Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2007 Jul 16.
Published in final edited form as: Oncogene. 2005 Apr 21;24(18):3020–3027. doi: 10.1038/sj.onc.1208547

Ultraviolet irradiation-induced K+ channel activity involving p53 activation in corneal epithelial cells

Ling Wang 1, Wei Dai 2, Luo Lu 1,*
PMCID: PMC1920501  NIHMSID: NIHMS22482  PMID: 15750624

Abstract

Recent studies from our lab found that ultraviolet (UV) irradiation induces a voltage-gated potassium (Kv) channel activation and subsequently activates JNK signaling pathway resulting in apoptosis. The present study in rabbit corneal epithelial (RCE) cells is to investigate mechanisms of UV irradiation-induced Kv channel activity involving p53 activation in parallel to DNA damage-induced signaling pathway. UV irradiation-induced signaling events were characterized by measurements of JNK activation and further downstream p53 phosphorylation. UV irradiation elicited an early response in the cell membrane through activation of Kv channels to activate the JNK signaling pathway and p53 phosphorylation. Exposure of RCE cells to UV irradiation within a few min resulted in JNK and p53 activations that were markedly inhibited by suppression of Kv channel activity. However, suppression of Kv channel activity failed to prevent p53 activation induced by extended DNA damages through prolonging UV exposure time (more than 15 min). In addition, caffeine inhibited UV-induced activation of SEK, an upstream MAPK kinase of JNK, resulting in suppression of both Kv channel-involved and DNA damage-induced p53 activation. Our results indicate in these cells that UV irradiation induces earlier and later intracellular events that link to activation of JNK and p53. The early event in response to UV irradiation is initiated by activating Kv channels in the cell membrane, and the later event is predominated by UV irradiation-caused DNA damage.

Keywords: gene expression, Kv channel blocker, DNA damage, apoptosis

Introduction

The corneal epithelium absorbs ultraviolet (UV) wavelengths shorter than 310 nm thereby acting as a filter and protecting the lens and retina from UV-induced damage (Ringvold, 1998). UV irradiation-induced corneal epithelial injury can affect corneal epithelial barrier function and in turn increase susceptibility to infection, and the development of corneal opacity. UV irradiation triggers a series of cellular events that include eliciting stress-induced signaling pathways and DNA damage. Both activation of cellular signaling pathways and DNA damage can change the phosphorylation levels of p53 protein resulting in cell cycle arrest and apoptosis. The most common DNA damage is forming cyclobutane pyrimidine dimmers (CPD) mutations that stimulate increases in p53 phosphorylation levels through activating chks (van Kranen et al., 1995; Ziegler et al., 1996). In addition, Chk activation induced by DNA damage, caused by ionizing radiation (IR), reactive oxygen species (ROS), and UV irradiation, increases p53 phosphorylation levels at various sites and is mediated by activation of Polo-like kinase (PLK), (Xie et al., 2001a, b, 2002). Recent studies suggest that MAP kinases including JNK, Erk, and p38 phosphorylate and activate p53, leading to p53-mediated cellular responses (Fuchs et al., 1998b; Bulavin et al., 1999; Wu, 2004). UV irradiation-induced activations of JNK and Erk result in phosphorylations of p53 at Thr81 and Ser15 sites, respectively (Buschmann et al., 2001; Melnikova et al., 2003). In previous studies, we found that UV irradiation also initiates membrane events to cause K+ channel activation (Wang et al., 1999). Voltage-gated potassium (Kv) channels are universally present in the cell membrane and are essential in maintaining the normal cell function. UV irradiation-induced apoptosis in corneal epithelial cells is mediated by activation of Kv channels in the cell membrane (Lu et al., 2003; Wang et al., 2003). Suppression of Kv channel activity using specific Kv channel blockers effectively inhibited JNK activation and prevents these cells from UV irradiation-induced death.

It is known that p53 is multifunctional in various cell types. One of its roles is to act as a tumor suppressor protein that senses the DNA damage and acts as a guardian of genome stability (Chernova et al., 1995; Blaise et al., 2001; Gentiletti et al., 2002). The phosphorylation status of p53 determines the stability of the protein and controls cell cycle progression, which serves as a master-switch for promoting apoptosis (Ginsberg et al., 1991; Ashcroft and Vousden, 1999; Colman et al., 2000; Asher et al., 2001; Sogame et al., 2003). Alterations of p53 protein, such as missense mutations and loss of its expression caused by nonsense or frame-shift mutations, can result in carcinogenesis (Hussain and Harris, 1998; Medina et al., 2002; Shirai et al., 2002; Nishikawa et al., 2003; Hofseth et al., 2004). In some cases, p53 becomes a molecular signature based on the type of cancer (Hussain et al., 2000). The multiple functions of p53 include: (1) to regulate cell cycle progression; (2) to survey cellular stress; and (3) to induce apoptosis in extreme cases. Although the mechanisms underlying p53-dependent apoptotic responses remain incompletely characterized, p53 is known to be involved in both the extrinsic and the intrinsic pathways of apoptosis by initiating apoptosis through mitochondrial depolarization and sensitizing cells to inducers of apoptosis (Wang et al., 1998; Wang, 1999; Haupt et al., 2003; Tang et al., 2003). For example, active p53 stimulates the apoptotic infrastructure by increasing the expression of apoptotic protease-activating factor 1 (APAF-1), a crucial component of the apotosome (Kinzler and Vogelstein, 1997; Levine, 1997). The signaling pathways underlying the cellular response to DNA damage (genotoxic stress) consist of sensors, signal transducers, and effectors (Zhou and Elledge, 2000). Although the identities of the damage sensors remain unknown, the molecular entities responsible for transducing the damage signals to specific effectors are relatively well characterized. Ataxia telangiectasia mutated (ATM) and its homolog (ATR) function early in the signaling pathways and are central to the DNA damage response (Zhou and Elledge, 2000; Goodarzi et al., 2003; Dodson et al., 2004; Irarrazabal et al., 2004; Kastan, 2004; Yang et al., 2004). Downstream targets (substrates) of ATM/ATR include the checkpoint protein kinases Chk1 and Chk2 (Matsuoka et al., 1998; Sanchez et al., 1999; Tominaga et al., 1999). Chk1 and Chk2 are thought to mediate different types of DNA damage to the specific cellular responses. Further downstream, p53 is a major molecule executing UV-induced DNA damage (Abrahams et al., 1995; Smirnova et al., 2001).

