Advertisement

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*

Open AccessPublished:September 02, 2003DOI:https://doi.org/10.1074/jbc.M300574200
      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. (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ). 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 (
      • Shea R.
      • Parrish J.
      ).
      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) (
      • Weedon D.
      • Searle J.
      • Kerr J.
      ). 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 (
      • Ziegler A.
      • Jonason A.
      • Leffell D.
      • Simon J.
      • Sharma H.
      • Kimmelman J.
      • Remington L.
      • Jacks T.
      • Brash D.
      ,
      • Leverkus M.
      • Yaar M.
      • Gilchrest B.A.
      ,
      • Aragane Y.
      • Kulms D.
      • Metze D.
      • Wilkes G.
      • Poppelmann B.
      • Luger T.
      • Schwarz T.
      ) as well as in Hela cells (
      • Kulms D.
      • Schwarz T.
      ). UV-induced DNA damage leads to p53-mediated apoptosis (
      • Ziegler A.
      • Jonason A.
      • Leffell D.
      • Simon J.
      • Sharma H.
      • Kimmelman J.
      • Remington L.
      • Jacks T.
      • Brash D.
      ). Upon severe DNA damage, p53 up-regulates Bax, a pro-apoptotic Bcl-2 family member (
      • Miyashita T.
      • Reed J.
      ). Bax binds to the mitochondrial membrane and induces cytochrome c release, which subsequently activates caspase-9 and caspase-3 leading to downstream apoptotic responses (
      • Finucane D.
      • Bossy-Wetzel E.
      • Waterhouse N.
      • Cotter T.
      • Green D.
      ,
      • Jurgensmeier J.
      • Xie Z.
      • Deveraux Q.
      • Ellerby L.
      • Bredesen D.
      • Reed J.
      ). The other pathway by which UV induces apoptosis is through activation of the membrane death receptors Fas (CD95) (
      • Leverkus M.
      • Yaar M.
      • Gilchrest B.A.
      ,
      • Aragane Y.
      • Kulms D.
      • Metze D.
      • Wilkes G.
      • Poppelmann B.
      • Luger T.
      • Schwarz T.
      ), which initiates apoptosis by activation of caspase-8 followed by activation of caspase-3 (
      • Boldin M.
      • Varfolomeev E.
      • Pancer Z.
      • Mett I.
      • Camonis J.
      • Wallach D.
      ,
      • Boldin M.
      • Goncharov T.
      • Goltsev Y.
      • Wallach D.
      ).
      Phosphatidylinositol 3′-OH kinase (PI3K)
      The 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.
      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 (
      • Stein R.
      • Waterfield M.
      ). 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 (
      • Stein R.
      • Waterfield M.
      ). The phospholipid products of PI3K activates phosphoinositide-dependent kinase 1 (PDK1) and recruits Akt to plasma membrane through their pleckstrin homology domains (
      • Stein R.
      • Waterfield M.
      ). 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 (
      • Bellacosa A.
      • Testa J.
      • Staal S.
      • Tsichlis P.
      ). 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 (
      • Alessi D.
      • James S.
      • Downes C.
      • Holmes A.
      • Gaffney P.
      • Reese C.
      • Cohen P.
      ,
      • Alessi D.
      • Deak M.
      • Casamayor A.
      • Caudwell F.
      • Morrice N.
      • Norman D.
      • Gaffney P.
      • Reese C.
      • MacDougall C.
      • Harbison D.
      • Ashworth A.
      • Bownes M.
      ) and integrin-linked kinase (
      • Persad S.
      • Attwell S.
      • Gray V.
      • Mawji N.
      • Deng J.T.
      • Leung D.
      • Yan J.
      • Sanghera J.
      • Walsh M.P.
      • Dedhar S.
      ). Mutagenesis studies have revealed that phosphorylation of threonine 308 and serine 473 is required for Akt activation and mimicking phosphorylation partially activates Akt (
      • Alessi D.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.
      ). Akt was first demonstrated to promote cell survival in neurons by Dudek in 1997 (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ), and later in dorsal ganglion cell line (
      • Goswami R.
      • Kilkus J.
      • Dawson S.A.
      • Dawson G.
      ), fibroblasts (
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ), epithelial cell line (
      • Lee J.W.
      • Juliano R.L.
      ), and endothelial cell line (
      • Fujio Y.
      • Walsh K.
      ,
      • Hermann C.
      • Assmus B.
      • Urbich C.
      • Zeiher A.M.
      • Dimmeler S.
      ).
      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 (
      • Madrid L.
      • Mayo M.
      • Reuther J.
      • Baldwin Jr., A.
      ,
      • Ozes O.
      • Mayo L.
      • Gustin J.
      • Pfeffer S.
      • Pfeffer L.
      • Donner D.
      ,
      • Romashkova J.
      • Makarov S.
      ,
      • Yuan Z.-Q.
      • Feldman R.
      • Sun M.
      • Olashaw N.
      • Coppola D.
      • Sussman G.
      • Shelley S.
      • Nicosia S.
      • Cheng J.
      ,
      • Deveraux Q.
      • Reed J.
      ) and that Akt is a downstream target of NF-κB (
      • Meng F.
      • Liu L.
      • Chin P.
      • D'Mello S.
      ). 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 (
      • Fisher G.
      • Talwar H.
      • Lin J.
      • Lin P.
      • McPhillips F.
      • Wang Z.
      • Li X.
      • Wan Y.
      • Kang S.
      • Voorhees J.
      ,
      • Peus D.
      • Vasa R.
      • Meves A.
      • Beyerle A.
