December 2003
Volume 44, Issue 12
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Cornea  |   December 2003
UV-Induced Signaling Pathways Associated with Corneal Epithelial Cell Apoptosis
Author Affiliations
  • Luo Lu
    From the Division of Molecular Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine, University of California, Los Angeles, Torrance, California.
  • Ling Wang
    From the Division of Molecular Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine, University of California, Los Angeles, Torrance, California.
  • Beth Shell
    From the Division of Molecular Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine, University of California, Los Angeles, Torrance, California.
Investigative Ophthalmology & Visual Science December 2003, Vol.44, 5102-5109. doi:10.1167/iovs.03-0591
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      Luo Lu, Ling Wang, Beth Shell; UV-Induced Signaling Pathways Associated with Corneal Epithelial Cell Apoptosis. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5102-5109. doi: 10.1167/iovs.03-0591.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. UV-C irradiation of corneal epithelial cells elicits K+ channel activation, which in turn causes them to undergo apoptosis. In the present study, the intermediary role played by mitogen-activated protein kinase (MAPK) signaling pathways in mediating this response was investigated.

methods. Western blot and kinase assays were used to measure UV-induced activation (i.e., phosphorylation) of c-Jun NH2-terminal kinase (JNK)/SEK, extracellular signal-regulated kinase (ERK), and p38. Corneal epithelial apoptosis was determined by measuring caspase 3 activity.

results. UV-irradiation–induced increases in cell membrane K+ channel activity resulted in activations of JNK, SEK upstream of JNK, and caspase 3 downstream of JNK. Suppression of K+ channel activity with specific K+ channel blockers significantly inhibited UV-irradiation–induced activation of JNK cascades. However, suppression of K+ channel activity did not prevent hyperosmotic-stress–induced JNK activation. In addition, UV-irradiation–induced SEK/JNK activation was unaffected by removal of extracellular free Ca2+ with EGTA.

conclusions. UV-irradiation–induced corneal epithelial cell apoptosis is mediated through activation of the SEK/JNK signaling pathway. Such activation is dependent on increases in K+ channel activity, which play an important role in the early events that result in activation of this pathway.

