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• W2002153971 abstract "AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H2O2-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H2O2 induce AMPK activation (Thr172 phosphorylation) in cultured human skin keratinocytes. UV and H2O2 also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal growth factor receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK 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 signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC2-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-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 synthesis, and PFK2, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV- and H2O2-induced apoptosis via multiple mechanisms in human skin keratinocytes and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer. AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H2O2-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H2O2 induce AMPK activation (Thr172 phosphorylation) in cultured human skin keratinocytes. UV and H2O2 also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal growth factor receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK 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 signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC2-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-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 synthesis, and PFK2, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV- and H2O2-induced apoptosis via multiple mechanisms in human skin keratinocytes and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer. Ultraviolet (UV) spectrum is divided into UVC (200–280 nm), UVB (280–320 nm), and UVA (320–400 nm). UVB and UVA are of environmental significance and social concern, because UVC is filtered through the ozone layer. UV penetrates into 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 functionally undergoing apoptosis upon UV radiation (1Wang S. El-Deiry W.S. Oncogene. 2003; 22: 8628-8633Crossref PubMed Scopus (687) Google Scholar, 2Wan Y.S. Wang Z.Q. Shao Y. Voorhees J.J. Fisher G.J. Int. J. Oncol. 2001; 18: 461-466PubMed Google Scholar). Previous studies in human keratinocytes in vitro and in human skin in vivo have demonstrated that UV response comprises trans-activation of cell surface growth factor receptors, such as EGFR, 3The abbreviations used are: EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; MEF, mouse embryonic fibroblasts; MTT, 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; FACS, fluorescence-activated cell sorter; NAC, N-acetyl-l-cysteine; siRNA, small interfering RNA; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AICAR, 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside; S6K, S6 kinase. 3The abbreviations used are: EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; MEF, mouse embryonic fibroblasts; MTT, 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; FACS, fluorescence-activated cell sorter; NAC, N-acetyl-l-cysteine; siRNA, small interfering RNA; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AICAR, 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside; S6K, S6 kinase. and their attendant downstream signal transduction machinery such as MAPK and phosphatidylinositol 3-kinase/AKT (2Wan Y.S. Wang Z.Q. Shao Y. Voorhees J.J. Fisher G.J. Int. J. Oncol. 2001; 18: 461-466PubMed Google Scholar, 3Xu Y. Voorhees J.J. Fisher G.J. Am. J. Pathol. 2006; 169: 823-830Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4Li Y. Bi Z. Yan B. Wan Y. Int. J. Mol. Med. 2006; 18: 713-719PubMed Google Scholar, 5Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 6Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2293) Google Scholar). Although MAPK, including JNK and p38, is responsible for UV-induced cell apoptosis and skin aging, other cellular signals such as AKT (also known as protein kinase B) serve as survival signals in skin cells to fight against UV-induced widespread cell death (1Wang S. El-Deiry W.S. Oncogene. 2003; 22: 8628-8633Crossref PubMed Scopus (687) Google Scholar, 2Wan Y.S. Wang Z.Q. Shao Y. Voorhees J.J. Fisher G.J. Int. J. Oncol. 2001; 18: 461-466PubMed Google Scholar, 3Xu Y. Voorhees J.J. Fisher G.J. Am. J. Pathol. 