      • Pittelkow M.
      ,
      • Rosette C.
      • Karin M.
      ). 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 (
      • Piette J.
      • Piret B.
      • G B.
      • Schoonbroodt S.
      • Merville M.
      • Legrand-Poels S.
      • Bours V.
      ,
      • Huang T.
      • Feinberg S.
      • Suryanarayanan S.
      • Miyamoto S.
      ). We have investigated UV-induced PI3K/Akt activation, and the role of Akt versus NF-κB, in protecting keratinocytes against UV-induced apoptosis.

      EXPERIMENTAL PROCEDURES

      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 (
      • Fisher G.
      • Tavakkol A.
      • Leach K.
      • Burns D.
      • Basta P.
      • Loomis C.
      • Griffiths C.
      • Cooper K.
      • Reynolds N.
      • Elder J.
      • Livneh E.
      • Voorhees J.J.
      ). 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 (
      • Li L.
      • Lorenzo P.
      • Bogi K.
      • Blumberg P.
      • Yuspa S.
      ). 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 (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ). 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 (
      • Wang H.
      • Smart R.
      ). 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 (
      • Fujio Y.
      • Walsh K.
      ,
      • Kennedy S.
      • Kandel E.
      • Cross T.
      • Hay N.
      ). 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 (
      • Gerard R.
      • Meidell R.
      ). 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 (
      • Li L.
      • Lorenzo P.
      • Bogi K.
      • Blumberg P.
      • Yuspa S.
      ). Twenty-four hours later, growth media were removed, and growth factor-free medium was added. Cells were utilized the following day.

      RESULTS

      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).
      Figure thumbnail gr1
      Fig. 1Ultraviolet (UV) irradiation-induced Akt phosphorylation is mediated by PI3K and EGFR. A, time course of UV-induced Akt phosphorylation in human keratinocytes by Western blot analysis. Human keratinocytes at 70-80% confluence exposed to 50 mJ/cm2 UV. Keratinocytes were collected at indicated times after UV irradiation, whole cell homogenates were prepared, and total and serine 472-phosphorylated Akt were determined by Western analysis. Inset shows representative Western blot. *, p < 0.05 versus non-irradiated control keratinocytes; n = 4. B, UV dose response for Akt phosphorylation in human keratinocytes. Human keratinocytes at 70-80% confluence exposed to indicated doses of UV, and keratinocytes were collected 15 min post-UV irradiation. Whole cell homogenates were prepared and total and serine 472-phosphorylated Akt were determined by Western analysis. Inset shows representative Western blot. *, p < 0.05 versus non-irradiated control keratinocytes; n = 4. C, PI3K inhibitors wortmannin (WM) and LY294002 (LY), block UV-induced Akt phosphorylation in human keratinocytes. Human keratinocytes at 70-80% confluence treated with LY 20 μm, WM 500 nm for 1 h, prior to UV irradiation (50 mJ/cm2). Keratinocytes were collected 15 min post-UV irradiation and whole cell homogenates were prepared. Phospho-Akt (open bars) and total Akt (closed bars) were analyzed by Western analysis. Inset shows representative Western blot. Bar heights are the means ± S.E. fold increase in Akt protein levels relative to levels in untreated control cells (Ctrl); *, p < 0.05 versus UV irradiated KCs; n = 4. D, UV irradiation activation of Akt is mediated by the EGF receptor. Top panel, human keratinocytes at 70-80% confluence were exposed to 50mJ/cm2 UV. Keratinocytes were collected at the indicated times post-UV irradiation, whole cell homogenates prepared, and p85 subunit of PI3K immunoprecipitated. Immunoprecipitates were analyzed for p85 and EGF receptor by Western analysis. Inset shows representative Western blot. Bottom panel, keratinocytes were treated with PD169540 (PD, 100 nm) for 1 h prior to exposure to UV irradiation (50mJ/cm2). Keratinocytes were collected 15 min post-UV irradiation, whole cell homogenates were prepared, and total and serine 473-phosphorylated Akt determined by Western analysis. Inset shows representative Western blot; n = 4.
      UV has been shown to activate EGFR in human keratinocytes by a ligand independent mechanism (
      • Fisher G.
      • Talwar H.
      • Lin J.
      • Lin P.
      • McPhillips F.
      • Wang Z.
      • Li X.
      • Wan Y.
      • Kang S.
      • Voorhees J.
      ,
      • Peus D.
      • Vasa R.
      • Meves A.
      • Beyerle A.
      • Pittelkow M.
      ). Ligand activation of EGFR stimulates PI3K activation and Akt activities (
      • Stein R.
      • Waterfield M.
      ,
      • Okano J.
      • Gaslightwala I.
      • Birnbaum M.
      • Rustgi A.
      • Nakagawa H.
      ,
      • Lin J.
      • Adam R.M.
      • Santiestevan E.
      • Freeman M.
      ,
      • Kohn A.
      • Kovacina K.
      • Roth R.
      ,
      • Burgering B.
      • Coffer P.
      ). 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 (
      • Stover D.
      • Becker M.
      • Leiebetanz J.
      • Lydon N.
      ). Binding of p85 to EGFR is a hallmark of PI3K activation (
      • Tiganis T.
      • Kemp B.
      • Tonks N.
      ). 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 (
      • Fry 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.
      ) 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 (
      • Datta S.
      • Brunet A.
      • Greenberg M.
      ). 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.
