The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP1 Activation...

The Role of JNK and p38 MAPK Activities in UVA-InducedSignaling Pathways Leading to AP-1 Activation andc-Fos Expression1

Amy L. Silvers, Michael A. Bachelor and G. Timothy Bowden

Department of Radiation Oncology, Arizona Cancer Center, The University of Arizona, Tucson, AZ, USA

Abstract

To further delineate ultraviolet A (UVA) signaling

pathways in the human keratinocyte cell line HaCaT,

we examined the potential role of mitogen-activated

protein kinases (MAPKs) in UVA-induced activator

protein-1 (AP-1) transactivation and c-Fos expres-

sion. UVA-induced phosphorylation of p38 and c-Jun

N-terminal kinase (JNK) proteins was detected im-

mediately after irradiation and disappeared after

approximately 2 hours. Conversely, phosphorylation

of extracellular signal-regulated kinase was signifi-

cantly inhibited for up to 1 hour post-UVA irradi-

ation. To examine the role of p38 and JNK MAPKs in

UVA-induced AP-1 and c-fos transactivations, the

selective pharmacologic MAPK inhibitors, SB202190

(p38 inhibitor) and SP600125 (JNK inhibitor), were

used to independently treat stably transfected HaCaT

cells in luciferase reporter assays. Both SB202190 and

SP600125 dose-dependently inhibited UVA-induced

AP-1 and c-fos transactivations. SB202190 (0.25–

0.5 MM) and SP600125 (62–125 nM) treatments also

primarily inhibited UVA-induced c-Fos expression.

These results demonstrated that activation of both

JNK and p38 play critical role in UVA-mediated AP-1

transactivation and c-Fos expression in these human

keratinocyte cells. Targeted inhibition of these MAPKs

with their selective pharmacologic inhibitors may be

effective chemopreventive strategies for UVA-induced

nonmelanoma skin cancer.

Neoplasia (2003) 5, 319 – 329

Keywords: UVA; AP-1; MAPK; c-Fos; HaCaT.

Introduction

Exposure to sunlight is the major risk factor for the develop-

ment of nonmelanoma skin cancer [1]. Because strato-

spheric ozone completely absorbs short-wavelength

ultraviolet C (<280 nm), the relevant carcinogenic compo-

nents of sunlight that reach the Earth’s surface are ultraviolet

B (UVB) (280–320 nm) and ultraviolet A (UVA) (320–400

nm) [2,3]. Although UVA is magnitudes less carcinogenic

than UVB [4,5], chronic UVA exposure has been shown to

induce photoaging [6] and skin tumors (papillomas and squ-

amous cell carcinomas) [5,7,8] in experimental animals.

The signaling pathways involved in UVA-induced photo-

aging and skin tumorigenesis are subjects of intense interest

and have not been fully elucidated. UVA has been shown to

alter the expression of numerous mammalian genes, such as

heme oxygenase-1 [9], intercellular adhesion molecule-1

(ICAM-1) [10], and matrix metalloproteinase-1 [11], through

the generation of reactive oxygen intermediates, specifically

singlet O2 [12]. UVA irradiation has also been reported to

activate several transcription factors, including activator pro-

tein-1 (AP-1) [13–15], activator protein-2 (AP-2) [10], nuclear

factor kappa-B (NFnB) [16,17], and signal transducer and

activator of transcription (STAT) 1 and 3 [18,19]. Both UVA-

induced AP-1 and AP-2 activations were mediated through

singlet oxygen [10,14].

AP-1 is an important regulatory protein involved in cell

growth, differentiation, transformation, and apoptosis, and

may also contribute to inflammatory and immune responses

[20,21]. It can be induced by growth factors, cytokines, 12-O-

tetradecanoylphorbol-13-acetate (TPA), UV radiation, and

transforming oncoproteins [20]. The AP-1 complex consists

of heterodimers of Fos (c-Fos, Fra-1, Fra-2, and FosB) and Jun

(c-Jun, JunB, and JunD) family members, or homodimers and

heterodimers of Jun family members that bind to TPA response

elements (TREs) in AP-1–inducible gene promoters, contribu-

ting to transcriptional activity or repression of these genes [20].

