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Epidermal Growth Factor Receptor-dependent, NF-κB-independent Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Inhibits Ultraviolet Irradiation-induced Caspases-3, -8, and -9 in Human Keratinocytes

2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês

10.1074/jbc.m300574200

1083-351X

Hui Qin Wang, Taihao Quan, Tianyuan He, Thomas Franke, John J. Voorhees, Gary J. Fisher,

PI3K/AKT/mTOR signaling in cancer

Both phosphatidylinositol 3-kinase (PI3K)/Akt and NF-κB pathways function to promote cellular survival following stress. Recent evidence indicates that the anti-apoptotic activity of these two pathways may be functionally dependent. Ultraviolet (UV) irradiation causes oxidative stress, which can lead to apoptotic cell death. Human skin cells (keratinocytes) are commonly exposed to UV irradiation from the sun. We have investigated activation of the PI3K/Akt and NF-κB pathways and their roles in protecting human keratinocytes (KCs) from UV irradiation-induced apoptosis. This activation of PI3K preceded increased levels (3-fold) of active/phosphorylated Akt. UV (50 mJ/cm2 from UVB source) irradiation caused rapid recruitment of PI3K to the epidermal growth factor receptor (EGFR). Pretreatment of KCs with EGFR inhibitor PD169540 abolished UV-induced Akt activation/phosphorylation, as did the PI3K inhibitors LY294002 or wortmannin. This inhibition of Akt activation was associated with a 3-4-fold increase of UV-induced apoptosis, as measured by flow cytometry and DNA fragmentation ELISA. In contrast to Akt, UV irradiation did not detectably increase nuclear localization of NF-κB, indicating that it was not strongly activated. Consistent with this observation, interference with NF-κB activation by adenovirus-mediated overexpression of dominant negative IKK-β or IκB-α did not increase UV-induced apoptosis. However, adenovirusmediated overexpression of constitutively active Akt completely blocked UV-induced apoptosis observed with PI3K inhibition by LY294002, whereas adenovirus mediated overexpression of dominant negative Akt increased UV-induced apoptosis by 2-fold. Inhibition of UV-induced activation of Akt increased release of mitochondrial cytochrome c 3.5-fold, and caused appearance of active forms of caspase-9, caspase-8, and caspase-3. Constitutively active Akt abolished UV-induced cytochrome c release and activation of caspases-9, -8, and -3. These data demonstrate that PI3K/Akt is essential for protecting human KCs against UV-induced apoptosis, whereas NF-κB pathway provides little, if any, protective role. Both phosphatidylinositol 3-kinase (PI3K)/Akt and NF-κB pathways function to promote cellular survival following stress. Recent evidence indicates that the anti-apoptotic activity of these two pathways may be functionally dependent. Ultraviolet (UV) irradiation causes oxidative stress, which can lead to apoptotic cell death. Human skin cells (keratinocytes) are commonly exposed to UV irradiation from the sun. We have investigated activation of the PI3K/Akt and NF-κB pathways and their roles in protecting human keratinocytes (KCs) from UV irradiation-induced apoptosis. This activation of PI3K preceded increased levels (3-fold) of active/phosphorylated Akt. UV (50 mJ/cm2 from UVB source) irradiation caused rapid recruitment of PI3K to the epidermal growth factor receptor (EGFR). Pretreatment of KCs with EGFR inhibitor PD169540 abolished UV-induced Akt activation/phosphorylation, as did the PI3K inhibitors LY294002 or wortmannin. This inhibition of Akt activation was associated with a 3-4-fold increase of UV-induced apoptosis, as measured by flow cytometry and DNA fragmentation ELISA. In contrast to Akt, UV irradiation did not detectably increase nuclear localization of NF-κB, indicating that it was not strongly activated. Consistent with this observation, interference with NF-κB activation by adenovirus-mediated overexpression of dominant negative IKK-β or IκB-α did not increase UV-induced apoptosis. However, adenovirusmediated overexpression