      Figure thumbnail gr2
      Fig. 2PI3K inhibitors wortmannin and LY294002, and EGFR inhibitor PD169540 increased UV-induced apoptosis in human keratinocytes. Human keratinocytes were treated with wortmannin (WM, 500 nm), LY294002 (LY,1 μm,5 μm,20 μm), or PD169540 (PD, 100 nm) for 1 h, and irradiated with 50 mJ/cm2 UV. Four hours post-UV irradiation, keratinocytes were collected, and the relative number of apoptotic cells was determined by flow cytometry (A) and the DNA fragmentation ELISA (B). C, human keratinocytes were also treated with inhibitors alone for 1 h, and collected 4 h later for DNA fragmentation ELISA. Data are shown as the means ± S.E.; *, p < 0.05 versus UV-irradiated keratinocytes; n = 4.
      NF-κB Does Not Protect against UV-induced Apoptosis in Human Keratinocytes—NF-κB, like Akt, interferes with apoptotic pathways. Recent evidence suggests that the anti-apoptotic activity of Akt may be dependent on NF-κB (
      • Hatano E.
      • Brenner D.
      ) and that NF-κB may act through Akt to promote cell survival (
      • Yang C.
      • Murti A.
      • Pfeffer S.
      • Kim J.
      • Donner D.
      • Pfeffer L.
      ). Therefore, we next investigated the role of NF-κB in protection against UV-induced apoptosis. NF-κB was inhibited by overexpression of IκB-α or IKK-β dominant negative mutants, using adenovirual vectors, and the effect of this inhibition on UV-induced apoptosis determined by measurement of DNA fragmentation. Fig. 3 demonstrates the effects of UV irradiation and interleukin-1 (IL-1, used as positive control) on NF-κB/p65 nuclear localization, the hallmark of NF-κB activation. In untreated keratinocytes, p65 (and p50, data not shown) is primarily localized in the cytoplasm (Fig. 3A). IL-1 treatment causes rapid (within 15 min) and marked localization of p65 (and p50, data not shown) in the nucleus (Fig. 3B). In contrast, UV irradiation had no detectable effect on p65 intracellular localization at 15 min (Fig. 3C), 30 min, 1 h, 3 h, or 5 h (data not shown) post-treatment. Overexpression of dominant negative IKK-β (Fig. 3D) or dominant negative IκB-α (Fig. 3E) substantially prevented IL-1 induced p65 (and p50, data not shown) nuclear localization, indicating functional blockade of NF-κB activation. This inhibition of NF-κB activation did not cause increased apoptosis in keratinocytes following exposure to UV irradiation. In three independent experiments, there was no significant difference in UV-induced DNA fragmentation in cells infected with empty virus compared with dominant negative IKK-β or dominant negative IκB-α (Fig. 3F). To confirm the apparent lack of NF-κB activation by UV irradiation, we utilized a luciferase reporter gene assay. In these assays, in contrast to IL-1β and TNF-α, UV irradiation failed to induce detectable functional activation of NF-κB (Fig. 3G). The lack of effect of NF-κB inhibition on UV-induced apoptosis is in contrast to the substantial increase in UV-induced apoptosis observed with inhibition of Akt activation by PI3K inhibitors described above. These data suggest that PI3K/Akt is the major pathway that protects human keratinocytes from UV irradiation-induced apoptosis, and that Akt functions independent of NF-κB in this capacity. This lack of NF-κB involvement in UV-induced, Akt-mediated anti-apoptotic signaling in human keratinocytes differs from the reported interdependence of Akt and NF-κB function observed in response to exposure to TNF-α (
      • Ozes O.
      • Mayo L.
      • Gustin J.
      • Pfeffer S.
      • Pfeffer L.
      • Donner D.
      ), PDGF (
      • Romashkova J.
      • Makarov S.
      ), LPS (
      • Madrid L.
      • Mayo M.
      • Reuther J.
      • Baldwin Jr., A.
      ), and cellular stress (
      • Yuan Z.-Q.
      • Feldman R.
      • Sun M.
      • Olashaw N.
      • Coppola D.
      • Sussman G.
      • Shelley S.
      • Nicosia S.
      • Cheng J.
      ) in other cell types. In keratinocytes, UV irradiation activates Akt, without detectably activating NF-κB. This apparent independence of Akt and NF-κB may be specific to keratinocytes, since NF-κBin keratinocytes appears to have a unique and critical role in regulation of cellular maturation (
      • Delhase M.
      • Karin M.
      ,
      • Seitz C.
      • Lin Q.
      • Deng H.
      • Khavari P.
      ,
      • Seitz C.
      • Freiberg R.
      • Hinata K.
      • Khavari P.
      ).
      Figure thumbnail gr3
      Fig. 3UV irradiation does not induce NF-κB nuclear localization in human keratinocytes. Keratinocytes infected with adenovirus (2000 particles per cell) and treated with IL-1 (10 mg/ml) or UV irradiation (50mJ/cm2) 24 h postinfection. Cells were harvested 30 min after treatment and stained for p65 by immunofluorescence. A, empty adenovirus, no treatment; B, empty adenovirus plus IL-1; C, UV treatment; D, dominant negative mutant IKK-β plus IL-1; E, dominant negative mutant IκB-α plus IL-1. Results are representative of three experiments. F, human keratinocytes infected with adenovirus were exposed to UV irradiation 24 h postinfection. Four hours post-UV irradiation, keratinocytes were harvested, and the relative number of apoptotic cells determined by DNA fragmentation ELISA. Results are expressed as fold change compared with empty adenovirus, no UV control. Results are means + S.E. of experiments. G, human keratinocytes transiently transfected with pNF-κB-Luc were treated with UV (50mJ/cm2), IL-1β, or TNFα 16 h before harvesting. NF-κB activity is reflected by changes in luciferase activity. Results are means + S.E. for four experiments. *, p < 0.05 versus control.
