Estradiol-17β stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes

Abstract

Although the importance of estradiol-17β (E₂) in many physiological processes has been reported, to date no researchers have investigated the effects of E₂ on embryonic stem (ES) cell proliferation. Therefore, in the present study, we have examined the effect of E₂ on the DNA synthesis of murine ES (ES-E14TG2a) cells and its related signaling pathways. The results of this study show that E₂ (10⁻⁹ M) significantly increased [³H]thymidine incorporation at >4 h and that E₂ (>10⁻¹² M) induced an increase of [³H]thymidine incorporation after 8-h incubation. Moreover, E₂ (>10⁻¹² M) also increased 5′-bromo-2′-deoxyuridine (BrdU) incorporation and cell number. Indeed, E₂ stimulated estrogen receptor (ER)-α and -β protein levels and increased mRNA expression levels of protooncogenes (c- fos, c- jun, and c- myc). Tamoxifen (antiestrogen) completely inhibited E₂-induced increases in [³H]thymidine incorporation. In addition, estradiol-6- O-carboxymethyl oxime-BSA (E₂-BSA; 10⁻⁹ M) increased [³H]thymidine incorporation at >1 h, and E₂-BSA (>10⁻¹² M) increased [³H]thymidine incorporation after 1-h incubation. E₂-BSA-induced increase in BrdU incorporation also occurred in a dose-dependent manner. Tamoxifen had no effect on E₂-BSA-induced increase of [³H]thymidine incorporation. Also, E₂ and E₂-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 and 5 min, respectively. E₂ increased cyclins D1 and E as well as cyclin-dependent kinase (CDK)2 and CDK4. In contrast, E₂ decreased the levels of p21^cip1 and p27^kip1 (CDK-inhibitory proteins). Increases of these cell cycle regulators were blocked by 10⁻⁵ M PD-98059 (MEK inhibitor). Moreover, E₂-induced increase of [³H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor). In conclusion, estradiol-17β stimulates the proliferation of murine ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes.