AMP-activated Protein Kinase Contributes to UV and H2O2-induced Apoptosis in Human Skin...

AMP-activated Protein Kinase Contributes to UV- andH2O2-induced Apoptosis in Human Skin Keratinocytes*

Received for publication, May 30, 2008, and in revised form, August 15, 2008 Published, JBC Papers in Press, August 20, 2008, DOI 10.1074/jbc.M804144200

Cong Cao‡§1, Shan Lu‡1, Rebecca Kivlin‡, Brittany Wallin‡, Elizabeth Card‡, Andrew Bagdasarian‡,Tyrone Tamakloe‡, Wen-ming Chu§, Kun-liang Guan¶, and Yinsheng Wan‡2

From the ‡Department of Biology, Providence College, Providence, Rhode Island 02918, the §Department of MolecularMicrobiology and Immunology, Brown University, Providence, Rhode Island 02903, and the ¶Department of Pharmacology,University of California, San Diego, California 90293

AMP-activated protein kinase or AMPK is an evolutionarilyconserved sensor of cellular energy status, activated by a varietyof cellular stresses that deplete ATP. However, the possibleinvolvement of AMPK in UV- and H2O2-induced oxidativestresses that lead to skin aging or skin cancer has not been fullystudied. We demonstrated for the first time that UV and H2O2induce AMPK activation (Thr172 phosphorylation) in culturedhuman skin keratinocytes. UV and H2O2 also phosphorylateLKB1, an upstream signal of AMPK, in an epidermal growthfactor receptor-dependent manner. Using compound C, a spe-cific inhibitor of AMPK and AMPK-specific small interferingRNA knockdown as well as AMPK activator, we found thatAMPK serves as a positive regulator for p38 and p53 (Ser15)phosphorylation induced by UV radiation and H2O2 treatment.We also observed that AMPK serves as a negative feedback sig-nal against UV-induced mTOR (mammalian target of rapamy-cin) activation in a TSC2-dependent manner. Inhibiting mTORand positively regulating p53 and p38 might contribute to thepro-apoptotic effect of AMPK on UV- or H2O2-treated cells.Furthermore, activation of AMPK also phosphorylates acetyl-CoA carboxylase or ACC, the pivotal enzyme of fatty acid syn-thesis, and PFK2, the key protein of glycolysis in UV-radiatedcells. Collectively, we conclude that AMPK contributes to UV-and H2O2-induced apoptosis via multiple mechanisms inhuman skin keratinocytes and AMPK plays important roles inUV-induced signal transduction ultimately leading to skin pho-toaging and even skin cancer.

Ultraviolet (UV) spectrum is divided into UVC (200–280nm), UVB (280–320 nm), and UVA (320–400 nm). UVB andUVA are of environmental significance and social concern,becauseUVC is filtered through the ozone layer. UV penetratesinto the papillary area of the dermis (�0.2 mm) and induces

DNA damages of residing keratinocytes and dendritic cells.They are perturbed both phenotypically and functionallyundergoing apoptosis upon UV radiation (1, 2). Previous stud-ies in human keratinocytes in vitro and in human skin in vivohave demonstrated that UV response comprises trans-activa-tion of cell surface growth factor receptors, such as EGFR,3 andtheir attendant downstream signal transduction machinerysuch as MAPK and phosphatidylinositol 3-kinase/AKT (2–6).Although MAPK, including JNK and p38, is responsible forUV-induced cell apoptosis and skin aging, other cellular signalssuch as AKT (also known as protein kinase B) serve as survivalsignals in skin cells to fight against UV-induced widespread celldeath (1–4). However, the possible involvement of other sig-nals, AMP-activated protein kinase or AMPK, for example, inUV-induced cell apoptosis leading to skin aging or cancer hasnot been fully studied.AMPK is a heterotrimeric serine-threonine kinase that

senses depletion of intracellular energy and responds by stim-ulating catabolic pathways that generate ATP (5, 6). Onemech-anism for sensing cellular energy levels involves allosteric acti-vation of AMPK. Under conditions in which cellular energydemands are increased (such as enhanced cellular activities orcellular stresses) or when fuel availability is decreased (becauseof a reduced rate of glucose uptake), intracellular ATP isreduced and AMP levels rise. AMP then allosterically activatesAMPK. In addition to allosteric activation, AMPK activity canbe regulated by a mechanism involving covalent modificationthrough the addition of a phosphate group by other moleculessuch as LKB1 and CaMK or calmodulin-dependent proteinkinase (5–10). A number of stimuli (11, 12) have been foundthat can induce AMPK activation. However, the question howUV radiation, the major cause of skin aging and skin cancer,activates AMPK remains unknown.It is well established that the key function of AMPK is to

regulate the energy balance within the cell. Once activated,AMPK phosphorylates downstream substrates, the overalleffect of which is to switch off ATP consuming pathways (e.g.* This work was supported, in whole or in part, by National Institutes of Health

Grant P20 RR016457 from the INBRE Program of the National Center forResearch Resources. This work was also supported by grants for biomedi-cal research from the Rhode Island Foundation and the Slater Center forEnvironmental Biotechnology. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 Both authors contributed equally to this paper.2 To whom correspondence should be addressed: Providence College, 549

River Ave., Providence, RI 02918-0001. Tel.: 401-865-2507; Fax: 401-865-1438; E-mail: [email protected].

3 The abbreviations used are: EGFR, epidermal growth factor receptor;MAPK, mitogen-activated protein kinase; AMPK, AMP-activated proteinkinase; mTOR, mammalian target of rapamycin; MEF, mouse embryonicfibroblasts; MTT, 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide; ROS, reactive oxygen species; FACS, fluorescence-acti-vated cell sorter; NAC, N-acetyl-L-cysteine; siRNA, small interfering RNA;PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonic acid; AICAR, 5-aminoimidazole-4-carbox-amide-1-�-4-ribofuranoside; S6K, S6 kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 43, pp. 28897–28908, October 24, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

OCTOBER 24, 2008 • VOLUME 283 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 28897

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fatty acid synthesis and cholesterol synthesis) and to switch oncatabolic pathways that generate ATP (e.g. fatty acid oxidationand glycolysis). Activation of AMPK requires phosphorylationofThr172 in the activation loop of� subunit by upstreamAMPKkinase (6, 13–15). AMPK activation also triggers a phosphoryl-ation cascade that regulates the activity of various downstreamtargets, including transcription factors, enzymes, and otherregulatory proteins, such asmTORpathways (16), p53 (17), andp38 (18). However, the possible role of AMPK in UV-inducedsignal transduction and skin aging or cancer remains to beelucidated.In this study, we found for the first time that UV and H2O2

induce AMPK activation and downstream ACC and PFK2phosphorylation in cultured human skin keratinocytes andreactive oxygen species (ROS)-mediated EGFR trans-activationis involved in LKB1/AMPK activation. Using AMPK inhibitor(Compound C), AMPK� siRNA knockdown as well as AMPKactivator AICAR, we found that AMPK is actively involved inUV- and H2O2-induced signal transduction and skin cell dam-age probably by positively regulating downstreamp38, p53 acti-vation, and inhibiting mTOR activation. Our study providesnew insights into understanding the cellular and molecularmechanisms involved in UV-induced skin cell damage leadingto skin aging and skin cancer.

