title: Epidermal growth factor receptor-dependent, NF-kappaB-independent activation of the phosphatidylinositol 3-kinase/Akt pathway inhibits ultraviolet irradiation-induced caspases-3, -8, and -9 in human keratinocytes.
abstract: Both phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB 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-kappaB 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-kappaB, indicating that it was not strongly activated. Consistent with this observation, interference with NF-kappaB activation by adenovirus-mediated overexpression of dominant negative IKK-beta or IkappaB-alpha did not increase UV-induced apoptosis. However, adenovirus-mediated 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-kappaB pathway provides little, if any, protective role.
authors.affiliation: Department of Dermatology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0609, USA.
authors.forename: Hui Qin
authors.initials: HQ
authors.lastname: Wang
authors.forename: Taihao
authors.initials: T
authors.lastname: Quan
authors.forename: Tianyuan
authors.initials: T
authors.lastname: He
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Franke
authors.forename: John J
authors.initials: JJ
authors.lastname: Voorhees
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Fisher
mesh_terms: D000178:Acrylamides / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011799:Quinazolines / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D000178:Acrylamides; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; C000601814:N-(4-(3-bromophenylamino)-7-(3-(4-morpholinyl)propoxy)-6-quinazolinyl)-2-propenamide; D016328:NF-kappa B; D011799:Quinazolines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases