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 ₂O₂ 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 [³H]-thymidine incorporation and cell counting. Hypoxia also increased the level of [³H]-arachidonic acid release and inhibition of cPLA₂ reduced hypoxia-induced increase in levels of the cell-cycle regulatory proteins and [³H]-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 [³H]- thymidine incorporation. Indeed, the percentage of cells in S phase, levels of cell cycle regulatory proteins, and [³H]-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 [³H]-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 [³H]- 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 G₁ phase cyclins D₁ and E, and CDK 2 and 4, in mouse embryonic stem cells through the cooperation of PI3K/Akt/mTOR, MAPK and cPLA₂-mediated signal pathways.