EXPERIMENTAL PROCEDURES

UV Light Apparatus—As previously reported (19, 20), theUV-irradiation apparatus used in this study consisted of fourF36T12 EREVHO UV tubes. A Kodacel TA401/407 filter wasmounted 4 cm in front of the tubes to remove wavelengths�290 nm. Irradiation intensity was monitored using an IL443phototherapy radiometer and a SED240/UV/Wphotodetector.Before UV irradiation, cells were washed with 1 ml of phos-phate-buffered saline (PBS) and changed to fresh 0.5 ml of PBSfor each well. Cells were irradiated at the desired intensitywithout a plastic dish lid. After UV irradiation, cells werereturned to incubation in basal medium with treatments forvarious time points prior to harvest. Mouse skin dendriticcells (XS 106 cell line) were cultured in 10% fetal bovineserum in RPMI 1640 with granulocyte-macrophage colony-stimulating factor (Sigma).Chemicals and Reagents—PD153035, AG1478, SB203580,

andAMPK inhibitor (AMPKi, compoundC)were fromCalbio-chem (San Diego, CA). EGFR (1005) antibody, goat anti-rabbitIgG-horseradish peroxidase, and goat anti-mouse IgG-horse-radish peroxidase antibodywere purchased fromSantaCruzBio-technology (SantaCruz,CA).Monoclonalmouseanti-�-actinwasobtained from Sigma. Phospho-S6K (Thr389), phospho-4E-BP1(Ser65), total-S6K, phospho-EGFR (Tyr1068), phospho-EGFR(Tyr1045), phospho-mTOR (Ser2448), mTOR, phospho-AMPK(Thr172), phospho-p38 (Thr180/Tyr182), phospho-LKB1 (Ser428),p38 antibody, AMPK, LKB1, and AKT antibody were from CellSignaling Technology (Danvers, MA).Cell Culture—Spontaneously immortalized human keratino-

cytes (HaCaT cell line) (21) and human skin fibroblasts werecultured as previously reported (20, 22). EGFR wild type MEFs(mouse embryonic fibroblasts) and EGFR knock-out MEFs (4)were from Dr. Zhigang Dong; p53 wild type MEFs, p53 knock-

out MEFs, TSC2 wild type, and TSC2 knock-out MEFs werefrom Dr. Kun-liang Guan (8). Cells were maintained in a Dul-becco’s modified Eagle’s medium (Sigma) supplemented with10% fetal bovine serum (Invitrogen), penicillin/streptomycin(1:100, Sigma), and 4 mM L-glutamine (Sigma), in a CO2 incu-bator at 37 °C. ForWestern blot analysis, cells were reseeded in6-well plates at a density of 0.2 � 106 cells/ml with fresh com-plete culture medium.Western Blot Analysis—As reported previously (20, 22), cul-

tured cells with and without treatments were washed with coldPBS and harvested by scraping into 150 �l of RIPA buffer withprotease inhibitors. 20–40 �g of proteins were separated bySDS-PAGE and transferred onto polyvinylidene difluoridemembrane (Millipore, Bedford, MA). After blocking with 10%milk in Tris-buffered saline, membranes were incubated withspecific antibodies in a dilution buffer (2% bovine serum albu-min in Tris-buffered saline) overnight at 4 °C followed byhorseradish peroxidase-conjugated anti-rabbit or anti-mouseIgG at appropriate dilutions and incubated at room tempera-ture for 1 h. Antibody binding was detected using a enhancedchemiluminescence (ECL) detection system from GE Health-care following the manufacturer’s instructions and visualizedby fluorography with Hyperfilm.In Vitro Kinase Assay formTORActivity—The in vitro kinase

assay formTOR activity was performed as described previously(23). Briefly, HaCaT cells (2 � 106) were lysed in 200 �l of lysisbuffer containing 40 mM HEPES, pH 7.5, 120 mM NaCl, 0.3%CHAPS, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM

�-glycerophosphate, 1 mM Na3VO4, and protease inhibitormixture. Cell lysate (500 �g) was incubated with anti-mTORantibody (SantaCruz Biotechnology, SantaCruz, CA) and 30�lof Protein A/G-agarose beads at 4 °C for 3 h. Following theincubation, beads were washed 3 times with lysis buffer and 2times with kinase buffer containing 25 mM HEPES, pH 7.4, 50mMKCl, 20% glycerol, 10 mMMgCl2, 4 mMMnCl2, 1 mM dithi-othreitol, 1mM glycerophosphate, and 1mMNa3VO4. After thefinal wash, beads were assayed for kinase activity against puri-fied 4E-BP1 substrate by adding 4E-BP1 substrate (Santa CruzBiotechnology, Santa Cruz, CA) to the beads for 30 min at30 °C. The reactions were terminated by boiling in the presenceof 1� SDS sample buffer. The samples were subjected to SDS-PAGE, and phosphorylation of 4E-BP1 was detected by West-ern blotting using anti-phospho-4E-BP1 (Ser65) antibody.AMPK� RNA Interference Experiments—As described previ-

ously (22), siRNA for AMPK�1/�2 (sc-45312) was purchasedfrom Santa Cruz Biotechnology. HaCaT cells were cultured in acomplete medium that did not contain antibiotics for 4 days.50 � 10s cells were seeded in a 6-well plate 1 day prior to trans-fection and cultured to 60–70% confluence the following day.For RNA interference experiments, 6.25�l of LipofectamineTMLTX together with 2.5 �l of PLUSTM Reagent (Invitrogen) wasdiluted in 90 �l of Dulbecco’s modified Eagle’s medium for 5min at room temperature. Then, 8 �l of AMPK� siRNA wasmixed with Dulbecco’s modified Eagle’s medium containingLipofectamine together with PLUS reagent and incubated for30min at room temperature for complex formation. Finally, thecomplexwas added to thewells containing 2ml ofmediumwith