of constitutively active Akt completely blocked UV-induced apoptosis observed with PI3K inhibition by LY294002, whereas adenovirus mediated overexpression of dominant negative Akt increased UV-induced apoptosis by 2-fold. Inhibition of UV-induced activation of Akt increased release of mitochondrial cytochrome c 3.5-fold, and caused appearance of active forms of caspase-9, caspase-8, and caspase-3. Constitutively active Akt abolished UV-induced cytochrome c release and activation of caspases-9, -8, and -3. These data demonstrate that PI3K/Akt is essential for protecting human KCs against UV-induced apoptosis, whereas NF-κB pathway provides little, if any, protective role. Human skin, unlike all other organs, is continuously and directly exposed to environmental influences. Ultraviolet (UV) radiation from the sun is among the most ubiquitous damaging environmental factors from which human skin must protect itself. (1Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5512) Google Scholar). Solar UV radiation that reaches the surface of the earth is subdivided into three wavelength ranges: UVB (290-320 nm), UVA2 (320-340 nm), and UVA1 (340-400 nm). UVB wavelengths are the most energetic, and are responsible for sunburn. UVB is directly absorbed by DNA and protein, and as such accounts for much of the damaging biological effects of UV irradiation including cancer and premature skin aging (2Shea R. Parrish J. Goldsmith L. Physiology, Biochemistry, and Molecular Biology of the Skin. Vol. II. Oxford University Press, New York1991: 910-927Google Scholar). Sunburned cells, which are induced in human skin by UVB irradiation, bear characteristic apoptotic cell morphology, and are the first evidence that UVB triggers apoptosis in skin cells (keratinocytes) (3Weedon D. Searle J. Kerr J. Am. J. Dermatopathol. 1979; 1: 133-144Crossref PubMed Scopus (163) Google Scholar). UV-induced apoptosis is an important process for eliminating UV-damaged skin cells that potentially could become cancerous. Two signaling pathways have been demonstrated to contribute to UV-induced apoptosis in keratinocytes (4Ziegler A. Jonason A. Leffell D. Simon J. Sharma H. Kimmelman J. Remington L. Jacks T. Brash D. Nature. 1994; 372: 773-776Crossref PubMed Scopus (1367) Google Scholar, 5Leverkus M. Yaar M. Gilchrest B.A. Exp. Cell Res. 1997; 232: 255-262Crossref PubMed Scopus (173) Google Scholar, 6Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (433) Google Scholar) as well as in Hela cells (7Kulms D. Schwarz T. Photodermatol. Photoimmunol. Photomed. 2000; 16: 195-201Crossref PubMed Scopus (260) Google Scholar). UV-induced DNA damage leads to p53-mediated apoptosis (4Ziegler A. Jonason A. Leffell D. Simon J. Sharma H. Kimmelman J. Remington L. Jacks T. Brash D. Nature. 1994; 372: 773-776Crossref PubMed Scopus (1367) Google Scholar). Upon severe DNA damage, p53 up-regulates Bax, a pro-apoptotic Bcl-2 family member (8Miyashita T. Reed J. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar). Bax binds to the mitochondrial membrane and induces cytochrome c release, which subsequently activates caspase-9 and caspase-3 leading to downstream apoptotic responses (9Finucane D. Bossy-Wetzel E. Waterhouse N. Cotter T. Green D. J. Biol. Chem. 1999; 274: 2225-2233Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 10Jurgensmeier J. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1385) Google Scholar). The other pathway by which UV induces apoptosis is through activation of the membrane death receptors Fas (CD95) (5Leverkus M. Yaar M. Gilchrest B.A. Exp. 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Phosphatidylinositol 3′-OH kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3′-OH kinase; PDK1, 3-phosphoinositide-dependent kinase; KCs, keratinocytes; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; TNF, tumor necrosis factor; RIPA, radioimmune precipitation assay buffer. is a heterodimeric lipid kinase that consists of a p85 regulatory subunit and a p110 catalytic subunit. It is activated by the interaction of p85 subunit with phosphorylated tyrosine residues on activated growth factor receptors such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor, and insulin-like growth factor receptor (13Stein R. Waterfield M. Mol. Med. Today. 