br Material and methods br Results br

Material and methods
Results
Discussion The expression of SVCT transporter has been studied in osteoblast (Tsukaguchi et al., 1999), MC3T3-E1 (Fujita et al., 2001), and articular cartilage (Clark et al., 2002; McNulty et al., 2005) but there are no data until now on ascorbic MDV3100 manufacturer uptake or SVCT expression in BMSCs under undifferentiated conditions as well as during osteogenic differentiation. This report presents the first study of the characterization of ascorbic acid uptake in BMSCs. These studies show convincingly that BMSCs exhibit robust ascorbic acid uptake activity and that the uptake process is Na+-dependent. Our molecular studies reveal that BMSCs express only SVCT2 isoform of the Na+-coupled ascorbic acid transporter. SVCT1, another isoform of ascorbic acid transporter that is also capable of Na+-coupled transport, is not expressed in BMSCs. It has been reported that osteoblasts (Tsukaguchi et al., 1999) and MC3T3-E1 (mouse osteoblastic cell line) (Fujita et al., 2001) express SVCT2 but not SVCT1. The characteristics of ascorbic acid uptake in BMSCs include absolute requirement for Na+, saturability with high affinity for its substrate, specific for ascorbic acid, and a Na+:ascorbic acid stoichiometry of 2:1. The Michaelis constant for the uptake process was ~56μM. This value is in the range of previously reported data for SVCT2 in different cell types in different species (Rajan et al., 1999; Liang et al., 2001; Manfredini et al., 2002; Savini et al., 2008). We also investigated changes in the expression of SVCT2 in BMSCs during their osteogenic differentiation. Ascorbic acid uptake increases within 24h after addition of the osteogenic medium to the culture. This effect persisted for 6days but decreased at longer periods of time. Type I collagen expression was also upregulated in the initial phase of osteogenesis. Similar findings have been reported for MC3T3-El mouse calvaria-derived cell line, showing dramatically increased procollagen type I mRNA within 24h during osteogenesis and the decreased effect at longer treatment times (Franceschi, 1992). As expected, bone-specific markers BMP-2 and Runx-2 were also upregulated during differentiation of BMSCs into osteoblasts and this was accompanied with increased mineralization. Wu et al. (2004) showed that MC3T3-E1 osteoblasts SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of osteogenic media. Our results match up with the Wu et al. (2004) finding that SVCT2 plays important role in osteogenesis. Our findings suggest that SVCT2 is upregulated during the initial phase of differentiation of BMSCs into osteoblasts, which helps to promote collagen synthesis. This is followed by increased mineralization. There are reports that SVCT2 expression and ascorbic acid uptake are regulated by zinc, calcium and phosphate in murine osteoblastic cell line MC3T3-E1 during osteogenesis (Wu et al., 2003a,b). Analysis of the differential effects of the three important components of the osteogenic medium reveals that dexamethasone is primarily responsible for the observed increase in ascorbic acid uptake. Fujita et al. (2001) also reported that dexamethasone induces SVCT2 mRNA expression and uptake of AA in a mouse osteoblastic cell line MC3T3-E1. To further clarify the role of SVCT2 in the osteogenic differentiation of BMSCs, we knockdown SVCT2 expression utilizing lentivirus-based shRNA system and osteogenic analysis was performed. The knockdown of SVCT2 transporter significantly inhibits osteogenesis which clarifies the importance of this transporter in bone formation. We also investigated the relationship between SVCT2 and oxidative stress in undifferentiated BMSCs. It is generally accepted that osteoporosis and aging is associated with oxidative stress. Oxidative stress stimulates osteoclast differentiation through receptor activator of nuclear factor-kappaB ligand (RANKL) and induces bone loss (Lee et al., 2005; Wauquier et al., 2009). However, there are no data available concerning the impact of oxidative stress on SVCT2 regulation in BMSCs. In this study, we show that BMSCs cells modulated the AA uptake according to their redox balance. The AA uptake was down regulated in Sin-1-treated BMSCs, while Sin-1 along with antioxidant supplementation compensates AA uptake. Completely opposite findings were previously reported in other cell types, where oxidative stress induced SVCT-2 expression (Kannan et al., 2001; Savini et al., 2007; May et al., 2010; Portugal et al., 2012) and antioxidant supplementation reversed this effect (Savini et al., 2007). Our study and previous studies show that antioxidant treatment ablates the effect that oxidative stress imposes upon SVCT2 expression. The inconsistent AA uptake regulation among these cell types may be attributable to differences in cellularity, but it seems that AA uptake is in fact an adaptive response to redox reaction. We also found that Sin-1 treatment (1000μM) substantially reduced cell viability and AA protected cells from cytotoxicity of Sin-1.