AMPK Activation in UV-induced Apoptosis

28898 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 43 • OCTOBER 24, 2008

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the final AMPK� siRNA concentra-tion of 100 nM. AMPK� proteinexpression was determined byWestern blot.Cell Viability Assay (MTT Dye

Assay)—Cell viability was measuredby the 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) method (20). Briefly,cells were collected and seeded in96-well plates at a density of 2� 105cells/cm2. Different seeding densi-ties were optimized at the beginningof the experiments (data notshown). After incubation for 24 h,cells were exposed to fresh mediumcontaining reagents at 37 °C. Afterincubation for up to 24 h, 20 �l ofMTT tetrazolium (Sigma) salt dis-solved in Hank’s balanced salt solu-tion at a concentration of 5 mg/mlwas added to each well and incu-bated in a CO2 incubator for 4 h.Finally, the medium was aspiratedfrom each well and 150 �l of di-methyl sulfoxide (Sigma) was addedto dissolve formazan crystals andthe absorbance of each well wasobtained using a Dynatech MR5000plate reader at a test wavelength of490 nmwith a reference wavelengthof 630 nm.Assessment of the Percentage of

Apoptotic Cells—To detect apopto-tic cells (20), cells were stained withDNA binding dye Hoechst 33342(Sigma). After the cells wereexposed to UV and the test com-pounds for the allotted time peri-ods, they were fixed with 4% form-aldehyde in PBS for 10 min at 4 °C,and then washed with PBS. To stainthe nuclei, cells were incubated for20 min with 20 �g/ml of Hoechst33342. After washing with PBS, thecells were observed under a fluores-cence microscope (Zeiss Axiophoto2, Carl Zeiss, Germany). Cellsexhibiting condensed chromatinand fragmented nuclei were scoredas apoptotic cells. A minimum of200 cells were scored from eachsample.Measurement of Keratinocytes

Mitochondrial Membrane Poten-tial—HaCaT cell mitochondrialmembrane potential (��m) wasassessed with fluorescent probe

FIGURE 1. UV and H2O2 induce AMPK activation in cultured skin cells. HaCaT cells were treated with differ-ent doses of UV (0, 5, 10, 15, and 25 mJ/cm2) and cultured for 60 min (A and B) or treated with UV (25 mJ/cm2)and cultured for 15, 30, 60, and 120 min (C and D). p-AMPK� (Thr172) and T-AMPK� were detected by Westernblot. AMPK activation was quantified. HaCaT cells were also treated with different doses of H2O2 (0, 50, 100, 150,and 250 �M) and cultured for 60 min (E and F) or treated with H2O2 (250 �M) and cultured for 0, 15, 30, 60, and120 min (G and H). p-AMPK� (Thr172) and T-AMPK� were detected by Western blot. AMPK activation wasquantified. HaCaT cells were treated with different doses of AICAR (0, 0.1, 0.5, 1.0, and 2.0 mM) and cultured for120 min (I and J) or treated with AICAR (2 mM) and cultured for 0, 15, 30, 60, and 120 min (K and L). p-AMPK�(Thr172) and T-AMPK� were detected by Western blot. AMPK activation was quantified. The expression ofAMPK� and AMPK� in HaCaT cells with the indicated treatments (control, control siRNA, AMPK� siRNA, trans-fection control, and EGFR siRNA) were detected by Western blot (M). HaCaT cells with or without AMPK� siRNAwere treated with UV (25 mJ/cm2) and H2O2 (250 �M) for 1 h, p-AMPK� (Thr172), T-AMPK�, and �-actin weredetected by Western blot (N). Cultured human skin fibroblasts (O) and mouse dendritic cells (XS 106 cell line) (P)were treated with UV (25 mJ/cm2) for the indicated time points, p-AMPK� (Thr172) and T-AMPK� were detectedby Western blot. *, p � 0.05 versus control group. Data are presented as the mean � S.E. for three independentexperiments.



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JC-1 (20). At 490 nm, cells with depolarizedmitochondria con-tained JC-1 predominantly in monomeric form and fluorescedgreen. Cells with polarized mitochondria predominantly con-tain JC-1 in aggregate form, and mitochondria fluoresce red–orange. HaCaT cells were incubated with 5 �M JC-1 (Invitro-gen) for 30 min at 37 °C, washed, and fluorescent images werevisualized by a Zeiss fluorescencemicroscopewith excitation at490 nm and emission at 520 nm (monomeric form for depolar-ized ��m) and 590 nm (aggregate form for polarized ��m).HaCaT cells with polarized mitochondria were seen with dis-tinct mitochondria fluorescing red–orange, and with depolar-ized mitochondria, cell cytoplasm and mitochondria appearedgreen. The aquired signal was analyzed with image analysissoftware (20). A minimum of six fields were selected and aver-age intensity for each region was quantified. The ratio of J-ag-gregate to JC-1 monomer intensity for each region was calcu-lated. A decrease in this ratio was interpreted as loss of ��m,whereas an increase in the ratiowas interpreted as gain in��m.ROSDetection—ROS generationwas detected by FACS anal-

ysis as described previously (24, 25). Briefly, cultured humanskin keratinocytes (HaCaT cells) were loaded with 1 �M fluo-rescent dye dihydrorhodamine 2 h before UV radiation, whichreacts with ROS in cells and results in a change of fluorescence.After being treated with UV with or without reagents for thedesired time points, keratinocytes were trypsinized, suspendedin ice-cold PBS, and fixed in 70% ethyl alcohol in �20 °C. Thechanges in fluorescence in drug-treated cells were quantified byFACS analysis. Induction of ROS generation was expressed inarbitrary units.Statistical Analysis—The values in the figures are expressed

as themean� S.E. The figures in this study were representativeof more than 3 different experiments. Statistical analysis of thedata between the control and treated groups was performed bya Student’s t test. Values of p � 0.05 were considered statisti-cally significant.