2000; 6: 347-357Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Activated PI3K phosphorylates phosphotidylinositol (PI), PI 4-P, or PI 4,5-P2 at the 3′-position of the inositol head group to produce PI 3-P, PI 3,4-P2, and PI 3,4,5-P3, respectively (13Stein R. Waterfield M. Mol. Med. Today. 2000; 6: 347-357Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The phospholipid products of PI3K activates phosphoinositide-dependent kinase 1 (PDK1) and recruits Akt to plasma membrane through their pleckstrin homology domains (13Stein R. Waterfield M. Mol. Med. Today. 2000; 6: 347-357Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Akt, also known as protein kinase B, is a serine/threonine protein kinase, originally identified as the oncogene transduced by the acute transforming retrovirus AKT8 (14Bellacosa A. Testa J. Staal S. Tsichlis P. Science. 1991; 254: 274-277Crossref PubMed Scopus (807) Google Scholar). In quiescent cells, Akt resides within the cytosol in an inactive state. After growth factor or cytokine stimulation, Akt translocates to the inner surface of the plasma membrane where PI 3 kinase-generated 3′-phosphoinositides reside, and Akt is phosphorylated at threonine 308 and serine 473 by PDK1 (15Alessi D. James S. Downes C. Holmes A. Gaffney P. Reese C. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 16Alessi D. Deak M. Casamayor A. Caudwell F. Morrice N. Norman D. Gaffney P. Reese C. MacDougall C. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar) and integrin-linked kinase (17Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). Mutagenesis studies have revealed that phosphorylation of threonine 308 and serine 473 is required for Akt activation and mimicking phosphorylation partially activates Akt (18Alessi D. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2559) Google Scholar). Akt was first demonstrated to promote cell survival in neurons by Dudek in 1997 (19Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2243) Google Scholar), and later in dorsal ganglion cell line (20Goswami R. Kilkus J. Dawson S.A. Dawson G. J. Neurosci. Res. 1999; 57: 884-893Crossref PubMed Scopus (64) Google Scholar), fibroblasts (21Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (985) Google Scholar, 22Kulik G. Klippel A. Weber M.J. Mol. Cell Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (968) Google Scholar), epithelial cell line (23Lee J.W. Juliano R.L. Mol. Biol. Cell. 2000; 11: 1973-1987Crossref PubMed Scopus (143) Google Scholar), and endothelial cell line (24Fujio Y. Walsh K. J. Biol. Chem. 1999; 274: 16349-16354Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 25Hermann C. Assmus B. Urbich C. Zeiher A.M. Dimmeler S. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 402-409Crossref PubMed Scopus (202) Google Scholar). Resistance to stress-induced apoptosis is also mediated by the NF-κB pathway. NF-κB is a ubiquitously expressed transcription factor that regulates many genes involved in inflammation and immunity. NF-κB is regulated by exclusion from the nucleus by interaction with its inhibitor IκB. Exposure to a variety of stimuli, including cytokines/oxidative stress and UV irradiation stimulates phosphorylation by IκB by IκB kinase (IKK) complexes. Phosphorylation of IκB results in increase susceptibility to ubiquitin/proteasome degradation, thereby allowing NF-κB to localize to the nucleus and stimulates transcription of target genes. Recently it has been demonstrated that NF-κB stimulates expression of certain members of the Inhibitor of Apoptosis (IAP) family of genes. IAP binds to and inhibits certain caspases and thereby blocks apoptosis. Recent evidence indicates that Akt can activate the NF-κB pathway through phosphorylation of IKK (26Madrid L. Mayo M. Reuther J. Baldwin Jr., A. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 27Ozes O. Mayo L. Gustin J. Pfeffer S. Pfeffer L. Donner D. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1923) Google Scholar, 28Romashkova J. Makarov S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1685) Google Scholar, 29Yuan Z.-Q. Feldman R. Sun M. Olashaw N. Coppola D. Sussman G. Shelley S. Nicosia S. Cheng J. J. Biol. Chem. 2002; 277: 29973-29982Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 30Deveraux Q. Reed J. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2303) Google Scholar) and that Akt is a downstream target of NF-κB (31Meng F. Liu L. Chin P. D'Mello S. J. Biol. Chem. 2002; 277: 29674-29680Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). This observation raises the possibility that the anti-apoptotic activities of the PI3K/Akt and NF-κB pathways may be interdependent. UV irradiation has been demonstrated to induce phosphorylation/activation of EGFR (32Fisher G. Talwar H. Lin J. Lin P. McPhillips F. Wang Z. Li X. Wan Y. Kang S. Voorhees J. J. Clin. Investig. 1998; 101: 1432-1440Crossref PubMed Scopus (336) Google Scholar, 33Peus D. Vasa R. Meves A. Beyerle A. Pittelkow M. Photochem. Photobiol. 2000; 72: 135-140Crossref PubMed Scopus (74) Google Scholar, 34Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (947) Google Scholar). UV activation of EGFR may stimulate PI3K and, consequently, Akt. Since PI3K/Akt is downstream of EGFR, UV irradiation has also been reported to activate NF-κB (35Piette J. Piret B. G B. Schoonbroodt S. Merville M. Legrand-Poels S. Bours V. Biol. Chem. 1997; 378: 1237-1245PubMed Google Scholar, 36Huang T. Feinberg S. Suryanarayanan S. Miyamoto S. Mol. Cell. Biol. 2002; 22: 5813-5825Crossref PubMed Scopus (99) Google Scholar). We have investigated UV-induced PI3K/Akt activation, and the role of Akt versus NF-κB, in protecting keratinocytes against UV-induced apoptosis. Primary Human Keratinocyte and UV Irradiation—All procedures involving human subjects were approved by the University of Michigan Institutional Review Board and all subjects provided written informed consent. Primary human keratinocytes were established from normal adult human skin, as previously described (37Fisher G. Tavakkol A. Leach K. Burns D. Basta P. Loomis C. Griffiths C. Cooper K. Reynolds N. Elder J. Livneh E. Voorhees J.J. J. Investig. Dermatol. 1993; 101: 553-559Abstract Full Text PDF PubMed Scopus (82) Google Scholar). Cells were maintained in modified MCDB 153 (EpiLife, Cascade Biologics, Inc., Portland, OR). Cells at passage 3-5 were used for study. For UV irradiation, human keratinocytes in 100-mm dishes were placed in growth factor-free medium for 24 h, washed once with PBS, 3 ml of PBS added, and were irradiated with the dish lid removed. The light source was a Daavlin Spectra Lamp containing six FS24T12 UVB-HO fluorescent tubes. Kodacel TA 401/407 sheet was used to remove wavelengths below 290 nm (UVC). The resulting output consisted of 40% UVB, 27% UVA2, 19% UVA1, and 14% visible light as analyzed by Spectroradiometry OL 754 system (Optronic Laboratories, Orlando, FL). Irradiation intensity was monitored by IL1400A Radiometer/photometer and SEL 240/UVB/W photodetector (International Light Inc, Newburyport, MA). After irradiation, PBS was removed, and the original media were put back into the plates. In selected experiments, cells were pretreated with PI3K inhibitors LY294002 or wortmannin (Sigma), or EGFR inhibitor PD169540 (Pfizer, Ann Arbor, MI), or caspase-8 inhibitor II (Z-IETD-FMK), caspase-9 inhibitor I (Z-LEHD-FMK), or caspase-3 inhibitor (Z-VAD-FMK) (Calbiochem, San Diego, CA) for 1 h prior to UV irradiation. Preparation of Whole Cell Homogenates or Cytosolic Protein—Whole cell homogenates or cytosolic lysates were prepared as previously described (38Li L. Lorenzo P. Bogi K. Blumberg P. Yuspa S. Mol. Cell Biol. 1999; 19: 8547-8558Crossref PubMed Google Scholar). For whole cell homogenates, keratinocytes were washed once in PBS, and scraped into whole cell lysis buffer (25 mm Hepes, pH 7.7, 75 mm NaCl, 2.5 mm MgCl2·6H2O, 0.2 mm EDTA, 0.1% Triton X-100, 0.5 mm dithiothreitol, 20 