Ultraviolet-induced junD Activation and Apoptosis In Myeloblastic Leukemia ML-1 Cells*

The exposure of mammalian cells to UV irradiation induces the expression of immediate early genes such as c-jun and c-fos and activates the transcription factors AP-1 and NF-κB. JunD is one of the three members of the Jun family and shares some functional characteristics with c-Jun. In the present study, we found that the exposure of myeloblastic leukemia ML-1 cells to UV light (UVC) caused a significant increase injunD mRNA expression within 5 min that persisted for a period of 3 h. The activation of protein kinase C (PKC) with 12-O-tetradecaoylphorbol-13-acetate (TPA) also induced increases in junD expression similar to those of UV irradiation. In addition, UV irradiation- and TPA-induced increases injunD expression were completely abolished by GF-109203X, a PKC-specific inhibitor. UV irradiation activated intracellular signaling pathways including extracellular regulated kinase-2 (Erk-2), c-Jun N-terminal kinases-1 (JNK-1), and p38. However, TPA-induced activation of PKC affected only Erk-2 activity, and GF-109203X (a PKC inhibitor) markedly suppressed UV-induced Erk-2 activation. To further investigate the effect of UV-induced Erk-2 activation on the expression of junD mRNA, cDNA encoding mitogen-activated protein kinase kinase (MEK1) was overexpressed in ML-1 cells. The overexpression of MEK1 enhanced substantially junD expression in response to UV or TPA. In contrast, the suppression of Erk activation with PD98059, a specific inhibitor of MEK1, inhibited UV- and TPA-induced junD mRNA expression, UV-induced increases in caspase-3 activities, and cell death. In addition, the overexpression of junD enhanced the UV irradiation-induced increases in caspase-3 activity and cell death. We conclude that UV irradiation-induced increases in junD expression in ML-1 cells are mediated through activation of the PKC-coupled Erk-2 signaling pathway and play an important role in ML-1 cell apoptosis.

The exposure of mammalian cells to UV light elicits a rapid transcriptional activation of immediate-early genes such as c-jun and c-fos (1). New products of c-jun and c-fos genes form a multimer known as transcriptional factor AP-1 1 (activator protein 1) (2)(3)(4). The activated AP-1 regulates expressions of its target genes including c-jun itself. The multimer of AP-1 is not only composed of c-Jun and c-Fos but also other members of the Jun and Fos family such as JunB (5), JunD (6,7), and FosB. Similar to c-jun, the transcription of junB is activated when the cells are exposed to UV irradiation (8). However, little is known about either the effects of UV irradiation on junD expression or the involvement of specific cell signaling pathways in mediating this response.
Earlier studies suggest that the JunD protein shares some functional similarities with other members of the Jun family. First, this protein can form a heterodimeric complex with c-Fos and homodimeric complexes with other jun products (4,6,7). Second, it has the ability to bind to the cAMP response element (CRE) and to AP-1 consensus DNA sequences (4). Finally, some stimuli such as ionizing radiation, hypoxic-ischemic injury, okadaic acid, insulin-like growth factors, and alkylating mutagens can activate c-jun and junB expressions as well as junD expression (8 -11). Our previous studies have demonstrated that the activation of protein kinase C (PKC) by TPA can induce junD expression (12). PKC isoforms have been divided into three categories on the basis of their structure and biochemical properties, including conventional PKC (cPKC), novel PKC (nPKC) and atypical PKC (aPKC). Recently, it has been shown that UV-induced cellular responses involve PKC activation (13,14). Therefore, it is important for us to examine whether UV irradiation can cause overexpression of junD.