RESULTS

UV Radiation and H2O2 Induce AMPK Activation in Cul-tured Human Skin Keratinocytes—To investigate the role ofAMPK in UV signaling, we first tested whether UV or H2O2induces AMPK activation using cultured human skin keratino-cytes (HaCaT cells). The results showed that UV radiationinducesAMPK� phosphorylation in a dose (Fig. 1,A andB) andtime (Fig. 1, C and D)-dependent manner. Similarly, H2O2

induces AMPK� activation in a dose- and time-dependentmanner (Fig. 1, E–H). Furthermore, as expected, AMPK activa-tor 5-aminoimidazole-4-carboxamide-1-�-4-ribofuranoside(AICAR) also induces AMPK activation in a dose- (Fig. 1, I andJ) and time (Fig. 1, K and L)-dependent manner. AMPK�-spe-cific siRNA down-regulates AMPK� expression and largelyinhibits UV- and H2O2-induced AMPK activation (Fig. 1, Mand N). UV also induces AMPK activation in cultured humanskin fibroblasts (Fig. 1O) and dendritic cells (XS 106 cell line)(Fig. 1P).ROS-mediated EGFR Activation Is Involved in UV-induced

LKB1/AMPK Activation—The data above show that both UVand H2O2 induce AMPK activation (AMPK� phosphorylationat Thr172) in HaCaT cells. However, the cellular signalsinvolved in this AMPK activation are not fully studied. Previousstudies using human skin keratinocytes and dendritic cells haverevealed a important role of EGFR in UV-induced cellular sig-nals (3, 26), studies also show a critical role of LKB1 for AMPKactivation. As expected, in this study, we observed that bothUVandH2O2 induce EGFR activation in HaCaT cells (Fig. 2,A andB). Interestingly, UV radiation also induces LKB1 phosphoryl-ation in a time- and dose-dependent manner in HaCaT cells(Fig. 2, C and D). EGFR inhibitor PD 153035 and AG 1478inhibit UV-induced AMPK and LKB1 activation (Fig. 2E).EGFR ligand, EGF, also induces LKB1/AMPK activation, whichis blocked by PD 153035 (Fig. 2F). To further confirm the keyrole of EGFR in UV-induced AMPK activation, EGFR knock-out MEFs were used. As shown in Fig. 2G, UV induces LKB1/AMPK activation in wild type but not in EGFR knock-outMEFs. Furthermore, the induction ofAMPK is also inhibited bypretreatmentwith the antioxidantNACand pyrrolidine dithio-carbamate (Fig. 2H) in UV-treated HaCaT cells. AntioxidantNAC and EGFR inhibitor PD 153035 also reduce H2O2-in-duced LKB1/AMPK activation (Fig. 2I). As expected, UV- andH2O2-induced ROS production is inhibited by NAC pre-treat-ment (Fig. 2J). Collectively, our data suggest that ROS-medi-ated EGFR trans-activation is involved in UV-induced LKB1/AMPK activation.AMPK Is Involved in UV- and H2O2-induced p38 Activation—

Because previous studies have suggested that p38 MAPK is adownstream signal of AMPK upon various stimuli (18, 27, 28),and UV induces p38 activation (24), next we tested the possiblerole of AMPK in UV- and H2O2-induced p38 activation. As

FIGURE 2. ROS-mediated EGFR activation is involved in UV-induced LKB1/AMPK activation. HaCaT cells were pre-treated with EGFR inhibitor PD 153035(1 �M), followed by UV radiation (25 mJ/cm2) for different time points (0, 2, 5, 15, and 30 min), p-EGFR (Tyr1068), p-EGFR (Tyr1045), and T-EGFR were detected byWestern blot (A). HaCaT cells were also treated with H2O2 (250 �M) and cultured for 0, 5, 15, 30, and 60 min or treated with different doses of H2O2 (0, 50, 100,150, and 250 �M) and cultured for 5 min. p-EGFR (Tyr1068) and T-EGFR were detected by Western blot (B). HaCaT cells were treated with UV (25 mJ/cm2) andcultured for 30, 60, 120, 180, 240, and 360 min. p-AMPK� (Thr172), p-LKB1 (Ser428), T-LKB1, and T-AMPK� were detected by Western blot. AMPK phosphorylationwas quantified and normalized to T-AMPK (C). HaCaT cells were also treated with different doses of UV (0, 15, 25, 35, and 45 mJ/cm2) for 4 h, p-AMPK� (Thr172),p-LKB1 (Ser428), T-LKB1, and T-AMPK� were detected by Western blot. AMPK phosphorylation was quantified (D). HaCaT cells were pre-treated with EGFRinhibitor PD 153035 (1 �M) or AG1478 (1 �M) for 1 h, followed by UV radiation (25 mJ/cm2) for different time points (15, 30, and 60 min), p-AMPK� (Thr172),p-LKB1 (Ser428), and T-AMPK� were detected by Western blot (E). HaCaT cells were pre-treated with PD 153035 (1 �M) for 1 h, followed by EGF treatment (100ng/ml), p-AMPK� (Thr172), p-LKB1 (Ser428), and T-AMPK� were detected by Western blot (F). Wild type and EGFR knock-out MEFs were treated with UV (25mJ/cm2), p-AMPK� (Thr172), p-LKB1 (Ser428), T-EGFR, and T-AMPK� were detected by Western blot (G). HaCaT cells were pre-treated with the antioxidantpyrrolidine dithiocarbamate (PDTC, 100 �M) or N-acetyl-L-cysteine (NAC, 400 �M) for 1 h, followed by UV radiation (25 mJ/cm2) for different time points (15, 30,and 60 min), p-AMPK� (Thr172), p-LKB1 (Ser428), and T-AMPK� were detected by Western blot (H). HaCaT cells were pre-treated with the EGFR inhibitor PD153035 (1 �M) or antioxidant NAC (400 �M) for 1 h, followed by H2O2 (250 �M) for different time points (15, 30, and 60 min), p-AMPK� (Thr172), p-LKB1 (Ser428),and T-AMPK� were detected by Western blot (I). HaCaT cells were pre-treated with 400 �M NAC for 1 h, followed by UV (25 mJ/cm2) or H2O2 (250 �M) for 1 h,ROS production was detected by FACS as mentioned above (J). *, p � 0.05 versus untreated group. #, p � 0.05 versus same the time point of the UV- orH2O2-treated group. Data are presented as the mean � S.E. for three independent experiments.