2006; 169: 823-830Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4Li Y. Bi Z. Yan B. Wan Y. Int. J. Mol. Med. 2006; 18: 713-719PubMed Google Scholar). However, the possible involvement of other signals, AMP-activated protein kinase or AMPK, for example, in UV-induced cell apoptosis leading to skin aging or cancer has not been fully studied.AMPK is a heterotrimeric serine-threonine kinase that senses depletion of intracellular energy and responds by stimulating catabolic pathways that generate ATP (5Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 6Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2293) Google Scholar). One mechanism for sensing cellular energy levels involves allosteric activation of AMPK. Under conditions in which cellular energy demands are increased (such as enhanced cellular activities or cellular stresses) or when fuel availability is decreased (because of a reduced rate of glucose uptake), intracellular ATP is reduced and AMP levels rise. AMP then allosterically activates AMPK. In addition to allosteric activation, AMPK activity can be regulated by a mechanism involving covalent modification through the addition of a phosphate group by other molecules such as LKB1 and CaMK or calmodulin-dependent protein kinase (5Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 6Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2293) Google Scholar, 7Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1408) Google Scholar, 8Inoki K. Ouyang H. Zhu T. Lindvall C. Wang Y. Zhang X. Yang Q. Bennett C. Harada Y. Stankunas K. Wang C.Y. He X. MacDougald O.A. You M. Williams B.O. Guan K.L. Cell. 2006; 126: 955-968Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 9Jaswal J.S. Gandhi M. Finegan B.A. Dyck J.R. Clanachan A.S. Am. J. Physiol. 2007; 292: H1978-H1985Crossref PubMed Scopus (34) Google Scholar, 10Park Y. Lee S.W. Sung Y.C. J. Immunol. 2002; 168: 5-8Crossref PubMed Scopus (89) Google Scholar). A number of stimuli (11Yoon H. Oh Y.T. Lee J.Y. Choi J.H. Lee J.H. Baik H.H. Kim S.S. Choe W. Yoon K.S. Ha J. Kang I. Biochem. Biophys. Res. Commun. 2008; 371: 495-500Crossref PubMed Scopus (32) Google Scholar, 12Yamauchi M. Kambe F. Cao X. Lu X. Kozaki Y. Oiso Y. Seo H. Mol. Endocrinol. 2008; 22: 893-903Crossref PubMed Scopus (56) Google Scholar) have been found that can induce AMPK activation. However, the question how UV 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 overall effect of which is to switch off ATP consuming pathways (e.g. fatty acid synthesis and cholesterol synthesis) and to switch on catabolic pathways that generate ATP (e.g. fatty acid oxidation and glycolysis). Activation of AMPK requires phosphorylation of Thr172 in the activation loop of α subunit by upstream AMPK kinase (6Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2293) Google Scholar, 13Arad M. Seidman C.E. Seidman J.G. Circ. Res. 2007; 100: 474-488Crossref PubMed Scopus (272) Google Scholar, 14Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1039) Google Scholar, 15Ruderman N.B. Keller C. Richard A.M. Saha A.K. Luo Z. Xiang X. Giralt M. Ritov V.B. Menshikova E.V. Kelley D.E. Hidalgo J. Pedersen B.K. Kelly M. Diabetes. 2006; 55: S48-S54Crossref PubMed Scopus (142) Google Scholar). AMPK activation also triggers a phosphorylation cascade that regulates the activity of various downstream targets, including transcription factors, enzymes, and other regulatory proteins, such as mTOR pathways (16Inoki K. Zhu T. Guan K.L. Cell. 2003; 115: 577-590Abstract Full Text Full Text PDF PubMed Scopus (2943) Google Scholar), p53 (17Jones R.G. Plas D.R. Kubek S. Buzzai M. Mu J. Xu Y. Birnbaum M.J. Thompson C.B. Mol. Cell. 2005; 18: 283-293Abstract Full Text Full Text PDF PubMed Scopus (1277) Google Scholar), and p38 (18Yoon M.J. Lee G.Y. Chung J.J. Ahn Y.H. Hong S.H. Kim J.B. Diabetes. 2006; 55: 2562-2570Crossref PubMed Scopus (424) Google Scholar). However, the possible role of AMPK in UV-induced signal transduction and skin aging or cancer remains to be elucidated.In this study, we found for the first time that UV and H2O2 induce AMPK activation and downstream ACC and PFK2 phosphorylation in cultured human skin keratinocytes and reactive oxygen species (ROS)-mediated EGFR trans-activation is involved in LKB1/AMPK activation. Using AMPK inhibitor (Compound C), AMPKα siRNA knockdown as well as AMPK activator AICAR, we found that AMPK is actively involved in UV- and H2O2-induced signal transduction and skin cell damage probably by positively regulating downstream p38, p53 activation, and inhibiting mTOR activation. Our study provides new insights into understanding the cellular and molecular mechanisms involved in UV-induced skin cell damage leading to skin aging and skin cancer.EXPERIMENTAL PROCEDURESUV Light Apparatus—As previously reported (19Fisher G.J. Talwar H.S. Lin J. Lin P. McPhillips F. Wang Z. Li X. Wan Y. Kang S. Voorhees J.J. J. Clin. Investig. 1998; 101: 1432-1440Crossref PubMed Scopus (323) Google Scholar, 20Cao C. Healey S. Amaral A. Lee-Couture A. Wan S. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2007; 212: 252-263Crossref PubMed Scopus (41) Google Scholar), the UV-irradiation apparatus used in this study consisted of four F36T12 EREVHO UV tubes. A Kodacel TA401/407 filter was mounted 4 cm in front of the tubes to remove wavelengths <290 nm. Irradiation intensity was monitored using an IL443 phototherapy radiometer and a SED240/UV/W photodetector. Before UV irradiation, cells were washed with 1 ml of phosphate-buffered saline (PBS) and changed to fresh 0.5 ml of PBS for each well. Cells were irradiated at the desired intensity without a plastic dish lid. After UV irradiation, cells were returned to incubation in basal medium with treatments for various time points prior to harvest. Mouse skin dendritic cells (XS 106 cell line) were cultured in 10% fetal bovine serum in RPMI 1640 with granulocyte-macrophage colony-stimulating factor (Sigma).Chemicals and Reagents—PD153035, AG1478, SB203580, and AMPK inhibitor (AMPKi, compound C) were from Calbiochem (San Diego, CA). EGFR (1005) antibody, goat anti-rabbit IgG-horseradish peroxidase, and goat anti-mouse IgG-horseradish peroxidase antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal mouse anti-β-actin was obtained 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 Cell Signaling Technology (Danvers, MA).Cell Culture—Spontaneously immortalized human keratinocytes (HaCaT cell line) (21Cao C. Wan S. Jiang Q. Amaral A. Lu S. Hu G. Bi Z. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2008; 215: 506-516Crossref PubMed Scopus (78) Google Scholar) and human skin fibroblasts were cultured as previously reported (20Cao C. Healey S. Amaral A. Lee-Couture A. Wan S. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2007; 212: 252-263Crossref PubMed Scopus (41) Google Scholar, 22Cao C. Sun Y. Healey S. Bi Z. Hu G. Wan S. Kouttab N. Chu W. Wan Y. Biochem. J. 2006; 400: 225-234Crossref PubMed Scopus (114) Google Scholar). EGFR wild type MEFs (mouse embryonic fibroblasts) and EGFR knock-out MEFs (4Li Y. Bi Z. Yan B. Wan Y. Int. J. Mol. Med. 2006; 18: 713-719PubMed Google Scholar) were from Dr. Zhigang Dong; p53 wild type MEFs, p53 knock-out MEFs, TSC2 wild type, and TSC2 knock-out MEFs were from Dr. Kun-liang Guan (8Inoki K. Ouyang H. Zhu T. Lindvall C. Wang Y. Zhang X. Yang Q. Bennett C. Harada Y. Stankunas K. Wang C.Y. He X. MacDougald O.A. You M. Williams B.O. Guan K.L. Cell. 2006; 126: 955-968Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). Cells were maintained in a Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Invitrogen), penicillin/streptomycin (1:100, Sigma), and 4 mm l-glutamine (Sigma), in a CO2 incubator at 37 °C. For Western blot analysis, cells were reseeded in 6-well plates at a density of 0.2 × 106 cells/ml with fresh complete culture medium.Western Blot Analysis—As reported previously (20Cao C. Healey S. Amaral A. Lee-Couture A. Wan S. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2007; 212: 252-263Crossref PubMed Scopus (41) Google Scholar, 22Cao C. Sun Y. Healey S. Bi Z. Hu G. Wan S. Kouttab N. Chu W. Wan Y. Biochem. J. 2006; 400: 225-234Crossref PubMed Scopus (114) Google Scholar), cultured cells with and without treatments were washed with cold PBS and harvested by scraping into 150 μl of RIPA buffer with protease inhibitors. 