title: 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: 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-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-1WTmutant) and assessed NO-mediated osteogenic differentiation and the relationship with the Wnt signaling pathway.
  
  
  
  NO production was enhanced by lentiviral vector co-delivery of eNOS and CAV-1F92Ato 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.
  
  
  
  NO production as measured by nitrite was significantly increased in eNOS and CAV-1F92Atransduced 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-1WTtransgenes resulted in lower NO production. Canonical Wnt signaling pathway-associated Wnt3a and Wnt8a gene expressions were increased in eNOS-CAV-1F92Acells 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.
  
  
  
  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.
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.|O'Brien Institute Department, St. Vincent's Institute of Medical Research, Fitzroy, VIC, 3065, Australia.
authors.forename: Nadeeka
authors.initials: N
authors.lastname: Bandara
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.|School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.
authors.forename: Saliya
authors.initials: S
authors.lastname: Gurusinghe
authors.affiliation: O'Brien Institute Department, St. Vincent's Institute of Medical Research, Fitzroy, VIC, 3065, Australia.|Department of Surgery, St. Vincent's Hospital, University of Melbourne, Melbourne, VIC, 3002, Australia.
authors.forename: Shiang Yong
authors.initials: SY
authors.lastname: Lim
authors.affiliation: Department of Cardiology, Liaocheng People's Hospital and Affiliated Liaocheng People's Hospital of Shandong University, Liaocheng, Shandong, 252000, China.
authors.forename: Haying
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Cardiology, Liaocheng People's Hospital and Affiliated Liaocheng People's Hospital of Shandong University, Liaocheng, Shandong, 252000, China.
authors.forename: Shuangfeng
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Cardiology, Liaocheng People's Hospital and Affiliated Liaocheng People's Hospital of Shandong University, Liaocheng, Shandong, 252000, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Wang
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.
authors.forename: Bryan
authors.initials: B
authors.lastname: Hilbert
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.|Department of Cardiology, Liaocheng People's Hospital and Affiliated Liaocheng People's Hospital of Shandong University, Liaocheng, Shandong, 252000, China.
authors.forename: Le-Xin
authors.initials: LX
authors.lastname: Wang
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia. pstrappe@csu.edu.au.
authors.forename: Padraig
authors.initials: P
authors.lastname: Strappe
mesh_terms: D017667:Adipocytes / Q000378:metabolism / Q000502:physiology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D057809:HEK293 Cells; D006736:Horses; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010012:Osteogenesis / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013234:Stem Cells / Q000378:metabolism / Q000502:physiology*; D060449:Wnt Signaling Pathway / Q000502:physiology*; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D050659:Core Binding Factor Alpha 1 Subunit; D051176:beta Catenin; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D000469:Alkaline Phosphatase; D002118:Calcium