      Overexpression of Myristoylated and Dominant Negative Mutant Forms of Akt Alter UV-induced Apoptosis in Human Keratinocytes—Given the primary importance of the PI3K/Akt pathway in protecting keratinocytes from UV-induced apoptosis, we further investigated the anti-apoptotic mechanisms of PI3K/Akt signaling. PI3K has multiple effectors, including Ras, PKA, PKC, Erk, JNK, and p38 in addition to Akt, that participate in regulation of cell survival (
      • Vanhaesebroeck B.
      • Waterfield M.
      ). The observed augmentation of UV-induced apoptosis by PI3K inhibition could result from blocking activation of other PI3K downstream effectors in addition to Akt. Therefore, we overexpressed myristoylated Akt, which is not dependent on PI3K for activation, to specifically determine the role of Akt in countering UV-induced apoptosis. Infection of keratinocytes with myristoylated Akt (Adeno-myr-Akt 5000 particles per cell) increased Akt protein levels 10-fold, while infection with empty adenovirus did not alter endogenous Akt levels as shown by Western blot analysis (Fig. 4A). Overexpression of myristoylated Akt substantially reduced UV-induced apoptosis and LY294002 augmentation of UV-induced apoptosis (Fig. 4A). Myristoylated Akt was capable of reducing UV-induced apoptosis up to a UV dose of 150mJ/cm2 (Fig. 4B). At higher UV doses, myristoylated Akt no longer was able to protect against UV-induced apoptosis. This lack of protection was likely due to large amount of cellular damage caused by exposure to high levels of UV irradiation. These data indicate that Akt rather than other PI3K effectors is primarily responsible for protecting human keratinocytes against UV-induced apoptosis. We next examined the effect of overexpression of dominant negative Akt-308T/A-473S/A in which threonine 308 and serine 473, which are sites of activating phosphorylations, were mutated to alanine. Infection of human keratinocytes with adenovirus expressing dominant negative Akt-308T/A-473S/A (5000 particles/cell) resulted in 10-fold increase in the Akt protein while infection with empty virus did not alter endogenous Akt protein levels as shown by Western blot analysis (Fig. 5). UV-induced DNA fragmentation was 2-fold greater in Akt-308T/A-473S/A-infected keratinocytes compared with keratinocytes infected with empty adenovirus (Fig. 5). This modest increase of UV-induced apoptosis was consistent with the modest ability (2-fold) of Akt-308T/A-473S/A to inhibit UV activation of endogenous Akt (data not shown). It is not known why inhibition of endogenous Akt by Akt-308T/A-473S/A in human keratinocytes is relatively weak. These data provide further support that Akt functions to block UV-induced apoptosis in human keratinocytes.
      Figure thumbnail gr4
      Fig. 4Constitutively active Akt blocks PI3K inhibitor LY294002-induced increase in UV-induced apoptosis in human keratinocytes. A, human keratinocytes were infected with empty adenovirus or adenovirus expressing constitutively active Akt (Adenovirus-Myr-Akt) at 5000 particles/cell in growth factor-free media for 1 h. Virus-containing media were removed, and keratinocytes were re-fed with fresh growth media. Twenty-four hours later, keratinocytes were exposed to UV irradiation (50mJ/cm2). Where indicated keratinocytes were treated with LY294002 (LY, 20 μm) 1 h prior to UV irradiation. Four hours later, keratinocytes were collected, and apoptosis was measured by DNA fragmentation ELISA. Inset shows a representative Western blot for total Akt after adenovirus myr-Akt or empty adenovirus infection. Data are shown as the means ± S.E.; *, p < 0.05 versus UV-treated empty virus infected keratinocytes, n = 3. B, human keratinocytes infected with empty virus (open bars) or Adenovirus-Myr-Akt (closed bars) were exposed to the indicated doses of UV. Data are shown as means ± S.E. Representative of two independent experiments.
      Figure thumbnail gr5
      Fig. 5Dominant negative Akt increases UV-induced apoptosis in human keratinocytes. Human keratinocytes were infected with control empty adenovirus or adenovirus expressing dominant negative-Akt (Adenovirus dn-Akt) at 5000 particles/cell in growth factorfree media for 1 h. Virus containing media were removed, and keratinocytes were re-fed with fresh growth media. Twenty-four hours later, keratinocytes were exposed to 50 mJ/cm2 UV irradiation. Four-hours after UV irradiation, keratinocytes were collected, and apoptosis was determined by DNA fragmentation ELISA. Inset shows a representative Western blot for total Akt after adenovirus dn-Akt or empty adenovirus infection. Data are shown as the means ± S.E.; *, p < 0.05 versus UV-treated empty virus infected keratinocytes, n = 3.
      Overexpression of Myristolyated Akt Blocks PI3K Inhibitor LY294002-induced Increase in UV-induced Mitochondrial Cytochrome c Release in Human Keratinocytes—Apoptosis is mediated by mitochondrial and death receptor pathways (
      • Kulms D.
      • Schwarz T.
      ). In mitochondrial-mediated death pathway, release of cytochrome c from mitochondria triggers the caspase cascade that is critical for the apoptotic process. UV-induced apoptosis has been shown to be reduced in a cytochrome c-deficient cell line, indicating that UV-induced mitochondrial cytochrome c release apoptosis plays an important role (
      • Li K.
      • Li Y.
      • Shelton J.
