title: Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1.
abstract: Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network.
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312 kim.tran@dmu.edu.
authors.forename: Quang-Kim
authors.initials: QK
authors.lastname: Tran
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Rachel
authors.initials: R
authors.lastname: Firkins
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Giles
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Sarah
authors.initials: S
authors.lastname: Francis
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Vahe
authors.initials: V
authors.lastname: Matnishian
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Phuong
authors.initials: P
authors.lastname: Tran
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Mark
authors.initials: M
authors.lastname: VerMeer
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Jake
authors.initials: J
authors.lastname: Jasurda
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Michelle Ann
authors.initials: MA
authors.lastname: Burgard
authors.affiliation: From the Department of Physiology and Pharmacology, College of Osteopathic Medicine, Des Moines University, Des Moines, Iowa 50312.
authors.forename: Briana
authors.initials: B
authors.lastname: Gebert-Oberle
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism*; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism; D013552:Swine
chemical_list: D002147:Calmodulin; D047628:Estrogen Receptor alpha; D004967:Estrogens; D004958:Estradiol; D052250:Nitric Oxide Synthase Type III; D053499:Plasma Membrane Calcium-Transporting ATPases
keywords: GPER/GPR30; calmodulin (CaM); endothelial nitric-oxide synthase; estrogen; estrogen receptor; estrogen receptor alpha; nitric-oxide synthase; plasma membrane calcium-transporting ATPase 4 (ATP2B4)