Ultraviolet (UV) irradiation can induce DNA damage, which may result in programmed cell death (apoptosis). This response may also be mediated through different pathways subsequent to the stimulation of cell membrane cytokine receptors, resulting in activation of specific signaling pathways. 1 2 At the cell membrane level, UV irradiation induces clustering and internalization of the membrane receptors for epidermal growth factor (EGF), tumor necrosis factor (TNF), and interleukin (IL)-1. 1 Subsequently, the UV response is elicited in numerous different cell types by activation of intracellular signaling pathways. UV irradiation activates c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), resulting in cellular apoptotic responses. In contrast, UV irradiation modestly increases extracellular signal-regulated kinase (ERK1/2) activity, as opposed to transient more intense activation by growth factors such as EGF. 1 3 4 5 6 UV-induced stimulation of signaling pathways that are upstream of MAP kinases includes activation of RAS, a guanosine triphosphate (GTP)–binding protein and Src, a nonreceptor tyrosine kinase. 7 8 9 10 11 12 In addition, the membrane-associated protein tyrosine phosphatases can be inhibited with UV irradiation by targeting an essential −SH group in the tyrosine phosphatase, resulting in inhibition of dephosphorylation and enhancement of autophosphorylation of cytokine receptors. 13 Farther downstream, the UV response is characterized by activation of the transcription factor genes including NF-κB and immediate early genes such as, c-fos and c-jun. 14 15 16  
K+ channel activity is widely distributed in the cell membrane, to stabilize cell membrane electrophysiological properties. In many cell types, including myeloblasts, fibroblasts, and neurons, cytokine-mediated changes in activity are involved in cell proliferation and apoptosis. 6 17 18 19 20 As in other tissue types, we found that there is a 4-aminopyridine (4-AP)–sensitive K+ channel in corneal epithelial cells. We showed that this channel is involved in UV-irradiation–induced corneal epithelial cell death by using the nystatin-perforated whole-cell technique to elicit a K+ current with an amplitude that markedly increased on exposure to UV-C light for as little time as 1 minute. Single-channel recording using the cell-attached mode revealed that exposure to UV-C irradiation (45 μJ/cm2) strongly stimulated K+ channel activity within 30 seconds. Quick loss of intracellular K+ ions can cause membrane fluctuations and cell shrinkage, resulting in activation of surface receptors and signaling pathways. It has been shown that the loss of intracellular K+ activates interleukin (IL)-1β–converting enzyme (ICE). 17 21 22 Recent evidence suggests that ICE can affect upstream events in the JNK pathway at the JNK level. 23 UV-irradiation–induced activation of ICE, and JNK-1 could occur subsequent to the stimulation of K+ channel activity and the loss of intracellular K+. This mechanism has been implicated in apoptosis in neuronal cells. 24 25 In addition, suppression of K+ channel activity can protect corneal epithelial cells from UV-irradiation–induced DNA fragmentation and nuclear death. However, suppression of K+ channel activity does not prevent corneal epithelial apoptosis induced by etoposide, because etoposide directly inhibits topoisomerase II activity in the nucleus rather than affecting UV-induced cell membrane events. Currently, in corneal epithelial cells the signaling pathways that link UV-irradiation–induced K+ channel activation in the membrane to the nuclear events resulting in corneal epithelial cell apoptosis are unknown. In the present study, we investigated in human and rabbit corneal epithelial cells the role of the ERK, JNK/SAPK, and p38 branches of the MAP kinase superfamily in mediating UV-C–induced apoptosis. We found that UV irradiation strongly stimulated JNK-1 activity and caused corneal epithelial apoptosis. In contrast, suppression of cell membrane K+ channel activity inhibited UV-C induced JNK-1 activation and prevented cells from undergoing apoptosis. 
Materials and Methods
Culture of Corneal Epithelial Cells
SV40-transformed rabbit corneal epithelial (RCE), primary cultured rabbit corneal epithelial (PRCE), and human corneal epithelial (HCE) cells were a generous gift from Kaoru Araki-Sasaki (Tane Memorial Eye Hospital) and characterized in our laboratory. 26 PRCE cells were cultured using a protocol from Reinach’s laboratory. Briefly, isolated corneal wedges were placed epithelial side down in a dish containing serum-free DMEM/F-12 with 5% dispase II at 37°C for 30 to 40 minutes. Afterward, the dispase medium was replaced with DMEM/F-12 culture medium containing 10% FBS. The loosened epithelial cells were scraped off and further dispersed by passing them through a syringe attached to a 23-gauge needle. Cells were grown in DMEM/F12 (1:1) culture medium containing 10% fetal bovine serum and 5 μg/mL insulin, and maintained in an incubator supplied with 95% air and 5% CO2 at 37°C. Culture medium was replaced every 2 days. Corneal epithelial cells were detached by treatment with 0.05% trypsin-EDTA and passaged at a seeding density of 105/mL. For UV irradiation experiments, confluent corneal epithelial cells were placed in a tissue culture hood at a distance of 60 inches from the UV-C light source and exposed at an intensity of 45 μJ/cm2. Cell nuclear staining was performed to detect nuclear DNA condensation by adding a dye mixture containing ethidium bromide and acridine orange (EB/AO), each present at 100 μg/mL, to a cell culture dish containing a cell density of 3 × 105/mL. Cell populations were scored according to their color by UV fluorescence microscope (Nikon, Tokyo, Japan). 
Immunoblot Analysis
The cells (2 × 105) were washed once with ice-cold PBS and lysed in sodium dodecyl sulfate (SDS) polyacrylamide sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol (DTT), 0.01% (wt/vol) bromophenol blue or phenol red. The resultant suspensions were boiled for 5 minutes. After fractionation of cell lysates by with 12% PAGE, proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes. They were exposed to blocking buffer containing 5% nonfat milk in TBS-0.1% Tween 20 (TBS-T) for 1 hour at room temperature (RT, 22°C), and then incubated overnight or for 1 hour with the antibodies of interest at 4°C. After three washes with TBS-T buffer, the membrane was incubated with alkaline phosphatase (AP)–linked secondary antibody for 1 hour at RT. The proteins were detected by Western blot analysis (Phototope-Star Western Blot Detection kit; Cell Signaling Technology, Beverly, MA). 
Immunoprecipitation and Kinase Assay
Immunocomplex kinase assays to detect activities of ERK-2, JNK-1, and p38 were performed as described previously. 6 27 In brief, 5 × 106 corneal epithelial cells were exposed to UV-C (45 μJ/cm2) in the presence or absence of the K+ channel blockers 4-aminopyridine (4-AP), tetraethylammonium (TEA), or barium (Ba2+); washed once with ice cold PBS; and lysed with 0.8 mL of lysis buffer containing 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 10 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/mL leupeptin, and 10 μg/mL aprotinin. Cell lysates were incubated on ice for 10 minutes and then were precleared by centrifugation at 13,000g for 25 minutes. JNK-1, p38, or ERK-2 proteins were immunoprecipitated with 0.5 μg of rabbit polyclonal antibody against JNK-1, p38, or ERK-2, respectively (Santa Cruz Biotechnology, Santa Cruz, CA). The immunocomplex was washed three times with lysis buffer and twice with kinase buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl2, 25 mM β-glycerophosphate, 100 mM sodium orthovanadate, and 2 mM DTT and resuspended in 60 μL kinase buffer. Substrates (1 μg each) were added to 30 μL of the immunocomplex. The kinase reaction was initiated by adding 2 μL of adenosine triphosphate (ATP) mixture containing 20 μM ATP and 10 μCi of [γ-32P]-ATP (Amersham Pharmacia Biotech, Piscataway, NJ). The reaction proceeded at RT for 5 minutes before it was terminated by adding 30 μL of 2× Laemmli buffer. Phosphorylation of substrates was visualized by autoradiography after SDS-PAGE. Intensities of phosphorylation were quantified by densitometry. 
Determination of Caspase 3 Activity
Caspase 3 activity was determined by a caspase 3 assay kit (Promega, Madison, WI). In brief, an equal amount of cells were washed once with ice-cold PBS and lysed with cell lysis buffer containing 1% Triton X-100, 50 mM-HCl (pH 7.4), 1 mM EDTA, and 1 mM PMSF and by freezing and thawing three times. Lysates were incubated on ice for 15 minutes and centrifuged at 15,000g for 20 minutes at 4°C. The supernatant fractions were collected and incubated with the caspase 3 substrate, acetyl- Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD pNA) at 37°C for 4 hours. Caspase 3 activity was assessed by measuring the absorbance at a wavelength of 405 nm with a fluorometer. 
Results
Effect of UV Irradiation on K+ Channel and JNK Activity
To determine in corneal epithelial cells the effect of UV irradiation on specific signaling pathways, we measured changes in UV-irradiation–induced JNK-1 activities in this tissue. After UV irradiation, JNK-1 activities were markedly increased for up to 60 minutes in RCE, HCE, and primary cultured RCE cells compared with control groups (Figs. 1A 1B 1E , respectively). UV-irradiation–induced JNK-1 activation was initially observed within 5 to 15 minutes after the end of the exposure period. There was a delay for the JNK activity to reach its peak level in primary cultured RCE cells. To determine whether a change in K+ channel activity is associated in these cells with UV-irradiation–induced JNK activity, we inhibited K+ channel activity using a specific K+ channel blocker, 4-AP. A dose–response curve demonstrated that 4-AP effectively inhibited JNK activity with a median inhibitory concentration (IC50) of 177 ± 26 μM (n = 4, Fig. 1C ). This IC50 is very close to the concentration that is required for 4-AP to block the K+ current in patch-clamp experiments. 28 Blockade of K+ channel activity using 4-AP (1 mM) significantly prevented UV-irradiation–induced JNK-1 activation in RCE and primary cultured RCE cells (Figs. 1D 1F) . These results indicate that UV irradiation stimulates JNK-1 activities in corneal epithelial cells and that this activation is associated with UV-irradiation–evoked increases in cell membrane K+ channel activity. 
Effect of UV Irradiation on Increases in K+ Channel and SEK Activation
SEK is a MAPK kinase immediately upstream of JNK in the JNK signaling pathway. Activation of SEK results in the phosphorylation and activation of JNK. 6 29 SEK activity was determined by measuring the phosphorylation status of SEK. SEK-1 protein was strongly phosphorylated in response to UV irradiation after 60 minutes, detected by a specific antibody against the phospho-SEK (Fig. 2A) . Suppression of K+ channel activity with 4-AP effectively prevented UV-irradiation–induced SEK-1 phosphorylation (Fig. 2B) . This result indicates that UV-irradiation–induced increases in K+ channel activity are an early event upstream from SEK in the JNK signaling pathway. 
Dependence of JNK Activation on K+ Efflux and Ca2+ Influx