However, there is very little information up to date about the expression and function of p53 in corneal epithelial cells. In the present study, we for the first time demonstrate that there are high expression levels of p53 protein in corneal epithelial cells. UV irradiation in these cells induces two stages (earlier and later) of cellular events that subsequently involve activation of p53. Suppression of Kv channel activity inhibits UV irradiation-induced JNK activation and p53 phosphorylation occurring in the early stage. However, UV irradiation-induced DNA damage increases p53 phosphorylation levels in both earlier and later stages.

Results

UV irradiation-induced activation of p53

Activation of p53 in response to a variety of cellular stresses including UV irradiation is a crucial component of cellular mechanism. Western analysis was used to measure expression levels of p53 protein in both primary cultured and SV-40-transformed rabbit corneal epithelial (RCE) cells. We found that p53 proteins in a hypophosphorylated state were highly expressed in RCE cells (Figure 1a). The protein amount and phosphorylation status of p53 in the cellular level were not altered by passages in the cultured condition in both types of RCE cells. UV irradiation-induced increases in phosphorylation levels of p53 were studied in RCE cells using antibodies specific to phospho-serine residues in p53 following a time course (Figure 1b). UV irradiation clearly induced an increase in phorsphorylation levels of p53 at Ser15 and Ser20, while there was no change in phosphorylation of p53ser46, indicating that activation of p53 in response to UV irradiation in RCE cells is phophorylation site-specific. On the other hand, UV irradiation-induced p53ser15 phosphorylation occurred earlier than the site of p53ser20 (Figure 1b). Immunostaining experiments were performed to determine the effect of UV irradiation on p53 phosphorylation and localization (Figure 1c). UV irradiation increased phosphorylation of p53ser15 in the nuclei, but total p53 protein levels were not changed in UV irradiation-induced and control RCE cells. Results in this figure suggest that there are high levels of p53 expressed in primary cultured and SV40-transformed RCE cells, and UV irradiation activates p53 by phosphorylating both ser15 and ser20 sites.

Figure 1.

Figure 1

Open in a new tab

UV irradiation induced the phosphorylation of p53 in RCE cell. (a) Expression of p53 in primary and SV40-transformed RCE cells. Expression of p53 and phosphorylated p53 (pp53) were detected by Western analysis. High expression levels of p53 were found in both primary and SV40-transformed RCE cells; however, phosphorylation levels of p53 protein were rather low in these proliferative cells. β-Actin was detected as the internal control. (b) Site-specific analysis of UV-irradiation-induced p53 phosphorylation. UV-irradiation-induced phosphorylations of p53 were analysed with site-specific antibodies in Western blots. RCE cells were exposed to UV lights, and then whole cell lysates were fractionated on a PAGE gel. Antibodies against pp53ser15, pp53ser20, pp53ser46, and total p53 protein were used in Western analysis. (c) Immunostaining of p53 and pp53ser15 induced by UV irradiation. RCE cells were grown on glass slide. Cellular expressions of p53 and pp53ser15 were detected by specific antibodies. Pictures were taken by a digital camera under Nikon fluorescent microscope (× 60 power lens)

Effect of suppressing Kv channels on JNK and p53 activities

We have previously reported that suppression of Kv channels prevents UV-induced apoptosis in RCE cells. UV irradiation-induced apoptotic response in these cells is through eliciting Kv channel activity and activation of JNK cascades (Lu et al., 2003). To test the further downstream p53 phosphorylation in the JNK signaling pathway, phosphorylation levels of JNK and p53ser15 were measured in RCE cells following a time course after UV induction. It appeared that phosphorylation levels of JNK1/2 were rapidly increased within 5 min after UV irradiation, while phosphorylation of p53ser15 was also increased in a later time between 5 and 15 min (Figure 2a). To further test the notion, JNK1 proteins were immunoprecipitated from UV irradiation-induced RCE cells and hypophosphorylated GST-p53 fusion protein was used as a substrate in immunocomplex kinase assays (Figure 2b). UV irradiation-activated JNK1 from RCE cells effectively phosphorylated GST-p53 and the control substrate ATF2. In the other group study, 4-aminopyridine (4-AP) that specifically blocks Kv channel activity in RCE cells was applied to the cells 15 min prior to exposure of UV irradiation. A dose–response relationship between suppressing Kv channel activity by 4-AP and UV irradiation-induced JNK phosphorylation levels were observed in Figure 2C. To test the effect of Kv channel activity on p53 in JNK signaling pathway in RCE cells, the interaction between JNK and p53 was determined by immunocoprecipitation and immunocomplex kinase assays. JNK1 and p53 proteins were obtained in RCE cells by immunocoprecipitation using specific JNK1 and p53 antibodies. Inhibition of Kv channels with 1 mM 4-AP effectively prevented UV irradiation-induced JNK1 activation and phosphorylation of p53 (Figure 2d). Furthermore, the dose–response relationship between suppressing Kv channel activity by 4-AP and UV irradiation-induced p53ser15 phosphorylation levels were also examined in RCE cells (Figure 3a). UV irradiation-induced phosphorylation of p53ser15 was markedly suppressed by 4-AP in a similar dose–response pattern compared with the effect of 4-AP on UV irradiation-induced JNK1/2 phosphorylation. The inhibitory effect of 4-AP (1 mM) on UV irradiation-induced p53ser15 phosphorylation was sustained up to 8 h in the testing period post to UV irradiation (Figure 3b). Effects of other Kv channel inhibitors on UV irradiation-induced p53ser15 were demonstrated in Figure 3c. Besides 4-AP (1 mM) effectively inhibited about 50% UV irradiation-induced p53ser15 phosphorylation, other blockers in the group, including Ba2+ (5 mM), α-dendrotoxin (α-DTX at 200 nM) and blood depressing substance-I (BDS-I at 400 nM), also partially inhibited the effect of UV irradiation on p53ser15 phosphorylation. These are consistent with the previous patch clamp study results because these blockers are effective to Kv channels in RCE cells. The results suggest that p53 phosphorylation results from UV irradiation-induced activation of Kv channel and JNK cascades, and p53 is a downstream component in JNK signaling pathway.