The activation of distinct cellular signaling pathways in many different cell types usually regulates gene expression through the activation of transcription factors. Recent studies have focused on identifying signaling molecules that are involved in UV irradiation-induced increases in transcription factor expressions. It has been shown in different cell types that UV irradiation activates three intracellular signaling cascades, e.g. extracellular regulated kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (15)(16)(17). JNK has been found to play a major role in transmitting signaling events from the cell membrane to the nucleus in a UV-induced rise in c-jun expression. JNK activation is much stronger than that of Erk or p38 in response to UV irradiation (16, 18 -20). UV-induced activation of JNK results in an increase in c-jun gene expression and activation of the c-Jun protein (21)(22)(23). Erk is also involved in UV-induced immediateearly gene activation because the UV-induced increase in c-jun expression can be inhibited by pretreatment of cells with PD98059, a specific inhibitor of the Erk signaling pathway (24). Thus, it is necessary to identify which signaling pathway(s) mediates UV irradiation-induced increases in junD expression. In the present study, we investigated the signaling pathway in myeloblastic ML-1 cells that mediates increases in junD mRNA expression in response to UV irradiation and TPA stimulation. Our results indicate that the PKC-coupled Erk-2 signaling pathway is responsible for the transmission of UV-induced signaling events resulting in a high level of expression of junD mRNA.

EXPERIMENTAL PROCEDURES
Cell Culture and Treatment-Human myeloblastic leukemia ML-1 cells were cultured in conditions as described (12). Briefly, ML-1 cells were cultured in RPMI 1640 medium containing 7.5% heat-inactivated fetal bovine serum (Invitrogen) in a humidified incubator supplied with 5% CO 2 at 37°C. Cells were passed at a seeding density of 3 ϫ 10 5 cells/ml. Before treatment with UV irradiation or drugs, cells were synchronized by serum-deprivation in RPMI 1640 medium containing 0.3% fetal bovine serum for 36 h. They were then exposed to UVC light (40 J/cm 2 ). After UV irradiation (5,30,60, and 180 min), the cells were collected and rinsed with phosphate-buffered saline (PBS). Total RNA was isolated, and immunoprecipitation/protein kinase assays were performed. In some experiments, the cells were treated with 1 nM 12-Otetradecanoylphorbol-13-acetate (TPA) (Sigma) for 5, 30, 60, and 180 min. To determine the roles of PKC and MAPK in UV-and TPA-induced junD expression, either GF-109203X (1 M) or PD98059 (25 M) (Calbiochem) was added to the cell medium 20 min prior to UV or TPA treatment, respectively.
RNA Extraction and Northern Blot Analysis-RNAs from ML-1 cells were extracted with a guanidium thiocyanate procedure (12,25). Briefly, 1 ϫ 10 7 cells were collected for sample preparation at each time point. Following rinsing with ice-cold PBS, the cells were immediately lysed with 1 ml of guanidium solution (5 M guanidium thiocyanate, 50 mM Tris-HCl, pH 8, 0.5% N-lauroylsarcosine, and 100 mM ␤-mercaptoethanol). The lysates were extracted three times with phenol/chloroform (50:50). Finally, RNAs were precipitated by centrifugation at 12,000 rpm for 20 min after pre-incubating with ethanol at Ϫ80°C. Samples of RNAs (20 g each) were loaded in agarose gel (1%) denatured with 2.2 M formaldehyde. The fractionated RNA was transferred onto a nylon membrane that was subsequently hybridized with a ␣-32 Plabeled junD cDNA probe using a random primer labeling kit (New England Biolabs, Beverly, MA).
Kinase Assay and Western Blot-Immunocomplexes were washed twice with a kinase buffer (20 mM MgCl 2 , 25 mM ␤-glycerophosphate, 100 mM sodium orthovanadate, 2 mM dithiothreitol, and 20 mM HEPES, pH 7.6) and then resuspended in 100 l of the kinase buffer. GST-ATF-2 (Santa Cruz Biotechnology) was used as a substrate in kinase activity assay for JNK-1 and p38, and GST-MBP (Santa Cruz Biotechnology) was used as a substrate in the Erk-2 kinase activity assay. The kinase reaction was initiated by adding 2 l of an ATP mixture containing 20 M ATP and 10 Ci of [␥-32 P]ATP (Amersham Biosciences). The reaction was performed at room temperature for 5 min and terminated by adding 30 l of 2ϫ Laemmli buffer. Phosphorylations of ATF-2 and MBP were resolved on PAGE gels and visualized by autoradiography.