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demonstrated in Fig. 3, A and B, compound C (AMPKi), anAMPK inhibitor, largely impairs the activation of p38 MAPKupon UV radiation, whereas SB203580, a p38 inhibitor, doesnot affect AMPK activation. Furthermore, AMPK also posi-tively regulates H2O2-induced p38 activation, because AMPKiinhibits H2O2-induced p38 activation, whereas AICAR, anAMPK activator, enhances it (Fig. 3, C and D). AICAR alonealso induces p38 activation, which is reversed by compound C(Fig. 3, E and F). To further confirm the role of AMPK in UV-

FIGURE 3. AMPK is involved in UV- and H2O2-induced p38 activation.HaCaT cells were pre-treated with AMPK inhibitor compound C (AMPKi, 10�M) or p38 inhibitor SB 203580 (10 �M) for 1 h, followed by UV radiation fordifferent time points (15, 30, and 60 min), p-AMPK� (Thr172), p-p38 (Thr180/Tyr182), and T-p38 were detected by Western blot (A), and p38 phosphoryla-tion was quantified (B). HaCaT cells were pre-treated with AMPK activatorAICAR (2 mM) or inhibitor compound C (AMPKi, 10 �M) for 1 h, followed byH2O2 (250 �M) for different time points (15, 30, and 60 min), p-AMPK� (Thr172),p-p38 (Thr180/Tyr182), and T-p38 were detected by Western blot (C), and p38phosphorylation was quantified (D). HaCaT cells were pre-treated with AMPKinhibitor compound C (AMPKi, 10 �M) for 1 h, followed by AMPK activatorAICAR (2 mM) for different time points (30, 60, 90, and 120 min), p-AMPK�(Thr172), p-p38 (Thr180/Tyr182), and T-p38 were detected by Western blot (Eand F). HaCaT cells with or without AMPK� siRNA or control siRNA weretreated with UV (25 mJ/cm2) and H2O2 (250 �M) for 1 h, p-p38 (Thr180/Tyr182)

and T-p38 were detected by Western blot (G). *, p � 0.05 versus the same timepoint as the UV-treated group. Data are presented as the mean � S.E. forthree independent experiments.

FIGURE 4. AMPK positively regulates UV-induced phosphorylation ofp53. HaCaT cells were pre-treated with EGFR inhibitor PD 153035 (1 �M), AG1478 (1 �M), or the antioxidant NAC (400 �M) for 1 h, followed by UV radiation(25 mJ/cm2) or H2O2 (250 �M) (A) for 2 h, p-p53 (Ser15) and T-p53 weredetected by Western blot. p53 phosphorylation was quantified (B). HaCaTcells were also pre-treated with AMPK inhibitor compound C (AMPKi, 10 �M)or p38 inhibitor SB 203580 (10 �M) for 1 h, followed by UV radiation for differ-ent time points (0.5, 1.0, and 2 h), p-p53 (Ser15) and T-p53 were detected byWestern blot. p53 phosphorylation was quantified (D). HaCaT cells with orwithout AMPK� siRNA were treated with UV radiation (25 mJ/cm2) or H2O2(250 �M) for 1 h, p-p53 and T-p53 were detected by Western blot (E). *, p �0.05 versus untreated group. #, p � 0.05 versus same time point as the UV-treated group. Data are presented as the mean � S.E. for three independentexperiments.



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FIGURE 5. UV and H2O2 induce phosphorylation of ACC and PFK2. HaCaT cells were treated with different doses of UV (5, 15 and 25 mJ/cm2) and culturedfor 120 min) (A) or treated with 25 mJ/cm2 of UV and cultured for the indicated time points (0, 30, 60, and 120 min) (C). p-ACC (Ser79) and �-actin were detectedby Western blot. ACC phosphorylation was quantified (B and D). HaCaT cells were also treated with different doses of H2O2 (50, 125, and 250 �M) and culturedfor 120 min (E) or treated with 250 �M H2O2 and cultured for the indicated time points (G). p-ACC (Ser79) and �-actin were detected by Western blot. ACCphosphorylation was quantified (F and H). HaCaT cells were pre-treated with AMPK activator AICAR (2 mM) or inhibitor compound C (AMPKi, 10 �M) for 1 h,followed by UV (25 mJ/cm2) (I and J) or H2O2 (250 �M) (K and L) for different time points (30, 60, and 120 min), p-ACC (Ser79) and �-actin were detected byWestern blot. HaCaT cells were also treated with UV and H2O2 for the indicated time points, p-PFK2 (Ser466) and �-actin were detected by Western blot (M).HaCaT cells were pre-treated with AMPK inhibitor compound C or AICAR for 1 h, followed by UV radiation, p-PFK2 and �-actin were detected by Western blot(N). HaCaT cells were treated with compound C for 1 h, followed by AMPK activator AICAR (2 mM) for different time points (30, 60, 90, and 120 min), p-PFK2(Ser466) and �-actin were detected by Western blot (O). *, p � 0.05 versus untreated group. **, p � 0.05 versus the same time point as the UV-treated group. Dataare presented as the mean � S.E. for three independent experiments.



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induced p38 activation, AMPK�siRNA was used. As demonstratedin Fig. 3G, activation of p38 inresponse to UV or H2O2 is inhibitedin AMPK� siRNA-treated HaCaTcells. Collectively, our data suggestthat AMPK is involved in UV- andH2O2-induced p38 activation.AMPK Positively Regulates UV-

and H2O2-induced Phosphorylationof p53—p53 tumor suppressorexerts anti-proliferative effects,including growth arrest, apoptosis,and cell senescence, in response toUV radiation (29). Its activationform (both phosphorylation andacetylation) is regulated by a num-ber of proteins such as ATM (30),Chk1/2 (30), M2M, p300 (31), andSIRT1 (15, 32). However, whetherAMPK also affects UV-induced p53activation is unknown. As shown inFig. 4, A and B, UV- and H2O2-in-duced phosphorylation of p53 isinhibited by EGFR inhibitors (PDand AG) and antioxidant NAC, sug-gesting that the upstream compo-nents of AMPK regulate p53 activa-tion. Furthermore, AMPK inhibitorcompound C (AMPKi) and p38inhibitor SB 203580 inhibit UV-in-duced phosphorylation p53 (Fig. 4,C and D), suggesting the positiveregulatory role of AMPK activationin UV-induced p53 phosphoryla-tion, which is consistent with recentstudies demonstrating that AMPKdirectly phosphorylates p53 at aspecific site (33). To further confirmthis notion, siRNA-mediatedAMPK� knockdown was used. Asshown in Fig. 4E, phosphorylationof p53 in AMPK� knockdown cellsis impaired, compared with controlsiRNA-treated cells. Collectively,our data suggest that AMPK posi-tively regulates UV- and H2O2-in-duced phosphorylation of p53.UV Radiation Induces ACC and