Figure 2.

Figure 2

Open in a new tab

UV irradiation-induced JNK activation and phosphorylation of p53 at Ser15. (a) UV irradiation-induced time-dependent activation of JNK and phosphorylation of p53ser15. (b) Phosphorylation of p53 fusion protein by UV activates JNK in vitro. UV-activated JNK was purified by immunoprecipitation (IP), and GST-ATF fusion protein was used as the positive control. (c) Dose-dependent inhibition of 4-AP in UV irradiation-induced activation of JNK. Total JNK proteins were detected in Western blots as the controls. (d) Inhibitory effect of 4-AP on UV irradiation-induced p53 phosphorylation following the time course. RCE cells were pretreated with 4-AP, a specific Kv channel blocker, 30 min prior to UV irradiation. Phosphorylation of p53 as analysed by Western blot

Figure 3.

Figure 3

Open in a new tab

Effect of suppressing Kv channel activity on UV irradiation-induced p53 phosphorylation. (a) Concentration-dependent effect of 4-AP on inhibition of UV irradiation-induced p53 phosphorylation. Total p53 proteins were detected with anti-p53 antibody. Phosphorylated p53ser15 proteins were determined by Western analysis using anti-phospho-p53 antibody. (b) Time course of inhibiting UV irradiation-induced phosphorylation of p53ser15 by 4-AP. RCE cells were treated with 1 mM 4-AP, an inhibitor of Kv channels, 30 min prior to UV irradiation. UV irradiation-activated p53 in RCE cells was determined by Western analysis. (c) Effects of suppressing Kv channel activity with various blockers on UV irradiation-induced phosphorylation of p53ser15. RCE cells were pretreated with specific Kv channel blockers, including 1 mM 4-AP, 5 mM Ba2+, 200 nM α-DTX, or 400 nM BDS-I, 30 min prior to UV irradiation. Phosphorylation of p53 as analysed by Western blot. Symbols ‘*’ represent significant difference analysed by one-way ANOVA and multiple comparison test, P<0.05

Effect of drug-induced DNA damage on phosphorylation of p53

Melphalan, a DNA damage agent that induces DNA intrastrand crosslinks, was used to study effects of DNA damage-induced increases in phosphorylation levels of p53. Treatment of RCE cells with melphalan induced phosphorylation of p53ser15 following a dose-dependent pattern (Figure 4a). The effect of melphalan (100 μg/ml) on phosphorylation of p53ser15 was also observed in RCE cells following a time course of the treatment (Figure 4b). Melphalan-induced phosphorylation of p53ser15 was markedly suppressed by various concentrations of caffeine (Figure 4c). However, suppression of Kv channel with different dosages of 4-AP (up to 2 mM) failed to inhibit melphalan-induced phosphorylation of p53ser15 (Figure 4d). These results support the notion that suppression of Kv channels in the membrane inhibits UV irradiation-induced JNK activation, subsequently suppressing phosphorylation of p53ser15. Blockade of Kv channel does not affect melphalan-induced phosphorylation of p53ser15 through DNA damage-evoked pathways.

Figure 4.

Figure 4

Open in a new tab

Effect of melphalan-induced DNA damages on phophorylation of p53ser15 in RCE cells. (a) Dose-dependent response of p53ser15 phosphorylation induced by melphalan. (b) Time-dependent response of p53ser15 phosphorylation induced by melphalan. (c) Effect of caffeine on melphalan-induced p53 phosphorylation. Different concentrations of caffeine were added in RCE cells prior to melphalan induction. (d) Effect of suppressing Kv channel activity with 4-AP on melphalan-induced p53 phosphorylation. Different concentrations of 4-AP were added in RCE cells prior to melphalan induction. In this group of experiments, DNA damage was induced by 100 μg/ml melphalan and phosphorylation of p53ser15 was analysed by Western analysis with a specific antibody against phospho-p53ser15 and total amount of p53 protein was detected by an anti-p53 antibody as the loading control