Protein levels of JNK-1, Erk-2, and p38 were determined by Western blotting. Briefly, an equal volume of 2ϫ Laemmli buffer was added to 30 l of the immunocomplex. After fractionation of proteins on a 12% PAGE gel, the proteins were transferred to a polyvinylidene difluoride membrane. The membrane was then incubated with the same antibodies as used for immunoprecipitation with dilutions of 1:2000 for JNK-1, 1:5000 for Erk-2, and 1:2000 for p38. Following incubations with goat anti-rabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (1:1000) (Santa Cruz Biotechnology), the protein bands were visualized with a Phototope-Star Western blot detection kit (New England Biolabs).
Transfection of ML-1 Cells-To establish a model for exogenous transient gene expression in ML-1 cells, pEGFP (CLONTECH) and ␤-galactosidase control vectors (CLONTECH) were employed as reporter genes. Both genes were introduced into ML-1 cells by electroporation as described below. ML-1 cells were washed twice with cold PBS and resuspended in 0.8 ml of cold PBS at 10 7 cells/ml. For each transfection, DNA samples (10 g per sample) were added into 0.8 ml of cell suspension. This mixture was shocked in a 0.4-cm gap cuvette at 300 V. Reporter gene expressions were detected every 24 h following electroporation. The expression of green fluorescent protein (GFP) was directly observed under a fluorescence microscope. The expression of ␤-galactosidase was determined with a detection assay kit (CLONTECH). Briefly, transfected ML-1 cells were lysed at 24, 48, and 72 h postelectroporation by repeated freezing and thawing 3 times. Cell lysates were centrifuged at 12,000 ϫ g for 5 min at 4°C. To determine ␤-galactosidase activity, samples (30 l of the supernatant of each) were added into a white opaque 96-well plate with a flat bottom. Each well contained 200 l of the reaction buffer mixture (4 l of reaction substrate, 196 l of reaction buffer). After incubation at room temperature for 1 h, the plate was directly exposed onto x-ray film for 15-30 min, and then all reaction mixtures were transferred into 0.5-ml tubes, and chemiluminescence intensity was determined in a scintillation counter. After characterizing optimal conditions for the electroporation of ML-1 cells, three exogenous genes, full-length human MEK1 (FL-MEK1-EE, in pcDNA III), constitutively activated JNKK1 kinase domain (ƒJNKK1-KD, in pcDNA III) and junD (in pcDNA III), were introduced into ML-1 cells by electroporation.
Cell Death Assay and Caspase-3 Activity Detection-Cell death was analyzed by evaluating trypan blue exclusion. The cells were exposed to trypan blue (0.2% final concentration in PBS), and the percentage of trypan blue-positive dead cells was determined in fields of at least 200 cells. Caspase-3 activity in ML-1 cells was determined using a colorimetric CaspACE TM Assay system (Promega, Madison, WI). ML-1 cells (10 7 ) were washed twice with cold PBS and lysed in 100 l of lysis buffer by three cycles of freeze thawing. Following centrifugation at 15,000 ϫ g for 15 min at 4°C, 50-l lysates were added into a 96-well plate containing a reaction mix to measure caspase-3 activity. After incubation for 4 h at 37°C, A 405 of each sample was measured with a microplate reader (Molecular Devices Corporation, Sunnyvale, CA). Based on a standard curve obtained with p-nitroaniline standards, p-nitroaniline produced in the reaction was calculated as specific caspase activity. Activities are expressed as pmol/10 6 cell.
