Molecular control of nitric oxide synthesis through eNOS and caveolin-1 interaction regulates osteogenic differentiation of adipose-derived stem cells by modulation of Wnt/β-catenin signaling

Abstract

BackgroundNitric oxide (NO) plays a role in a number of physiological processes including stem cell differentiation and osteogenesis. Endothelial nitric oxide synthase (eNOS), one of three NO-producing enzymes, is located in a close conformation with the caveolin-1 (CAV-1WT) membrane protein which is inhibitory to NO production. Modification of this interaction through mutation of the caveolin scaffold domain can increase NO release. In this study, we genetically modified equine adipose-derived stem cells (eASCs) with eNOS, CAV-1WT, and a CAV-1F92A (CAV-1WT mutant) and assessed NO-mediated osteogenic differentiation and the relationship with the Wnt signaling pathway.MethodsNO production was enhanced by lentiviral vector co-delivery of eNOS and CAV-1F92A to eASCs, and osteogenesis and Wnt signaling was assessed by gene expression analysis and activity of a novel Runx2-GFP reporter. Cells were also exposed to a NO donor (NONOate) and the eNOS inhibitor, l-NAME.ResultsNO production as measured by nitrite was significantly increased in eNOS and CAV-1F92A transduced eASCs +(5.59 ± 0.22 μM) compared to eNOS alone (4.81 ± 0.59 μM) and un-transduced control cells (0.91 ± 0.23 μM) (p < 0.05). During osteogenic differentiation, higher NO correlated with increased calcium deposition, Runx2, and alkaline phosphatase (ALP) gene expression and the activity of a Runx2-eGFP reporter. Co-expression of eNOS and CAV-1WT transgenes resulted in lower NO production. Canonical Wnt signaling pathway-associated Wnt3a and Wnt8a gene expressions were increased in eNOS-CAV-1F92A cells undergoing osteogenesis whilst non-canonical Wnt5a was decreased and similar results were seen with NONOate treatment. Treatment of osteogenic cultures with 2 mM l-NAME resulted in reduced Runx2, ALP, and Wnt3a expressions, whilst Wnt5a expression was increased in eNOS-delivered cells. Co-transduction of eASCs with a Wnt pathway responsive lenti-TCF/LEF-dGFP reporter only showed activity in osteogenic cultures co-transduced with a doxycycline inducible eNOS. Lentiviral vector expression of canonical Wnt3a and non-canonical Wnt5a in eASCs was associated with induced and suppressed osteogenic differentiation, respectively, whilst treatment of eNOS-osteogenic cells with the Wnt inhibitor Dkk-1 significantly reduced expressions of Runx2 and ALP.ConclusionsThis study identifies NO as a regulator of canonical Wnt/β-catenin signaling to promote osteogenesis in eASCs which may contribute to novel bone regeneration strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0442-9) contains supplementary material, which is available to authorized users.

High Expression of CD200 and CD200R1 Distinguishes Stem and Progenitor Cell Populations within Mammary Repopulating Units

“…Among the numerous pathways supporting the higher metabolic activity of MRU not CD200/CD200R1 , the most highly expressed ones were involved in differentiation and function of progenitors, suggesting that the MRU not CD200/CD200R1 are not stem cells ( Figure S4 ). For example, eNOS and nitric oxide signaling have been associated with osteogenic differentiation through activation of the downstream canonical WNT/β-catenin signaling pathway ( Bandara et al., 2016 ). Higher levels of reactive oxygen species have been recently identified as a marker of progenitors when compared with stem cells in the mammary gland ( Diehn et al., 2009 ).…”

Uptake and transport of pullulan acetate nanoparticles in the BeWo b30 placental barrier cell model

“…Caveolin-1 is a key negative regulator of endothelial nitric oxide synthase (eNOS) activation. 28 Under physiological conditions, caveolin-1 can bind to eNOS, leading to eNOS inhibition and reduced NO production. 29 eNOS binds to the caveolin-1 scaffolding domain and remains inactive when bound.…”

Endothelial nitric oxide synthase (−786T>C) polymorphism and migraine susceptibility

“…Nitric oxide (NO) is a signaling molecule with a short half-life, which is synthesized from L-arginine by 3 isozymes of nitric oxide synthase (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). [ 6 ] It exerts a variety of physiological effects such as regulating blood pressure via smooth muscle relaxation, and functioning as a neurotransmitter. [ 7 ] NO in the migraine pathogenesis has been reported in several studies.…”