Wang Hui Qin, Quan Taihao, He Tianyuan, Franke Thomas F, Voorhees John J, Fisher Gary J
Department of Dermatology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0609, USA.
J Biol Chem. 2003 Nov 14;278(46):45737-45. doi: 10.1074/jbc.M300574200. Epub 2003 Sep 2.
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.
磷脂酰肌醇3激酶（PI3K）/Akt和核因子κB（NF-κB）信号通路在应激后均发挥促进细胞存活的作用。最近的证据表明，这两条信号通路的抗凋亡活性可能在功能上相互依赖。紫外线（UV）照射会引起氧化应激，进而导致细胞凋亡。人类皮肤细胞（角质形成细胞）经常暴露于来自太阳的紫外线照射下。我们研究了PI3K/Akt和NF-κB信号通路的激活情况及其在保护人类角质形成细胞（KCs）免受紫外线照射诱导的细胞凋亡中的作用。PI3K的这种激活先于活性/磷酸化Akt水平升高（3倍）。紫外线（来自UVB光源的50 mJ/cm2）照射导致PI3K迅速募集到表皮生长因子受体（EGFR）。用EGFR抑制剂PD169540预处理角质形成细胞可消除紫外线诱导的Akt激活/磷酸化，PI3K抑制剂LY294002或渥曼青霉素也有同样效果。通过流式细胞术和DNA片段化ELISA检测发现，这种对Akt激活的抑制与紫外线诱导的细胞凋亡增加3至4倍相关。与Akt不同，紫外线照射未检测到NF-κB的核定位明显增加，表明其未被强烈激活。与此观察结果一致，通过腺病毒介导的显性负性IKK-β或IkappaB-α的过表达干扰NF-κB激活并未增加紫外线诱导的细胞凋亡。然而，腺病毒介导的组成型活性Akt的过表达完全阻断了LY294002抑制PI3K时观察到的紫外线诱导的细胞凋亡，而腺病毒介导的显性负性Akt的过表达使紫外线诱导的细胞凋亡增加了2倍。抑制紫外线诱导的Akt激活使线粒体细胞色素c的释放增加了3.5倍，并导致半胱天冬酶-9、半胱天冬酶-8和半胱天冬酶-3的活性形式出现。组成型活性Akt消除了紫外线诱导的细胞色素c释放以及半胱天冬酶-9、-8和-3的激活。这些数据表明，PI3K/Akt对于保护人类角质形成细胞免受紫外线诱导的细胞凋亡至关重要，而NF-κB信号通路几乎没有（如果有的话）保护作用。
