Hypoosmotic stress stimulates growth in HepG2 cells via protein kinase B-dependent activation of activator protein-1

Although hypoosmotic stress-induced cell swelling activates phosphatidylino sitol-3-kinase, its impact on the downstream signal protein kinase B and ce ll growth is unknown. Activator protein-1 is in part phosphatidylinositol-3 -kinase dependent, and is important in proliferation. We hypothesized that cell swelling modulates proliferation in HepG2 cells via the protein kinase B-dependent activation of activator protein-1. HepG2 cells pretreated with or without LY294002 were exposed for up to 30 minutes to hypoosmotic mediu m (160 mOsm/L. Tumor necrosis factor-alpha (1.4 nmol/L) or normoosmolar med ium (270 mOsm/L) served as positive and negative controls, respectively. We stern immunoblots measured cytoplasmic phosphorylated and total protein kin ase B. Electromobility shift assays measured nuclear activator protein-1. M ethylene blue assays measured cell proliferation at 24, 48, and 72 hours af ter stimulation. Hypoosmotic stress phosphorylated protein kinase B by 10 m inutes. Subsequently, hypoosmotic exposure stimulated activator protein-1 b y 30 minutes. Pulse exposure to hypoosmotic stress potentiated HepG2 prolif eration by 72 hours as compared to both negative controls and LY-inhibited cells (n = 4 per group, P = 0.009 and 0.004, respectively; P < 0.001 analys is of variance. All three activation events were abolished with LY294002 pr etreatment. In HepG2 cells, hypoosmotic stress-induced swelling stimulates proliferation via protein kinase B-mediated activation of activator protein -1. These data delineate a possible mechanism linking changes in cell volum e to growth in human liver cancer.