Abbreviations: AP-1, activator protein-1; AP-2, activator protein-2; ATF-2, activating

transcription factor-2; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol;

EDTA, ethylenediamine-tetraacetic acid; EGTA, ethylene glycol-bis(h-aminoethly ether)-

N,N,N V,N V-tetraacetic acid; ERK, extracellular signal-regulated kinase; HRP, horseradish

peroxidase; ICAM-1, intercellular adhesion molecule-1; JNK, c-Jun NH2-terminal kinase;

MAPK, mitogen-activated protein kinase; MAPKAPK2, MAPK-activated protein kinase-2;

MEF, MADS box transcription enhancer factor; MEK, MAPK/ERK kinase; NFnB, nuclear

factor kappa-B; PBS, phosphate-buffered saline; SAP, serum response factor accessory

protein; SDS, sodium dodecyl sulfate; SRE, serum response element; STAT, signal

transducer and activator of transcription; TBST, Tris-buffered saline with 0.05% Tween

20; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA response element; UVA,

ultraviolet A

Address all correspondence to: G. Timothy Bowden, Arizona Cancer Center, Room 4999,

1515 North Campbell Avenue, Tucson, AZ 85724, USA. E-mail: [email protected] work was supported, in part, by the National Cancer Institute – funded grant

R25CA78447 through the Cancer Prevention and Control Program at the Arizona Cancer

Center, Tucson, AZ. This work was also supported by the National Institutes of Health grants

CA27502 and CA23074.

Received 9 February 2003; Revised 18 June 2003; Accepted 18 June 2003.

Copyright D 2003 Neoplasia Press, Inc. All rights reserved 1522-8002/03/$25.00

Neoplasia . Vol. 5, No. 4, July/August 2003, pp. 319 – 329 319

www.neoplasia.com

RESEARCH ARTICLE

Deregulated expression of the AP-1 complex has been

shown to play a prominent role in skin tumor promotion. Loss

of AP-1 DNA-binding activity resulted in loss of proliferative

potential and induction of differentiation in human keratino-

cytes [22]. Dong et al. [23] demonstrated that anchorage-

independent growth of JB6 cells required the transactivation

of AP-1.

Deregulated expression of individual AP-1 complex com-

ponents has also been shown to induce malignant trans-

formation in vivo. Transfection of v-fos into murine papilloma

cell lines expressing an activated Ha-ras oncogene resulted

in the malignant conversion of these cells [24]. Similarly, the

development of malignant skin tumors in v-Ha-ras transgenic

mice following TPA treatment was inhibited in c-fos �/� mice

[25]. Young et al. [26] also reported that TPA-mediated

promotion in a two-stage skin carcinogenesis mouse model

was suppressed by the stable expression of an epidermis-

targeted dominant negative c-jun transgene. Similarly,

Thompson et al. [27] demonstrated that the expression of

the same epidermis-targeted dominant negative c-jun trans-

gene inhibited okadaic acid–mediated skin tumor promotion.

The mitogen-activated protein kinase (MAPK) family of

proteins include p38, c-Jun N-terminal kinase (JNK), and

extracellular signal-regulated kinase (ERK). MAPKs are pro-

line-directed serine/threonine kinases that are activated by

dual phosphorylation on threonine and tyrosine residues in

response to a wide variety of extracellular stimuli [28]. They

mediate signal transduction from the cell surface to

the nucleus. Activation of ERK is primarily involved in

growth factor– and phorbol ester–stimulated responses.

Responses to proinflammatory cytokines, UV radiation,

and other stresses are mostly dependent on JNK and p38

activation [21,29]. MAPK signaling pathways have been

shown to affect AP-1 activity by direct phosphorylation

of AP-1 proteins and by influence on the abundance of

individual AP-1 components in a cell [29,30].

c-Jun is directly phosphorylated by JNK at N-terminal

serines 63 and 73, resulting in increased stability and trans-

activation potential [31,32]. c-Jun is also phosphorylated by

ERK1/2 on C-terminal inhibitory sites [33,34]. Although the in

vivo relevance is still unclear, ERK1/2 can also phosphor-

ylate c-Fos and ATF-2 [28,35].

MAPKs also increase the abundance of AP-1 complex

components by transcriptionally activating their promoters.

The ternary complex factors Elk-1 (a substrate of ERK,

p38, and JNK) and serum response factor accessory

protein (SAP) 1a and 2 (substrates of ERK and p38) form

complexes with dimeric serum response factors at the

serum response element (SRE) in the c-fos promoter

[28,30]. Additionally, activation of STAT1 and STAT3 by

JNK [18] and possibly ERK [30,36] at the cis-inducible

element in the c-fos promoter may act in cooperation

with the SRE to influence c-fos expression [30]. Induction

of c-jun expression is predominantly mediated by two

TREs that preferentially bind c-Jun and ATF-2 hetero-

dimers. These proteins are activated by phosphorylation

in their transactivation domains [29]. JNK and p38 phos-

phorylate and activate ATF-2, whereas JNK phosphorylates

the c-Jun activation domain [37,38]. Additionally, ERK and

p38 activation may contribute to c-jun expression through

phosphorylation of MEF2 proteins—transcription factors

that also bind to the c-jun promoter [39,40].

Although a few studies have addressed the potential role

of MAPK activation in UVA signaling, results are not con-

sistent. Klotz et al. [12] reported a rapid and transient

induction of p38 and JNK activity, but not ERK activity, in

human skin fibroblasts. In contrast, UVA irradiation stimu-

lated the activation of all three MAPKs in the NCTC 2544

human keratinocyte cell line [41] and in the mouse epidermal

JB6 promotion-sensitive Cl 41 cell line [42,43]. Additionally,

Djavaheri-Mergny and Dubertret [14] provided evidence that

UVA-induced AP-1 activation required the Raf/ERK pathway

in NCTC 2544 keratinocytes.

Our previous results demonstrated that UVA irradiation of

the human immortalized keratinocyte cell line HaCaT

induced the expression of several AP-1 family members,

including c-Fos and c-Jun, and potentiated the transactiva-

tion of the c-fos promoter and the AP-1–binding site in the

collagenase-1 gene promoter [15]. To further delineate the

UVA signaling pathway(s) in these keratinocytes, we exam-

ined the potential role of MAPKs in UVA-induced AP-1

transactivation and c-fos expression through the use of

specific pharmacologic MAPK inhibitors. We report, for the

first time, that p38 and JNK MAPKs contributed to UVA-

induced AP-1 activation and UVA-induced c-fos transactiva-

tion as well as UVA-induced c-Fos protein expression. The

use of SB202190 and SP600125 to selectively inhibit their

respective UVA-induced stress-activated protein kinases

may be a useful chemopreventive strategy for UVA-induced

nonmelanoma skin cancer.

Materials and Methods

Cell Culture

The human keratinocyte cell line, HaCaT, was stably

transfected with a sequence from the human collagenase-1

gene promoter (�73 to +63) containing one endogenous

AP-1 – binding site driving a luciferase reporter gene

(HCL14 cells), as reported previously [44]. HaCaT cells

were also independently stably transfected with a sequence

from the human c-fos promoter (�404 to +41) driving a

luciferase reporter gene (FL30 cells), as reported previously

[44]. These cells were cultured in Dulbecco’s modified

Eagle’s medium (DMEM) supplemented with 10% fetal

bovine serum and 100 U/ml penicillin/streptomycin at

37jC and 5% CO2. Cells were grown to near confluence

and then serum-starved for 24 to 28 hours prior to treat-

ment. Treatment of cells with selective pharmacologic

MAPK inhibitors involved a 1-hour preirradiation incubation

and variable postirradiation incubations. Each compound

was diluted in serum-free medium prior to use.

Pharmacologic Inhibitors of MAPKs

SB202190 (Calbiochem, San Diego, CA) is a potent, cell-

permeable, selective, and reversible inhibitor of p38a and

320 UVA-Induced Signaling Pathways Silvers et al.

Neoplasia . Vol. 5, No. 4, 2003

h1 isoforms. Inhibition is competitive with ATP and requires

substrates to accommodate the fluorophenyl ring structure of

the pyridinyl imidazole in their ATP-binding pocket. Struc-

tures of other p38 isoforms, JNKs, and ERK1/2 do not allow

inhibitor binding at equivalent positions [45].

SP600125 (Calbiochem) is a potent, cell-permeable,

selective, and reversible inhibitor of JNK1, JNK2, and

JNK3 isoforms. Inhibition is competitive with ATP and may

involve the interaction of the nitrogen-containing ring system

of the anthrapyrazolone with key residues in the kinase

active site of substrates [46].

PD98059 (Alexis Biochemicals, Carlsbad, CA) is a selec-

tive, cell-permeable inhibitor of MEK1, MEK2, and MEK5 that

does not directly inhibit kinase activity but rather prevents the

activation of the kinase [45,47,48]. It has also been shown to

directly inhibit cyclooxygenase-1 and cyclooxygenase-2

enzyme activities [49].

UVA Irradiation

Cells were irradiated with a bank of four F20T12/BL/HO

UVA bulbs (National Biological, Twinsburg, OH) that were

powered by an Advance electronic ballast REL-4P32-RH-TP

(120 V, 60 Hz, 90 A) (Advance Transformer Co., Chicago, IL).

Spectral emission was reported previously [15]. A UVX

radiometer equipped with a UVX-36 sensor (UVP, Upland,

CA) was used to measure radiation doses. Plate glass

(4 mm) was used to filter wavelengths below 320 nm.

Irradiation was performed in a sterile, well-ventilated laminar

flow hood to eliminate thermal stimulation. Cells were irradi-

ated in phosphate-buffered saline (PBS) supplemented with

0.01% MgCl2 and 0.01% CaCl2 at room temperature. Control

cells were mock-irradiated under similar conditions. Cultures

were continued in serum-free DMEM until harvest.

Luciferase Assay for AP-1 and c-fos Transactivations

Total cellular protein from stably transfected HaCaT

cells was extracted in lysis buffer (15 mM MgSO4, 25 mM

glycylglycine, 4 mM EGTA, 1% vol/vol Triton X-100, and 1

mM DTT) and was quantitated using the Bio-Rad Dc

Protein Assay kit (Bio-Rad Laboratories, Hercules, CA).

Luciferase activity of 20 to 30 Ag of total cellular protein

was measured using the TD-20/20 Luminometer (Turner

Designs, Sunnyvale, CA).

Western Analysis

Total cellular protein was extracted in lysis buffer (150 mM

NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 2.5 mM

Na4P2O7, 1 mM h-glycerol phosphate, 1 mM Na3VO4, 1 Ag/

ml leupeptin, and 1% Triton X-100) supplemented with addi-

tional phosphatase inhibitors (1 mM Na3VO4, 2 Ag/ml leu-

peptin, and 10 Ag/ml aprotinin) and was quantitated using the

Bio-Rad Dc Protein Assay kit (Bio-Rad Laboratories).

Lysates (40 Ag) were resolved on 12.5% sodium dodecyl

sulfate (SDS) polyacrylamide gels and were then transferred

to Immobilon-P nylon membranes (Millipore, Bedford, MA).

Membranes were blocked with 5% evaporated milk in 1�TBS/0.05% Tween 20 (TBST) for 2 hours at room temper-

ature. Primary antibodies against AP-1 family members

Figure 1. Western time course for UVA-induced MAPK activation. HCL14 cells were mock-irradiated or irradiated with 250 kJ/m2 UVA and harvested at the

appropriate time points postirradiation. Forty micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P

membranes, and immunodetected using optimal primary and secondary antibody concentrations for each MAPK. Western blot data are representative of at least

two independent experiments. (A) p38; (B) JNK; and (C) ERK.

UVA-Induced Signaling Pathways Silvers et al. 321


(Santa Cruz Biotechnology, Santa Cruz, CA) were diluted

at 1:1000 (c-Fos) or 1:500 (c-Jun), primary antibody against

a-tubulin (Oncogene Research Products, Boston, MA) was

diluted at 1:2500, and primary antibodies against p38, JNK,

and ERK MAPKs (Cell Signaling, Beverly, MA) were diluted at

1:2000 in 5% evaporated milk/TBST and were incubated

with membranes for 2 hours at room temperature. Primary

antibodies against phospho-MAPKs (Cell Signaling)

and phospho-c-Jun (Cell Signaling) were diluted at 1:800

(phospho-p38) or 1:1000 in 5% evaporated milk/TBST

and were incubated with membranes overnight at 4jC.

Anti– rabbit HRP–conjugated secondary antibodies (Cell

Signaling) were diluted at 1:2000, whereas anti–mouse

HRP–conjugated secondary antibodies (Santa Cruz

Biotechnology) were diluted at 1:5000 (c-Fos) and 1:8000

(a-tubulin) in 5% evaporated milk/TBST and were incubated

with membranes for 1 hour at room temperature. Membranes

were washed three times for 10 minutes in TBST after each

antibody incubation. Protein bands were visualized using the

ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).

In Vivo p38 Activity Assay

The in vivo p38 activity assay was reported previously

[50]. Briefly, total cellular protein was extracted in lysis buffer

(150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM

EGTA, 2.5 mM Na4P2O7, 1 mM h-glycerol phosphate, 1 mM

Na3VO4, 1 Ag/ml leupeptin, and 1% Triton X-100) supple-

mented with additional phosphatase inhibitors (1 mM

Na3VO4, 2 Ag/ml leupeptin, and 10 Ag/ml aprotinin) and

was quantitated using the Bio-Rad Dc Protein Assay kit

(Bio-Rad Laboratories). Lysates (10 Ag) were resolved on

12.5% SDS polyacrylamide gels overnight at 4jC and

Figure 2. Target specificity of SB202190. HCL14 cells were pretreated with SB202190 in serum-free medium for 1 hour, mock-irradiated or irradiated with

250 kJ/m2 UVA, and then continued in serum-free medium supplemented with SB202190 until each desired time point. Ten micrograms (MAPKAPK2) or

40 lg (phospho-JNK and phospho-ERK) of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes,

and immunodetected using optimal primary and secondary antibody concentrations for the target proteins. Western blot data are representative of at least two

independent experiments. (A) Effect of SB202190 on p38 activity through detection of changes in MAPKAPK2 phosphorylation; (B) effect of SB202190 on

JNK activation; and (C) effect of SB202190 on ERK activation.



were then transferred to Immobilon-P nylon membranes

(Millipore). The membrane was blocked with 5% evaporated

milk/1� TBS/0.1% Tween 20 for 1 hour at room temperature

and was then washed three times for 5 minutes in 1� TBS/

0.1% Tween 20. MAPK-activated protein kinase-2 (MAP-

KAPK2) primary antibody (Cell Signaling) was diluted at

1:1000 in 5% BSA/1� TBS/0.1% Tween 20 and was incu-

bated with the membrane overnight at 4jC. HRP-conjugated

secondary antibody (Cell Signaling) was diluted at 1:2000 in

5% evaporated milk/1� TBS/0.1% Tween 20 and was incu-

bated with the membrane for 1 hour at room temperature.

The membrane was washed three times for 5 minutes in 1�TBS/0.1% Tween 20 after each antibody incubation. Protein

bands were visualized using the ECL kit (Amersham Phar-

macia Biotech). Both phosphorylated and unphosphorylated

bands were detected.

Results

Effects of UVA Irradiation on the Activation of MAPKs

As reported previously, maximal AP-1 transactivation in

the stably transfected HaCaT cells was achieved with a dose

of 250 kJ/m2 UVA. This dose of UVA increased AP-1 trans-

activation levels between 2 and 8 hours post-UVA [15].

Therefore, time courses for Western analyses to detect the

Figure 3. Target specificity of SP600125. HCL14 cells were pretreated with a SP600125 in serum-free medium for 1 hour, mock-irradiated or irradiated with

250 kJ/m2 UVA, and then continued in serum-free medium supplemented with SP600125 until each desired time point. Forty micrograms of total cell lysate was

electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using optimal primary and secondary antibody

concentrations for the target proteins. Western blot data are representative of at least two independent experiments. (A) Effect of SP600125 on JNK activity as

detected by changes in c-Jun phosphorylation and expression; (B) effect of SP600125 on p38 activation; and (C) effect of SP600125 on ERK activation.



UVA-induced activation of MAPKs were performed on these

cells using 250 kJ/m2 UVA, beginning immediately after

irradiation and spanning 8 hours post-UVA irradiation

(Figure 1).

Phosphorylation of p38 was detected immediately after

UVA irradiation and persisted for approximately 1 hour, with

only very weak activation detected at 2, 4, and 8 hours

postirradiation (Figure 1A). Total p38 levels were unaffected

by UVA irradiation. Increased phosphorylation of JNK was

detected immediately after irradiation and disappeared after

approximately 2 hours, with maximal expression between 15

and 30 minutes postirradiation (Figure 1B). UVA irradiation

did not affect basal levels of JNK. Interestingly, UVA irradi-

ation transiently but consistently inhibited the activation of

ERK. This inhibition was detected immediately after irradi-

ation and persisted for up to 1 hour post-UVA (Figure 1C).

Conversely, ERK was transiently activated in mock-treated

cells. This activation may have been due to the environ-

mental change experienced by the cells after irradiation,

specifically the switch from supplemented PBS to serum-

free medium. Despite this activation, ERK phosphorylation

was consistently lower in irradiated lysates compared to

controls. Basal levels of ERK expression were unaffected

by UVA irradiation.

Target Specificity of SB202190

Concentrations of SB202190 as high as 15 AM have

been used in HaCaT cells to assess the role of p38 a/hisoforms in UV-induced signaling pathways [50–53]. How-

ever, much lower doses of this compound (2 AM) have

been shown to inhibit UVA-induced p38 activity [53]. In

vivo p38 activity assays were performed using even lower

doses of SB202190 (0.25 and 0.5 AM) in HCL14 cells to

ensure that these doses were also inhibitory. At 30 minutes

post-UVA irradiation, the p38 MAPK substrate MAPKAPK2

was primarily phosphorylated, whereas MAPKAPK2 in

Figure 4. Effects of SB202190 and SP600125 on UVA-induced AP-1 transactivation. HCL14 cells were pretreated with a MAPK inhibitor in serum-free medium for

1 hour, mock-irradiated or irradiated with 250 kJ/m2 UVA, and then continued in serum-free medium supplemented with the respective doses of inhibitor for 4 hours

postirradiation. Thirty micrograms of total cell lysate was analyzed for each luciferase reporter assay. Fold inductions with respect to mock-treated controls are

listed above their respective bars for each dose. Each bar represents the meanFSD of triplicate samples. Data are representative of at least two independent

experiments. (A) SB202190; and (B) SP600125.



mock-treated cells was unphosphorylated. Treatment with

a combination of UVA and SB202190 resulted in detection

of both phosphorylated and unphosphorylated forms of

MAPKAPK2, indicating that SB202190 reduced the

capacity of p38 to phosphorylate and activate MAPKAPK2

(Figure 2A).

To determine the target specificity of SB202190, a time

point was selected for each MAPK that would allow for the

detection of inhibition of UVA-mediated forms of the

kinases by the pharmacologic inhibitor. No effects on

JNK phosphorylation were detected with 0.25 or 0.5 AM

SB202190 at 30 minutes post-UVA irradiation (Figure 2B).

Minimal effects of SB202190 on ERK phosphorylation were

observed, with band intensity decreasing slightly in both

unirradiated and irradiated groups at 15 minutes postirra-

diation (Figure 2C).

Target Specificity of SP600125

The anthrapyrazolone SP600125 has been recently mar-

keted as a potent, selective, and reversible inhibitor of all

three JNK isoforms. The inhibitory activity of SP600125 in

Jurkat T cells and primary human monocytes required IC50

doses between 5 and 10 AM [46]. However, in HaCaT cells,

much lower doses were sufficient to inhibit UVA-induced

JNK activity and c-Jun expression. We previously showed

that c-Jun was biphasically activated by UVA irradiation.

Preexisting c-Jun protein was phosphorylated at early time

points, followed by maximal c-Jun expression between 2 and

4 hours after UVA irradiation [15]. The JNK inhibitor

SP600125 diminished UVA-induced JNK activity as well as

c-Jun expression with a dose of 125 nM at 2 hours postirra-

diation (Figure 3A). The effect on total c-Jun levels might be

a consequence of c-Jun autoregulation through the TRE-

binding sites in the c-Jun promoter [54]. SP600125 treatment

of these human keratinocytes in combination with UVA

resulted in the appearance of a slower-migrating, unidenti-

fied band that may represent a modified form of c-Jun. These

doses of SP600125 did not significantly affect p38 or ERK1/2

phosphorylation at 15 minutes postirradiation (Figure 3, B

and C, respectively).

Figure 5. Effects of SB202190 and SP600125 on UVA-induced c-fos transactivation. FL30 cells were pretreated with a MAPK inhibitor in serum-free medium for

1 hour, mock-irradiated or irradiated with 250 kJ/m2 UVA, and then continued in serum-free medium supplemented with the respective doses of inhibitor for 4 hours

postirradiation. Thirty micrograms of total cell lysate was analyzed for each luciferase reporter assay. Fold inductions with respect to mock-treated controls are

listed above their respective bars for each dose. Each bar represents the meanFSD of triplicate samples. Data are representative of at least two independent

experiments. (A) SB202190; and (B) SP600125.



Effects of SB202190 and SP600125 on UVA-Induced AP-1

Transactivation

As previously reported, maximal AP-1 transactivation was

achieved with a dose of 250 kJ/m2 at 4 hours post-UVA

irradiation [15]. Therefore, these experimental conditions

were used to examine the downstream effects of p38 and

JNK MAPK inhibition. Independent dose responses with

SB202190 and SP600125 were performed to determine

the potential involvement of p38 and/or JNK in UVA-induced

AP-1 transactivation using HaCaT cells stably transfected

with the collagenase promoter driving a luciferase reporter

gene (HCL14 cells). As shown in Figure 4A, UVA-induced

AP-1 transactivation was significantly and dose-dependently

blocked with SB202190 treatment at 4 hours post-UVA

irradiation. Doses as low as 0.25 AM inhibited UVA-induced

AP-1 transactivation by as much as 64% compared to

controls. SP600125 also significantly and dose-dependently

blocked UVA-induced AP-1 transactivation at this time point

(Figure 4B). A 78-nM dose of SP600125 resulted in an

approximately 51% reduction in UVA-induced AP-1 trans-

activation compared to controls. The increased AP-1 activity

in unirradiated cells between 62 and 109 nM SP600125 in

this representative experiment was not consistently

observed. Basal levels of AP-1 activity tended to bobble

around control levels in additional experiments.

Effects of SB202190 and SP600125 on UVA-Induced c-fos

Transactivation

To determine whether p38 and JNK were involved in UVA-

induced c-fos transactivation, HaCaT cells that were stably

transfected with the human c-fos promoter driving a lucifer-

ase reporter gene (FL30 cells) were treated with varying

doses of their pharmacologic inhibitors in combination with

250 kJ/m2 UVA. As shown in Figure 5A, UVA-induced c-fos

transactivation was significantly and dose-dependently

blocked with SB202190 at 4 hours post-UVA irradiation.

Figure 6. Effects of SB202190 and SP600125 on UVA-induced c-Fos protein expression. HCL14 cells were pretreated with a MAPK inhibitor in serum-free medium

for 1 hour, mock-irradiated or irradiated with 250 kJ/m2 UVA, and then continued in serum-free medium supplemented with the respective inhibitor for 1 hour. Forty

micrograms of total cell lysate was electrophoresed on 12.5% SDS polyacrylamide gels, transferred to Immobilon-P membranes, and immunodetected using

optimal primary and secondary antibody concentrations for c-Fos. Western blot data are representative of two independent experiments. (A) SB202190; and

(B) SP600125.