The effect of the inhibition of K+ efflux on JNK activation was studied by determining whether UV-C–induced stimulation of its activity is suppressed by the K+ channel blockers 4-AP, TEA, and Ba2+. In this group of blockers, 4-AP was the most effective in suppressing JNK-1 activation (Fig. 3A) . Because changes in K+ channel activity can alter the membrane potential and cause increases in Ca2+ influx, a rise in Ca2+ influx may play a role in activating intracellular signaling pathways that mediate UV-C-irradiation–induced corneal epithelial cell apoptosis. To determine whether such an effect is dependent on an increase in Ca 2+ influx, the effect of UV-C irradiation on JNK activation was determined in a nominally Ca2+-free condition with 5 mM EGTA. We found that UV irradiation had the same effect on JNK activation in a Ca2+-free medium, whereas suppression of K+ channel activity with 4-AP prevented UV-C-irradiation–induced JNK activation under this condition (Fig. 3B) . Therefore, Ca2+ influx does not apparently play a role in the UV-C-irradiation–mediated activation of the JNK cascade in RCE cells. 
To determine whether the UV-C irradiation induced increase in K+ efflux is mimicked by the K+ ionophore valinomycin, RCE cells were exposed to this ionophore. Exposure of the cells to valinomycin markedly increased JNK activity in a dose-dependent manner (Fig. 3C) . Inhibition of K+ channel activity using 4-AP did not affect valinomycin-induced JNK activation in these cells (Fig. 3D) . Our finding that valinomycin dose dependently stimulated JNK activity provides further evidence that the rapid loss of intracellular K+ ions caused by K+ channel activation constitutes an early event in the UV irradiation-activated JNK signaling pathway, resulting in cell apoptosis. To test whether 4-AP inhibition of JNK enzyme activity was mediated by a mechanism other than suppression of K+ channel activity, the direct effect of 4-AP on purified in vitro JNK activity was examined (Fig. 3E) . Inhibition of JNK-1 activity did not occur with 3 mM 4-AP, which supports the notion that UV-irradiation–induced activation of JNK-1 is dependent on an increase in cell membrane K+ channel activity. 
Dependence of ERK and p38 Pathway Activation on UV-Irradiation–Induced K+ Channel Activation
In many different cell types, UV irradiation can induce increases in the activity of two other branches of the MAP kinase signaling cascade: ERK and p38. 12 To investigate whether UV irradiation induces ERK activity, the time-dependent effects of UV irradiation on activation of ERK-2 were measured (Fig. 4A) . ERK-2 activity increased 5 minutes after UV irradiation, and its activation reached a plateau at 15 minutes after irradiation. These effects were significantly inhibited by suppression of K+ channel activity using 4-AP (1 mM), suggesting that K+ channel activity is required for UV-irradiation–induced activation of the ERK cascade (Fig. 4B) . UV irradiation induced time-dependent, but weak activation of the p38 branch. However, suppression of K+ channel activity by 4-AP did not block this effect of UV irradiation (Figs. 4C 4D)
Nondependence of Hypertonic Stress–Induced Apoptosis on K+ Channel Activity
Hyperosmotic stress induces cell apoptosis by activating JNK and p38 signaling pathways in various cell types. In RCE cells, the effect of hyperosmotic stress on apoptosis was characterized by measuring cell viability and activations of JNK and p38 signaling pathways. Cell viability was determined based on nuclear EB/AO staining. RCE cell viability was significantly decreased after an increase in sorbitol concentration, which created hyperosmotic stress (Fig. 5A) . To further support the specific role of K+ channel activity in UV-irradiation–induced activation of the JNK cascade, we examined the effect of 4-AP on JNK-1 activation induced by such a stress. The time-dependent effects of exposure to a hypertonic stress of 600 mM sorbitol on JNK-1 activity are shown in Figure 5B . This challenge markedly evoked JNK-1 activity, but 4-AP had no inhibitory effect on the activation (Fig. 5C) , indicating that sorbitol-induced JNK activation is not dependent on activation of the 4-AP–sensitive K+ channel. In addition, sorbitol induced a strong activation of p38. Similarly, inhibition of K+ channel activity by 4-AP had no effect on sorbitol-induced p38 activation (Fig. 5D) . These results further indicate that UV-irradiation–induced JNK activation is probably mediated by activation of a 4-AP–sensitive cell membrane K+ channel. 
Dependence of UV-Irradiation–Induced Caspase 3 Activation on K+ Channel Stimulation
To further understand the role played by K+ channel stimulation in mediating UV-C–induced corneal epithelial cell apoptosis, we measured downstream activation of caspase 3. Specific caspase 3–like protease activity was determined by using the specific substrates Acetyl-Asp-Val-Asp p-nitroanilide (Ac-DEVD pNA). Caspase 3 protease activity was markedly increased after exposure of RCE cells to UV irradiation. Suppression of K+ channel activity with 4-AP reduced UV-C-irradiation–induced caspase 3 activity to the control level (Fig. 6) . This result further demonstrates and explains why prevention of UV-C-irradiation–induced K+ channel activation can protect corneal epithelial cells from undergoing apoptosis. 
Discussion
The present study focuses on UV-C-irradiation–induced corneal epithelial cell apoptosis. It has important physiological relevance to corneal epithelial wound healing, because the doses of UV-C irradiation used in this study are similar to those experienced in daily living. Recent studies show that UV irradiation induces a conformational change in the cell membrane. 30 31 32 UV irradiation causes membrane receptors to form multimers and activates signaling pathways, such as Src, Ras, Raf, and the MAP kinases (ERK, JNK, and p38), resulting in the activation of the transcription factors AP-1 and NF-κB and immediate early genes, such as c-fos and c-jun. 15 33 34 35 36 37 38 39 Furthermore, activation of cell surface receptors by UV-C irradiation is independent of ligand binding and DNA synthesis. 1 40 However, these effects do not explain why UV irradiation activates different receptors in a similar pattern. The unanswered question is which cell membrane phenomenon acts as a sensor to absorb UV energy and in turn elicits receptor stimulation as a result of forming multimers. 
K+ channels are universally present in the cell membrane and are essential in maintaining normal functions of the cell. We found that changes in K+ channel activity are involved in growth factor–stimulated cell growth and differentiation, suggesting that there is cross talk between growth factor receptor–linked signaling pathways and K+ channel activity in the cell membrane. 6 27 28 In the present study, suppression of K+ channel activity prevented UV-induced SEK, JNK, and caspase 3 activation, resulting in protection of corneal epithelial cells from undergoing apoptosis. Our results also suggest that this cell membrane channel protein may be the initial targeting site for absorption of UV-C irradiation. UV-C irradiation triggers excessive opening of K+ channels and increases K+ efflux. If UV-irradiation–induced K+ channel activation is an early event in eliciting activation of downstream signaling pathways, suppression of UV-irradiation–induced K+ channel activation should prevent UV-irradiation–induced JNK activation. We, therefore, evaluated the role of K+ channel activity in UV-irradiation–induced JNK activation. Exposure of corneal epithelial cells to UV irradiation also induced ERK activation. Our data showed that UV irradiation induced a weaker activation of the ERK than the JNK/SAPK branch, which is consistent with previous studies in other cell types. 35 40 41 Relative to JNK/SAPK activation, there was a much weaker activation of p38 in RCE cells in response to UV irradiation. It is not known why UV irradiation of RCE cells elicited a smaller increase in p38 activity than in other cell types. Suppression of K+ channel with the specific K+ channel blocker 4-AP markedly inhibited UV-irradiation–induced activation of JNK and ERK, indicating that UV-irradiation–induced K+ channel activation plays an important role in eliciting activation of downstream kinase cascades. In contrast, UV irradiation moderately and stably activated the ERK branch in these cells. It has been shown that ERK activation by UV irradiation promotes cell survival in A431 cells. 42 Further evidence to verify the role of K+ channel in the UV-irradiation–induced JNK signaling pathway is that suppression of K+ channel activity diminished UV-irradiation–induced SEK phosphorylation directly upstream from JNK. 
We further demonstrated the specificity of K+ channel activation in mediating the activation of the UV-irradiation–induced JNK signaling pathway. First, there was a close correspondence between the concentrations of several K+ channel inhibitors necessary to block K+ channel activity effectively and to suppress UV-irradiation–induced JNK activation (Fig. 1) . Second, a much higher concentration of 4-AP had no effect on isolated, purified JNK activity (Fig. 2B) . Third, another type of stress, osmotic shock, which perturbs the cell membrane by shrinking cell volume, stimulated growth factor receptor tyrosine phosphorylation and JNK. 1 43 44 Similarly, a hypertonic solution (600 mOsM sorbitol) strongly activated JNK in RCE cells. However, suppression of K+ channel activity by 4-AP failed to prevent sorbitol-induced activation of JNK (Fig. 3B) . The failure of osmotic stress to activate K+ channel activity is consistent with the finding in these cells that such a challenge instead increases the activity and at later times gene and protein expression of the Na,K,2Cl cotransporter (NKCC). 45 Finally, JNK was activated by application of valinomycin to increase efflux of K+ ions across the cell membrane. This response mimics the effect of UV-induced K+ channel activation on JNK activity. Activation of JNK by valinomycin occurred in a dose-dependent manner. Suppression of K+ channel activation with 4-AP did not affect valinomycin-induced JNK activation (Figs. 3C 3D) . These results support the idea that K+ channels are specifically involved in the events preceding UV-induced JNK activation. 
Previous studies have evaluated whether Ca2+ is necessary for the activation of JNK. 46 47 Some of these studies suggest that the response is Ca2+ independent. 6 47 In rabbit corneal epithelial cells, cell membrane hyperpolarization results in an increase in plasma membrane calcium influx through relatively nonselective plasma membrane channels. 48 Our results indicate that increases in calcium influx are not needed for JNK activation by UV-C in these cells. However, these experiments do not rule out the possibility that mobilization of calcium from intracellular stores may be sufficient to activate JNK. Other studies have shown that extracellular Ca2+ influx is involved in the activation of JNK. 29 49 UV-stimulated K+ channel hyperactivity could increase the electrical driving force for Ca2+ influx, which may stimulate activation of JNK. We placed 5 mM EGTA in the culture solution to remove extracellular free Ca2+ and to prevent Ca2+ influx. Our results indicate that extracellular Ca2+ did not play a role in UV-irradiation–induced activation of JNK, because we found (1) UV-irradiation–induced JNK activation still occurred when extracellular Ca2+ was removed, and (2) suppression of K+ channel activity effectively inhibited UV-irradiation–induced JNK activation in Ca2+-free medium. In conclusion, our results revealed that hyperactivity of a 4-AP–sensitive K+ channel stimulated by UV irradiation is an early event in UV-irradiation–induced activation of JNK. 
 
