Exposure to asphalt fumes activates activator protein-1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in mouse epidermal cells

Occupational exposure to asphalt fumes may pose a health risk. Experimental studies using animal and in vitro models indicate that condensates from asphalt fumes are genotoxic and can promote skin tumorigenesis. Enhanced activity of activator protein-1 (AP-1) is frequently associated with the promotion of skin tumorigenesis. The current study investigated the effect of exposure to asphalt fumes on AP-1 activation in mouse JB6 P+ epidermal cells and the skin of transgenic mice expressing the AP-1 luciferase reporter gene. Asphalt fumes were generated from a dynamic generation system that simulated road-paving conditions. Exposure to asphalt fumes significantly increased AP-1 activity in JB6 P+ cells as well as in cultured keratinocytes isolated from transgenic mice expressing AP-1 reporter. In addition, topical application of asphalt fumes by painting the tail skin of mice increased AP-1 activity by 14-fold. Exposure to asphalt fumes promoted basal as well as epidermal growth factor-stimulated anchorage-independent growth of JB6 P+ cells in soft agar. It activated phosphatidylinositol 3-kinase and induced phosphorylation of Akt at Ser-473/Thr-308, and concurrently activated downstream p70 S6 kinase as well as glycogen synthase kinase-3beta. Asphalt fumes transiently activated c-Jun NH2-terminal kinases without affecting extracellular signal-regulated kinases and p38 mitogen-activated protein kinases. Further study indicated that blockage of phosphatidylinositol 3-kinase activation eliminated asphalt fume-stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. This is the first report showing that exposure to asphalt fumes can activate AP-1 and intracellular signaling that may promote skin tumorigenesis, thus providing important evidence on the potential involvement of exposure to asphalt fumes in skin carcinogenesis.     

Cholecystokinin activates pancreatic calcineurin-NFAT signaling in vitro and in vivo

Elevated endogenous cholecystokinin (CCK) release induced by protease inhibitors leads to pancreatic growth. This response has been shown to be mediated by the phosphatase calcineurin, but its downstream effectors are unknown. Here we examined activation of calcineurin-regulated nuclear factor of activated T-cells (NFATs) in isolated acinar cells, as well as in an in vivo model of pancreatic growth. Western blotting of endogenous NFATs and confocal imaging of NFATc1-GFP in pancreatic acini showed that CCK dose-dependently stimulated NFAT translocation from the cytoplasm to the nucleus within 0.5-1 h. This shift in localization correlated with CCK-induced activation of NFAT-driven luciferase reporter and was similar to that induced by a calcium ionophore and constitutively active calcineurin. The effect of CCK was dependent on calcineurin, as these changes were blocked by immunosuppressants FK506 and CsA and by overexpression of the endogenous protein inhibitor CAIN. Parallel NFAT activation took place in vivo. Pancreatic growth was accompanied by an increase in nuclear NFATs and subsequent elevation in expression of NFAT-luciferase in the pancreas, but not in organs unresponsive to CCK. The changes also required calcineurin, as they were blocked by FK506. We conclude that CCK activates NFATs in a calcineurin-dependent manner, both in vitro and in vivo.     

Activation of NFAT signal by p53-K120R mutant

The tumor suppressor p53 is activated by phosphorylation and/or acetylation. We constructed 14 non-phosphorylated, 11 phospho-mimetic, and 1 non-acetylated point p53 mutations and compared their transactivation ability in U-87 human glioblastoma cells by the luciferase reporter assay. Despite mutations at the phosphorylation sites, only the p53-K120R and p53-S9E mutants had marginally reduced activities. On the other hand, the Nuclear factor of activated T-cells (NFAT)-luciferase reporter was more potently activated by p53-K120R than by wild-type p53 and other mutants in glioblastoma, hepatoma and esophageal carcinoma cells. This suggests that acetylation at Lys-120 of p53 negatively regulates a signaling pathway leading to NFAT activation.