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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.

Authors :
Bandara, Nadeeka
Gurusinghe, Saliya
Shiang Yong Lim
Haying Chen
Shuangfeng Chen
Dawei Wang
Hilbert, Bryan
Le-Xin Wang
Strappe, Padraig
Source :
Stem Cell Research & Therapy; 12/7/2016, Vol. 7, p1-15, 15p
Publication Year :
2016

Abstract

Background: Nitric 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-1<superscript>WT</superscript>) 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-1<superscript>WT</superscript>, and a CAV-1<superscript>F92A</superscript> (CAV-1<superscript>WT</superscript> mutant) and assessed NO-mediated osteogenic differentiation and the relationship with the Wnt signaling pathway. Methods: NO production was enhanced by lentiviral vector co-delivery of eNOS and CAV-1<superscript>F92A</superscript> 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. Results: NO production as measured by nitrite was significantly increased in eNOS and CAV-1<subscript>F92A</subscript> 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 mMLNAME resulted in reduced Runx2, ALP, and Wnt3a expressions, whilst Wnt5a expression was increased in eNOSdelivered 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. Conclusions: This 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. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
17576512
Volume :
7
Database :
Complementary Index
Journal :
Stem Cell Research & Therapy
Publication Type :
Academic Journal
Accession number :
120088494
Full Text :
https://doi.org/10.1186/s13287-016-0442-9