br Author Contributions br Acknowledgments

Author Contributions
Acknowledgments
Introduction Osteoporosis is a common bone disease that frequently leads to disruption of the alveolar and systemic bones with age. Novel osteoanabolic strategies to treat osteoporotic bone loss or augment fracture repair efficiently are desired. Since bone metabolism is maintained through bone formation and resorption by osteoblasts and osteoclasts, it is necessary to understand the mechanisms of cell proliferation and differentiation in bone metabolism [1]. Recent studies have demonstrated that application of mechanical stress and electrical stimulation promoted osteogenic differentiation [[2], [3], [4], [5]]. As a common pathway, activation of calcium channels in osteoblasts has been suggested to play a role in osteogenic differentiation [6]. The intracellular signaling pathway of calcium/calmodulin, calcineurin, and nuclear factor of activated T Alisol B 23-acetate (NFAT) is known to be related to osteogenic differentiation [[7], [8], [9]]. Ca2+ oscillations, rhythmical fluctuation of calcium concentration [10], were observed in mesenchymal stem cells during osteogenic differentiation [11]. However, how the changes in intracellular Ca2+ can affect cellular differentiation is unknown. In this study, to examine whether Ca2+ oscillation alone is sufficient to promote differentiation of osteoblasts, we applied optical stimulation to an osteogenic differentiation model cell line, MC3T3-E1 [12], which was genetically engineered to express blue light-activated Ca2+ channel switch (BACCS) [13]. BACCS is an optogenetic tool composed of fusion proteins comprising light-sensitive domain LOV2-Jα and Ca2+ sensor stromal interaction molecule-1 (STIM1) protein. When conformational changes in BACCS are induced by irradiation with 488 nm blue light, STIM1 of BACCS binds to the store-operated Ca2+ channel ORAI1 on the cell membrane. BACCS can control intracellular Ca2+ signaling with high sensitivity and good temporal resolution. We aimed to evaluate optical control with BACCS as a simple method to regulate osteoblast differentiation without further physical and biochemical treatment. Spatiotemporal control of Ca2+ signaling may facilitate intracellular interrogation of osteogenesis and extend the application of BACCS to physiological and pathological study of not only excitable cells/tissue but also non-excitable specimens.
Materials & methods
Results
Discussion In this study, we manipulated intracellular Ca2+ spikes in MC3T3-E1 cells using the optogenetic tool BACCS for the first time. Using this method, we enhanced the differentiation of osteoblasts. These results supported our hypothesis that manipulating intracellular Ca2+ oscillation promotes differentiation of MC3T3-E1 cells. Previous studies showed that flow-induced mechanical stress or electrical stimulation increased the intracellular Ca2+ responses of osteoblasts [4,17]. However, because these types of stimulation did not activate only Ca2+, it was unclear whether the changes in intracellular Ca2+ concentration promoted differentiation of osteoblasts. In previous studies, to elucidate the molecular mechanism of osteoblast differentiation, optogenetic tools were used. The earliest optogenetic tool, channelrhodopsin (ChR), could control the membrane voltage in response to light [18,19]. Therefore, ChR is not specific for Ca2+ signaling. To directly control Ca2+ signaling, we engineered BACCS [13]. Using BACCS, we evaluated whether intracellular Ca2+ changes from the outside of the cells promote osteoblast differentiation. As shown here, Ca2+ promoted osteoblast differentiation. However, the increase in Ca2+ signaling by normal differentiation as well as the subsequent expression of Col1a1 and Osx was modest. It is possible that a different stimulation protocol, such as with faster repetitive stimulation or longer stimulation, could enhance the differentiation. Furthermore, in addition to Ca2+ signaling, we suspect that other signaling pathways are necessary for full osteoblast differentiation. Indeed, osteoblast differentiation requires key transcription factors [20]. Then, we could find the responsible signals by optogenetics to further corroborate our findings here. This study will contribute to understand the pathogenesis of osteoporosis by reconstructing Ca2+ environment, and hopefully provide a novel therapeutic agent to the field.