Background: Nitric oxide (NO) plays a role in a number of physiological processes including stem cell differentiation\nand osteogenesis. Endothelial nitric oxide synthase (eNOS), one of three NO-producing enzymes, is located in a close\nconformation with the caveolin-1 (CAV-1WT) membrane protein which is inhibitory to NO production. Modification of\nthis interaction through mutation of the caveolin scaffold domain can increase NO release. In this study, we genetically\nmodified equine adipose-derived stem cells (eASCs) with eNOS, CAV-1WT, and a CAV-1F92A (CAV-1WT mutant) and\nassessed NO-mediated osteogenic differentiation and the relationship with the Wnt signaling pathway.\nMethods: NO production was enhanced by lentiviral vector co-delivery of eNOS and CAV-1F92A to eASCs, and\nosteogenesis and Wnt signaling was assessed by gene expression analysis and activity of a novel Runx2-GFP reporter.\nCells were also exposed to a NO donor (NONOate) and the eNOS inhibitor, L-NAME.\nResults: NO production as measured by nitrite was significantly increased in eNOS and CAV-1F92A transduced eASCs\n+(5.59 �± 0.22 �¼M) compared to eNOS alone (4.81 �± 0.59 �¼M) and un-transduced control cells (0.91 �± 0.23 �¼M) (p < 0.05).\nDuring osteogenic differentiation, higher NO correlated with increased calcium deposition, Runx2, and alkaline\nphosphatase (ALP) gene expression and the activity of a Runx2-eGFP reporter. Co-expression of eNOS and CAV-1WT\ntransgenes resulted in lower NO production. Canonical Wnt signaling pathway-associated Wnt3a and Wnt8a gene\nexpressions were increased in eNOS-CAV-1F92A cells undergoing osteogenesis whilst non-canonical Wnt5a was\ndecreased and similar results were seen with NONOate treatment. Treatment of osteogenic cultures with 2 mMLNAME\nresulted in reduced Runx2, ALP, and Wnt3a expressions, whilst Wnt5a expression was increased in eNOSdelivered\ncells. Co-transduction of eASCs with a Wnt pathway responsive lenti-TCF/LEF-dGFP reporter only showed\nactivity in osteogenic cultures co-transduced with a doxycycline inducible eNOS. Lentiviral vector expression of\ncanonical Wnt3a and non-canonical Wnt5a in eASCs was associated with induced and suppressed osteogenic\ndifferentiation, respectively, whilst treatment of eNOS-osteogenic cells with the Wnt inhibitor Dkk-1 significantly\nreduced expressions of Runx2 and ALP.\nConclusions: This study identifies NO as a regulator of canonical Wnt/�²-catenin signaling to promote osteogenesis in\neASCs which may contribute to novel bone regeneration strategies.
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