Effect of increased intensity of UV exposure on phosphorylation of p53ser15

In order to distinguish the effect of UV irradiation-induced membrane signaling pathway activation from the effect of UV irradiation-induced DNA damage on phosphorylation of p53, RCE cells were extensively exposed to UV irradiation using a protocol (protocol B) by extending UV exposure time up to 30 min instead of the fixed UV dosage that has been used in previous studies. In previous studies, we have reported that the effect of UV irradiation at a dosage of 40 μJ/cm2 on Kv channel activity and subsequent JNK activation can be completely blocked by 1 mM 4-AP. In the present study, Kv channel activity was blocked by application of 1 mM 4-AP in RCE cells during entire extended period of UV irradiation. Suppression of Kv activation by 4-AP partially inhibited UV irradiation-induced phosphorylation of p53ser15 up to 15 min; however, suppression of Kv channel activity was not able to inhibit the effect of extensive UV exposure on phosphorylation of p53ser15 after 15 min of exposure period (Figure 5a). On the other hand, caffeine (5 mM) was applied in RCE cells during the testing period of UV irradiation. Caffeine effectively inhibited the effect of extensive UV exposure on phosphorylation of p53ser15 during the entire 30 min testing period (Figure 5b). It was noticed that UV irradiation-induced early phosphorylation of p53ser15 was completely inhibited by caffeine, suggesting that caffeine may also interact with signaling components at JNK level. To test this notion, the effect of caffeine on UV irradiation-induced phosphorylation of SEK, an MAPK kinase immediately upstream from JNK, was studied by incubation of cells with different concentrations of caffeine for 20 min prior to UV irradiation using the protocol A. Applications of 1–20 mM caffeine effectively suppressed UV irradiation-induced SEK phosphorylation (Figure 5c). UV irradiation-induced JNK phosphorylation was also inhibited by caffeine in a similar dosage range (data not shown). Results presented in Figure 5 indicate that there are two different signaling pathways mediating UV irradiation-induced p53 activation through membrane Kv channel activation and DNA damages.

Figure 5.

Figure 5

Open in a new tab

Effects of 4-AP and caffeine on UV irradiation-induced phosphorylation of p53ser15 and SEK. (a) Effect of suppressing Kv channel activity with 4-AP on extended UV irradiation-induced phosphorylation of p53ser15. Phosphorylation levels of p53ser15 in the absence and presence of 1 mM 4-AP were plotted as a function of extended time of UV exposures. (b) Effect of caffeine on extended UV irradiation-induced phosphorylation of p53ser15. Phosphorylation levels of p53ser15 in the absence and presence of 5 mM caffeine were plotted as a function of extended time of UV exposures. For UV treatments, RCE cells were exposed to UV irradiation with an extended time for 3, 5, 10, 15, 20, and 30 min (protocol B) instead of a standard protocol (protocol A) of 40 μJ/cm2. Phosphorylation levels of p53ser15 were detected by Western analysis in RCE cells in the absence and presence of 4-AP or caffeine. (c) Effect of caffeine on UV irradiation-induced SEK activation. Inhibitory effect of caffeine on SEK phosphorylation was detected in a dose-dependent manner. Statistical analysis demonstrated significant inhibition of UV irradiation-induced SEK phosphorylation by caffeine (P<0.01, n = 3)

Discussion

In the present study, we report that exposure of RCE cells to UV irradiation significantly increases phosphorylation levels of p53ser15. The pattern of this increase is linked to both components including: (1) membrane Kv channel activity and JNK signaling pathway activation and (2) nuclear DNA damage. Our previous studies indicate that UV irradiation induces hyperactivity of Kv channels in RCE cells leading to activation of JNK cascades (Wang et al., 1999; Lu et al., 2003). Suppression of Kv channel activity effectively prevents UV irradiation-induced apoptotic response in these cells. In the present study, we found that expression level of p53 is rather high in the primary RCE cells and RCE cell line. UV irradiation induced increases in p53 phosphorylation at multiple levels. Specifically, UV irradiation induced phosphorylation of p53ser15 and p53ser20, but not ser46 analysed with Western blots with phosphorylation site-specific anti-p53 antibodies. Upon exposure of RCE cells to UV irradiation, phosphorylated p53ser15 proteins are rapidly increases in the nuclei, suggesting that phosphorylation of p53ser15 plays an important role in UV irradiation-induced signaling events leading to RCE cell apoptosis.

Our results indicate that there is a correlation between JNK activation and p53 phosphorylation in response to UV irradiation in RCE cells. To investigate the signal transduction of UV irradiation-induced phosphorylation of p53, the interaction between JNK and p53 was determined by several approaches. First, time courses of UV irradiation-induced increase in phosphorylation levels of JNK and p53 proteins demonstrate that UV irradiation-induced JNK phosphorylation occurs earlier than phosphorylation of p53ser15, suggesting that phosphorylation of p53ser15 is a further downstream event in the pathway (Figure 2a). Second, JNK1 and p53 proteins can be pulled down in UV irradiation-induced RCE cells in immnunocoprecipitation experiments by anti-JNK1 antibodies (Figure 2d). In addition, we examined the ability of UV-activated JNK kinase to catalyse phosphorylation of p53 fusion protein in vitro by using immunocomplex kinase assay (Figure 2b). In fact, JNK1 purified by immunoprecipitation from UV irradiated RCE cells is able to catalyse phosphorylation of GST-p53 at ser15 (Figure 2b). The activation of JNK by UV irradiation is confirmed by using ATF-2 fusion protein as the substrate of JNK. Our data provide additional evidence that p53 is a substrate protein of JNK and are consistent to previous studies that describe the interaction between JNK and p53 in other cell types (Fuchs et al., 1998a, b; Pluquet et al., 2003).