Figure 1.
 
Effect of suppressing K+ channel activity on UV-C-irradiation–induced JNK activation. (A) Time course of UV-induced JNK-1 activation in RCE cells. (B) Time course of UV-induced JNK-1 activation in HCE cells. (C) Dose-dependent inhibition of UV-irradiation–induced JNK-1 activation by suppression of K+ channel activity. RCE cells were incubated with various concentration of 4-AP before exposure to UV irradiation. (D) Inhibition of UV-induced JNK-1 activation by suppression of K+ channel activity. UV-irradiation–induced JNK activation was significantly blocked by 4-AP (1 mM) in RCE cells (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. (E) Time course of UV-induced JNK-1 activation in primary RCE cells. (F) Prevention of JNK-1 activation in primary RCE cells. Blockade of K+ channel activity with 4-AP (1 mM) significantly inhibited UV-irradiation–induced JNK activation (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. In all the experiments, HCE, RCE, and primary RCE cells were pretreated or untreated with K+ channel blockers for 30 minutes, or cells were exposed to UV light (45 μJ/cm2). After an additional 30-minute incubation, the cells were harvested to measure JNK-1 activity and JNK protein concentrations by mmunocomplex kinase assay and Western blot, respectively.
Figure 1.
 
Effect of suppressing K+ channel activity on UV-C-irradiation–induced JNK activation. (A) Time course of UV-induced JNK-1 activation in RCE cells. (B) Time course of UV-induced JNK-1 activation in HCE cells. (C) Dose-dependent inhibition of UV-irradiation–induced JNK-1 activation by suppression of K+ channel activity. RCE cells were incubated with various concentration of 4-AP before exposure to UV irradiation. (D) Inhibition of UV-induced JNK-1 activation by suppression of K+ channel activity. UV-irradiation–induced JNK activation was significantly blocked by 4-AP (1 mM) in RCE cells (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. (E) Time course of UV-induced JNK-1 activation in primary RCE cells. (F) Prevention of JNK-1 activation in primary RCE cells. Blockade of K+ channel activity with 4-AP (1 mM) significantly inhibited UV-irradiation–induced JNK activation (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. In all the experiments, HCE, RCE, and primary RCE cells were pretreated or untreated with K+ channel blockers for 30 minutes, or cells were exposed to UV light (45 μJ/cm2). After an additional 30-minute incubation, the cells were harvested to measure JNK-1 activity and JNK protein concentrations by mmunocomplex kinase assay and Western blot, respectively.
Figure 2.
 
