The Glycolytic Enzyme PFKFB3 Is Involved in Estrogen-Mediated Angiogenesis via GPER1

Abstract

The endogenous estrogen 17-estradiol (E2) is a key factor in promoting endothelial healing and angiogenesis. Recently, proangiogenic signals including vascular endothelial growth factor and others have been shown to converge in endothelial cell metabolism. Because inhibition of the glycolytic enzyme activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) reduces pathologic angiogenesis and estrogen receptor (ER) signaling stimulates glucose uptake and glycolysis by inducing PFKFB3 in breast cancer, we hypothesized that E2 triggers angiogenesis in endothelial cells via rapid ER signaling that requires PFKFB3 as a downstream effector. We report that treatment with the selective G protein-coupled estrogen receptor (GPER1) agonist G-1 (10 to 10 M) mimicked the chemotactic and proangiogenic effect of E2 as measured in a number of short-term angiogenesis assays in human umbilical vein endothelial cells (HUVECs); in addition, E2 treatment upregulated PFKFB3 expression in a time- and concentration-dependent manner. Such an effect peaked at 3 hours and was also induced by G-1 and abolished by pretreatment with the GPER1 antagonist G-15 or GPER1 siRNA, consistent with engagement of membrane ER. Experiments with the PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one showed that PFKFB3 activity was required for estrogen-mediated HUVEC migration via GPER1. In conclusion, E2-induced angiogenesis was mediated at least in part by the membrane GPER1 and required upregulation of the glycolytic activator PFKFB3 in HUVECs. These findings unravel a previously unrecognized mechanism of estrogen-dependent endocrine-metabolic crosstalk in HUVECs and may have implications in angiogenesis occurring in ischemic or hypoxic tissues.

Endothelial and non-endothelial responses to estrogen excess during development lead to vascular malformations

“…Polarized activation of Cdc42, Rac1 and RhoA is required for directional cell migration 3,4 . In agreement with previous studies 14,17,45–48 , our pharmacological data shows excess estrogen signaling promotes an increased activation of PI3K and Rho GTPase signaling. Importantly, it also indicates that hyperactivation of these pathways induces cell-cell disconnections within the intersegmental and axial vessels.…”

“…Our study shows that excess estrogens predominantly signal via Esr1 and Gper1, albeit a cooperative synergism with Esr2a and Esr2b may contribute to the etiology of the vascular defects. In addition to promoting Vegfa expression, ESR1 and GPER1 signaling from the plasma membrane promotes EC migration via activation of PI3K/AKT signaling and downstream effectors in the endothelium 14,17,45–48 . We show that endothelial Gper1 overexpression is sufficient to induce cell-cell disconnections and excessive migration, demonstrating for the first time a cell-autonomous role for Gper1 signaling in the etiology of vascular malformations.…”

Non‐genomic mechanisms in the estrogen regulation of glycolytic protein levels in endothelial cells

“…We previously established that E2 promotes angiogenesis via GPER-mediated rapid signaling and up-regulation of the key glycolytic protein PFKFB3 as a downstream effector. 17 Herein we showed that treatment with the selective GPER agonist G1 39 increased PFKFB3 protein levels in a time-dependent manner without affecting mRNA levels. PFKFB3 expression is tightly controlled at genomic and non-genomic levels to trigger glycolysis in a time-and spatial-specific way.…”

“…16,31 Conversely, we recently established that E2 promotes endothelial cell metabolism and angiogenesis by increasing PFKFB3 protein but not its mRNA levels in HUVECs. 17 To further investigate the mechanism of estrogen PFKFB3 regulation in HUVECs, we analyzed at different time points the effect of the selective GPER agonist G1. Similarly to what previously reported for E2, treatment with G1 (100 nM) increased PFKFB3 and the effect was maximum at 3 hours ( Figure 1A).…”

Estradiol Metabolism: Crossroads in Pulmonary Arterial Hypertension

“…The effects of estrogens on metabolic changes in pulmonary vasculature are unknown. Nonetheless, E2, via ERα-mediated upregulation of glycolytic enzymes, not only stimulates pathologic angiogenesis in breast cancer [186], but also boosts a mitogenic response in highly proliferative human endometrial cells and umbilical vein endothelial cells [187,188]. In contrast, because 2ME is a strong HIF-1α inhibitor, 2ME would be expected to inhibit metabolic reprograming in PAH.…”