The most important finding of the present study is that phosphorylation of p53ser15 in UV irradiation-induced RCE cells results from two different signaling pathways, including UV irradiation-activated membrane Kv channels and nuclear DNA damage. There are two UV irradiation protocols used in this study: (1) protocol A uses a fixed UV dosage of 40 μJ/cm2 that equals to 3 min exposure time; and (2) protocol B is extensive exposure of cells to UV irradiation by extending exposure time to increase UV dosage up to 30 min. From previous study in these cells, we observed that the fixed dose UV irradiation activates a Kv channel in the membrane resulting in activation of JNK cascades and apoptosis. In the previous studies, we found that there are several channel blockers that are effective to inhibit Kv channel activity in RCE cells (Wang et al., 2004). The linkage between UV-induced hyperactivation of Kv channels and JNK activation has not been established yet. It is possible that UV-induced Kv channel hyperactivity can cause a fast loss of intracellular K+ ions, resulting in cell volume shrinkage. Recent studies suggest that scaffold protein MEKK1, an upstream MAPKK kinase of JNK cascades, is associated with cytoskeleton reorganization and activated in response to cell volume changes (Kwan et al., 2001; Cross and Templeton, 2004; Lieber et al., 2004).

Blockade of Kv channel activity with these Kv channel blockers suppress activation of JNK and prevent apoptosis in UV irradiation-induced RCE cells (Wang et al., 1999; Lu et al., 2003). However, blockade of Kv channel in RCE cells that were extensive exposure to UV irradiation only partially prevent phosphorylation of p53ser15 in 15 min, and failed to prevent phosphorylation of p53ser15 after 15 min continuous exposure of these cells to UV irradiation. In fact, we observed that blockade of Kv channel activity has no effect on phosphorylation of p53ser15 induced by a nuclear DNA damage reagent, melphalan, which mimics UV irradiation-induced DNA damage. It has been shown that ATM and ATR are DNA damage sensors in response to DNA damage resulting in phosphorylation of p53ser15 (Xie et al., 2001a; Ye et al., 2001). Caffeine can specifically block DNA damage-induced ATM and ATR responses and phosphorylation of p53ser15 (Ito et al., 2003; Costanzo et al., 2003). In the present case, DNA damage-induced phosphorylation of p53ser15 in both UV irradiation-induced and melphalan-treated RCE cells can be suppressed by caffeine. In addition, we have for the first time observed the new effect of caffeine that can inhibit UV irradiation-induced JNK signaling pathway by suppressing SEK phosporylation (Figure 5c). Apparently, the multi effects of caffeine on UV irradiation-induced signaling pathways provide new leads and tools for future stress-related signal pathway studies. It also requires further investigation to understand the pharmacological effect of caffeine in the JNK signaling pathway. There is another interesting observation in the study that suppression of Kv channels with 4-AP (1 mM) can completely prevent UV irradiation-induced JNK activation, but only partially prevent UV irradiation-evoked phosphorylation of p53ser15. Taken together, our data indicate that UV irradiation-induced phosphorylation of p53ser15 results from two signaling pathways. One of the pathways is the membrane Kv channel activity-mediated activation of JNK cascades. Suppression of this pathway with Kv channel blocker 4-AP can prevent UV irradiation-induced phosphorylation of p53ser15 in the early stage. The other pathway is DNA damage-induced signaling pathway. Suppression of DNA damage-induced signaling pathway with caffeine can effectively prevent UV irradiation-induced phosphorylation of p53ser15 in RCE cells. However, caffeine is able to effectively suppress UV irradiation-evoked phosphorylation of p53ser15, suggesting that there may be other factors that are involved in the p53 modulation (Figure 5). We therefore can conclude that UV-activated Kv channel activity mediates UV-induced phosphorylation of p53ser15 through JNK pathway, and plays an early role in RCE cell apoptotic pathways. In cells that are exposed to extensive UV irradiation, phosphorylation of p53ser15 is mainly due to UV irradiation-induced DNA damage. These findings extend our knowledge about the cellular signaling mechanisms mediating UV irradiation-induced apoptosis.

Materials and methods

Corneal epithelial cell culture

Primary RCE cells were isolated from the cornea by using an established protocol in our lab (Roderick et al., 2003; Wang et al., 2004). The cornea was removed from rabbit eyeballs and cut into small pieces. Corneal tissues were placed upside down in culture dishes containing DMEM/F12 medium supplemented with 10% FBS, 5 mg/ml EGF, 5 μg/ml insulin and 10 000 U/ml penicillin/10 000 μg/ml streptomycin. After corneal epithelial cells growing out within 5 days, cornea tissues were removed to allow RCE cells to continue growing to confluence. RCE cells were passed at a ratio of 1 to 4. RCE cells were maintained in an incubator supplied with 95% air and 5% CO2 at 37°C. The medium was replaced every 2 days and cells were passed using 0.05% Trypsin-EDTA.

For UV irradiation induction, two different protocols were designed. (1) Protocol A is designed to study UV irradiation-induced signaling response. In protocol A, corneal epithelial cells were exposed to UV-C light with a standard protocol that used a fixed UV dosage that was calculated at an intensity of 40 μJ/cm2 (equivalent to 3 min exposure time of protocol B). (2) Protocol B was used for DNA damage studies. In protocol B, RCE cells were extensively exposed to UV irradiation by extending UV exposure time up to 30 min. After UV irradiation, cells were collected and rinsed with phosphate-buffered saline (PBS). K+ channel blockers including 4-AP, BaCl2, DTX and BDS-I were obtained from Sigma and Alomone Labs, respectively.