Effect of UV irradiation on SEK-1 activation. (A) Phosphorylation of SEK-1 induced by UV irradiation. RCE cells were exposed to UV irradiation and then incubated in culture medium for indicated periods. (B) Prevention of UV-induced SEK-1 phosphorylation by suppression of K+ channel activity. RCE cells were incubated with 1 mM 4-AP for 30 minutes before UV irradiation. SEK-1 activation was determined with anti-phospho-SEK-1 antibody by Western blot analysis.
Figure 2.
 
Effect of UV irradiation on SEK-1 activation. (A) Phosphorylation of SEK-1 induced by UV irradiation. RCE cells were exposed to UV irradiation and then incubated in culture medium for indicated periods. (B) Prevention of UV-induced SEK-1 phosphorylation by suppression of K+ channel activity. RCE cells were incubated with 1 mM 4-AP for 30 minutes before UV irradiation. SEK-1 activation was determined with anti-phospho-SEK-1 antibody by Western blot analysis.
Figure 3.
 
Effects of K+ influx and extracellular Ca2+ on JNK-1 activity. (A) Effects of selective K+ channel blockers on activation of JNK-1 induced by UV irradiation. Selective K+ channel blockers, 4-AP (1 mM), TEA (10 mM), and Ba2+ (5 mM), were applied to inhibit UV-irradiation–induced JNK activation in RCE cells. Data are expressed as the mean ± SE. *Significant difference after application of blockers (n = 4, P < 0.01). (B) Effect of extracellular Ca2+ on UV-induced JNK activation. RCE cells were exposed to UV irradiation in the presence and absence of 1 mM 4-AP. Extracellular Ca2+ was removed by preincubation of RCE cells in medium containing 5 mM EGTA for 30 minutes before UV exposure. (C) Effect of valinomycin on JNK-1 activation. RCE cells were treated with valinomycin (1–50 nM) for 20 minutes before JNK activity was measured. (D) Effect of 4-AP on valinomycin-induced JNK-1 activation. RCE cells were incubated with 50 nM valinomycin for 20 minutes in the presence and absence of 3 mM 4-AP. (E) Direct effect of 4-AP on purified JNK activity. JNK-1 protein was immunoprecipitated from UV-induced RCE cells and JNK-1 activity was measured with or without 3 mM 4-AP in vitro. JNK-1 activity was measured by using immunocomplex kinase assay using GST-ATF2 fusion protein as a substrate. Total JNK protein levels were determined by Western blot.
Figure 3.
 
Effects of K+ influx and extracellular Ca2+ on JNK-1 activity. (A) Effects of selective K+ channel blockers on activation of JNK-1 induced by UV irradiation. Selective K+ channel blockers, 4-AP (1 mM), TEA (10 mM), and Ba2+ (5 mM), were applied to inhibit UV-irradiation–induced JNK activation in RCE cells. Data are expressed as the mean ± SE. *Significant difference after application of blockers (n = 4, P < 0.01). (B) Effect of extracellular Ca2+ on UV-induced JNK activation. RCE cells were exposed to UV irradiation in the presence and absence of 1 mM 4-AP. Extracellular Ca2+ was removed by preincubation of RCE cells in medium containing 5 mM EGTA for 30 minutes before UV exposure. (C) Effect of valinomycin on JNK-1 activation. RCE cells were treated with valinomycin (1–50 nM) for 20 minutes before JNK activity was measured. (D) Effect of 4-AP on valinomycin-induced JNK-1 activation. RCE cells were incubated with 50 nM valinomycin for 20 minutes in the presence and absence of 3 mM 4-AP. (E) Direct effect of 4-AP on purified JNK activity. JNK-1 protein was immunoprecipitated from UV-induced RCE cells and JNK-1 activity was measured with or without 3 mM 4-AP in vitro. JNK-1 activity was measured by using immunocomplex kinase assay using GST-ATF2 fusion protein as a substrate. Total JNK protein levels were determined by Western blot.
Figure 4.
 
Effect of UV irradiation on ERK and p38 activation. (A) Time course of UV-induced ERK activation in RCE cells. (B) Inhibition of UV-induced ERK activation in RCE cells by suppression of K+ channel activity with 4-AP. (C) Time course of UV-induced p38 activation in RCE cells. (D) Effect of suppressing K+ channel activity with 4-AP (2 mM) on UV-irradiation–induced p38 activation in RCE cells. ERK-2 and p38 activities in RCE cells were measured by using immunocomplex kinase assay. Total protein levels of ERK-2 and p38 were determined by Western blot.
Figure 4.
 
Effect of UV irradiation on ERK and p38 activation. (A) Time course of UV-induced ERK activation in RCE cells. (B) Inhibition of UV-induced ERK activation in RCE cells by suppression of K+ channel activity with 4-AP. (C) Time course of UV-induced p38 activation in RCE cells. (D) Effect of suppressing K+ channel activity with 4-AP (2 mM) on UV-irradiation–induced p38 activation in RCE cells. ERK-2 and p38 activities in RCE cells were measured by using immunocomplex kinase assay. Total protein levels of ERK-2 and p38 were determined by Western blot.
Figure 5.
 
Effect of suppressing K+ channel activity on hyperosmotic-stress–induced apoptotic response. (A) Hyperosmotic-stress–induced RCE cell apoptosis. Cell viability was determined by nuclear EB/OA staining. (B) Time course of hyperosmotic-stress–induced activation of JNK. JNK was activated by application of 600 mM sorbitol and measured in RCE cells up to 60 minutes. (C) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced JNK activation. (D) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced p38 activation. Hyperosmotic-stress–induced JNK and p38 activations were measured by immunocomplex assay 5 minutes after exposure to 600 mM sorbitol in the absence and presence of 4-AP (2 mM).
Figure 5.
 
Effect of suppressing K+ channel activity on hyperosmotic-stress–induced apoptotic response. (A) Hyperosmotic-stress–induced RCE cell apoptosis. Cell viability was determined by nuclear EB/OA staining. (B) Time course of hyperosmotic-stress–induced activation of JNK. JNK was activated by application of 600 mM sorbitol and measured in RCE cells up to 60 minutes. (C) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced JNK activation. (D) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced p38 activation. Hyperosmotic-stress–induced JNK and p38 activations were measured by immunocomplex assay 5 minutes after exposure to 600 mM sorbitol in the absence and presence of 4-AP (2 mM).
Figure 6.
 
Inhibition of UV-induced caspase 3 activation by suppression of K+ channel activity. RCE cells were preincubated with and without 1 mM 4-AP before UV irradiation. Caspase 3 activity in RCE cells was determined by using a caspase assay kit. Ac-DEVD pNA was used as substrate for caspase 3. Caspase 3 activity was assessed by measuring fluorescence at 405 nm wavelength with a fluorometer.
Figure 6.
 
