Vacuolar ATPases V ATPases are large

Vacuolar ATPases (V-ATPases) are large multi-subunit complexes that are organized into V0 and V1 domains, which operate by a rotary mechanism (Forgac, 2007). V-ATPase-driven proton pumping and organellar acidification are essential for vesicular trafficking along both the exocytotic and endocytotic pathways of eukaryotic cells. In Wnt producing cells, vacuolar acidification is required for Wnt signaling (Cruciat et al., 2010; Coombs et al., 2010). The secretion of Wnt3a protein into the cell culture medium was shown to be dependent on vacuolar pH. Moreover, acidification inhibitor was shown to decrease secreted and increase cell-associated Wnt3a. The inhibition of V-ATPase blocks Wnt3a secretion and inhibits Wnt/β-catenin signaling both in cultured human MK-8669 Supplier and in vivo (Coombs et al., 2010). In the present study, we found that HBO increased cell proliferation, LRP6 phosphorylation, and cyclin D1 expression in osteogenically differentiated BMSCs. HBO increased the osteogenic differentiation of BMSCs via regulation of Wnt3a signaling as well as increased the TCF-dependent transcription and Runx2 promoter/Luc gene activity. Because Wnt/β-catenin signaling is an upstream activator of BMP2 expression in osteoblasts, we found that HBO dose dependently increased the BMP2 and osterix production. Since endosomal acidification is an essential function of the Wnt secretion pathway, we further demonstrated that HBO increased the expression of V-ATPases to stimulate Wnt3a secretion. Finally, we showed the beneficial effects of HBO on bone formation via Wnt/β-catenin signaling regulation in a rabbit model.
Materials and methods
Results
Discussion Human BMSCs cultured in hypoxia show greater proliferation than those cultured in normoxic conditions (Grayson et al., 2006; Fehrer et al., 2007). However, both inhibitory and enhancing effects of hypoxia on osteogenic differentiation have been reported (Grayson et al., 2006; Fehrer et al., 2007; Pattappa et al., 2011). Because HBO increases the oxygen tension in vivo (Ueng et al., 1998; Korhonen et al., 1999) and in vitro (Ueng et al., 2013; Niu et al., 2013), we used HBO to alter the hypoxic microenvironment for cell proliferation and differentiation and activate the oxygen sensitive pathways. Our findings support those of previous studies, which suggest that undifferentiated BMSCs and committed BMSCs could respond differently to oxygen signals (Fehrer et al., 2007). HBO decreases cell proliferation when undifferentiated BMSCs are cultured in complete medium (Fig. 2A). However, increased levels of cell proliferation were induced by HBO treatment when the BMSCs were committed to the osteoblast lineage (Fig. 2B). These findings were further validated by the evaluated expression levels of cyclin D1 after HBO treatment (Fig. 3A). Although the responses of osteoblasts to HBO have been documented (Wu et al., 2007; Hsieh et al., 2010), the direct effects of HBO on human BMSCs that are induced to differentiate down the osteoblastic pathway have, to the best of our knowledge, not been previously investigated. Oxygen availability regulates stem cells via Wnt/β-catenin signaling (Mazumdar et al., 2010). Because HBO has stimulatory effects on cell growth (Fig. 2B), we wanted to identify the molecular mechanisms involved by assessing the Wnt/β-catenin pathway. Our data showed that the protein levels of Wnt3a, phosphorylated LRP6, and cyclin D1 were upregulated after culturing for 7d with HBO treatment (Fig. 3A). A key step after Wnt stimulation is the phosphorylation of the LRP6 intracellular domain. This phosphorylation event stabilizes the Wnt signaling transducer β-catenin (Bilic et al., 2007). Activation of the Wnt3a pathway results in enhanced expression of the Wnt3a target gene, cyclin D1, which is required for G1/S phase traversal (Xiong et al., 1997). Osteoblasts were induced to enter the S and G2/M phases of the cell cycle after HBO treatment (Hsieh et al., 2010). HBO increases the proliferation of BMSCs that are beginning to differentiate down the osteoblastic pathway via Wnt3a signaling (Fig. 3), which was in contrast to previous observations that hypoxia selectively activates Wnt/β-catenin signaling in undifferentiated neural stem cells but not in differentiated neurons (Mazumdar et al., 2010).