20–40 μg of proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking with 10% milk in Tris-buffered saline, membranes were incubated with specific antibodies in a dilution buffer (2% bovine serum albumin in Tris-buffered saline) overnight at 4 °C followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG at appropriate dilutions and incubated at room temperature for 1 h. Antibody binding was detected using a enhanced chemiluminescence (ECL) detection system from GE Healthcare following the manufacturer's instructions and visualized by fluorography with Hyperfilm.In Vitro Kinase Assay for mTOR Activity—The in vitro kinase assay for mTOR activity was performed as described previously (23Bai X. Ma D. Liu A. Shen X. Wang Q.J. Liu Y. Jiang Y. Science. 2007; 318: 977-980Crossref PubMed Scopus (306) Google Scholar). Briefly, HaCaT cells (2 × 106) were lysed in 200 μl of lysis buffer containing 40 mm HEPES, pH 7.5, 120 mm NaCl, 0.3% CHAPS, 1 mm EDTA, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, and protease inhibitor mixture. Cell lysate (500 μg) was incubated with anti-mTOR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 30 μl of Protein A/G-agarose beads at 4 °C for 3 h. Following the incubation, beads were washed 3 times with lysis buffer and 2 times with kinase buffer containing 25 mm HEPES, pH 7.4, 50 mm KCl, 20% glycerol, 10 mm MgCl2, 4 mm MnCl2, 1 mm dithiothreitol, 1 mm glycerophosphate, and 1 mm Na3VO4. After the final wash, beads were assayed for kinase activity against purified 4E-BP1 substrate by adding 4E-BP1 substrate (Santa Cruz Biotechnology, Santa Cruz, CA) to the beads for 30 min at 30 °C. The reactions were terminated by boiling in the presence of 1× SDS sample buffer. The samples were subjected to SDS-PAGE, and phosphorylation of 4E-BP1 was detected by Western blotting using anti-phospho-4E-BP1 (Ser65) antibody.AMPKα RNA Interference Experiments—As described previously (22Cao C. Sun Y. Healey S. Bi Z. Hu G. Wan S. Kouttab N. Chu W. Wan Y. Biochem. J. 2006; 400: 225-234Crossref PubMed Scopus (114) Google Scholar), siRNA for AMPKα1/α2 (sc-45312) was purchased from Santa Cruz Biotechnology. HaCaT cells were cultured in a complete medium that did not contain antibiotics for 4 days. 50 × 10s cells were seeded in a 6-well plate 1 day prior to transfection and cultured to 60–70% confluence the following day. For RNA interference experiments, 6.25 μl of Lipofectamine™ LTX together with 2.5 μl of PLUS™ Reagent (Invitrogen) was diluted in 90 μl of Dulbecco's modified Eagle's medium for 5 min at room temperature. Then, 8 μl of AMPKα siRNA was mixed with Dulbecco's modified Eagle's medium containing Lipofectamine together with PLUS reagent and incubated for 30 min at room temperature for complex formation. Finally, the complex was added to the wells containing 2 ml of medium with the final AMPKα siRNA concentration of 100 nm. AMPKα protein expression was determined by Western blot.Cell Viability Assay (MTT Dye Assay)—Cell viability was measured by the 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (20Cao C. Healey S. Amaral A. Lee-Couture A. Wan S. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2007; 212: 252-263Crossref PubMed Scopus (41) Google Scholar). Briefly, cells were collected and seeded in 96-well plates at a density of 2 × 105 cells/cm2. Different seeding densities were optimized at the beginning of the experiments (data not shown). After incubation for 24 h, cells were exposed to fresh medium containing reagents at 37 °C. After incubation for up to 24 h, 20 μl of MTT tetrazolium (Sigma) salt dissolved in Hank's balanced salt solution at a concentration of 5 mg/ml was added to each well and incubated in a CO2 incubator for 4 h. Finally, the medium was aspirated from each well and 150 μl of dimethyl sulfoxide (Sigma) was added to dissolve formazan crystals and the absorbance of each well was obtained using a Dynatech MR5000 plate reader at a test wavelength of 490 nm with a reference wavelength of 630 nm.Assessment of the Percentage of Apoptotic Cells—To detect apoptotic cells (20Cao C. Healey S. Amaral A. Lee-Couture A. Wan S. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2007; 212: 252-263Crossref PubMed Scopus (41) Google Scholar), cells were stained with DNA binding dye Hoechst 33342 (Sigma). After the cells were exposed to UV and the test compounds for the allotted time periods, they were fixed with 4% formaldehyde in PBS for 10 min at 4 °C, and then washed with PBS. To stain the nuclei, cells were incubated for 20 min with 20 μg/ml of Hoechst 33342. After washing with PBS, the cells were observed under a fluorescence microscope (Zeiss Axiophoto 2, Carl Zeiss, Germany). Cells exhibiting condensed chromatin and fragmented nuclei were scored as apoptotic cells. A minimum of 200 cells were scored from each sample.Measurement of Keratinocytes Mitochondrial Membrane Potential—HaCaT cell mitochondrial membrane potential (ΔΨm) was assessed with fluorescent probe JC-1 (20Cao C. Healey S. Amaral A. Lee-Couture A. Wan S. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2007; 212: 252-263Crossref PubMed Scopus (41) Google Scholar). At 490 nm, cells with depolarized mitochondria contained JC-1 predominantly in monomeric form and fluoresced green. Cells with polarized mitochondria predominantly contain JC-1 in aggregate form, and mitochondria fluoresce red–orange. HaCaT cells were incubated with 5 μm JC-1 (Invitrogen) for 30 min at 37 °C, washed, and fluorescent images were visualized by a Zeiss fluorescence microscope with excitation at 490 nm and emission at 520 nm (monomeric form for depolarized ΔΨm) and 590 nm (aggregate form for polarized ΔΨm). HaCaT cells with polarized mitochondria were seen with distinct mitochondria fluorescing red–orange, and with depolarized mitochondria, cell cytoplasm and mitochondria appeared green. The aquired signal was analyzed with image analysis software (20Cao C. Healey S. Amaral A. Lee-Couture A. Wan S. Kouttab N. Chu W. Wan Y. J. Cell. Physiol. 2007; 212: 252-263Crossref PubMed Scopus (41) Google Scholar). A minimum of six fields were selected and average intensity for each region was quantified. The ratio of J-aggregate to JC-1 monomer intensity for each region was calculated. A decrease in this ratio was interpreted as loss of ΔΨm, whereas an increase in the ratio was interpreted as gain in ΔΨm.ROS Detection—ROS generation was detected by FACS analysis as described previously (24Zhang Q.S. Maddock D.A. Chen J.P. Heo S. Chiu C. Lai D. Souza K. Mehta S. Wan Y.S. Int. J. Oncol. 2001; 19: 1057-1061PubMed Google Scholar, 25Fisher G.J. Kang S. Varani J. Bata-Csorgo Z. Wan Y. Datta S. Voorhees J.J. Arch. Dermatol. 2002; 138: 1462-1470Crossref PubMed Scopus (1195) Google Scholar). Briefly, cultured human skin keratinocytes (HaCaT cells) were loaded with 1 μm fluorescent dye dihydrorhodamine 2 h before UV radiation, which reacts with ROS in cells and results in a change of fluorescence. After being treated with UV with or without reagents for the desired time points, keratinocytes were trypsinized, suspended in ice-cold PBS, and fixed in 70% ethyl alcohol in –20 °C. The changes in fluorescence in drug-treated cells were quantified by FACS analysis. Induction of ROS generation was expressed in arbitrary units.Statistical Analysis—The values in the figures are expressed as the mean ± S.E. The figures in this study were representative of more than 3 different experiments. Statistical analysis of the data between the control and treated groups was performed by a Student's t test. Values of p < 0.05 were considered statistically significant.RESULTSUV Radiation and H2O2 Induce AMPK Activation in Cultured Human Skin Keratinocytes—To investigate the role of AMPK in UV signaling, we first tested whether UV or H2O2 induces AMPK activation using cultured human skin keratinocytes (HaCaT cells). The results showed that UV radiation induces AMPKα phosphorylation in a dose (Fig. 1, A and B) and time (Fig. 1, C and D)-dependent manner. Similarly, H2O2 induces AMPKα activation in a dose- and time-dependent manner (Fig. 1, E–H). Furthermore, as expected, AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) also induces AMPK activation in a dose(Fig. 1, I and J) and time (Fig. 1, K and L)-dependent manner. AMPKα-specific siRNA down-regulates AMPKα expression and largely inhibits UV- and H2O2-induced AMPK activation (Fig. 1, M and N). UV also induces AMPK activation in cultured human skin 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 UV and H2O2 induce AMPK activation (AMPKα phosphorylation at Thr172) in HaCaT cells. However, the cellular signals involved in this AMPK activation are not fully studied. Previous studies using human skin keratinocytes and dendritic cells have revealed a important role of EGFR in UV-induced cellular signals (3Xu Y. Voorhees J.J. Fisher G.J. Am. J. Pathol. 2006; 169: 823-830Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 26Cao C. Lu S. Jiang Q. Wang W.J. Song X. Kivlin R. Wallin B. Bagdasarian A. Tamakloe T. Chu W.M. Marshall J. Kouttab N. Xu A. Wan Y. Cell Signal. 