Western analysis and immunochemistry

Western blot assay was performed as described previously (Xu et al., 1999). In brief, 5 × 105 cells were harvested in 0.5 ml lysate buffer containing (mM): 137 NaCl, 1.5 MgCl2, 2 EDTA, 10 sodium pyrophosphate, 25 β-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 Na-orthovanadate, 1 phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 20 Tris, pH 7.5. Cell lysates were rinsed by PBS and precleared by centrifugation at 13 000 g for 20 min. Samples were denatured by adding an equal volume of 2 × Laemmli buffer and boiling for 5 min, and loaded into 12% SDS–PAGE gel. After fractionation by electrophoresis, proteins were electrotransferred to PVDF membrane. The membrane was exposed to blocking buffer (5% nonfat milk in TBS-0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), and then incubated with respective antibodies overnight at 4°C or for 1 h at RT. After three washes with TBS-T buffer, the membrane was incubated with Horse Radish Peroxydase (HRP)-linked secondary antibody for 1 h at RT. Expression of proteins was detected with a Western blot detection kit (Santa Cruz Biotechnology).

For immunostaining experiments, RCE cells were grown on glass slides. After different treatments, RCE cells were rinsed twice with PBS and fixed for 15 min in 4% paraformaldehyde. RCE cells on slides were treated to be membrane permeable for 30 min with 0.1 M PBS plus 0.1% Triton X-100 (PBS-T), blocked for 1 h with 10% normal horse serum (NHS), and/or 10% normal goat serum in PBS-T at RT. RCE cell samples were incubated overnight at 4°C with primary antibody in PBS-T. After washing three times in PBS, the slides were incubated with fluorescein isothiocyanated (FITC)-conjugated donkey anti-goat IgG antibody (1 : 100, Jackson ImmunoR-esearch Lab) or Cy3-conjugated goat anti-mouse IgG antibody for 1.5 h at RT. The slides were washed and mounted in Vectashield (Vector Laboratories) for analasis under Olympus epifluorescence microscope or confocal microscopy (Olympus Fluoview: × 60 oil immersion objective lens, NA 1.4).

Immunoprecipitation and kinase assay

Protein kinase activity was measured by using the immunocomplex kinase assay as described previously (Xu et al., 1999). In brief, 5 × 106 RCE cells were exposed to UV-C irradiation (42 μJ/cm2) in the absence and presence of Kv channel blockers. Cells were washed once with ice-cold PBS, and then lysed with 0.8 ml of lysis buffer containing (mM): 20 Tris–HCl, 137 NaCl, 1.5 MgCl2, 2 EDTA, 10 sodium pyrophoisphate, 25 β-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 sodium orthovanadate, 1 phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and pH 7.5. Cell lysates were incubated on ice for 10 min, and then were precleared by centrifugation at 13 000 g for 10 min. JNK-1 proteins were immunoprecipitated by 0.5 μg of rabbit polyclonal antibody against JNK-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and protein A-sepharose beads (Sigma). The immunocomplex was washed three times with lysis buffer and twice with a kinase buffer containing (mM): 20 HEPES, 20 MgCl2, 25 β-glycerophosphate, 100 sodium orthovanadate, 2 dithiothreitol, and pH 7.6. The immunocomplex was resuspended in 60 μl of kinase buffer. GST-ATF-2 (Cell signaling technology) or GST-p53 (Santa cruz Biotechonlogy) was added into each 30 μl volume of the immunocomplex. Kinase reactions were initiated by adding 2 μl ATP mixture (20 μm ATP and 10 μCi of [γ-32P]ATP into each tube (Amersham Pharmacia Biotech)). The reaction proceeded at RT for 5–15 min before it was terminated by adding 30 μl 2 × Laemmli buffer. Phosphorylation of substrate proteins was visualized by autoradiography after fractionalized by electrophoresis on SDS–PAGE. ATF-2 and p53 phosphorylation were quantified by a densitometry. Kinase assay was also used to detect site-specific phosphorylation of p53 at Ser15 site. Immunocomplex beads of JNK were washed twice with the kinase buffer. Immunocomplex of JNK was incubated with 1 μg/ml full-length GST-p53 fusion protein for 30 min at 37°C in the kinase buffer containing 200 μM ATP. The reaction was terminated by adding SDS buffer. Reaction protein samples were fractionalized by electrophoresis on 12% SDS–PAGE. Western blot was used to analyse phosphorylation levels of p53ser15 with application of phospho-p53ser15 antibody in the reaction (Cell Signaling Technology).

Acknowledgments

This study was supported by NIH Grants EY12953 and EY15282 to LL and CA 79229 to WD.

