells. A high level of PKC/ dominant negative mutant protein was detected by antiserum against PKC/ in Xenopus PKC/ mass 1 cells (Fig. 7). The results observed from these stable transfectants are consistent with those from transient transfectants at all doses and times studied (Fig. 8). We have performed these experiments with rat PKC dominant negative mutant (39), and the results indicated that the PKC dominant negative mutant also inhibits UVB-induced AP-1 activity (data not shown). All of these data suggested that aPKC is involved in UVB-induced AP-1 activation.
The mechanism behind the tumor-promoting ability of UV is not well understood. This is especially true in the study of UVB, which is very active and is responsible for most of the carcinogenic effect of sun exposure (12). Most of our understanding of the UV-induced signal transduction is based on the studies of UVC using HeLa cells, a fully transformed, cervix-derived carcinoma cell line (9). The UVC-induced signal transduction pathway is believed to originate at the cell membrane and is mediated by EGFR and other growth factor receptors but not by PKC (9, 10). The signal is then subsequently transferred to the nucleus via a signaling cascade involving Src-like tyrosine kinase, Ras/Raf kinase, mitogen-activated protein kinase in- FIG. 5. Pretreatment of cells with TPA blocks TPA-induced but not UVB-induced AP-1 activity. 5 ϫ 10 3 JB6 AP-1 reporter stable transfected P ϩ 1-1 cells suspended in 5% FBS MEM were added to each well of a 96-well plate. After an overnight culture at 37°C, the cells were starved by replacing the medium with 0.1% FBS MEM for 12-20 h. Afterward, the cells were first treated either with or without 20 ng/ml TPA for 30 h and then exposed either to TPA (20 ng/ml) or UVB (2.0 kJ/m 2 ). After a 24-h culture, the AP-1 activity was measured by luciferase activity assay as described previously (9). The results were presented as relative luciferase units (RLU). Each bar indicates the mean and standard deviation of nine assay wells from three independent experiments.
FIG. 6. Inhibition of UVB-induced AP-1 activity by transient overexpression of dominant negative Xenopus PKC/. 5 ϫ 10 4 of JB6 P ϩ Cl 41 cells in 5% FBS MEM were seeded to each well of a 6-well plate. After being cultured at 37°C for 12-14 h, the cells of each well were transiently transfected with 2 g of AP-1 luciferase reporter (Col-Luc) plasmid and 0.3 g of ␤-galactosidase plasmid together with 6 g of dominant negative Xenopus PKC/ plasmid (CMV-Zn PKC/ mutant (mut)). After 12 h, the medium was replaced with 5% FBS MEM. The cells were cultured at 37°C overnight and starved in 0.1% FBS MEM for 12 h. Then the cells were or were not exposed to UVB (1.5 kJ/m 2 ) or 20% FCS. The cells were extracted with lysis buffer 12 h later, and the luciferase activity was measured according to the manufacturer's instructions (Promega). The luciferase activity was corrected with ␤-galactosidase activity, and the results were shown with relative AP-1 activity as described previously (9). Each bar indicates the mean and standard deviation of four assay wells from two independent experiments.

FIG. 7.
Overexpression of dominant negative PKC/ in stable transfectants. 2 ϫ 10 5 of JB6 Cl 41 transfectants as indicated were cultured in each well of a 6-well plate with 5% FBS MEM. After cells reached 95% of confluency, the cells were washed once with ice-cold phosphate-buffered saline and extracted with SDS-sample buffer. The cell extracts were separated on an 8% polyacrylamide-SDS gel and then transferred and probed with the rabbit polyclonal IgG against PKC/ using ECL detection system. cluding c-Jun N-terminal kinase, resulting in the activation of transcription factors such as AP-1 and TCF/Elk-1 (21)(22)(23)(24). Evidence supporting this model has been derived mainly from pharmacological inhibitors and dominant negative mutants of EGFR, Src, Ras, and Raf (9,(21)(22)(23)(24). In this study we have provided the first clear and strong evidence that the pathway whereby UVB activates AP-1 activity requires aPKC but not the EGF receptor. Though the upstream effector of aPKC in the UVB signal transduction cascade is not known, there are a number of known activators of PKC/ and PKC. Native PKC/ or PKC is known to be activated by lipid second messengers such as phosphatidic acid, phosphatidylinositol 3,4,5-P 3 , and ceramide (32, 35, 36, 40 -42). In terms of inflammatory cytokines such as interleukin-1 or tumor necrosis factor-␣ (43,44), signaling involves the binding and activation of PKC/ and PKC by ceramide. Birt et al. (45,46) have reported that PKC may play certain roles in the process of tumor promotion. Very recently, Verheij et al. (47) demonstrated that UVC irradiation rapidly induces an increase in ceramide in a dose-dependent manner and that exposure of cells to C2-ceramide induced concentration-dependent c-Jun N-terminal kinase activation, which is well known as a key component in UVCinduced signal cascades, to as much as 40-fold of control. Moreover, several groups demonstrated that PKC isotypes have a different substrate specificity in vitro (33,37,48). Whether these substrates are involved in UVB-induced signal transduction in cells has yet to be tested. Further study on the precise mechanism by which UV irradiation triggers signal transduction pathways should help us in understanding the basis of UVB-induced skin diseases such as cancer and aging.
FIG. 8. Inhibition of UVB-induced AP-1 activity by stable overexpression of dominant negative Xenopus PKC/. 5 ϫ 10 3 JB6 P ϩ Cl 41 cells from AP-1 reporter stable transfectant (AP-1 mass 1 ) or AP-1 reporter and Xenopus PKC/ stable transfectants (Xenopus PKC/ -mass 1 ) were seeded into each well of a 96-well plate. After overnight culture at 37°C, the cells were starved for 12 h by replacing medium with 0.1% FBS MEM. For the dose response study (A), cells were treated with the indicated dose of UVB and incubated for 24 h before assaying for AP-1 activity. For the time course study (B), the cells were or were not exposed to UVB (1.5 kJ/m 2 ). The AP-1 activity was determined in time as indicated, and the results were presented as described in the legend to Fig. 2. Each bar indicates the mean and standard deviation of ten assay wells from three independent experiments.