Statistical Analysis-For Northern and Western analysis, autoradiograph films were scanned, and relative densities of each signal band were analyzed using an NIH Image program. The results are reported as mean Ϯ S.E., and statistical significance was determined with the paired Student's t test at p Ͻ 0.05 or by one-way analysis of variance (ANOVA; F Ͻ 0.05).

RESULTS
UV Irradiation-induced junD mRNA Expression-We first determined whether UV irradiation affected junD mRNA expression in ML-1 cells following serum deprivation for 36 h, which synchronized the cell cycle at the G 1 phase. This was done because previous studies revealed that junD expression level was very low under this condition (12). Using Northern analysis, junD expression was detected at 5, 30, 60, and 180 min after UV exposure. UV irradiation markedly increased junD mRNA expression compared with that in the control cells (Fig. 1).
Role of PKC in UV-induced junD Expression-Upon application of TPA (1 nM), a strong activator of cPKC and nPKC, junD expression significantly increased at 5 min (data not shown) and was continuously maintained at a high level for up to 180 min ( Fig. 2A). The pattern of the time-dependent stimulation by TPA of junD expression was very similar to that induced by UV irradiation. This similarity suggests that UV-induced rise in junD expression may require activation of a PKC-linked signaling pathway. To make this determination, the effect of UV on junD expression was measured during exposure to GF-109203X, a selective inhibitor of cPKC. In both cases, UV-and TPA-induced junD expressions fell close to the basal level in cells pre-incubated with 1 M GF-109203X (Fig. 2, B and C), indicating that PKC activation is a component of the signaling pathway mediating UV-and TPA-induced increases in junD expression.
Effects of UV Irradiation and TPA on Intracellular Signal Pathways-To further characterize downstream events involved in a UV-induced rise in junD expression, JNK, Erk, and p38 kinase activities were measured in response to UV-irradiation. We found that all three limbs of the MAP kinase cascade were activated by UV irradiation. JNK-1, ERK-2, and p38 Kinase activities quickly increased within 5 min after UV irradiation. However, the time courses of activation were variable. JNK-1 and p38 reached peak levels at 30 min followed by declines lasting from 1 to 3 h (Fig. 3, A and B). However, UV increased Erk-2 activity to a high level that remained stable during the entire observation period (Fig. 3C). It should be noted that the time courses for UV-induced Erk-2 activation and increases in junD expression are very similar to one another ( Fig. 1).
Previous studies showed that PKC activation by TPA stimulates JNK, Erk, and p38 signaling pathways. Unlike UV irradiation, TPA induces in some cell types a much stronger activation of Erk kinase than that of JNK or p38 (13). To determine whether a specific MAP kinase pathway mediates UV-induced junD expression, the difference was determined between TPA-and UV-induced MAP kinase activations. When ML-1 cells were treated with 1 nM TPA, Erk-2 activity dramatically increased throughout the entire observation period (Fig.  4A) with a pattern very similar to that induced by UV (Fig. 3C). However, TPA had a much weaker activation effect on both JNK-1 and p38 (Fig. 4, B and C). These results strongly suggest that the MAPK signaling pathway containing Erk-2 kinase is possibly a mediator of UV-induced increases in junD expression situated downstream from PKC stimulation.
Effect of Suppressing PKC Activity on MAP Kinases-To confirm that Erk-2 is a downstream mediator of PKC in the UV-induced signaling pathway, we next determined whether inhibition of PKC activation by GF-109203X affected UV stimulation of Erk-2, JNK, and p38 activities. ML-1 cells were pretreated with 1 M GF-109203X for 20 min prior to UV irradiation. As shown in Fig. 5A, the inhibition of PKC activity by GF-109203X completely suppressed UV-induced Erk-2 activation. GF-109203X had a similar inhibitory effect on TPA-induced Erk-2 activation (Fig. 5B). However, UV-induced JNK-1 and p38 activation were unaffected by the inhibition of PKC activity by GF-109203X (Fig. 5, C and D). These results further indicate that PKC (possibly cPKC) plays an important role in mediating UV-induced Erk activation in ML-1 cells.
