Short‐period hypoxia increases mouse embryonic stem cell proliferation through cooperation of arachidonic acid and PI3K/Akt signalling pathways

.  Hypoxia plays important roles in some early stages of mammalian embryonic development and in various physiological functions. This study examined the effect of arachidonic acid on short‐period hypoxia‐induced regulation of G 1 phase cell‐cycle progression and inter‐relationships among possible signalling molecules in mouse embryonic stem cells. Hypoxia increased the level of hypoxia‐inducible factor‐1α (HIF‐1α) expression and H 2 O 2 generation in a time‐dependent manner. In addition, hypoxia increased the levels of cell‐cycle regulatory proteins (cyclin D 1 , cyclin E, cyclin‐dependent kinase 2 (CDK2) and CDK4). Maximum increases in the level of these proteins and retinoblastoma phosphorylation were observed after 12–24 h of exposure to hypoxic conditions, and then decreased. Alternatively, the level of the CDK inhibitors, p21 Cip1 and p27 Kip1 were decreased. These results were consistent with the results of [ 3 H]‐thymidine incorporation and cell counting. Hypoxia also increased the level of [ 3 H]‐arachidonic acid release and inhibition of cPLA 2 reduced hypoxia‐induced increase in levels of the cell‐cycle regulatory proteins and [ 3 H]‐thymidine incorporation. The level of cyclooxygenase‐2 (COX‐2) was also increased by hypoxia and inhibition of COX‐2 decreased the levels of cell‐cycle regulatory proteins and [ 3 H]‐thymidine incorporation. Indeed, the percentage of cells in S phase, levels of cell cycle regulatory proteins, and [ 3 H]‐thymidine incorporation were further increased in hypoxic conditions with arachidonic acid treatment compared to normoxic conditions. Hypoxia‐induced Akt and mitogen‐activated protein kinase (MAPK) phosphorylation was inhibited by vitamin C (antioxidant, 10 −3 M). In addition, hypoxia‐induced increase of cell‐cycle regulatory protein expression and [ 3 H]‐thymidine incorporation were attenuated by LY294002 (PI3K inhibitor, 10 −6 M), Akt inhibitor (10 −6 M), rapamycin (mTOR inhibitor, 10 −9 M), PD98059 (p44/42 inhibitor, 10 −5 M), and SB203580 (p38 MAPK inhibitor, 10 −6 M). Furthermore, hypoxia‐induced increase of [ 3 H]‐arachidonic acid release was blocked by PD98059 or SB203580, but not by LY294002 or Akt inhibitor. In conclusion, arachidonic acid up‐regulates short time‐period hypoxia‐induced G 1 phase cyclins D 1 and E, and CDK 2 and 4, in mouse embryonic stem cells through the cooperation of PI3K/Akt/mTOR, MAPK and cPLA 2 ‐mediated signal pathways.