Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H(2)O(2)-induced proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10(-4) M H(2)O(2) for 1 h. In addition, H(2)O(2) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen-activated protein kinase (MAPK), and JNK/SAPK. Moreover, H(2)O(2) induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H(2)O(2)-induced cytosolic phospholipase A(2) (cPLA(2)) activation and AA release. H(2)O(2) increased NF-kappaB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)-2 proteins. Subsequently, H(2)O(2) stimulated PGE(2) synthesis, which was reduced by the inhibition of NF-kappaB activation. Moreover, each H(2)O(2) or PGE(2) increased DNA synthesis and the number of cells. However, H(2)O(2)-induced increase in DNA synthesis was inhibited by the suppression of cPLA(2) pathway. In conclusion, H(2)O(2) increased AA release and PGE(2) production by the upregulation of cPLA(2) and COX-2 via Ca(2+)/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells.

Epinephrine is a catecholamine that plays important roles in regulating a wide variety of physiological systems by acting through the adrenergic receptors (ARs). The cellular responses to AR stimulation are mediated through various signaling pathways. Therefore, this study examined the effects of epinephrine on DNA synthesis and related signaling molecules in mouse embryonic stem cells (ESCs). Epinephrine increased DNA synthesis in a dose- and time-dependent manner, as determined by the level of [(3)H]-thymidine incorporation. AR subtypes (alpha1(A), alpha2(A), beta1, beta2, and beta3) were expressed in mouse ESCs and their expression levels were increased by epinephrine. In this experiment, epinephrine increased cAMP levels, intracellular Ca(2+) concentration ([Ca(2+)](i)), and translocation of protein kinase C (PKC) from the cytosol to the membrane compartment. In addition, we observed Akt phosphorylation in response to epinephrine; this was stimulated by phosphorylation of the epidermal growth factor receptor (EGFR). Epinephrine also induced phosphorylation of ERK1/2 (p44/42 MAPKs), while inhibition of PKC or Akt blocked this phosphorylation. Epinephrine increased the mRNA levels of proto-oncogenes (c-fos, c-jun, c-myc), while inhibition of ERK1/2 decreased these mRNA levels. In experiments aimed at examining the involvement of cell cycle regulatory proteins, epinephrine increased the levels of cyclin E/cyclin-dependent kinase 2 (CDK2) and cyclin D1/cyclin-dependent kinase 4 (CDK4). In conclusion, epinephrine stimulates DNA synthesis via ERK1/2 through cAMP, Ca(2+)/PKC, and PI3K/Akt signaling pathways in mouse ESCs.

Hydrogen peroxide (H₂O₂) has been shown to act as a signaling molecule that is involved in many cellular functions. This study investigated the effect of H₂O₂ on the [³H]-2-deoxyglucose (2-DG) uptake and its related signaling pathways in mouse embryonic stem (ES) cells. H₂O₂ significantly increased the level of 2-DG uptake in a time- (> 4 hr) and concentration- (>10^-4 M) dependent manner due to an increase in V_max but not K_m. Indeed, H₂O₂ increased the mRNA and protein level of glucose transporter 1 (GLUT 1). PD 98059 (a p44/42 MAPKs inhibitor, 10^-5 M), SB 203580 (a p38 MAPK inhibitor, 10^-6 M), and SP 600125 (a SAPK/JNK inhibitor, 10^-6 M) blocked the H₂O₂-induced increase in 2-DG uptake. H₂O₂ also increased phosphorylation of p44/42 mitogen activated protein kinases (MAPKs), p38 MAPK, and stress-activated protein kinase/Jun-N-terminal kinase (SAPK/JNK). In addition, H₂O₂ stimulated the translocation of cytosolic phospholipase A₂ (cPLA₂) from the cytosolic fraction to the membrane fraction, the release of arachidonic acid, and the activation of NF-κB. AACOCF₃ or mepacrine (cPLA₂ inhibitors, 10^-6 M), SN 50 (NF-κB nucleus translocation inhibitor, 500 ng/ml) or Bay11-7082 (a IκB-α phosphorylation inhibitor, 2x10^-5 M) blocked the H₂O₂-induced increase in 2-DG uptake. H₂O₂ increased the protein level of glucose transporter 1 (GLUT 1), which was blocked by PD 98059, SB 203580, SP 600125, mepacrine, or Bay11-7082. In conclusion, H₂O₂ increases the 2-DG uptake via MAPKs, cPLA₂, and NF-κB signaling pathways in mouse ES cells.