Abstract
Key points: The catechol metabolites of 17β-oestradiol (E2 β), 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate proliferation of pregnancy-derived ovine uterine artery endothelial cells (P-UAECs) through β-adrenoceptors (β-ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2-OHE2 - and 4-OHE2 -induced P-UAEC proliferation, as well as proliferation induced by the parent hormone E2 β and other β-AR signalling hormones (i.e. catecholamines). Conversely, although 2-OHE2 and 4-OHE2 rapidly activate phosphatidylinositol 3-kinase (PI3K), its activation is not involved in catecholoestradiol-induced P-UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol-induced P-UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2 β- and catecholamine-induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy. Previously we demonstrated that the biologically active metabolites of 17β-oestradiol, 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate pregnancy-specific proliferation of uterine artery endothelial cells derived from pregnant (P-UAECs), but not non-pregnant ewes. However, unlike 17β-oestradiol, which induces proliferation via oestrogen receptor-β (ER-β), the catecholoestradiols mediate P-UAEC proliferation via β-adrenoceptors (β-AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P-UAECs. P-UAECs were treated with 2-OHE2 and 4-OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24 h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)-AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist-induced P-UAEC proliferation. 2-OHE2 and 4-OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol-induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β-oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P-UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol-, 17β-oestradiol- and catecholamine-induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.