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

Abstract

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-1alpha (HIF-1alpha) expression and H2O2 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 cPLA(2) 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 [(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.