Bone Abstracts

Background: Autocrine and paracrine Wnt signaling operates in stem cell populations and regulates mesenchymal lineage specification. In Wnt producing cells, Wntless (GPR177) supports the transport of Wnt from the trans-Golgi network (TGN) to the cell surface in vesicles, from which Wnt is then released. The retromer complex (VPS35) interacts with Wntless and retrieves Wntless from endosomes back to TGN, thereby maintaining the normal levels of Wntless protein. V-ATPases-driven proton pumping and organellar acidification are essential for Wnt secretion. However, little is known about the effects of HBO on the Wnt processing, secretion, and signaling in MSCs.

Materials and methods: Cells were cultured in osteogenic induction medium. Control cells were maintained in 5% CO₂/95% air and the hyperoxic cells were exposed to 100% O₂ at 2.5 ATA (atmospheres absolute) in a hyperbaric chamber. To investigate the effects of HBO on Wnt processing and secretion, the protein levels of Wnt3a, GPR177, VPS35, ATP6V0, ATP6V1, and β-actin were detected by western blot. The secreted Wnt3a in the collected medium was quantified by ELISA. To investigate the effects of HBO on Wnt signaling, the mRNA and protein levels of Wnt3a, β-catenin, GSK-3β, Runx 2, as well as alkaline phosphatase activity and calcium deposition were analyzed after HBO treatment. The von Kossa staining was tested in osteogenetic differentiated MSCs after HBO treatment.

Results: Our data showed that HBO treatment increased the expression of Wnt3a, GPR17, VPS35, ATP6V0, and ATP6V1 to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment. The mRNA and protein levels β-catenin and Runx 2 were up-regulated, while GSK-3β was down-regulated after HBO treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs.

Conclusion: HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.