Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

Abstract

Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure alteration inhibits the function performing.

Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia

“…In particular, Dai et al . reported that actin microfilaments participate in BMP-2 activity in activating Runx-2 and that their disruption might be an important contributor to microgravity-induced inhibition on BMP-2 activity in osteogenic induction 49 . In the present study, we found that microgravity-induced abrogation of primary cilia may be another underlying mechanism for the microgravity inhibitory effect on osteogenesis.…”

Physiological Effects of Microgravity on Bone Cells

“…Actin microfilaments participate in BMP2 induction and Cbfa1 activity, and their disruption could contribute to the inhibition of the osteogenic functions of BMP2 under simulated microgravity [40]. MAPK signaling also plays a role in microfilament rearrangement during parabolic flight [62]. However, the exact mechanism underlying weightlessnessinduced microfilament reorganization is not clear.…”

Frizzled-9 activates YAP to rescue simulated microgravity induced osteoblasts dysfunction

“…PKC signal pathway and actin microfilament organization were sensitive to microgravity (Rijken et al, 1994). MAPK pathway was reported to alter the actin dynamics of osteoblast under microgravity (Dai et al, 2011). SMG also reduces intracellular-free calcium concentration by inhibiting calcium channels in primary mouse osteoblasts (Sun et al, 2019b).…”