Effect of Suppressing MEK on Erk-2 Activation and junD Expression-In the Erk signaling pathway, Erk is directly activated by MAPK kinase (MEK1), whereas MEK1 is activated  (Fig.  6, A and B). However, a blockade of MEK1 activation had no effect on the UV-induced rise in JNK-1 and p38 activities (Fig.   FIG. 3. UV irradiation-induced JNK-1, Erk-2, and p38 kinase activities. The effects of UV irradiation on JNK-1 (A), Erk-2 (B), and p38 (C) activities were studied following a time course up to 180 min after UV stimulation. JNK-1, Erk-2, and p38 proteins were collected from ML-1 cells by immunoprecipitation using antibodies against JNK-1, Erk-2, or p38, respectively. Kinase activities were determined by isotopic kinase assay using ATF-2 as substrates for JNK and p38 and MBP as a substrate for Erk. Protein concentrations in kinase assay experiments were determined by Western analysis (lower panels). * indicates a significant difference compared with the control (n ϭ 4, F Ͻ 0.05).

FIG. 4. Effects of TPA-induced PKC activation on MAP kinase activities.
Kinase activities of Erk (A), JNK (B), and p38 (C) in ML-1 cells were measured by immunoprecipitation and kinase assays. Serum-starved ML-1 cells were treated with 1 nM TPA and collected for immunoprecipitation and kinase assay at the indicated time points. Protein concentrations of JNK-1, Erk-2, or p38 were determined by Western analysis using specific antibodies (lower panels). * indicates a significant difference compared with the control at time 0 (n ϭ 4, F Ͻ 0.05). 6, C and D). These results are fairly consistent with previous data showing that the inhibition of PKC activity by GF-109203 did not affect JNK-1 and p38 activities. In addition, the inhibition of MEK1 activity by PD98059 completely suppressed UV-and TPA-induced junD expression (Fig. 6, E and F). These results provide additional support for the hypothesis that Erk-2 activation is a signaling event situated downstream from PKC stimulation in the signaling pathway that mediates the UV-induced rise in junD expression.
Effect of Overexpression of MEK1 on junD Expression-To confirm the role of the Erk signaling pathway in UV-induced increases in junD expression, an exogenous MEK1 gene was inserted and overexpressed in ML-1 cells. First, a ␤-galactosidase control vector was transfected into ML-1 cells by electroporation to determine the expression efficiency. Galactosidase activity in transfected ML-1 cells was significantly increased compared with control cells, indicating that the transfection method was appropriate for these cells (Fig. 7A). Thus, a fulllength MEK1 sequence contained in the pcDNA III (FL-MEK1-EE) vector was inserted with electroporation. Following transfection, basic Erk-2 activity was obviously elevated relative to that in non-transfected cells, and the baseline expression of junD was also significantly increased (Fig. 7, B and C). Moreover, UV-induced increases in Erk-2 activity and junD expression in MEK1-transfected cells were significantly larger than in non-transfected cells (Fig. 7, B and C). However, MEK1 overexpression did not affect JNK-1 activity (Fig. 7D) and p38 activity (data not shown). In comparison, the transfection of a constitutive positive JNKK1 gene (ƒJNKK1-KD, constructed in pcDNA III) into ML-1 cells resulted in the overexpression of JNKK1 directly upstream from JNK but had no significant effect on junD expression, although it increased the baseline activity of JNK-1 (Fig. 7, D and E). All of these results consistently showed that the overexpression of MEK1 selectively enhanced Erk-2 activity and junD expression. The results provide further evidence in ML-1 cells that the Erk signaling pathway is a downstream mediator of PKC activation in UVinduced junD expression.