Inhibition of UV-induced caspase 3 activation by suppression of K+ channel activity. RCE cells were preincubated with and without 1 mM 4-AP before UV irradiation. Caspase 3 activity in RCE cells was determined by using a caspase assay kit. Ac-DEVD pNA was used as substrate for caspase 3. Caspase 3 activity was assessed by measuring fluorescence at 405 nm wavelength with a fluorometer.
Rosette C, Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science. 1996;274:1194–1197. [CrossRef] [PubMed]
Kleiman NJ, Wang RR, Spector A. Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells. Curr Eye Res. 1990;9:1185–1193. [CrossRef] [PubMed]
Galcheva-Gargova Z, Derijard B, Wu IH, Davis RJ. An osmosensing signal transduction pathway in mammalian cells. Science. 1994;265:806–808. [CrossRef] [PubMed]
Kallunki T, Su B, Tsigelny I, et al. JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation. Genes Dev. 1994;8:2996–3007. [CrossRef] [PubMed]
Minden A, Lin A, Claret FX, Abo A, Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 1995;81:1147–1157. [CrossRef] [PubMed]
Wang L, Xu D, Dai W, Lu L. An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells. J Biol Chem. 1999;274:3678–3685. [CrossRef] [PubMed]
Kitagawa D, Tanemura S, Ohata S, et al. Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation: its implication in an anti-apoptotic function. J Biol Chem. 2002;277:366–371. [CrossRef] [PubMed]
Derijard B, Hibi M, Wu IH, et al. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994;76:1025–1037. [CrossRef] [PubMed]
Devary Y, Gottlieb RA, Smeal T, Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell. 1992;71:1081–1091. [CrossRef] [PubMed]
Engelberg D, Klein C, Martinetto H, Struhl K, Karin M. The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals. Cell. 1994;77:381–390. [CrossRef] [PubMed]
Hibi M, Lin A, Smeal T, Minden A, Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993;7:2135–2148. [CrossRef] [PubMed]
Radler-Pohl A, Sachsenmaier C, Gebel S, et al. UV-induced activation of AP-1 involves obligatory extranuclear steps including Raf-1 kinase. EMBO J. 1993;12:1005–1012. [PubMed]
Knebel A, Rahmsdorf HJ, Ullrich A, Herrlich P. Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents. EMBO J. 1996;15:5314–5325. [PubMed]
Buscher M, Rahmsdorf HJ, Litfin M, Karin M, Herrlich P. Activation of the c-fos gene by UV and phorbol ester: different signal transduction pathways converge to the same enhancer element. Oncogene. 1988;3:301–311. [PubMed]
Devary Y, Gottlieb RA, Lau LF, Karin M. Rapid and preferential activation of the c-jun gene during the mammalian UV response. Mol Cell Biol. 1991;11:2804–2811. [PubMed]
Herrlich P, Ponta H, Rahmsdorf HJ. DNA damage-induced gene expression: signal transduction and relation to growth factor signaling (review). Rev Physiol Biochem Pharmacol. 1992;119:187–223. [PubMed]
Bortner CD, Hughes FM, Jr, Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem. 1997;272:32436–32442. [CrossRef] [PubMed]
Duprat F, Guillemare E, Romey G, et al. Susceptibility of cloned K+ channels to reactive oxygen species. Proc Natl Acad Sci USA. 1995;92:11796–11800. [CrossRef] [PubMed]
Xu B, Wilson BA, Lu L. Induction of human myeloblastic ML-1 cell G1 arrest by suppression of K+ channel activity. Am J Physiol. 1996;271:C2037–C2042. [PubMed]
Yu SP, Yeh CH, Sensi SL, et al. Mediation of neuronal apoptosis by enhancement of outward potassium current. Science. 1997;278:114–117. [CrossRef] [PubMed]
Frisch SM, Vuori K, Kelaita D, Sicks S. A role for Jun-N-terminal kinase in anoikis; suppression by bcl-2 and crmA. J Cell Biol. 1996;135:1377–1382. [CrossRef] [PubMed]
Walev I, Reske K, Palmer M, Valeva A, Bhakdi S. Potassium-inhibited processing of IL-1 beta in human monocytes. EMBO J. 1995;14:1607–1614. [PubMed]
Cahill MA, Peter ME, Kischkel FC, et al. CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases. Oncogene. 1996;13:2087–2096. [PubMed]
Eldadah BA, Yakovlev AG, Faden AI. The role of CED-3-related cysteine proteases in apoptosis of cerebellar granule cells. J Neurosci. 1997;17:6105–6113. [PubMed]
Schulz JB, Weller M, Klockgether T. Potassium deprivation-induced apoptosis of cerebellar granule neurons: a sequential requirement for new mRNA and protein synthesis, ICE-like protease activity, and reactive oxygen species. J Neurosci. 1996;16:4696–4706. [PubMed]
Kang SS, Wang L, Kao WW, Reinach PS, Lu L. Control of SV-40 transformed RCE cell proliferation by growth-factor-induced cell cycle progression. Curr Eye Res. 2001;23:397–405. [CrossRef] [PubMed]
Xu D, Wang L, Dai W, Lu L. A requirement for K+-channel activity in growth factor-mediated extracellular signal-regulated kinase activation in human myeloblastic leukemia ML-1 cells. Blood. 1999;94:139–145. [PubMed]
Lu L, Yang T, Markakis D, Guggino WB, Craig RW. Alterations in a voltage-gated K+ current during the differentiation of ML-1 human myeloblastic leukemia cells. J Membr Biol. 1993;132:267–274. [PubMed]
Sanchez I, Hughes RT, Mayer BJ, et al. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature. 1994;372:794–798. [CrossRef] [PubMed]
Pizarro RA, Orce LV. Membrane damage and recovery associated with growth delay induced by near-UV radiation in Escherichia coli K-12. Photochem Photobiol. 1988;47:391–397. [CrossRef] [PubMed]
Petit E, Divoux D, Chancerelle Y, Kergonou JF, Nouvelot A. Immunological approach to investigating membrane cell damages induced by lipoperoxidative stress: application to far UV-irradiated erythrocytes. Biol Trace Elem Res. 1995;47:17–27. [CrossRef] [PubMed]
Schwarz T. UV light affects cell membrane and cytoplasmic targets. J Photochem Photobiol B. 1998;44:91–96. [CrossRef] [PubMed]
Gentz R, Rauscher FJ, III, Abate C, Curran T. Parallel association of Fos and Jun leucine zippers juxtaposes DNA binding domains. Science. 1989;243:1695–1699. [CrossRef] [PubMed]
Hirai SI, Ryseck RP, Mechta F, Bravo R, Yaniv M. Characterization of junD: a new member of the jun proto-oncogene family. EMBO J. 1989;8:1433–1439. [PubMed]
Li T, Dai W, Lu L. Ultraviolet-induced junD activation and apoptosis in myeloblastic leukemia ML-1 cells. J Biol Chem. 2002;277:32668–32676. [CrossRef] [PubMed]
Ryder K, Nathans D. Induction of protooncogene c-jun by serum growth factors. Proc Natl Acad Sci USA. 1988;85:8464–8467. [CrossRef] [PubMed]
Ryder K, Lanahan A, Perez-Albuerne E, Nathans D. jun-D: a third member of the jun gene family. Proc Natl Acad Sci USA. 1989;86:1500–1503. [CrossRef] [PubMed]
Ryseck RP, Bravo R. c-JUN, JUN B, and JUN D differ in their binding affinities to AP-1 and CRE consensus sequences: effect of FOS proteins. Oncogene. 1991;6:533–542. [PubMed]
Smeal T, Angel P, Meek J, Karin M. Different requirements for formation of Jun:Jun and Jun:Fos complexes. Genes Dev. 1989;3:2091–2100. [CrossRef] [PubMed]
Karin M, Liu Z, Zandi E. AP-1 function and regulation (review). Curr Opin Cell Biol. 1997;9:240–246. [CrossRef] [PubMed]
Lu ML, Sato M, Cao B, Richie JP. UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells. Proc Natl Acad Sci USA. 1996;93:8977–8982. [CrossRef] [PubMed]
Kitagawa D, Tanemura S, Ohata S, et al. Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation: its implication in an anti-apoptotic function. J Biol Chem. 2002;277:366–371. [CrossRef] [PubMed]
Klein JD, Lamitina ST, O’Neill WC. JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro. Am J Physiol. 1999;277:C425–C431. [PubMed]
Koh YH, Che W, Higashiyama S, et al. Osmotic stress induces HB-EGF gene expression via Ca(2+)/Pyk2/JNK signal cascades in rat aortic smooth muscle cells. J Biochem (Tokyo). 2001;130:351–358. [CrossRef]
Bildin VN, Yang H, Crook RB, Fischbarg J, Reinach PS. Adaptation by corneal epithelial cells to chronic hypertonic stress depends on upregulation of Na:K:2Cl cotransporter gene and protein expression and ion transport activity. J Membr Biol. 2000;177:41–50. [CrossRef] [PubMed]
Lewis RS, Cahalan MD. Potassium and calcium channels in lymphocytes (review). Ann Rev Immunol. 1995;13:623–653. [CrossRef]
Koh JY, Wie MB, Gwag BJ, et al. Staurosporine-induced neuronal apoptosis. Exp Neurol. 1995;135:153–159. [CrossRef] [PubMed]
Rich A, Rae JL. Calcium entry in rabbit corneal epithelial cells: evidence for a nonvoltage dependent pathway. J Membr Biol. 1995 Mar;144:177–184.
Yan M, Dai T, Deak JC, et al. Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature. 1994;372:798–800. [CrossRef] [PubMed]
Figure 1.
 