2008; 20: 1830-1838Crossref PubMed Scopus (33) Google Scholar), studies also show a critical role of LKB1 for AMPK activation. As expected, in this study, we observed that both UV and H2O2 induce EGFR activation in HaCaT cells (Fig. 2, A and B). Interestingly, UV radiation also induces LKB1 phosphorylation in a time- and dose-dependent manner in HaCaT cells (Fig. 2, C and D). EGFR inhibitor PD 153035 and AG 1478 inhibit UV-induced AMPK and LKB1 activation (Fig. 2E). EGFR ligand, EGF, also induces LKB1/AMPK activation, which is blocked by PD 153035 (Fig. 2F). To further confirm the key role 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-out MEFs. Furthermore, the induction of AMPK is also inhibited by pretreatment with the antioxidant NAC and pyrrolidine dithiocarbamate (Fig. 2H) in UV-treated HaCaT cells. Antioxidant NAC and EGFR inhibitor PD 153035 also reduce H2O2-induced LKB1/AMPK activation (Fig. 2I). As expected, UV- and H2O2-induced ROS production is inhibited by NAC pre-treatment (Fig. 2J). Collectively, our data suggest that ROS-mediated EGFR trans-activation is involved in UV-induced LKB1/AMPK activation.FIGURE 2ROS-mediated EGFR activation is involved in UV-induced LKB1/AMPK activation. HaCaT cells were pre-treated with EGFR inhibitor PD 153035 (1 μm), 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 by Western blot (A). HaCaT cells were also treated with H2O2 (250 μm) and cultured for 0, 5, 15, 30, and 60 min or treated with different doses of H2O2 (0, 50, 100, 150, and 250 μm) 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) and cultured for 30, 60, 120, 180, 240, and 360 min. p-AMPKα (Thr172), p-LKB1 (Ser428), T-LKB1, and T-AMPKα were detected by Western blot. AMPK phosphorylation was 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-AMPKα (Thr172), p-LKB1 (Ser428), T-LKB1, and T-AMPKα were detected by Western blot. AMPK phosphorylation was quantified (D). HaCaT cells were pre-treated with EGFR inhibitor PD 153035 (1 μm) or AG1478 (1 μm) for 1 h, followed by UV radiation (25 mJ/cm2) for different time points (15, 30, and 60 min), p-AMPKα (Thr172), p-LKB1 (Ser428), and T-AMPKα were detected by Western blot (E). HaCaT cells were pre-treated with PD 153035 (1 μm) for 1 h, followed by EGF treatment (100 ng/ml), p-AMPKα (Thr172), p-LKB1 (Ser428), and T-AMPKα were detected by Western blot (F). Wild type and EGFR knock-out MEFs were treated with UV (25 mJ/cm2), p-AMPKα (Thr172), p-LKB1 (Ser428), T-EGFR, and T-AMPKα were detected by Western blot (G). HaCaT cells were pre-treated with the antioxidant pyrrolidine dithiocarbamate (PDTC, 100 μm) or N-acetyl-l-cysteine (NAC, 400 μm) for 1 h, followed by UV radiation (25 mJ/cm2) for different time points (15, 30, and 60 min), p-AMPKα (Thr172), p-LKB1 (Ser428), and T-AMPKα were detected by Western blot (H). HaCaT cells were pre-treated with the EGFR inhibitor PD 153035 (1 μm) or antioxidant NAC (400 μm) for 1 h, followed by H2O2 (250 μm) for different time points (15, 30, and 60 min), p-AMPKα (Thr172), p-LKB1 (Ser428), and T-AMPKα were detected by Western blot (I). HaCaT cells were pre-treated with 400 μm NAC for 1 h, followed by UV (25 mJ/cm2) or H2O2 (250 μm) 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- or H2O2-treated group. Data are presented as the mean ± S.E. for three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AMPK Is Involved in UV- and H2O2-induced p38 Activation—Because previous studies have suggested that p38 MAPK is a downstream signal of AMPK upon various stimuli (18Yoon M.J. Lee G.Y. Chung J.J. Ahn Y.H. Hong S.H. Kim J.B. Diabetes. 2006; 55: 2562-2570Crossref PubMed Scopus (424) Google Scholar, 27Du J.H. Xu N. Song Y. Xu M. Lu Z.Z. Han C. Zhang Y.Y. Biochem. Biophys. Res. Commun. 2005; 337: 1139-1144Crossref PubMed Scopus (51) Google Scholar, 28Capano M. Crompton M. Biochem. J. 2006; 395: 57-64Crossref PubMed Scopus (109) Google Scholar), and UV induces p38 activation (24Zhang Q.S. Maddock D.A. Chen J.P. Heo S. Chiu C. Lai D. Souza K. Mehta S. Wan Y.S. Int. J. Oncol. 2001; 19: 1057-1061PubMed Google Scholar), next we tested the possible role of AMPK in UV- and H2O2-induced p38 activation. As demonstrated in Fig. 3, A and B, co" @default.
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