      • Richardson J.
      • Spencer E.
      • Chen Z.
      • Wang X.
      • Williams R.
      ). Akt has been shown to maintain mitochondria integrity and blocking cytochrome c release (
      • Kennedy S.
      • Kandel E.
      • Cross T.
      • Hay N.
      ). Therefore we examined the role of Akt in regulating UV-induced cytochrome c release. Keratinocytes were infected with Adeno-myr-Akt, or empty adenovirus, and pretreated with or without LY294002 for 1 h prior to irradiation with UV (50 mJ/cm2). In keratinocytes infected with empty adenovirus, UV irradiation caused significant release of mitochondrial cytochrome c. This release was substantially increased in the presence of LY294002 (Fig. 6). In contrast, overexpression of myristoylated Akt completely blocked UV-induced release of mitochondrial cytochrome c, in the absence or presence of the PI3K inhibitor LY294002 (Fig. 6).
      Figure thumbnail gr6
      Fig. 6Constitutively active Akt blocks PI3K inhibitor LY294002-induced increases in UV-induced mitochondria cytochrome c release in human keratinocytes. Human keratinocytes were infected with empty adenovirus or adenovirus expressing constitutively active Akt (Adenovirus-Myr-Akt) at 5000 particles/cell in growth factor-free media for 1 h. One hour later, virus containing media were removed, and keratinocytes were re-fed with fresh growth media. Twenty-four hours later, keratinocytes were pretreated with 20 μm LY294002 (LY) for 1 h and irradiated with 50 mJ/cm2 UV. Four hours after UV irradiation, keratinocytes were collected, and cytosolic proteins were isolated. 70 μg of cytosolic protein was separated on 14% Tris-glycine PAGE. Western analysis was performed with monoclonal anti-cytochrome c antibody (Clone 7H8.2C12). Inset shows a representative Western blot for cytochrome c. Bar heights are the means ± S.E. for fold increase in cytochrome c relative to levels in untreated control cells (Ctrl); *, p < 0.05 versus UV empty virus-infected keratinocytes, n = 3; †, p < 0.05 versus LY/UV-treated empty virus-infected keratinocytes, n = 3.
      The mechanism by which Akt blocks the mitochondria cytochrome c release could be mediated by Akt phosphorylation of a pro-apoptotic Bcl-2 family member Bad, which results in loss of Bad pro-apoptosis function. In fact, Bad has been shown to be phosphorylated by Akt at serine 136 and has been implicated in the mechanism by Akt mitigates mitochondrial cytochrome c release (
      • Datta S.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.
      ,
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ). However, we were unable to detect increased phosphorylated Bad in UV irradiated human keratinocytes (data not shown). Recently, Gottlob et al. (
      • Gottlob K.
      • Majewski N.
      • Kennedy S.
      • Kandel E.
      • Robey R.
      • Hay N.
      ) reported that coupling between glucose metabolism and mitochondria function is a prerequisite to Akt-mediated cell survival (
      • Hatano E.
      • Brenner D.
      ). It is sufficient for Akt to prevent mitochondrial cytochrome c release. Whether Akt-mediated increase of mitochondrial hexokinase activity, which catalyzes the first step of glycolysis, is found to be a mechanism(s) by which Akt blocks the mitochondria cytochrome c release from in human keratinocytes remains to be determined.
      Overexpression of Myristoylated Akt Blocks UV-induced Cleavage of Procaspase-9 and Procaspase-3 in Human Keratinocytes—Cytochrome c that is released into the cytosol complexes with apoptotic protease activating factor-1 (Apaf-1) and procaspase-9. Formation of this complex results in cleavage of procaspase-9 into active caspase-9, which in turn cleaves procaspase-3 into active caspase-3 (
      • Zou H.
      • Li Y.
      • Liu X.
      • Wang X.
      ,
      • Thornberry N.
      • Lazebnik Y.
      ). In keratinocytes infected with empty adenovirus, UV irradiation alone caused modest increase (25%) in procaspase-9 cleavage (Fig. 7A). UV induction of procaspase-9 cleavage was substantially increased (4-fold) in cells pretreated with LY294002. These UV-induced increases in cleavage of procaspase-9 were completely blocked in keratinocytes overexpressing myristoylated Akt (Fig. 7A). Similar results were observed for UV-induced cleavage of caspase-3. UV induction of caspase-3 cleavage was substantially enhanced by LY294002 pretreatment and blocked by overexpression of myristoylated Akt (Fig. 7B). These results indicate that UV activation of Akt opposes UV-induced activation of procaspase-9 and procaspase-3 in human keratinocytes.
      Figure thumbnail gr7
      Fig. 7Constitutively active Akt blocks PI3K inhibitor LY294002-induced increase in UV-induced cleavage/activation of procaspase-9 and -3 in human keratinocytes. Human keratinocytes were infected with empty adenovirus or adenovirus expressing constitutively active Akt (Adenovirus-Myr-Akt) at 5000 particles/cell in growth factor-free media for 1 h. One hour later, virus-containing media were removed, and keratinocytes were re-fed with fresh growth media. Twenty-four hours later, keratinocytes were pretreated with 20 μm LY294002 (LY) for 1 h and irradiated with 50 mJ/cm2 UV. Four hours later, keratinocytes were collected, and whole cell homogenate was prepared. 50 μg of protein were separated on 10 or 14% Tris-glycine PAGE. Western analysis was performed with polyclonal antibody against caspase-9 (A) and caspase-3 (B). Inset shows a representative Western blots for caspase-9 and -3. Bar heights are the means ± S.E. for fold increase in procaspase-9 and -3 and active caspase-9 and -3 relative to levels in untreated control cells (Ctrl). *, p < 0.05 versus LY/UV empty virus-infected keratinocytes, n = 4.