The effects of drug combined with UVA irradiation paral-

leled the effects observed with this combination treatment

on AP-1 transactivation, with a dose of 0.25 AM SB202190

reducing UVA-induced c-fos transactivation by 61% com-

pared to controls. SP600125 also significantly and dose-

dependently blocked UVA-induced c-fos transactivation

(Figure 5B). Although SP600125 dose-dependently inhibited

both AP-1 and c-fos transactivations, 62 nM SP600125

consistently blocked UVA-induced c-fos transactivation

(approximately 39% of control values) but did not effectively

block UVA-induced AP-1 transactivation. In fact, in some

experiments, this dose of SP600125 appeared to slightly

enhance AP-1 transactivation.

Effects of SB202190 and SP600125 on c-Fos Expression

To further explore the downstream effects of p38 and JNK

inhibition in irradiated and unirradiated keratinocytes,

changes in c-Fos expression were examined. At 1 hour

postirradiation, the dose of SB202190 that blocked both

AP-1 and c-fos transactivations also significantly inhibited

UVA-induced c-Fos expression and only marginally affected

basal c-Fos expression (Figure 6A). Doses of SP600125 that

reduced UVA-induced c-fos transactivation were also able to

block UVA-induced c-Fos expression, whereas basal c-Fos

expression was not affected by drug treatment (Figure 6B).

The observed effects of these MAPK inhibitors on activation

of the c-fos promoter and c-Fos protein expression were

highly correlative in these human keratinocytes.

Discussion

A prominent role for UVA in skin tumor promotion has

recently emerged, involving altered expression of mamma-

lian genes through activation of transcription factors such as

AP-1, AP-2, NFnB, and STATs [10,13–19]. UVA irradiation

was previously shown to induce AP-1 DNA binding and AP-1

transactivation in the human keratinocyte cell line HaCaT.

This induction correlated with UVA-induced c-fos promoter

activation and c-Fos protein expression [15]. To further

delineate the UVA signaling pathway(s) in these immortal-

ized keratinocytes, we examined potential roles for p38,

JNK, and ERK MAPKs in the mediation of UVA-induced

AP-1 and c-fos promoter activations as well as UVA-induced

c-Fos expression.

UVA irradiation (250 kJ/m2) rapidly and transiently

induced p38 and JNK activation but inhibited ERK activa-

tion. Although peak activation or inhibition of UVA-induced

MAPK activities differed slightly, all three kinases returned

to basal levels by 2 hours postirradiation. These combined

effects of UVA on MAPK activities may have temporarily

halted proliferative signaling and allowed the keratinocytes

to respond to the environmental stress through the p38

and JNK pathways before progressing through the cell

cycle. Interestingly, basal levels of ERK activity were

induced up to 15 minutes post-UVA irradiation. This induc-

tion may have been due to the environmental change

experienced by the cells after the long irradiation period.

Despite this induction, ERK activity was consistently lower

in UVA-irradiated samples compared to controls. Basal

levels of activated p38 and JNK were not affected by the

transition. Total MAPK protein levels were not affected by

UVA irradiation.

Studies that have addressed the potential roles of

MAPKs in UVA signaling have been inconsistent. In the

NCTC 2544 human keratinocyte cell line, UVA-induced

ERK, JNK, and p38 activation through the generation of

oxidative stress [14,41]. UVA-mediated AP-1 DNA binding

and transcriptional activity through the Raf/ERK cascade,

with dose-dependent ERK activation considerably declining

up to 240 kJ/m2 UVA [14]. In mouse epidermal JB6

promotion-sensitive Cl 41 cells, UVA also activated ERK,

JNK, and p38 MAPKs at doses between 40 and 160 kJ/m2

[42,43]. Klotz et al. examined MAPK activation patterns

induced by singlet oxygen and UVA in human skin fibro-

blasts. With 300 kJ/m2 UVA, the authors reported a rapid and

transient induction of p38 and JNK but no effect on ERK

activation [12,55]. Although UVA irradiation did not appear to

mediate ERK activity, unirradiated controls for each time

point were not reported. Interestingly, intracellularly gener-

ated singlet oxygen activated p38 and JNK but appeared to

inhibit ERK activity [12]. Similarly, Zhang et al. observed the

UVA-induced activation of p38 and JNK but no activation of

ERK in the normal human lymphoblast cell line JY. At

60 minutes post-UVA irradiation, however, these cells

appeared to have decreased levels of ERK phosphorylation

in comparison to controls [56].