PFK2Phosphorylation in anAMPK-dependent Manner—AMPK wasoriginally perceived as a regulator ofcellular energy balance that acts in acell-autonomous matter. AMPKappears to play as important a rolein whole body energy balance as itdoes in cellular energy balance.Once it is activated, it helps to

FIGURE 6. AMPK negatively regulates UV- and H2O2-induced mTOR activation. HaCaT cells were pre-treated with AMPK inhibitor (10 �M) or AICAR (2 mM) for 1 h, followed by UV radiation (25 mJ/cm2) (A and B) orH2O2 (250 �M) (C and D) for 30, 60, and 90 min, p-S6K (Thr389), p-4E-BP1 (Ser65), and T-S6K were detected byWestern blot. S6K phosphorylation was quantified. Wild type (WT) or TSC2 knock-out MEFs were treated UVradiation (25 mJ/cm2) (E and F) or H2O2 (250 �M) (G and H) for 30, 60, 120, and 180 min, p-S6K (Thr389) and T-S6Kwere detected by Western blot. S6K phosphorylation was quantified. HaCaT cells with or without AMPK� siRNAwere treated with UV radiation (25 mJ/cm2) or H2O2 (250 �M), p-S6K (Thr389) and T-S6K were detected byWestern blot (I and J). S6K phosphorylation was quantified. HaCaT cells were pre-treated with AMPK activatorAICAR (2 mM) or inhibitor compound C (AMPKi, 10 �M) for 1 h, followed by 25 mJ/cm2 of UV radiation for theindicated time points, p-mTOR (Ser2448) and T-S6K were detected by Western blot (K). mTOR activity wasanalyzed by detecting 4E-BP1 phosphorylation at Ser65 (L). HaCaT cells with or without AMPK siRNA weretreated with 25 mJ/cm2 of UV radiation for 1 h, mTOR activity was analyzed by detecting 4E-BP1 phosphoryl-ation at Ser65 (M), cell lysates were used for Western blot (WB), p-AMPK (Thr172), p-LKB1 (Ser428), p-4E-BP1(Ser65), T-AMPK, and T-S6K were detected by Western blot (N). *, p � 0.05 versus untreated group. ***, p � 0.05versus same time point as the UV-treated group. Data are presented as the mean � S.E. for three independentexperiments.



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restore the energy state of the cell by acutely and chronicallyenhancing processes that generate ATP, such as fatty acid oxi-dation by phosphorylation and inhibiting acetyl-CoA carboxyl-ase or ACC (6). AMPK also phosphorylates and activates PFK2to restoreATPunder anaerobic conditions (34).Wenext inves-tigated whether UV radiation also activates those two keyenzymes.As indicated in Fig. 5,A–H, bothUVandH2O2 induceACC phosphorylation in a time- and dose-dependent manner.Furthermore, whereas AMPK inhibitor compound C inhibitsUV-induced ACC phosphorylation, AICAR, AMPK activator,enhances it (Fig. 5, I–L). UV and H2O2 also induce PFK2 phos-phorylation (Fig. 5M), which is largely impaired by AMPKinhibitor pretreatment, but is enhanced by AICAR pretreat-ment (Fig. 5N). AICAR alone also induces PKF phosphoryla-tion, which in blocked by the AMPK inhibitor (Fig. 5O). Theseresults suggest that AMPK activation might also be involved inmetabolic regulation in skin keratinocytes in response to UVradiation.

AMPK Negatively Regulates UV-inducedmTORActivationinaTSC2-dependent Manner—AMPK hasbeen shown to serve as a negativesignal against mTOR activation in anumber of cell models by phospho-rylating activation of TSC2 leadingto mTOR inhibition (5, 8). Our pre-vious studies have demonstratedthatUV activatesmTOR in culturedhuman skin keratinocytes.4 How-ever, the possible role of AMPK inUV-induced mTOR activation isnot fully elucidated. In this study, asdemonstrated in Fig. 6,A–D, AMPKinhibitor enhances both UV- andH2O2-induced S6K and 4E-BP1phosphorylation (indicators ofmTOR activation), whereas AICARinhibits them. Furthermore,UVandH2O2 induce S6K activation in wildtype but not in TSC2 knock-outMEFs where S6K is overactivated(Fig. 6,E–H). To further confirm thenegative effects of AMPKonUV-in-duced mTOR activation, AMPKsiRNA was used. As demonstratedin Fig. 6, I and J, AMPK� knock-down enhances UV- and H2O2-in-duced S6K phosphorylation. Fur-thermore, compound c alsoenhancesUV-inducedmTORphos-phorylation at Ser2448 (Fig. 6K) andmTOR activity (Fig. 6L), whereasAICAR inhibits them and AMPKsiRNA enhances UV-inducedmTORactivity (Fig. 6,M andN).AMPK Activation Contributes to

UV-induced Skin Cell Damage—Because published studies have

demonstrated the critical role of AMPK activation in cell deathor apoptosis (33), we next examined the functional results ofAMPK activation in UV-treated cells. As shown in Fig. 7, A–D,although AMPK inhibitor compound C and AMPK� specificknockdown inhibit UV-induced Bcl-XL degradation and mito-chondrial membrane potential loss, AICAR pre-treatmentenhances it. Furthermore, AMPK� knockdown inhibits UV-induced HaCaT cells death (Fig. 7E) and apoptosis (Fig. 7F).Next we studied downstream signals of AMPK mediating pro-cell death effects. p38 inhibitor SB 203580 (Fig. 7G) and p53knock-out (Fig. 7H) partly inhibit UV- andAICAR-induced celldeath. Collectively, our data suggest that activation of p38 andp53 as well as inhibiting mTOR, which lies downstream ofAMPK, may contribute to AMPK-induced pro-apoptoticeffects in response to UV radiation.