Effect of suppressing K+ channel activity on UV-C-irradiation–induced JNK activation. (A) Time course of UV-induced JNK-1 activation in RCE cells. (B) Time course of UV-induced JNK-1 activation in HCE cells. (C) Dose-dependent inhibition of UV-irradiation–induced JNK-1 activation by suppression of K+ channel activity. RCE cells were incubated with various concentration of 4-AP before exposure to UV irradiation. (D) Inhibition of UV-induced JNK-1 activation by suppression of K+ channel activity. UV-irradiation–induced JNK activation was significantly blocked by 4-AP (1 mM) in RCE cells (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. (E) Time course of UV-induced JNK-1 activation in primary RCE cells. (F) Prevention of JNK-1 activation in primary RCE cells. Blockade of K+ channel activity with 4-AP (1 mM) significantly inhibited UV-irradiation–induced JNK activation (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. In all the experiments, HCE, RCE, and primary RCE cells were pretreated or untreated with K+ channel blockers for 30 minutes, or cells were exposed to UV light (45 μJ/cm2). After an additional 30-minute incubation, the cells were harvested to measure JNK-1 activity and JNK protein concentrations by mmunocomplex kinase assay and Western blot, respectively.
Figure 1.
 
Effect of suppressing K+ channel activity on UV-C-irradiation–induced JNK activation. (A) Time course of UV-induced JNK-1 activation in RCE cells. (B) Time course of UV-induced JNK-1 activation in HCE cells. (C) Dose-dependent inhibition of UV-irradiation–induced JNK-1 activation by suppression of K+ channel activity. RCE cells were incubated with various concentration of 4-AP before exposure to UV irradiation. (D) Inhibition of UV-induced JNK-1 activation by suppression of K+ channel activity. UV-irradiation–induced JNK activation was significantly blocked by 4-AP (1 mM) in RCE cells (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. (E) Time course of UV-induced JNK-1 activation in primary RCE cells. (F) Prevention of JNK-1 activation in primary RCE cells. Blockade of K+ channel activity with 4-AP (1 mM) significantly inhibited UV-irradiation–induced JNK activation (P < 0.01). Data were collected from four independent experiments and expressed as means ± SE. In all the experiments, HCE, RCE, and primary RCE cells were pretreated or untreated with K+ channel blockers for 30 minutes, or cells were exposed to UV light (45 μJ/cm2). After an additional 30-minute incubation, the cells were harvested to measure JNK-1 activity and JNK protein concentrations by mmunocomplex kinase assay and Western blot, respectively.
Figure 2.
 
Effect of UV irradiation on SEK-1 activation. (A) Phosphorylation of SEK-1 induced by UV irradiation. RCE cells were exposed to UV irradiation and then incubated in culture medium for indicated periods. (B) Prevention of UV-induced SEK-1 phosphorylation by suppression of K+ channel activity. RCE cells were incubated with 1 mM 4-AP for 30 minutes before UV irradiation. SEK-1 activation was determined with anti-phospho-SEK-1 antibody by Western blot analysis.
Figure 2.
 
