synthesis of collagen type I and Runx2. Interestingly, in opposite to MSC-cultures, when nicotinamide was added to pre-osteoblastic MC3T3-E1 cells, no significant effect was seen on formation of adipocytes and PPAR-c expression compared with MSCs and this was in a concentration-dependent manner. Moreover, pre-treatment of pre-osteoblastic MC3T3-E1 cells with resveratrol followed by stimulation with nicotinamide resulted in an inhibition of nicotinamideinduced effects on collagen type I production and Runx2 and downregulated PPAR-c in high-density cultures. However, 1 mM resveratrol could not completely inhibit the blocking effect of 100 mM nicotinamide on the synthesis of collagen type I and Runx2 in high-density culture. Taken together, these results indicate that adipocytes and osteoblasts share a common progenitor, i.e. MSCs expressing PPAR-c signaling can induce trans-differentiation of osteoblasts to adipocytes by inhibiting of Runx2, whereas, the pre-osteoblastic cells only have the capability to differentiate into osteoblasts. Resveratrol inhibits nicotinamide-induced downregulation of Sirt-1 during osteogenic differentiation of MSCs in vitro To investigate the possible mechanism for dedifferentiation of MSCs to adipocytes during osteogenesis, we investigated the effect of resveratrol on the expression of Sirt-1. As shown in Sirt-1 blocks adipogenesis by repressing PPAR-c activity and NCoR involvement in this process The nuclear receptor PPAR-c is known to regulate adipogenesis. It has also been shown that the nuclear receptor co-repressor, NCoR, binds to known PPAR-c sites of promoters of adipogenic genes in differentiated 3T3-L1 adipocytes. To test whether Sirt-1 is a PPAR-c co-repressor by means of NCoR, we pretreated the MSCs with resveratrol followed by stimulation with nicotinamide in high-density cultures, and co-immunoprecipitation assays. As shown in Expression of Sirt-1 in MSCs before and after osteoblastic differentiation in vitro The NAD-dependent protein deacetylase Sirt-1 has been shown to attenuate development of adipocytes from pre-adipocytes through inhibition of PPAR-c activity. Next, we wanted to evaluate whether phytochemicals known to regulate the activity of Sirt-1 could influence the formation of MSCs during osteoblast differentiation in vitro. First, we could demonstrate the expression of Sirt-1 in the MSCs derived from fat tissue. Whole cell lysate from MSCs in monolayer cultures treated with osteogenic induction medium for 0, 7, 14 and 21 days were analyzed by western blot with anti-Sirt-1 antibody. Sirt-1 was expressed in the MSCs before and after induction of osteoblastic differentiation. Resveratrol blocks nicotinamide-induced inhibition of the Relebactam web association of Sirt-1 proteins with the early osteogenic transcription factor Runx2 in 9528756 MSC highdensity cultures Resveratrol Promotes Osteogenesis of MSCs antibodies, the samples were probed by immunoblotting with antiRunx2. The results indicated that Runx2 was co-immunoprecipitated by anti-Sirt-1 antiserum but not by pre-immune serum in high-density cultures. This interaction of Sirt-1 with Runx2 was decreased with as little as 10 mM nicotinamide and indicates that the expression and association of Runx2 with Sirt-1 is concentration-dependent. Taken together, these results indicate that during osteogenesis resveratrol activates Sirt-1 and induces 25909282” Sirt-Runx2 complex formation, which activates the osteogenic pathway. Effect of resveratrol on nicotinamide-induced
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