      Overexpression of Myristoylated Akt Blocks UV-induced Cleavage and Activation of Procaspase-8 in Human Keratinocytes—The death receptor Fas has been shown to be activated by UV irradiation, and its activation contributes to UV-induced apoptosis (
      • Aragane Y.
      • Kulms D.
      • Metze D.
      • Wilkes G.
      • Poppelmann B.
      • Luger T.
      • Schwarz T.
      ,
      • Caricchio R.
      • Reap E.
      • Cohen P.
      ,
      • Hill L.
      • Ouhtit A.
      • Loughlin S.
      • Kripke M.
      • Ananthaswamy H.
      • Owen-Schaub L.
      ). Fas activation results in receptor clustering and recruitment of the adapter protein FADD, and further recruiting procaspase-8 and formation of death-inducing signaling complex. Complex formation results in cleavage and activation of procaspase-8 (
      • Boldin M.
      • Varfolomeev E.
      • Pancer Z.
      • Mett I.
      • Camonis J.
      • Wallach D.
      ,
      • Boldin M.
      • Goncharov T.
      • Goltsev Y.
      • Wallach D.
      ). Caspase-8 then activates caspase-3 (
      • Boldin M.
      • Varfolomeev E.
      • Pancer Z.
      • Mett I.
      • Camonis J.
      • Wallach D.
      ,
      • Boldin M.
      • Goncharov T.
      • Goltsev Y.
      • Wallach D.
      ). We investigated UV activation of caspase-8 and its regulation by Akt. This UV activation of caspase-8 was further enhanced by pretreatment of keratinocytes with LY294002, in a dose-dependent manner (Fig. 8A). In cells infected with empty adenovirus, pretreatment with LY294002 substantially stimulated UV-induced cleavage of procaspase-8 (Fig. 8B). This cleavage of caspase-8 was blocked by overexpression of myristoylated Akt. UV irradiation caused a modest (50%) increase in caspase-8 activity (Fig. 8A). One possible mechanism by which Akt blocks capsase-8 activation is increased expression of c-FLIP, which inhibits caspase-8 activity (
      • Panka D.J.
      • Mano T.
      • Suhara T.
      • Walsh K.
      • Mier J.W.
      ,
      • Suhara T.
      • Mano T.
      • Oliveira B.E.
      • Walsh K.
      ,
      • Scaffidi C.
      • Schmitz I.
      • Krammer P.
      • Peter M.
      ,
      • Micheau O.
      • Thome M.
      • Schneider P.
      • Holler N.
      • Tschopp J.
      • Nicholson D.
      • Briand C.
      • Grutter M.
      ). Alternatively, Akt may indirectly block caspase-8 activation by blocking activation of caspase-9 that may precede caspase-8 activation in UV-induced apoptosis pathways (
      • Sitailo L.
      • Tibudan S.
      • Denning M.
      ).
      Figure thumbnail gr8
      Fig. 8PI3K inhibitor LY294002 increases UV-induced caspase-8 activity in human keratinocytes. A, human keratinocytes at 70% confluence were placed in growth factor-free media for 24 h, pretreated with 5 μm or 20 μm LY294002 (LY) for 1 h, and exposed to 50 mJ/cm2 UV. Two hours later, keratinocytes were collected, and caspase-8 activity was measured with an ELISA-based assay; *, p < 0.05 versus control (Ctrl). B, human keratinocytes were infected with empty adenovirus or adenovirus expressing constitutively active Akt (Adenovirus-Myr-Akt) at 5000 particles/cell in growth factor-free media for 1 h. One hour later, virus-containing media were removed, and keratinocytes were re-fed with fresh growth media. Twenty-four hours later, keratinocytes were pretreated with 20 μm LY for 1 h, and irradiated with 50 mJ/cm2 UV. Four hours later, keratinocytes were collected, and whole cell homogenate was prepared. 50 μg of protein were separated on 10 or 14% Tris-glycine PAGE. Western analysis was performed with polyclonal antibody against Caspase-8 and Procaspase-8. *, p < 0.05 versus LY/UV-treated keratinocytes, n = 4. B, inset shows a representative Western blots for caspase-8 and procaspase-8. Data are shown as the means ± S.E. *, p < 0.05 versus LY/UV empty virusinfected keratinocytes, n = 4.

      DISCUSSION

      We have investigated activation of PI3K/Akt and NF-κB pathways by UV irradiation and the function of these pathways in protecting human keratinocytes from UV-induced apoptosis. We demonstrate that UV irradiation activates Akt in primary human keratinocytes, and this activation is mediated by EGFR/PI3K. In contrast, UV-irradiation does not detectably induce nuclear localization of NF-κB in human keratinocytes. Most importantly we demonstrate that Akt provides a critical pro-survival function, which is independent of NF-κB, that protects human keratinocytes from UV-induced apoptosis. Furthermore, we find that Akt blocks UV-induced apoptosis by blocking mitochondrial cytochrome c release, preventing the activation of procaspase-9, and -3, and that Akt also blocks cleavage and activation of procaspase-8.
      UV irradiation activates apoptotic pathways that are triggered by DNA damage or death receptor activation (
      • Kulms D.
      • Schwarz T.
