Roles of brain and muscle ARNT-like 1 and Wnt antagonist Dkk1 during osteogenesis of bone marrow stromal cells

ObjectivesMany studies have demonstrated that the clock gene, brain and muscle ARNT-like 1 (Bmal1), is directly related to bone ageing by affecting age-related changes to mesenchymal stem cells (MSCs). As a main developmental signal, Wnt may play an important role in this process. Here, we have aimed to elucidate whether Bmal1 positively regulates osteogenesi via Wnt pathways. Materials and methodsBone marrow stromal cells were cultured in basic and in osteo-induction medium with Wnt signalling inhibitor Dkk1 and Bmal1 transfection. Proliferation and osteogenesis of MSCs, expression of Bmal1 and activation of Wnt signalling were investigated by flow cytometry, senescence-associated -galactosidase (SA--gal) staining, real-time quantitative PCR and western blot analysis. ResultsExpression of Bmal1 (specially after 7days osteo-induction), activation of Wnt signalling and osteo-related factors fell significantly during osteo-induction after Dkk1 addition. When cellular Bmal1 was increased by transfection, osteogenesis inhibition by Dkk1 was rescued to a certain extent with activation of Wnt signalling. However, Dkk1 did not significantly affect proliferation or senescence of MSCs during early periods of culture. ConclusionThese findings demonstrated that Bmal1 and Wnt signalling may have a synergistic effect at a particular stage of osteogenesis. Inhibition of Wnt signalling did not greatly affect ageing of MSCs through early passages.