Effect of UV irradiation on SEK-1 activation. (A) Phosphorylation of SEK-1 induced by UV irradiation. RCE cells were exposed to UV irradiation and then incubated in culture medium for indicated periods. (B) Prevention of UV-induced SEK-1 phosphorylation by suppression of K+ channel activity. RCE cells were incubated with 1 mM 4-AP for 30 minutes before UV irradiation. SEK-1 activation was determined with anti-phospho-SEK-1 antibody by Western blot analysis.
Figure 3.
 
Effects of K+ influx and extracellular Ca2+ on JNK-1 activity. (A) Effects of selective K+ channel blockers on activation of JNK-1 induced by UV irradiation. Selective K+ channel blockers, 4-AP (1 mM), TEA (10 mM), and Ba2+ (5 mM), were applied to inhibit UV-irradiation–induced JNK activation in RCE cells. Data are expressed as the mean ± SE. *Significant difference after application of blockers (n = 4, P < 0.01). (B) Effect of extracellular Ca2+ on UV-induced JNK activation. RCE cells were exposed to UV irradiation in the presence and absence of 1 mM 4-AP. Extracellular Ca2+ was removed by preincubation of RCE cells in medium containing 5 mM EGTA for 30 minutes before UV exposure. (C) Effect of valinomycin on JNK-1 activation. RCE cells were treated with valinomycin (1–50 nM) for 20 minutes before JNK activity was measured. (D) Effect of 4-AP on valinomycin-induced JNK-1 activation. RCE cells were incubated with 50 nM valinomycin for 20 minutes in the presence and absence of 3 mM 4-AP. (E) Direct effect of 4-AP on purified JNK activity. JNK-1 protein was immunoprecipitated from UV-induced RCE cells and JNK-1 activity was measured with or without 3 mM 4-AP in vitro. JNK-1 activity was measured by using immunocomplex kinase assay using GST-ATF2 fusion protein as a substrate. Total JNK protein levels were determined by Western blot.
Figure 3.
 
Effects of K+ influx and extracellular Ca2+ on JNK-1 activity. (A) Effects of selective K+ channel blockers on activation of JNK-1 induced by UV irradiation. Selective K+ channel blockers, 4-AP (1 mM), TEA (10 mM), and Ba2+ (5 mM), were applied to inhibit UV-irradiation–induced JNK activation in RCE cells. Data are expressed as the mean ± SE. *Significant difference after application of blockers (n = 4, P < 0.01). (B) Effect of extracellular Ca2+ on UV-induced JNK activation. RCE cells were exposed to UV irradiation in the presence and absence of 1 mM 4-AP. Extracellular Ca2+ was removed by preincubation of RCE cells in medium containing 5 mM EGTA for 30 minutes before UV exposure. (C) Effect of valinomycin on JNK-1 activation. RCE cells were treated with valinomycin (1–50 nM) for 20 minutes before JNK activity was measured. (D) Effect of 4-AP on valinomycin-induced JNK-1 activation. RCE cells were incubated with 50 nM valinomycin for 20 minutes in the presence and absence of 3 mM 4-AP. (E) Direct effect of 4-AP on purified JNK activity. JNK-1 protein was immunoprecipitated from UV-induced RCE cells and JNK-1 activity was measured with or without 3 mM 4-AP in vitro. JNK-1 activity was measured by using immunocomplex kinase assay using GST-ATF2 fusion protein as a substrate. Total JNK protein levels were determined by Western blot.
Figure 4.
 
Effect of UV irradiation on ERK and p38 activation. (A) Time course of UV-induced ERK activation in RCE cells. (B) Inhibition of UV-induced ERK activation in RCE cells by suppression of K+ channel activity with 4-AP. (C) Time course of UV-induced p38 activation in RCE cells. (D) Effect of suppressing K+ channel activity with 4-AP (2 mM) on UV-irradiation–induced p38 activation in RCE cells. ERK-2 and p38 activities in RCE cells were measured by using immunocomplex kinase assay. Total protein levels of ERK-2 and p38 were determined by Western blot.
Figure 4.
 
Effect of UV irradiation on ERK and p38 activation. (A) Time course of UV-induced ERK activation in RCE cells. (B) Inhibition of UV-induced ERK activation in RCE cells by suppression of K+ channel activity with 4-AP. (C) Time course of UV-induced p38 activation in RCE cells. (D) Effect of suppressing K+ channel activity with 4-AP (2 mM) on UV-irradiation–induced p38 activation in RCE cells. ERK-2 and p38 activities in RCE cells were measured by using immunocomplex kinase assay. Total protein levels of ERK-2 and p38 were determined by Western blot.
Figure 5.
 
Effect of suppressing K+ channel activity on hyperosmotic-stress–induced apoptotic response. (A) Hyperosmotic-stress–induced RCE cell apoptosis. Cell viability was determined by nuclear EB/OA staining. (B) Time course of hyperosmotic-stress–induced activation of JNK. JNK was activated by application of 600 mM sorbitol and measured in RCE cells up to 60 minutes. (C) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced JNK activation. (D) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced p38 activation. Hyperosmotic-stress–induced JNK and p38 activations were measured by immunocomplex assay 5 minutes after exposure to 600 mM sorbitol in the absence and presence of 4-AP (2 mM).
Figure 5.
 
Effect of suppressing K+ channel activity on hyperosmotic-stress–induced apoptotic response. (A) Hyperosmotic-stress–induced RCE cell apoptosis. Cell viability was determined by nuclear EB/OA staining. (B) Time course of hyperosmotic-stress–induced activation of JNK. JNK was activated by application of 600 mM sorbitol and measured in RCE cells up to 60 minutes. (C) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced JNK activation. (D) Effect of suppressing K+ channel activity with 4-AP on hyperosmotic-stress–induced p38 activation. Hyperosmotic-stress–induced JNK and p38 activations were measured by immunocomplex assay 5 minutes after exposure to 600 mM sorbitol in the absence and presence of 4-AP (2 mM).
Figure 6.
 
Inhibition of UV-induced caspase 3 activation by suppression of K+ channel activity. RCE cells were preincubated with and without 1 mM 4-AP before UV irradiation. Caspase 3 activity in RCE cells was determined by using a caspase assay kit. Ac-DEVD pNA was used as substrate for caspase 3. Caspase 3 activity was assessed by measuring fluorescence at 405 nm wavelength with a fluorometer.
Figure 6.
 
Inhibition of UV-induced caspase 3 activation by suppression of K+ channel activity. RCE cells were preincubated with and without 1 mM 4-AP before UV irradiation. Caspase 3 activity in RCE cells was determined by using a caspase assay kit. Ac-DEVD pNA was used as substrate for caspase 3. Caspase 3 activity was assessed by measuring fluorescence at 405 nm wavelength with a fluorometer.
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