Mesenchymal stem cells (MSCs) have been isolated from numerous tissues such as adipose [ 1 ], heart [ 2 ], bone marrow [ 3 , 4 ], and blood [ 5 – 9 ], and have the potential to differentiate into different lineages, which includes osteoblasts, chondrocytes, and adipocytes [ 10 , 11 ]. The osteoblast differentiation program of MSCs is switched on by cellular recruitment, and timely expression of genes including Runx2, alkaline phosphatase (ALP), type I collagen (ColA1), plus osteocalcin (OC) followed by extracellular matrix mineralization [ 12 ]. This process can be induced by soluble molecules such as bone fragments morphogenetic proteins (BMPs) [ 13 ] or Wnts [ 14 – 16 ] that activate several pathways as well as other various downstream signals such as protein kinase [ 17 ] and growth factors [ 18 ] in order to trigger osteoblast differentiation of mesenchymal stem cells.
Nitric oxide (NO) is a signaling molecule with a short half-life [ 19 , 20 ]. It can respond within the cell where it is produced or penetrate cellular membranes to affect adjacent cells [ 21 ]. SIMPLY NO exerts a variety of physiological effects such as regulating blood pressure through smooth muscle relaxation [ 22 ], mediating immune reactions [ 23 ], controlling cell proliferation [ 24 ], modulating apoptosis [ 20 ], promoting growth factor-induced angiogenesis [ 4 , 21 ], accelerating wound healing [ 4 , 25 ], and working as a neurotransmitter [ 26 ]. These responses can be mediated through activating the primary NO effector soluble guanylyl cyclase to produce cGMP [ 27 ] by NO-based chemical substance modifications of proteins through S-nitrosylation [ 28 ] or through epigenetic modification [ 29 ]. NO is recognized to play an important role in bone homeostasis. It is produced by many cell types present in the bone environment, especially the osteoblast [ 30 ].
NO is synthesized from l -arginine by three isozymes of nitric oxide synthase (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS), and cytokine-inducible NOS (iNOS) [ 31 ]. Both iNOS [ 32 , 33 ] and eNOS [ 34 ] have been shown to play a role in osteoblast difference. Mice lacking eNOS have shown marked bone abnormalities because of impaired osteoblast differentiation resulting in poor maintenance of bone bulk [ 35 , 36 ]. Gene expression data from neonatal calvarial osteoblasts from eNOS – /– mice have shown downregulation of Runx2, Cbfa-1, and osteocalcin [ 37 ]. On the other hand, high concentrations of NO launched due to the pathological iNOS expression promote bone resorption by means of induced osteoclastogenesis [ 38 ]. Therefore , an optimum degree of NO is important to drive osteogenic differentiation of the MSCs.
In contrast with other NOS family members, eNOS is localized mainly in specific intracellular membrane domain names, including the Golgi apparatus [ 39 ] and lcd membrane caveolae [ 40 , 41 ]. A previously demonstrated immediate interaction of eNOS with wild-type caveolin-1 (CAV-1 WT ) [ 42 ] has suggested that CAV-1 WT functions being an endogenous negative regulator of eNOS [ 43 ]. In this particular context, eNOS binds to the caveolin-1 scaffolding domain (CSD; amino acids 82– 101) [ 44 ] and, additionally, Thr-90 and Thr-91 (T90 and T91), and Phe-92 (F92) were identified as critical residues for eNOS joining and inhibition [ 41 ]. Genetic modification of endothelial cells through overexpression of a mutated version of CAV-1 with a phenylalanine to alanine substitution at the amino acid placement 92 (CAV-1 F92A ) resulted in improved NO production, overcoming the inhibitory effect of CAV-1 WT [ 41 ].
In the present study, we tested the hypothesis that will molecular control of NO synthesis in equine adipose-derived come cells (eASCs), can promote osteogenic differentiation where endogenous eNOS is not available, by recreating the interaction in between eNOS and CAV-1 (CAV-1 WT and CAV-1 F92A ) regulates the particular osteogenic differentiation of eASCs. Our results indicate the optimum level of NO induces osteogenic differentiation through service of the downstream canonical Wnt/β -catenin signaling pathway.