References

  1. Abrahams PJ, Schouten R, van Laar T, Houweling A, Terleth C, van der Eb AJ. Mutat Res. 1995;336:169–180. doi: 10.1016/0921-8777(94)00049-c. [DOI] [PubMed] [Google Scholar]
  2. Ashcroft M, Vousden KH. Oncogene. 1999;18:7637–7643. doi: 10.1038/sj.onc.1203012. [DOI] [PubMed] [Google Scholar]
  3. Asher G, Lotem J, Cohen B, Sachs L, Shaul Y. Proc Natl Acad Sci USA. 2001;98:1188–1193. doi: 10.1073/pnas.021558898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blaise R, Masdehors P, Lauge A, Stoppa-Lyonnet D, Alapetite C, Merle-Beral H, Binet JL, Omura S, Magdelenat H, Sabatier L, Delic J. Leuk Lymphoma. 2001;42:1173–1180. doi: 10.3109/10428190109097742. [DOI] [PubMed] [Google Scholar]
  5. Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, Fornace AJ., Jr EMBO J. 1999;18:6845–6854. doi: 10.1093/emboj/18.23.6845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buschmann T, Potapova O, Bar-Shira A, Ivanov VN, Fuchs SY, Henderson S, Fried VA, Minamoto T, Alarcon-Vargas D, Pincus MR, Gaarde WA, Holbrook NJ, Shiloh Y, Ronai Z. Mol Cell Biol. 2001;21:2743–2754. doi: 10.1128/MCB.21.8.2743-2754.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chernova OB, Chernov MV, Agarwal ML, Taylor WR, Stark GR. Trends Biochem Sci. 1995;20:431–434. doi: 10.1016/s0968-0004(00)89094-5. [DOI] [PubMed] [Google Scholar]
  8. Colman MS, Afshari CA, Barrett JC. Mutat Res. 2000;462:179–188. doi: 10.1016/s1383-5742(00)00035-1. [DOI] [PubMed] [Google Scholar]
  9. Costanzo V, Shechter D, Lupardus PJ, Cimprich KA, Gottesman M, Gautier J. Mol Cell. 2003;11:203–213. doi: 10.1016/s1097-2765(02)00799-2. [DOI] [PubMed] [Google Scholar]
  10. Cross JV, Templeton DJ. Biochem J. 2004;381:675–683. doi: 10.1042/BJ20040591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dodson GE, Shi Y, Tibbetts RS. J Biol Chem. 2004;279:34010–34014. doi: 10.1074/jbc.C400242200. [DOI] [PubMed] [Google Scholar]
  12. Fuchs SY, Adler V, Buschmann T, Yin Z, Wu X, Jones SN, Ronai Z. Genes Dev. 1998a;12:2658–2663. doi: 10.1101/gad.12.17.2658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuchs SY, Adler V, Pincus MR, Ronai Z. Proc Natl Acad Sci USA. 1998b;95:10541–10546. doi: 10.1073/pnas.95.18.10541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gentiletti F, Mancini F, D’Angelo M, Sacchi A, Pontecorvi A, Jochemsen AG, Moretti F. Oncogene. 2002;21:867–877. doi: 10.1038/sj.onc.1205137. [DOI] [PubMed] [Google Scholar]
  15. Ginsberg D, Michael-Michalovitz D, Oren M. Mol Cell Biol. 1991;11:582–585. doi: 10.1128/mcb.11.1.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goodarzi AA, Block WD, Lees-Miller SP. Prog Cell Cycle Res. 2003;5:393–411. [PubMed] [Google Scholar]
  17. Haupt S, Berger M, Goldberg Z, Haupt Y. J Cell Sci. 2003;116:4077–4085. doi: 10.1242/jcs.00739. [DOI] [PubMed] [Google Scholar]
  18. Hofseth LJ, Robles AI, Yang Q, Wang XW, Hussain SP, Harris C. Chest. 2004;125:83S–85S. doi: 10.1378/chest.125.5_suppl.83s-a. [DOI] [PubMed] [Google Scholar]
  19. Hussain SP, Harris CC. Cancer Res. 1998;58:4023–4037. [PubMed] [Google Scholar]
  20. Hussain SP, Raja K, Amstad PA, Sawyer M, Trudel LJ, Wogan GN, Hofseth LJ, Shields PG, Billiar TR, Trautwein C, Hohler T, Galle PR, Phillips DH, Markin R, Marrogi AJ, Harris CC. Proc Natl Acad Sci USA. 2000;97:12770–12775. doi: 10.1073/pnas.220416097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Irarrazabal CE, Liu JC, Burg MB, Ferraris JD. Proc Natl Acad Sci USA. 2004;101:8809–8814. doi: 10.1073/pnas.0403062101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ito K, Nakazato T, Miyakawa Y, Yamato K, Ikeda Y, Kizaki M. J Cell Physiol. 2003;196:276–283. doi: 10.1002/jcp.10289. [DOI] [PubMed] [Google Scholar]
  23. Kastan MB. Biomed Pharmacother. 2004;58:72–73. [Google Scholar]
  24. Kinzler KW, Vogelstein B. Nature, 386. 1997;761:763. doi: 10.1038/386761a0. [DOI] [PubMed] [Google Scholar]
  25. Kwan R, Burnside J, Kurosaki T, Cheng G. Mol Cell Biol. 2001;21:7183–7190. doi: 10.1128/MCB.21.21.7183-7190.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Levine AJ. Cell. 1997;88:323–331. doi: 10.1016/s0092-8674(00)81871-1. [DOI] [PubMed] [Google Scholar]
  27. Lieber JG, Webb S, Suratt BT, Young SK, Johnson GL, Keller GM, Worthen GS. Blood. 2004;103:852–859. doi: 10.1182/blood-2003-04-1030. [DOI] [PubMed] [Google Scholar]