4 C. Cao, S. Lu, R. Kivlin, B. Wallin, E. Card, A. Bagdasarian, T. Tamakloe, W.-M.Chu, K.-l. Guan, and Y. Wan, unpublished data.

FIGURE 7. AMPK activation contributes to UV-induced skin cell death. HaCaT cells were pre-treated withmTOR inhibitor rapamycin (Rapa, 1 �M), AMPKi (10 �M), or AICAR (2 mM) for 1 h, followed by UV radiation (25mJ/cm2) (A and B) for 24 h, Bcl-XL and �-actin were detected by Western blot. HaCaT cells with or without AMPKsiRNA were treated with AMPK inhibitor (AMPKi), AICAR (2 mM) for 1 h, followed by UV radiation (25 mJ/cm2) for2 h (C and D), and mitochondrial membrane potential of cells were detected by the JC-1 fluorescence dye.HaCaT cells with or without AMPK siRNA were treated with UV radiation (25 mJ/cm2) or AICAR (2 mM), and cellviability was detected by MTT assay after 24 h (E), cell apoptosis was detected by Hoechst 33342 assay after 18 h(F). HaCaT cells were pretreated with p38 inhibitor (SB 203580, 10 �M) for 1 h, followed by UV radiation (25mJ/cm2) or AICAR (2 mM), cell viability was detected by MTT assay after 24 h (G). Wild type and p53 knock-outMEFs were treated with UV radiation (25 mJ/cm2) or AICAR (2 mM), cell viability was detected by MTT assay after24 h (H). *, p � 0.05 versus untreated group. #, p � 0.05 versus the same time point of UV or AICAR-treated group.Data are presented as the mean � S.E. for three independent experiments. For the fluorescence experiment, aminimum of six random fields and 200 cells per group were selected and average fluorescence intensity foreach group was quantified. Magnification: C, 1:400. For the apoptosis (Hoechst assay) (F) experiment, a mini-mum of 10 random fields and 500 cells were counted for the apoptotic death rate.



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DISCUSSION

As well as the numerous metabolic targets of AMPK forwhich it is best known, AMPK has many other downstreameffectors. Recent studies have revealed that AMPK inhibits cellgrowth and proliferation, and also positively regulates apopto-sis (33). These findings are not surprising, given that cellgrowth, DNA replication, and mitosis are all major consumersofATP, and also that the upstream regulator ofAMPK, LKB1, isknown to be a tumor suppressor. In this study, we discoveredthat UV and H2O2 induce AMPK activation in cultured skincells (Fig. 1). ROS-mediated EGFR trans-activation is involvedin UV-induced LKB1/AMPK activation (Fig. 2). We alsoobserved that AMPK activation contributes to UV- and H2O2-induced skin cell damage (Fig. 7) probably by positively regu-lating p38 (Fig. 3) and p53 phosphorylation (Fig. 4) and inhib-iting the mTOR pathway (Fig. 6). Collectively, we propose cellsignaling pathways involved in UV-induced AMPK activation,as depicted in Fig. 8.Although we found that UV-induced AMPK activation con-

sistently occurs in three different cell types (Fig. 1), our resultsseem inconsistent with what are mentioned in a recent study(35), which showed that UV radiation reduces AMPK activity(Thr172 phosphorylation) via down-regulation of LKB1 expres-sion. This may be due to the difference of tested time points

between previous work and ourstudy. Instead of time course (15, 30,60, 120, 180, 240, and 480 min as wetested in this study) (Figs. 1, C andD, and 2C), a previous studyreported the data collected at only4-h time points (35). We show herethat AMPK phosphorylation beginsat 0.5–1 h and lasts for at least 4–6 hafter 25 mJ/cm2 of UV radiation(Fig. 2C). Also we are unable to seeLKB1 expression change even at4–6 h after UV radiation (Fig. 2C).Instead, we see clearly LKB1 phos-phorylation after UV radiation atearlier time points in HaCaT cells(Fig. 2, C–E), and this LKB1 phos-phorylation lasts for about 4 h (Fig.2, C and D). Thus, LKB1 phospho-rylation might serve as a upstreamactivator of AMPKphosphorylationinduced by UV. We propose thatthis LKB1/AMPK phosphorylationis mediated by UV-induced ROSproduction, because ROS is knownas a strong AMPK activator asshown in a number of studies (36–38), and more importantly, in ourstudies, antioxidants such as NACand pyrrolidine dithiocarbamateinhibit UV-induced AMPK phos-phorylation (Fig. 2H). Anotherexplanation is that the dose of UVused in our study is slightly different

from a previous study (35) where as high as 37.5 mJ/cm2 of UVwas used, which might cause some nonspecific effects. Interest-ingly, in our study, at 4 h, UV-induced AMPK phosphorylation ismost pronounced at 25mJ/cm2 (Fig. 2D). However, even at a veryhigh dose of UV (35 and 45 mJ/cm2), we can still see enhancedAMPK/LKB1 phosphorylation (rather than decrease) (Fig. 2D).LKB1 phosphorylates and activates the catalytic�-subunit of

AMPK at its T-loop residue Thr172 in a cell-free system (7, 39).AMPK is minimally activated in cells that lack or havedecreased LKB1 expression and LKB1 forms a heterotrimericcomplex, with regulatory proteins termed STRAD and MO25,which are required for its activation and cytosolic targeting(40). Thus, LKB1 plays a crucial role in activatingAMPK. In thisstudy, we found that UV (Fig. 2, C and F) and H2O2 (Fig. 2G)induce LKB1 phosphorylation. However, because of limitationof experimental materials, we can only presume that LKB1 isupstream for AMPK activation upon UV and ROS stimulation.Further experiments using LKB1 deficiency cells are necessaryto confirm our hypothesis. Furthermore, the question whetherLKB1 activation plays other important roles in UV-inducedskin cell damage remains to be further addressed.p38 is involved in inflammation, cell growth and differentia-

tion, cell cycle, and cell death (41, 42). Recent studies have indi-cated that AMPK serves as an upstream signal for p38 activa-

FIGURE 8. The proposed signal pathways involved in AMPK activation in response to UV radiation.A, ROS-mediated EGFR trans-activation is involved in UV-induced LKB1/AMPK activation. B, AMPK activationnegatively regulates UV-induced mTOR activation in a TSC2-dependent manner. C, AMPK activation positivelyregulates UV-induced p38 and p53 phosphorylation. D, AMPK mediates ACC and PFK2 phosphorylation inresponse to UV radiation.