The pyridinyl imidazole SB202190 and the anthrapy-

razolone SP600125 were selected to implicate p38a/hand JNK, respectively, as components in the UVA-induced

signaling pathways leading to transcriptional activation of

AP-1–responsive genes. As potential modulators of AP-1

transactivation and c-Fos expression, these MAPK pharma-

cologic inhibitors could be clinically useful in skin cancer

chemopreventive strategies. As reported previously, UVA

irradiation of HaCaT cells significantly induced both AP-1

transactivation and c-fos transactivation [15]. Suppression

of JNK with SP600125 and of p38a/h with SB202190

resulted in the abrogation of both AP-1 and c-fos activities

in dose-dependent manners. Additionally, SB202190 and

SP600125 dose-dependently decreased UVA-induced lev-

els of c-Fos protein, the primary component in UVA-induced

AP-1 DNA binding in these cells [15]. The doses of

SB202190 and SP600125 that inhibited these downstream

targets were shown to specifically reduce their respective

UVA-induced MAPK activities.

The JNK inhibitor SP600125 consistently blocked c-fos

transactivation at a dose of 62 nM, whereas AP-1 trans-

activation consistently required doses of 125 nM. Although

c-Jun was a component of the UVA-induced AP-1 DNA-

binding complex in these cells, other Jun family members

that could compete for dimerization and AP-1 site binding

were also present [15]. We demonstrated that JNK inhibition

by SP600125 reduced not only c-Jun activity and expres-

sion, but also c-Fos expression. Preexisting c-Fos protein,

however, could still be activated by c-Fos–regulating kinase

[30,57]. Therefore, higher concentrations of inhibitors may



have been required to effectively inhibit AP-1 activation. In

contrast, JNK activates ternary complex factor proteins such

as Elk-1 and STAT proteins, which bind directly to transcrip-

tional control elements in the c-fos promoter [18,28,30].

Therefore, lower doses of SP600125 may be sufficient to

see an effect on this promoter construct.

SP600125 treatment of HaCaT cells with UVA irradia-

tion not only inhibited the phosphorylation of c-Jun on

serines 63 and 73 but also decreased total c-Jun protein

levels. The c-Jun promoter contains two AP-1–binding sites

that allow c-Jun protein to regulate its own transcription [54].

Therefore, inhibition of c-Jun phosphorylation may also

affect total c-Jun protein levels.

The doses of SP600125 that inhibited UVA-induced c-Jun

activity and expression were considerably lower than IC50

doses reported in primary human monocytes stimulated with

lipopolysaccharide and Jurkat T cells stimulated with phor-

bol-12-myristate-13-acetate plus anti-CD3 and anti-CD28

(5–10 AM) [46]. Additionally, although doses of SB202190

as high as 15 AM have been used in HaCaT cells to

determine the role of p38a/h in UV signaling, much lower

doses were capable of inhibiting UVA-induced p38 activity. In

addition to potentially diverse signaling pathways between

cell types, different stimuli can also alter the effects of an

inhibitor on cells. The combination of 250 kJ/m2 UVA with low

doses of either SB202190 or SP600125 resulted in dramatic

morphologic changes and increased cell death that were

visibly detectable approximately 4 hours post-UVA irradia-

tion (unpublished observations). We are currently exploring

the biologic consequences and significance of these syner-

gistic effects in HaCaT cells as well as in primary human

keratinocytes.

Although ERK activation did not play a role in the UVA-

induced AP-1 transactivation in these human keratino-

cytes, treatment with the MEK1/2 inhibitor PD98059

dose-dependently blocked both basal and UVA-induced

c-fos activation and c-Fos protein levels (data not shown).

The dose-dependent drop in c-fos activity observed in the

UVA-treated groups may have reflected the modulation of

basal c-fos activity. We suggest that ERK activity may

contribute to basal levels of c-fos expression in HaCaT cells.

Our increased elucidation and understanding of cellular

signaling pathways allow mechanistic approaches to chemo-

prevention and chemotherapy. The availability of potent,

selective inhibitors of specific signaling proteins may

allow researchers to further dissect cellular signaling path-

ways, to individually genetically tailor preventive strategies,

and to reduce adverse toxicologic responses elicited by the

screening of natural products with multiple mechanisms of

action [58,59].

Our results demonstrated for the first time that activa-

tion of the stress-activated kinases p38 and JNK played

important roles in UVA-mediated AP-1 transactivation and

c-fos transactivation, and that these kinases are involved in

UVA-induced c-Fos expression, a primary component of

the UVA-induced AP-1 DNA-binding complex [15].

Because deregulated expression of AP-1 has been shown

to play an important role in the neoplastic transformation

of epidermal cells in both cell culture and animal models

[23–26], targeted inhibition of the upstream kinases p38

and JNK with the pyridinyl imidazole SB202190 and the

anthrapyrazolone SP600125, respectively, may be effec-

tive chemopreventive strategies for UVA-induced nonme-

lanoma skin cancer.

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The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP1 Activation...

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