MACF1 Mediates the Impairment of Mechanical Unloading on Osteoblast Differentiation via F-Actin/ERK/Runx2 Axis

Decreased osteoblast differentiation contributes to bone loss induced by mechanical unloading. However, the underlying mechanism is still unclear. We previously found that microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal protein, plays an important role in regulating osteoblast differentiation, while the role of MACF1 in mechanical unloading suppressing osteoblast differentiation remains unclear. Here, an MACF1-knockdown (MACF1-KD) osteoblast cell line and primary osteoblasts were subjected to mechanical unloading conducted by a random positioning machine (RPM). Osteoblast differentiation was evaluated by alkaline phosphatase (ALP) staining and real-time PCR. F-actin distribution was examined by immunofluorescence staining. Western blot was adopted to detect the protein levels. Moreover, cytochalasin B and PD98059 were applied to disrupt F-actin and inhibit extracellular signal-regulated kinase (ERK) activity, respectively, to confirm the mechanism. The results show that MACF1 is significantly downregulated in osteoblasts by mechanical unloading together with decreased osteoblast differentiation. MACF1-KD osteoblasts exhibit reduced differentiation capacity and are insensitive to mechanical unloading. Mechanistically, MACF1 mediates the suppression of mechanical unloading on osteoblast differentiation by regulating F-actin distribution and the downstream ERK/Runx2 signaling. Furthermore, F-actin disruption and ERK inhibition assays confirm that MACF1 mediates the impairment of mechanical unloading on osteoblast differentiation via the F-actin/ERK/Runx2 axis. In conclusion, this study reveals MACF1 as a mechanotransduction mediator for mechanical unloading, inhibiting osteoblast differentiation via F-actin/ERK/Runx2, and contributes to a novel mechanistic insight of cell mechanotransduction.