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tion by increasing recruitment of p38MAPK to TAB1. And yet,other studies have yielded different conclusions (27, 28, 43).Our studies using AMPK inhibitor and AMPK� siRNA knock-down have demonstrated that inhibition of AMPK impairs p38activation in response to UV or H2O2 (Fig. 3, A–D and G). Weconclude that AMPK may serve as a positive regulator for p38activation upon UV radiation, which might contribute to thepro-apoptotic effect of AMPK on UV-treated cells (Fig. 7G).AMPK activation causes a G1/S phase cell cycle arrest in dif-

ferent cell lines, and this is associated with accumulation oftumor suppressor p53 and cyclin-dependent kinase inhibitorsp21 and p27, which act downstream of p53 (17, 44–46). Thisprocess involves phosphorylation of p53 at Ser15 (17, 44–46),although it has not been shown that there is a direct phos-phorylation. In this study, as demonstrated in Fig. 4, AMPKactivation serves as a positive regulator for UV-induced p53phosphorylation (Ser15). However, as seen in Fig. 4, C–E,UV- and H2O2-induced p53 phosphorylation is reduced (butnot largely impaired nor totally blocked) as a result of AMPKinhibition, which suggest that UV-induced p53 phosphoryl-ation is not solely dependent on AMPK activation, andAMPK may be one of many molecules that phosphorylatep53 upon UV radiation. We propose that this positive regula-tory effect of AMPK on p53 phosphorylation might contributeto its pro-apoptotic effect (Fig. 7H).AMPK activation may also inhibit cell growth and survival

because of its general effects on biosynthesis, including its abil-ity to inhibit fatty acid synthesis and the mTOR pathway (8, 47,48). AKT/PKBphosphorylates TSC2 at sites that are believed toinhibit its Rheb-GAP activity, whereas AMPK is thought tohave the opposite effect. Our previous studies have indicatedthat UV radiation induces mTOR activation in skin keratino-cytes in an AKT-dependentmanner.4 By using AMPK site-spe-cific mutation plasmids (8, 47, 48), our previous work also indi-cated that AMPK activates TSC2 by phosphorylation at Ser1345in response to UV radiation, which ultimately inhibits mTORactivation.4 This site-specific phosphorylation and activation ofTSC2 explains the inhibitory role of AMPK in UV-inducedmTOR activation.4 In this study, using AMPK inhibitor andspecific siRNA knockdown as well as AMPK activator AICAR-and TSC2-deficient cells, we found that AMPK serves as a neg-ative feedback signal against UV-induced mTOR activation(Fig. 6), whichmay also be involved in the pro-apoptotic effectsof AMPK (Fig. 7A).AMPK stimulates catabolic pathways that generate ATP,

including the fatty acid oxidation pathway. Most fatty acids areoxidized in mitochondria, and their entry into the organelle isblocked by malonyl-CoA, which inhibits carnitine palmitoyl-transferase-1, an enzyme required for the transport of fattyacids across the inner mitochondrial membrane. Malonyl-CoAis produced by one of the target enzymes for AMPK, acetyl-CoA carboxylase, which exists as two isoforms, ACC1 (�) andACC2 (�) (5). Both are phosphorylated and inactivated byAMPK. In this study, we also discovered that UV and H2O2induceACCphosphorylation and inactivation in anAMPK-de-pendent manner (Fig. 5), which might be responsible forreduced fat accumulation or adipogenesis (49). Furthermore,we also found that PFK2, the key protein of glycolysis is also

phosphorylated upon UV radiation in an AMPK-dependentmanner (Fig. 5). Based on these observations, together with thecritical role of AMPK in energy balancing and metabolism, wepropose that the metabolic systemmight be impaired after UVradiation in human skin cells, which might be another criticalmechanism responsible for skin cell damage. The fat accumu-lation and energy storage in human skin cells might also beimpaired (as the possible consequence of AMPK-dependentACC phosphorylation in keratinocytes). Thus, this study pro-vides novel insights into understanding the cellular mecha-nisms involved in UV-induced skin aging or skin cancer.

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VOLUME 281 (2006) PAGES 26865–26875DOI 10.1074/jbc.A109.513304

Toll-like receptor-2 is essential for the development ofpalmitate-induced insulin resistance in myotubes.Joseph J. Senn

PAGES 26868 and 26869:

After careful review of the files, Dr. Senn agrees that there was anerror in the MyD88 massWestern blot in Fig. 2A. It appears that it is infact the same scan as that used for the p38mass blot in Fig. 1B. This errorwas due to the similarity in molecular weight of these two molecules,which led to the inadvertent placement of the same scan when compos-ing the figures. The corrected Fig. 2 is shown below.

VOLUME 283 (2008) PAGES 28897–28908DOI 10.1074/jbc.A109.804144

AMP-activated protein kinase contributes to UV- andH2O2-induced apoptosis in human skin keratinocytes.Cong Cao, Shan Lu, Rebecca Kivlin, Brittany Wallin, Elizabeth Card,Andrew Bagdasarian, Tyrone Tamakloe, Wen-ming Chu, Kun-liang Guan,and Yinsheng Wan

This article has been withdrawn at the request of the authors.Reason: To demonstrate that the activation of AMPK is mediated by

EGFR activation, the authors included data in Fig. 2 (A, G, and H) thathad been used in a previous publication. After publication of the abovearticle, the authors weremade aware of this and are thereforewithdraw-ing this paper. Despite these errors, the authors stand by the reproduc-ibility of the experimental data and the conclusion.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 19, p. 14842, May 7, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

14842 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 19 • MAY 7, 2010

ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.org

We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the originalarticle. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carrynotice of these corrections as prominently as they carried the original abstracts.

Bagdasarian, Tyrone Tamakloe, Wen-ming Chu, Kun-liang Guan and Yinsheng WanCong Cao, Shan Lu, Rebecca Kivlin, Brittany Wallin, Elizabeth Card, Andrew

in Human Skin Keratinocytes-induced Apoptosis2O2AMP-activated Protein Kinase Contributes to UV- and H

doi: 10.1074/jbc.M804144200 originally published online August 20, 20082008, 283:28897-28908.J. Biol. Chem.

10.1074/jbc.M804144200Access the most updated version of this article at doi:

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AMP-activated Protein Kinase Contributes to UV and H2O2-induced Apoptosis in Human Skin...

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