  28. Lu L, Wang L, Shell B. Invest Ophthalmol Vis Sci. 2003;44:5102–5109. doi: 10.1167/iovs.03-0591. [DOI] [PubMed] [Google Scholar]
  29. Matsuoka S, Huang M, Elledge SJ. Science. 1998;282:1893–1897. doi: 10.1126/science.282.5395.1893. [DOI] [PubMed] [Google Scholar]
  30. Medina D, Ullrich R, Meyn R, Wiseman R, Donehower L. Environ Mol Mutagen. 2002;39:178–183. doi: 10.1002/em.10064. [DOI] [PubMed] [Google Scholar]
  31. Melnikova VO, Santamaria AB, Bolshakov SV, Ananthaswamy HN. Oncogene. 2003;22:5958–5966. doi: 10.1038/sj.onc.1206595. [DOI] [PubMed] [Google Scholar]
  32. Nishikawa T, Salim EI, Morimura K, Kaneko M, Ogawa M, Kinoshita A, Osugi H, Kinoshita H, Fukushima S. Cancer Lett. 2003;194:45–54. doi: 10.1016/s0304-3835(03)00057-0. [DOI] [PubMed] [Google Scholar]
  33. Pluquet O, North S, Bhoumik A, Dimas K, Ronai Z, Hainaut P. J Biol Chem. 2003;278:11879–11887. doi: 10.1074/jbc.M207396200. [DOI] [PubMed] [Google Scholar]
  34. Ringvold A. Acta Ophthalmol Scand. 1998;76:149–153. doi: 10.1034/j.1600-0420.1998.760205.x. [DOI] [PubMed] [Google Scholar]
  35. Roderick C, Reinach PS, Wang L, Lu L. J Membr Biol. 2003;196:41–50. doi: 10.1007/s00232-003-0623-1. [DOI] [PubMed] [Google Scholar]
  36. Sanchez Y, Bachant J, Wang H, Hu F, Liu D, Tetzlaff M, Elledge SJ. Science. 1999;286:1166–1171. doi: 10.1126/science.286.5442.1166. [DOI] [PubMed] [Google Scholar]
  37. Shirai N, Tsukamoto T, Yamamoto M, Iidaka T, Sakai H, Yanai T, Masegi T, Donehower LA, Tatematsu M. Carcinogenesis. 2002;23:1541–1547. doi: 10.1093/carcin/23.9.1541. [DOI] [PubMed] [Google Scholar]
  38. Smirnova IS, Yakovleva TK, Rosanov YM, Aksenov ND, Pospelova TV. Cell Biol Int. 2001;25:1101–1115. doi: 10.1006/cbir.2001.0709. [DOI] [PubMed] [Google Scholar]
  39. Sogame N, Kim M, Abrams JM. Proc Natl Acad Sci USA. 2003;100:4696–4701. doi: 10.1073/pnas.0736384100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tang ZH, Cai QF, Ye XA. Ai Zheng. 2003;22:1057–1061. [PubMed] [Google Scholar]
  41. Tominaga K, Morisaki H, Kaneko Y, Fujimoto A, Tanaka T, Ohtsubo M, Hirai M, Okayama H, Ikeda K, Nakanishi M. J Biol Chem. 1999;274:31463–31467. doi: 10.1074/jbc.274.44.31463. [DOI] [PubMed] [Google Scholar]
  42. van Kranen HJ, de Gruijl FR, de Vries A, Sontag Y, Wester PW, Senden HC, Rozemuller E, van Kreijl CF. Carcinogenesis. 1995;16:1141–1147. doi: 10.1093/carcin/16.5.1141. [DOI] [PubMed] [Google Scholar]
  43. Wang L, Fyffe RE, Lu L. Invest Ophthalmol Vis Sci. 2004;45:1796–1803. doi: 10.1167/iovs.03-1056. [DOI] [PubMed] [Google Scholar]
  44. Wang L, Li T, Lu L. Invest Ophthalmol Vis Sci. 2003;44:5095–5101. doi: 10.1167/iovs.03-0590. [DOI] [PubMed] [Google Scholar]
  45. Wang L, Xu D, Dai W, Lu L. J Biol Chem. 1999;274:3678–3685. doi: 10.1074/jbc.274.6.3678. [DOI] [PubMed] [Google Scholar]
  46. Wang LD, Zhou Q, Wei JP, Yang WC, Zhao X, Wang LX, Zou JX, Gao SS, Li YX, Yang C. World J Gastroenterol. 1998;4:287–293. doi: 10.3748/wjg.v4.i4.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang XW. Anticancer Res. 1999;19:4759–4771. [PubMed] [Google Scholar]
  48. Wu GS. Cancer Biol Ther. 2004;3:156–161. doi: 10.4161/cbt.3.2.614. [DOI] [PubMed] [Google Scholar]
  49. Xie S, Wang Q, Wu H, Cogswell J, Lu L, Jhanwar-Uniyal M, Dai W. J Biol Chem. 2001a;276:36194–36199. doi: 10.1074/jbc.M104157200. [DOI] [PubMed] [Google Scholar]
  50. Xie S, Wu H, Wang Q, Cogswell JP, Husain I, Conn C, Stambrook P, Jhanwar-Uniyal M, Dai W. J Biol Chem. 2001b;276:43305–43312. doi: 10.1074/jbc.M106050200. [DOI] [PubMed] [Google Scholar]
  51. Xie S, Wu H, Wang Q, Kunicki J, Thomas RO, Hollingsworth RE, Cogswell J, Dai W. Cell Cycle. 2002;1:424–429. doi: 10.4161/cc.1.6.271. [DOI] [PubMed] [Google Scholar]
  52. Xu D, Wang L, Dai W, Lu L. Blood. 1999;94:139–145. [PubMed] [Google Scholar]
  53. Yang J, Xu ZP, Huang Y, Hamrick HE, Duerksen-Hughes PJ, Yu YN. World J Gastroenterol. 2004;10:155–160. doi: 10.3748/wjg.v10.i2.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Ye R, Bodero A, Zhou BB, Khanna KK, Lavin MF, Lees-Miller SP. J Biol Chem. 2001;276:4828–4833. doi: 10.1074/jbc.M004894200. [DOI] [PubMed] [Google Scholar]
  55. Zhou BB, Elledge SJ. Nature. 2000;408:433–439. doi: 10.1038/35044005. [DOI] [PubMed] [Google Scholar]
  56. Ziegler A, Jonason A, Simon J, Leffell D, Brash DE. Photochem Photobiol. 1996;63:432–435. doi: 10.1111/j.1751-1097.1996.tb03064.x. [DOI] [PubMed] [Google Scholar]

RESOURCES