mm β-glycerophosphate) containing 1× complete protease inhibitors (Roche Applied Science) and 1 mm phenylmethylsulfonyl fluoride, and phosphatase inhibitors Na3VO4 (1 mm) and NaF (50 mm). Cell lysates were sonicated and centrifuged at 10,000 × g for 20 min at 4 °C. Supernatants were used as whole cell extracts for Western blot analysis. To prepare the cytosolic fractions, keratinocytes were trypsinized and pelleted by centrifugation. The cell pellet was resuspended in 250 μl of hypotonic buffer, containing 25 mm Tris (pH 7.4), 250 mm sucrose, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, and 1 mm dithiothreitol, 0.05% digitonin with 1× protease inhibitors and 1 mm phenylmethylsulfonyl fluoride, and homogenized 10 times with a Dounce homogenizer (1Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5512) Google Scholar). Unbroken cells and nuclei were removed by centrifugation at 750 × g for 10 min at 4 °C. Mitochondria were removed by centrifugation at 10,000 × g for 20 min at 4 °C. The supernatant was further centrifuged at 100,000 × g for 1 h at 4 °C, and the resulting supernatant was utilized as cytosolic fraction. Protein concentration was determined with Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as standard. Immunoprecipitation—After treatment, keratinocytes were washed with PBS once and 1.5 ml ice-cold RIPA buffer, 1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS containing [1× complete protease inhibitors and 1 mm phenylmethylsulfonyl fluoride, and phosphatase inhibitors Na3VO4 (1 mm) and NaF (50 mm)] were added to the cells and incubated at 4 °C for 10 min. Cells were disrupted by repeated aspiration through a 21-gauge needle and transferred to a 2-ml microcentrifuge tube. Cellular debris was pelleted by centrifugation at 10,000 × g for 10 min at 4 °C. Supernatants were precleared by adding 1.0 μg of normal rabbit IgG and protein G/A-agarose. The resulting supernatant was incubated with anti-p85 polyclonal antibody bound to agarose overnight at 4 °C. Immunoprecipitates were collected by centrifugation and washed four times with RIPA buffer. After final wash, pellets were resuspended in 40 μl of 1× electrophoresis sample buffer and analyzed by Western analysis. Western Blot Analysis—Western blot analysis was carried out as described (39Wang H. Smart R. J. Cell Sci. 1999; 112: 3497-3506PubMed Google Scholar). Briefly, whole cell extract proteins (50-100 μg) were separated on Tris-glycine polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to Immobilon™-P membrane (Millipore Corporation, Bedford, MA), blocked in 5% nonfat dry milk, 1% bovine serum albumin, and 0.1% Tween 20. Membranes were probed with polyclonal antibody against phospho-Akt (serine 473), total Akt, caspase-9 (Cell Signaling Technology, Beverly, MA), caspase-3, and caspase-8 (BD PharMingen, San Diego, CA). For cytochrome c Western blot analysis, 50 μg of cytosolic protein extract was separated on 14% Tris-glycine PAGE and transferred to Immobilon™-P membrane, and the membrane was probed with a monoclonal anti-cytochrome c antibody (Clone 7H8.2C12, BD PharMingen). Immunoreactive bands were visualized by enhanced chemifluorescence (ECF) (Amersham Biosciences, Piscataway, NJ) and quantified by STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Detection of Apoptosis by ELISA and Flow Cytometry—Apoptosis was examined by analysis of DNA fragmentation using the Cell Death Detection ELISA following the manufacturer's instructions (Roche Applied Science). For flow cytometry, keratinocytes were collected by trypsin and washed once with PBS. Cell pellets were resuspended in 50% cold ethanol and fixed at -20 °C. After fixation, cells were washed once with cold PBS, and incubated in 0.5 ml of PBS containing 100 μg/ml RNase A for 20 min at 37 °C. Keratinocytes were then pelleted by centrifugation, and 250 μl of PBS containing 50 μg/ml propidium iodide (PI) was added to the pellet. Thirty minutes later, flow cytometric analysis was carried out using Beckman Coulter Elite Esp. Cell Sorter in the Flow Cytometry Core