Differential responses of cultured MC3T3-E1 cells to dynamic and static stimulated effect of microgravity in cell morphology, cytoskeleton structure and Ca²⁺ signaling

Random positioning machine (RPM) and diamagnetic levitation are two essential ground-based methods used to stimulate the effect of microgravity in space life science research. However, the force fields generated by these two methods are fundamentally different, as RPM generates a dynamic force field acting on the surface in contact with supporting substrate, whereas diamagnetic levitation generates a static force field acting on the whole body volume of the object (e.g. cell). Surprisingly, it is hardly studied whether these two fundamentally different force fields would cause different responses in mammalian cells. Thus we exposed cultured MC3T3-E1 osteoblasts to either dynamically stimulated effect of microgravity (d-μg) with RPM or statically stimulated effect of microgravity (s-μg) with diamagnetic levitation, respectively, for 3 h. Subsequently, the cells were examined for changes in cell morphology, cytoskeleton (CSK) structure and Ca²⁺ signaling. The results show that compared to the condition of normal gravity (1g), both d-μg and s-μg resulted in decrease of cell area and disruption of the microfilaments and microtubules in MC3T3-E1 cells, but cells under d-μg were more smooth and round while those under s-μg exhibited more protrusions. The decrease of cell area and disruption of microfilaments and microtubules induced by d-μg but not s-μg were rescued by inhibition of the stretch-activated channel by gadolinium chloride (Gd). Inhibition of calmodulin (CaM) by inhibitor, W-7, promoted the effects of s-μg on cell area and CSK filaments, but inhibition of calmodulin-dependent protein kinase (CaMK) by inhibitor, KN-93, weakened d-μg-induced effects on cell area and cytoskeleton. In addition, both d-μg and s-μg decreased the CaM expression and CaMKII activity in MC3T3-E1 cells. Furthermore, s-μg resulted in decrease of the intracellular free Ca²⁺ concentration ([Ca²⁺]i) in MC3T3-E1 cells, which was reversed by disrupting microfilaments with cytochalasin B (CytB). Instead, d-μg induced increase of [Ca²⁺]i, which was inhibited by Gd. Taken together these data suggest that dynamic and static stimulated microgravity cause different responses in MC3T3-E1 cells. The dynamic force field acts on stretch-activated channels to induce microfilaments disruption and Ca²⁺ influx in MC3T3-E1 cells whereas the static force field directly induces microfilament disruption, which in turn decreases the [Ca²⁺]i in MC3T3-E1 cells. Such findings may have important implications to better understanding microgravity related cellular events and their applications.