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-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.
Lee
S H
SH
Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea.
Lee
M Y
MY
Han
H J
HJ
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001205:Ascorbic Acid / Q000494:pharmacology; D015687:Cell Hypoxia / Q000502:physiology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D019938:Cyclin D1 / Q000096:biosynthesis; D019927:Cyclin E / Q000096:biosynthesis; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005419:Flavonoids / Q000494:pharmacology; D016193:G1 Phase / Q000502:physiology; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism*; D054510:Phospholipases A2, Cytosolic / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D015854:Up-Regulation
D016718:Arachidonic Acid; D001205:Ascorbic Acid; D002867:Chromones; D019938:Cyclin D1; D019927:Cyclin E; D018844:Cyclin-Dependent Kinases; D005419:Flavonoids; D007093:Imidazoles; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D054510:Phospholipases A2, Cytosolic; D011494:Protein Kinases; D051057:Proto-Oncogene Proteins c-akt; D011725:Pyridines; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C546842:MTOR protein, human; C546843:mTOR protein, mouse; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one