Facility at University of Michigan. Cells with DNA content less than that in untreated cells in G0/G1 were considered apoptotic. Measurement of Caspase-8 Activity—Keratinocytes were collected by trypsinization, washed once with PBS, and cell pellets were resuspended in 250 μl of ice-cold lysis buffer (50 mm Hepes, pH 7.4, 100 mm NaCl, 0.1% CHAPS, 1 mm dithiothreitol, and 100 μm EDTA) and homogenized. Homogenates were centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatants were used for measuring caspase-8 activity using an ELISA-based assay, according to the manufacturer's instructions (Calbiochem). Measurement of NF-κB Activity—Human skin keratinocytes were transfected with pNF-κB-Luc (Clontech Laboratories, Inc., Palo Alto, CA) using Fugen 6 (Roche Applied Science) according to the manufacturer's protocol. Plasmid DNA containing the β-galactosidase gene (pCMVβ, Clontech Laboratories, Inc.) was used as an internal standard for transfection efficiency. Forty-eight hours after transfection, cells were sham- or UV-irradiated with UV (50 mJ/cm2) as described above. Alternatively, cells were treated with either IL-1β (10 ng/ml) or TNFα (10 ng/ml). Sixteen hours after treatment cells were harvested in lysis buffer (PharMingen International, San Diego, CA), and assayed for β-galactosidase activity. Luciferase activity was measured using an enhanced luciferase assay kit (PharMingen International) according to the manufacturer's protocol. Aliquots containing identical β-galactosidase activity were used for each luciferase assay. Infection of Human Keratinocytes with Adenovirus Constructs—Recombinant adenovirus constructs expressing constitutively active myristoylated Akt (myr-Akt), dominant negative Akt (dn-Akt, T308A, S473A), dominant negative IκB-α, and dominant negative IKK-β have been previously described (24Fujio Y. Walsh K. J. Biol. Chem. 1999; 274: 16349-16354Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 40Kennedy S. Kandel E. Cross T. Hay N. Mol. Cell Biol. 1999; 19: 5800-5810Crossref PubMed Scopus (600) Google Scholar). Dominant negative Akt was kindly provided by Dr. Kenneth Walsh (Tufts University School of Medicine, Boston, MA) and dominant negative IκB-α and IKK-β were generously provided by Michael Karin (University of California San Diego, La Jolla, CA). Adenovirus constructs were propagated in HEK 293 cells, and purified by CsCl gradient centrifugation, followed by Sepharose CL-4B column chromatography (41Gerard R. Meidell R. Hames B. Glover D. DNA Cloning: A Practical Approach. Oxford University Press, Oxford1995: 300-302Google Scholar). The amount of virus was estimated by absorbance at 260 nm. Human keratinocytes were infected with adenovirus at 5000 particles per cell in growth factor-free medium for 1 h. One hour later, media were removed and replaced with fresh growth medium (38Li L. Lorenzo P. Bogi K. Blumberg P. Yuspa S. Mol. Cell Biol. 1999; 19: 8547-8558Crossref PubMed Google Scholar). Twenty-four hours later, growth media were removed, and growth factor-free medium was added. Cells were utilized the following day. UV-induced Akt Phosphorylation Is Mediated by EGFR and PI3K in Human Keratinocytes—We initially characterized the kinetics and dose dependence of UV induction of Akt phosphorylation in primary human keratinocytes by Western blot analysis using an antibody that specifically recognizes activated Akt phosphorylated at serine 473. Levels of phospho-Akt increased (2.5-fold) within 10 min, and were maximal (3-fold) at 15 min after UV irradiation (Fig. 1A). Phospho-Akt levels decreased, but remained modestly elevated 20-30 min post-UV irradiation. Total Akt levels were not altered by UV irradiation (Fig. 1A). Maximum UV-induction of Akt phosphorylation was observed at a dose of 50 mJ/cm2 (Fig. 1B). Above this dose, Akt phosphorylation decreased, as a result of excessive cellular damage. To investigate if UV-induced Akt activation is dependent on PI3K, human keratinocytes were treated with two PI3K inhibitors LY294002 or wortmannin for 1 h prior to UV irradiation. LY294002 and Wortmannin abolished UV-induced Akt phosphorylation (Fig. 1C). UV has been shown to activate EGFR in human keratinocytes by a ligand independent mechanism (32Fisher G. Talwar H. Lin J. Lin P. McPhillips F. Wang Z. Li X. Wan Y. Kang S. Voorhees J. J. Clin. Investig. 