Convergent ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) signalling mediate catecholoestradiol‐induced proliferation of ovine uterine artery endothelial cells

Abstract

Previously we demonstrated that the biologically active metabolites of 17β-oestradiol, 2-hydroxyoestradiol (2-OHE ) and 4-hydroxyoestradiol (4-OHE ), 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-OHE and 4-OHE 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-OHE and 4-OHE 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.

Angiogenesis during pregnancy: all routes lead to MAPKs

“…in this issue of The Journal of Physiology demonstrates that E2β, 2‐hydroxyoestradiol, 4‐hydroxyoestradiol, norepinephrine, and epinephrine stimulate proliferation of uterine artery endothelial cells via activation of three convergent MAPK pathways (ERK1/2, p38 and JNK) (Landeros et al . ). The observations in the present and previous studies from the Magness laboratory are interesting.…”

Decisive role of P42/44 mitogen-activated protein kinase in Δ9-tetrahydrocannabinol-induced migration of human mesenchymal stem cells

“…Noteworthy, the mode of p42/44 MAPK activation by THC was found to be biphasic which is in line with cannabinoid-induced p42/44 MAPK activation in neuroglioma cells [ 28 ]. In the latter study, the endocannabinoid analogue R(+)-methanandamide was shown to confer an early peak after 15 min and a delayed peak after 4 and 8 h. Similar biphasic p42/44 MAPK activations were reported for doxorubicin in neuronal cells [ 29 ], for catechol metabolites of 17β-estradiol in pregnancy-derived ovine uterine artery endothelial cells [ 30 ] and uridine 5´-triphosphate-treated Schwannoma cells [ 31 ]. In the latter investigation biphasic p42/44 MAPK regulation was likewise associated with increased cellular migration [ 31 ].…”

Effects of the Catechol and Methoxy Metabolites of 17β-Estradiol on Nitric Oxide Production by Ovine Uterine Artery Endothelial Cells

“…All procedures were approved by the University of Wisconsin-Madison Research Animal Care and Use Committees for both the Medical School and the College of Agricultural and Life Sciences, following the recommendation from the American Veterinary Medicine Association guidelines for humane treatment and euthanasia of laboratory farm animals as described by Landeros et al 41 Briefly, UAECs were obtained from P ewes (gestational age ¼ 120-130; term ¼ 147 days) or luteal phase NP ewes of mixed Western and Polypay breeds and euthanized under sodium pentobarbital (50-70 mg/kg) anesthesia through bilateral thoracotomy and exsanguination by cardiac laceration. Uterine artery endothelial cells were isolated from both left and right uterine arteries by collagenase digestion and cultured in growth media, endothelial basal media (EBM), with 20% fetal bovine serum, 100 mg/mL penicillin, and 100 mg/mL streptomycin.…”