Effects of the Suppression of Erk Activation and the Overexpression of junD on UV-induced ML-1 Cell Death-The effect of the suppression of Erk activation on UV irradiation-induced cell death was studied by treating the cells with a MEK1 inhibitor. PD98059 (25 M) was added 20 min prior to UV irradiation. Cell viability was determined 8 h after UV irradiation using trypan blue exclusion to evaluate the rate of cell death. The inhibition of Erk activation markedly reduced cell death induced by UV irradiation (Fig. 8, A). In addition, cellular apoptotic response was evaluated based on measurements of caspase-3 activity. A time course of UV irradiation-induced caspase-3 activation is shown in Fig. 8B. The apoptotic response to UV irradiation in the presence and absence of Erk inhibition was determined 6 h after UV irradiation. UV-in- duced increases in caspase-3 activity were significantly decreased when cells were pretreated with the MEK inhibitor (Fig. 8C). The functional role of junD on UV irradiation-induced cell death was observed by the overexpression of fulllength cDNA encoding JunD. CMV-junD was inserted into ML-1 cells by electroporation, and the empty CMV vector was also transfected into ML-1 cells as a control. After culturing for 48 h, transfected cells were exposed to UV irradiation. The UV-induced death rate of junD transfected cells was markedly increased, and UV-induced caspase-3 activity was also signifi-cantly increased in junD transfected cells compared with empty vector transfected cells (Fig. 8, D and E). Results obtained from these experiments were consistent with the expression pattern of junD induced by UV irradiation, suggesting that Erk activation-linked increases in junD expression play an important role in UV irradiation-induced ML-1 cell apoptosis. DISCUSSION In the present study, we demonstrated that the exposure of ML-1 cells to UV irradiation significantly increased junD mRNA expression. The pattern of this increase is similar to the increases in junD expression resulting from PKC activation by TPA. Both UV-and TPA-induced junD expression can be blocked by the pretreatment of cells with a PKC inhibitor, GF-109203X. These results indicate that certain membranelinked PKC isozymes are components of UV-and stress-induced signaling pathways. PKC isoforms are divided into cPKC (including PKC␣, PKC␤I, PKC␤II, and PKC␥), nPKC (including PKC␦, PKC, PKC, and PKC⑀), and aPKC (including PKC and PKC/). Recent studies have shown that PKC is involved in UV-induced cellular responses. For example, UV irradiation can cause the translocation of some PKC subtypes (PKC⑀ and PKC␦) to the cell membrane, which is required for UV-induced activation of Erk and JNK (13). In addition, UVinduced AP-1 activation can be blocked by the expression of a dominant negative mutant PKC/ (14). Although the present study did not identify which PKC isoform(s) must be activated for UV exposure to induce activation of junD expression, both cPKC and nPKC (especially cPKC) seem to play an essential role in this event. First, the UV-induced junD expression pattern was similar to that in TPA-stimulated cells (Figs. 1 and 2A) (27,28). Second, pretreatment with GF-109203X selectively blocked cPKC activity and completely prevented UVinduced junD expression (Fig. 2C) (29). Finally, UV-induced Erk-2 activation was also blocked by the PKC inhibitor GF-109203X (Fig. 5A). In addition, the hypothesis is supported by a recent report that demonstrates that cPKC and nPKC activation are involved in mediating a cellular response to hyper-osmotic stress (28). However, further investigation is necessary to identify which PKC isoforms are involved in UV-induced junD expression.
Previous studies of c-jun gene expression suggest that UV irradiation can strongly activate the JNK signaling pathway resulting in increases in c-jun expression (17,30). JNK activation contributes in turn to both c-jun transcriptional activation and protein phosphorylation (28,31,32). Indeed, JNK, Erk, and p38 signaling pathways were activated by UV irradiation in ML-1 cells. However, our results indicate that JNK and p38 may not play roles in events downstream from PKC to mediate UV-induced activation of junD expression. In fact, the time course of UV-induced Erk activation matches the time course of UV-induced junD expression (Figs. 1 and 3C). On the other hand, even though a PKC inhibitor, GF-109203X, nearly completely blocked Erk activation induced by UV irradiation and TPA as well as junD expression, it had no inhibitory effect on UV-induced increases in JNK and p38 activity. Our evidence to support the hypothesis that Erk is the downstream mediator of PKC activation includes the following facts. 1) Down-regulation of PKC with GF-109203X selectively blocked Erk activation in response to UV and TPA stimulation. 2) Selective inhibition of the Erk signaling pathway by PD98059, a selective inhibitor of MEK (a MAPK kinase immediate upstream of Erk), suppressed Erk-2 activity to the basal level and prevented UVand TPA-induced increases in junD expression (Fig. 6). 3 control and UV-and TPA-induced cells (Fig. 7). In addition, the inhibition of Erk activation by PD98059 effectively suppressed UV irradiation-induced caspase-3 activity and ML-1 cell death (Fig. 8). These results consistently demonstrate that the Erk signaling pathway is located downstream from PKC and plays an important role in mediating UV-induced junD expression in ML-1 cells.
Our conclusion is consistent with recent studies showing the involvement of the Erk signaling pathway in c-jun activation. In NIH 3T3 cells the application of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocks UV-induced JNK activation without affecting UV-induced increases in c-jun expression (24). The inhibition of the Erk signaling pathway with PD98059 suppresses UV-induced increases in c-jun mRNA level and c-Jun protein expression (24). The overexpression of wild type Erk-2 causes 46 -140-fold increases in UV-induced AP-1 activity. Conversely, the introduction of a dominant negative Erk-2 into cells suppressed both UV-induced Erk and AP-1 activation (14).
A remaining question is how does PKC stimulation by UV result in Erk activation. The pathway that leads to Erk activation can be triggered by a variety of stimuli (including UV irradiation). It is known that membrane activation of Ras and PKC during UV exposure occurs earlier than Erk stimulation. Therefore, it is possible that UV irradiation induces Erk activation by either stimulating Ras or PKC or both. Our data imply that, in ML-1 cells, PKC activation instead of the Ras pathway may be an essential mediator in the transmission of UV stimulation to Erk because Erk activation by UV irradiation was completely abolished when PKC activity was inhibited by GF-109203X (Fig. 5A). This conclusion is consistent with other studies (27,33,34), indicating that PKC is involved in the transmission of other stress stimuli such as oxygen-stress (33,34) and hyperosmotic stress (27), resulting in activation of the Erk pathway.
As a member of the Jun protein family, JunD shows functional similarity to c-Jun (4, 6, 7). The overexpression of c-jun induces apoptosis in growth factor-deprived cells (35). The expression of the dominant negative mutant of c-Jun or the inhibition of c-Jun function by specific antibodies can protect neuronal cells from apoptosis induced by growth factor withdrawal (36,37). It is also known that UV irradiation induces c-jun activation and results in cell apoptosis (13,14,35,38). Interestingly, we did not find that either UV irradiation or TPA stimulation could activate c-jun expression in ML-1 cells (data not shown). We speculate that increases in junD expression of ML-1 cells may play a compensatory role in eliciting UV-induced apoptosis despite c-jun deficiency, although the effect of UV-induced junD expression on cellular function was not determined here. Nevertheless, this question warrants future investigation. We found that the overexpression of junD enhances UV-induced increases in caspase-3 activity and cell death (Fig. 8). This result indicates a functional role of increased junD expression in response to UV irradiation.
In conclusion, our results indicate that, in ML-1 cells, UV irradiation-induced activation of junD expression and its activation are mediated by a PKC-coupled Erk signaling pathway. The sequential responses to UV irradiation result in ML-1 cell death. These findings also extend our knowledge about the cellular signaling mechanisms that mediate UV-induced increases in immediate early gene expression.