1998; 101: 1432-1440Crossref PubMed Scopus (336) Google Scholar, 33Peus D. Vasa R. Meves A. Beyerle A. Pittelkow M. Photochem. Photobiol. 2000; 72: 135-140Crossref PubMed Scopus (74) Google Scholar). Ligand activation of EGFR stimulates PI3K activation and Akt activities (13Stein R. Waterfield M. Mol. Med. Today. 2000; 6: 347-357Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 42Okano J. Gaslightwala I. Birnbaum M. Rustgi A. Nakagawa H. J. Biol. Chem. 2000; 275: 30934-30942Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 43Lin J. Adam R.M. Santiestevan E. Freeman M. Cancer Res. 1999; 59: 2891-2897PubMed Google Scholar, 44Kohn A. Kovacina K. Roth R. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (323) Google Scholar, 45Burgering B. Coffer P. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1896) Google Scholar). We examined if UV-induced activation of PI3K and Akt is mediated by EGFR. Activated EGFR is phosphorylated at tyrosine residues, which function as binding sites for SH2 domain of p85 subunit of PI3K (46Stover D. Becker M. Leiebetanz J. Lydon N. J. Biol. Chem. 1995; 26: 15591-15597Abstract Full Text Full Text PDF Scopus (190) Google Scholar). Binding of p85 to EGFR is a hallmark of PI3K activation (47Tiganis T. Kemp B. Tonks N. J. Biol. Chem. 1999; 274: 27768-27775Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). UV irradiation induced binding of p85 to EGFR in human keratinocytes, as determined by Western analysis of p85 immunoprecipitates for EGFR (Fig. 1D). UV irradiation increased binding of p85 with EGFR within 5 min post-UV irradiation (Fig. 1D). The increased association of p85 with EGFR induced by UV irradiation was similar in magnitude to that observed following treatment of cells with EGFR (Fig. 1D). Furthermore, treatment of human keratinocytes with EGFR inhibitor PD169540 (48Fry D.W. Bridges A.J. Denny W.A. Doherty A. Greis K.D. Hicks J.L. Hook K.E. Keller P.R. Leopold W.R. Loo J.A. McNamara D.J. Nelson J.M. Sherwood V. Smaill J.B. Trumpp-Kallmeyer S. Dobrusin E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12022-12027Crossref PubMed Scopus (397) Google Scholar) for 1 h prior to UV irradiation abolished UV-induced Akt phosphorylation (Fig. 1D). These results indicate that UV-induced Akt phosphorylation is dependent on EGFR activation of PI3K in human keratinocytes. PI3K Inhibitor or EGFR Inhibitor Increases UV-induced Apoptosis in Human Keratinocytes—Akt has been shown to promote cell survival in a variety of cell types in response to growth factor or cytokine stimulation (49Datta S. Brunet A. Greenberg M. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3753) Google Scholar). In order to investigate the role of Akt activation in UV-induced apoptosis, keratinocytes were pretreated with or without PI3K inhibitors or EGFR inhibitor prior to UV irradiation. UV irradiation of keratinocytes with 50 mJ/cm2 resulted in apoptosis of ∼20% of the cells, as measured by flow cytometry (Fig. 2A). Pretreatment of keratinocytes with PI3K inhibitors LY294002 or wortmannin, or EGFR inhibitor PD169540 resulted in UV-induced apoptosis of 60-70% of the cells. DNA fragmentation is an early and characteristic event of apoptosis. Therefore we also examined UV-induced DNA fragmentation in human keratinocytes. UV-induced DNA fragmentation was substantially increased in keratinocytes by pretreatment with the PI3K inhibitors LY294002, in a dose-dependent manner (Fig. 2B) and wortmannin. PD169540 also significantly increased UV-induced DNA fragmentation (Fig. 2B). None of the inhibitors by themselves induced DNA fragmentation (Fig. 2C). The above data demonstrate that inhibition of EGFR or PI3K, which results in inhibition of Akt, increases UV-induced apoptosis. NF-κB Does Not Protect against UV-induced Apoptosis in Human K