      ). Our data demonstrate that UV irradiation activates anti-apoptotic pathways, in addition to apoptotic pathways. The final fate of keratinocytes after UV irradiation apparently will depend on the balance between pro- and anti-apoptotic pathways. Human skin consists of regenerative basal cells and non-proliferative suprabasal cells that form the protective barrier. Since human skin is chronically exposed to UV light, protection of suprabasal cells against apoptosis is critical for maintenance of epidermal integrity and barrier function. However, protection of basal keratinocytes against apoptosis is a double-edged sword, since in addition to preserving the pool of proliferative cells, it may foster survival of cells harboring oncogenic mutations, and thereby promote skin carcinogenesis. Selective targeting of the PI3K/Akt pathway could increase the sensitivity of keratinocytes to apoptotic signals, and thereby might be an effective strategy to prevent skin cancer.

      References

        • Brunet A.
        • Bonni A.
        • Zigmond M.
        • Lin M.
        • Juo P.
        • Hu L.
        • Anderson M.
        • Arden K.
        • Blenis J.
        • Greenberg M.
        Cell. 1999; 96: 857-868
        • Shea R.
        • Parrish J.
        Goldsmith L. Physiology, Biochemistry, and Molecular Biology of the Skin. Vol. II. Oxford University Press, New York1991: 910-927
        • Weedon D.
        • Searle J.
        • Kerr J.
        Am. J. Dermatopathol. 1979; 1: 133-144
        • Ziegler A.
        • Jonason A.
        • Leffell D.
        • Simon J.
        • Sharma H.
        • Kimmelman J.
        • Remington L.
        • Jacks T.
        • Brash D.
        Nature. 1994; 372: 773-776
        • Leverkus M.
        • Yaar M.
        • Gilchrest B.A.
        Exp. Cell Res. 1997; 232: 255-262
        • Aragane Y.
        • Kulms D.
        • Metze D.
        • Wilkes G.
        • Poppelmann B.
        • Luger T.
        • Schwarz T.
        J. Cell Biol. 1998; 140: 171-182
        • Kulms D.
        • Schwarz T.
        Photodermatol. Photoimmunol. Photomed. 2000; 16: 195-201
        • Miyashita T.
        • Reed J.
        Cell. 1995; 80: 293-299
        • Finucane D.
        • Bossy-Wetzel E.
        • Waterhouse N.
        • Cotter T.
        • Green D.
        J. Biol. Chem. 1999; 274: 2225-2233
        • Jurgensmeier J.
        • Xie Z.
        • Deveraux Q.
        • Ellerby L.
        • Bredesen D.
        • Reed J.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002
        • Boldin M.
        • Varfolomeev E.
        • Pancer Z.
        • Mett I.
        • Camonis J.
        • Wallach D.
        J. Biol. Chem. 1995; 270: 7795-7798
        • Boldin M.
        • Goncharov T.
        • Goltsev Y.
        • Wallach D.
        Cell. 1996; 85: 803-815
        • Stein R.
        • Waterfield M.
        Mol. Med. Today. 2000; 6: 347-357
        • Bellacosa A.
        • Testa J.
        • Staal S.
        • Tsichlis P.
        Science. 1991; 254: 274-277
        • Alessi D.
        • James S.
        • Downes C.
        • Holmes A.
        • Gaffney P.
        • Reese C.
        • Cohen P.
        Curr. Biol. 1997; 7: 261-269
        • Alessi 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: 776-789
        • Persad 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: 27462-27469
        • Alessi D.
        • Andjelkovic M.
        • Caudwell B.
        • Cron P.
        • Morrice N.
        • Cohen P.
        • Hemmings B.
        EMBO J. 1996; 15: 6541-6551
        • Dudek 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: 661-665
        • Goswami R.
        • Kilkus J.
        • Dawson S.A.
        • Dawson G.
        J. Neurosci. Res. 1999; 57: 884-893
        • Kennedy S.G.
        • Wagner A.J.
        • Conzen S.D.
        • Jordan J.
        • Bellacosa A.
        • Tsichlis P.N.
        • Hay N.
        Genes Dev. 1997; 11: 701-713
        • Kulik G.
        • Klippel A.
        • Weber M.J.
        Mol. Cell Biol. 1997; 17: 1595-1606
        • Lee J.W.
        • Juliano R.L.
        Mol. Biol. Cell. 2000; 11: 1973-1987
        • Fujio Y.
        • Walsh K.
        J. Biol. Chem. 1999; 274: 16349-16354
        • Hermann C.
        • Assmus B.
        • Urbich C.
        • Zeiher A.M.
        • Dimmeler S.
        Arterioscler. Thromb. Vasc. Biol. 2000; 20: 402-409
        • Madrid L.
        • Mayo M.
        • Reuther J.
        • Baldwin Jr., A.
        J. Biol. Chem. 2001; 276: 18934-18940
        • Ozes O.
        • Mayo L.
        • Gustin J.
        • Pfeffer S.
        • Pfeffer L.
        • Donner D.
        Nature. 1999; 401: 82-85
        • Romashkova J.
        • Makarov S.
        Nature. 1999; 401: 86-90
        • Yuan Z.-Q.
        • Feldman R.
        • Sun M.
        • Olashaw N.
        • Coppola D.
        • Sussman G.
        • Shelley S.
        • Nicosia S.
        • Cheng J.
        J. Biol. Chem. 2002; 277: 29973-29982
        • Deveraux Q.
        • Reed J.
        Genes Dev. 1999; 13: 239-252
        • Meng F.
        • Liu L.
        • Chin P.
        • D'Mello S.
        J. Biol. Chem. 2002; 277: 29674-29680
        • Fisher G.
        • Talwar H.
