Abstract
Fluid shear stress stimulates endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production through multiple kinases, including protein kinase A (PKA), AMP-activated protein kinase (AMPK), AKT and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an adaptor protein that stabilizes epithelial and endothelial cell-cell contacts. The aim of this study was to assess the unknown role of endothelial cell MAGI1 in response to fluid shear stress. We show constitutive expression and co-localization of MAGI1 with vascular endothelial cadherin (VE-cadherin) in endothelial cells at cellular junctions under static and laminar flow conditions. Fluid shear stress increases MAGI1 expression. MAGI1 silencing perturbed flow-dependent responses, specifically, Krüppel-like factor 4 (KLF4) expression, endothelial cell alignment, eNOS phosphorylation and NO production. MAGI1 overexpression had opposite effects and induced phosphorylation of PKA, AMPK, and CAMKII. Pharmacological inhibition of PKA and AMPK prevented MAGI1-mediated eNOS phosphorylation. Consistently, MAGI1 silencing and PKA inhibition suppressed the flow-induced NO production. Endothelial cell-specific transgenic expression of MAGI1 induced PKA and eNOS phosphorylation in vivo and increased NO production ex vivo in isolated endothelial cells. In conclusion, we have identified endothelial cell MAGI1 as a previously unrecognized mediator of fluid shear stress-induced and PKA/AMPK dependent eNOS activation and NO production.
Highlights
Nitric oxide (NO) is a key regulator of homeostasis and adaptive responses of the vascular system [1]
The main nitric oxide synthases (NOS) isoform expressed in endothelial cells is endothelial nitric oxide synthase (eNOS), which acts as an important regulator of essential vascular functions including vascular tone, angiogenesis, atheroprotection, anti-inflammatory and anti-thrombotic activities [3,4]
human umbilical vein endothelial cells (HUVEC) stimulation with vascular endothelial growth factors (VEGF) or thrombin, two physiological activators of endothelial cells, did not induce MAGI1 expression, suggesting that the observed induction is a rather specific response to shear stress. These results demonstrated that endothelial cell MAGI1 is induced by fluid shear stress and is involved in mediating endothelial cell response to it
Summary
Nitric oxide (NO) is a key regulator of homeostasis and adaptive responses of the vascular system [1]. NO is generated from L-arginine and molecular oxygen by a family of enzymes called nitric oxide synthases (NOS). There are three mammalian NOS isoforms: neuronal NOS (nNOS, NOS I), inducible NOS (iNOS, NOS II) and endothelial NOS (eNOS NOS III) [1]. They consist of an N-terminal oxygenase domain with binding sites for heme, l-arginine and tetrahydrobiopterin; Cells 2019, 8, 388; doi:10.3390/cells8050388 www.mdpi.com/journal/cells. The main NOS isoform expressed in endothelial cells is eNOS, which acts as an important regulator of essential vascular functions including vascular tone, angiogenesis, atheroprotection, anti-inflammatory and anti-thrombotic activities [3,4]. Regulation of eNOS is complex and occurs at transcriptional, post-transcriptional and post-translational levels [7,8]
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