Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β‐catenin signaling and osteoblast proliferation

Abstract

Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation.

MACF1 promotes preosteoblast migration by mediating focal adhesion turnover through EB1

“…MACF1 overexpressed preosteoblast was constructed as described previously (Yin et al, 2018). Briefly, MC3T3-E1 cells (1×10 7 per well) were electroplated (1800V and 30 ms) with the MACF1 overexpression plasmid pEGFP-C1A-ACF7 or control plasmid pEGFP-C1A using Neon Transfection System (Invitrogen) following the manufacturer's instructions.…”

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway

“…In previous studies, Macf1 has been found to participate in the regulation of osteoblast differentiation‐associated Wnt/β‐catenin signalling pathway . In our previous studies, it was demonstrated that Macf1 can regulate the proliferation, cell cycle progression and differentiation of MC3T3‐E1 osteoblastic cell line . However, it remains unknown whether Macf1 could regulate bone formation in vivo.…”

Recombinant Irisin Prevents the Reduction of Osteoblast Differentiation Induced by Stimulated Microgravity through Increasing β-Catenin Expression

“…In osteoblasts, mechanical stimulation causes a rapid, transient accumulation of active β-catenin in the cytoplasm and its translocation into the nucleus [34]. Furthermore, we reported that β-catenin reduction caused by simulated microgravity (Figure 5b) [14] and overexpression of β-catenin partially attenuated the negative effects of simulated microgravity on osteoblast differentiation ( Figure 6). Thus, we wonder whether β-catenin acts as a mechano-mediator in the processing of r-irisin, by regulating osteoblast differentiation under simulated microgravity.…”

“…According to reports, there are many osteoporosis risk factors such as cytokines (e.g., interleukin) [11,12], hormones (e.g., parathyroid hormone) [13], and signaling pathways molecules (e.g., wingless-type MMTV integration site family (Wnt), bone morphogenetic protein(BMP)) [14,15], regulating bone metabolism under microgravity environment. Skeletal muscles and bone, as neighboring tissues, are the two largest tissues in the musculoskeletal system [12].…”

“…Previous studies showed that Wnt/β-catenin signaling is a key signaling pathway in osteoblast differentiation and bone formation [29][30][31][32][33]. In osteoblasts, mechanical stimulation causes a rapid, transient accumulation of active β-catenin in the cytoplasm and its translocation into the nucleus [34,35], and β-catenin is also sensitive to simulated microgravity [14,36]. It was reported that irisin influences adipogenesis through regulating the Wnt/β-catenin signaling pathway [37], and it increases β-catenin expression during osteogenic differentiation [27].…”