        • Lin J.
        • Lin P.
        • McPhillips F.
        • Wang Z.
        • Li X.
        • Wan Y.
        • Kang S.
        • Voorhees J.
        J. Clin. Investig. 1998; 101: 1432-1440
        • Peus D.
        • Vasa R.
        • Meves A.
        • Beyerle A.
        • Pittelkow M.
        Photochem. Photobiol. 2000; 72: 135-140
        • Rosette C.
        • Karin M.
        Science. 1996; 274: 1194-1197
        • Piette J.
        • Piret B.
        • G B.
        • Schoonbroodt S.
        • Merville M.
        • Legrand-Poels S.
        • Bours V.
        Biol. Chem. 1997; 378: 1237-1245
        • Huang T.
        • Feinberg S.
        • Suryanarayanan S.
        • Miyamoto S.
        Mol. Cell. Biol. 2002; 22: 5813-5825
        • Fisher 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: 553-559
        • Li L.
        • Lorenzo P.
        • Bogi K.
        • Blumberg P.
        • Yuspa S.
        Mol. Cell Biol. 1999; 19: 8547-8558
        • Wang H.
        • Smart R.
        J. Cell Sci. 1999; 112: 3497-3506
        • Kennedy S.
        • Kandel E.
        • Cross T.
        • Hay N.
        Mol. Cell Biol. 1999; 19: 5800-5810
        • Gerard R.
        • Meidell R.
        Hames B. Glover D. DNA Cloning: A Practical Approach. Oxford University Press, Oxford1995: 300-302
        • Okano J.
        • Gaslightwala I.
        • Birnbaum M.
        • Rustgi A.
        • Nakagawa H.
        J. Biol. Chem. 2000; 275: 30934-30942
        • Lin J.
        • Adam R.M.
        • Santiestevan E.
        • Freeman M.
        Cancer Res. 1999; 59: 2891-2897
        • Kohn A.
        • Kovacina K.
        • Roth R.
        EMBO J. 1995; 14: 4288-4295
        • Burgering B.
        • Coffer P.
        Nature. 1995; 376: 599-602
        • Stover D.
        • Becker M.
        • Leiebetanz J.
        • Lydon N.
        J. Biol. Chem. 1995; 26: 15591-15597
        • Tiganis T.
        • Kemp B.
        • Tonks N.
        J. Biol. Chem. 1999; 274: 27768-27775
        • Fry 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: 12022-12027
        • Datta S.
        • Brunet A.
        • Greenberg M.
        Genes Dev. 1999; 13: 2905-2927
        • Hatano E.
        • Brenner D.
        Am. J. Physiol. Gastronintest. Liver Physiol. 2001; 1: G1357-G1368
        • Yang C.
        • Murti A.
        • Pfeffer S.
        • Kim J.
        • Donner D.
        • Pfeffer L.
        J. Biol. Chem. 2001; 276: 13756-13761
        • Delhase M.
        • Karin M.
        Cold Spring Harb. Symp. Quant. Biol. 1999; 64: 491-503
        • Seitz C.
        • Lin Q.
        • Deng H.
        • Khavari P.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2307-2312
        • Seitz C.
        • Freiberg R.
        • Hinata K.
        • Khavari P.
        J. Clin. Investig. 2000; 105: 253-260
        • Vanhaesebroeck B.
        • Waterfield M.
        Exp. Cell Res. 1999; 253: 239-254
        • Li K.
        • Li Y.
        • Shelton J.
        • Richardson J.
        • Spencer E.
        • Chen Z.
        • Wang X.
        • Williams R.
        Cell. 2000; 101: 389-399
        • Datta S.
        • Dudek H.
        • Tao X.
        • Masters S.
        • Fu H.
        • Gotoh Y.
        • Greenberg M.
        Cell. 1997; 91: 231-241
        • del Peso L.
        • Gonzalez-Garcia M.
        • Page C.
        • Herrera R.
        • Nunez G.
        Science. 1997; 278: 687-689
        • Gottlob K.
        • Majewski N.
        • Kennedy S.
        • Kandel E.
        • Robey R.
        • Hay N.
        Genes Dev. 2001; 15: 1406-1418
        • Zou H.
        • Li Y.
        • Liu X.
        • Wang X.
        J. Biol. Chem. 1999; 274: 11549-11556
        • Thornberry N.
        • Lazebnik Y.
        Science. 1998; 281: 1312-1316
        • Caricchio R.
        • Reap E.
        • Cohen P.
        J. Immunol. 1998; 161: 241-251
        • Hill L.
        • Ouhtit A.
        • Loughlin S.
        • Kripke M.
        • Ananthaswamy H.
        • Owen-Schaub L.
        Science. 1999; 285: 898-900
        • Panka D.J.
        • Mano T.
        • Suhara T.
        • Walsh K.
        • Mier J.W.
        J. Biol. Chem. 2001; 276: 6893-6896
        • Suhara T.
        • Mano T.
        • Oliveira B.E.
        • Walsh K.
        Circ. Res. 2001; 89: 13-19
        • Scaffidi C.
        • Schmitz I.
        • Krammer P.
        • Peter M.
        J. Biol. Chem. 1999; 274: 1541-1548
        • Micheau O.
        • Thome M.
        • Schneider P.
        • Holler N.
        • Tschopp J.
        • Nicholson D.
        • Briand C.
        • Grutter M.
        J. Biol. Chem. 2002; 277: 45162-45171
        • Sitailo L.
        • Tibudan S.
        • Denning M.
        J. Biol. Chem. 2002; 277: 19346-19352