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.
Highlights
Nitric oxide (NO) plays a role in a number of physiological processes including stem cell differentiation and osteogenesis
ENOS expression was absent in eASCWT (Fig. 1a), whereas strong Endothelial nitric oxide synthase (eNOS) expression was observed in eNOS-transduced cells (Fig. 1b)
ENOS expression was detected at the cytoplasm (Fig. 1e), whereas both the CAV-1F92A (Fig. 1f) and CAV-1WT (Fig. 1g) expressions were observed at the plasma membrane, confirming that F92A mutation of CAV-1 does not affect its cellular localization
Summary
Nitric oxide (NO) plays a role in a number of physiological processes including stem cell differentiation and osteogenesis. The osteoblast differentiation program of MSCs is switched on by cell recruitment, and timely expression of genes including Runx, alkaline phosphatase (ALP), type I collagen (ColA1), and osteocalcin (OC) followed by extracellular matrix mineralization [12] This process can be induced by soluble molecules such as bone morphogenetic proteins (BMPs) [13] or Wnts [14,15,16] that activate several pathways and other various downstream signals such as protein kinase [17] and growth factors [18] to trigger osteoblast differentiation of mesenchymal stem cells. It is generated by many cell types present in the bone environment, most notably the osteoblast [30]
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