MACF1 Overexpression by Transfecting the 21 kbp Large Plasmid PEGFP-C1A-ACF7 Promotes Osteoblast Differentiation and Bone Formation

Abstract

Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein known to have crucial roles in regulating cytoskeletal dynamics, cell migration, growth, and differentiation. However, its role and action mechanism in bone remain unclear. The present study investigated optimal conditions for effective transfection of the large plasmid PEGFP-C1A-ACF7 (∼21 kbp) containing full-length human MACF1 cDNA, as well as the potential role of MACF1 in bone formation. To enhance MACF1 expression, the plasmid was transfected into osteogenic cells by electroporation in vitro and into mouse calvaria with nanoparticles. Then, transfection efficiency, osteogenic marker expression, calvarial thickness, and bone formation were analyzed. Notably, MACF1 overexpression triggered a drastic increase in osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in vitro. Mouse calvarial thickness, mineral apposition rate, and osteogenic marker protein expression were significantly enhanced by local transfection. In addition, MACF1 overexpression promoted β-catenin expression and signaling. In conclusion, MACF1 overexpression by transfecting the large plasmid containing full-length MACF1 cDNA promotes osteoblast differentiation and bone formation via β-catenin signaling. Current data will provide useful experimental parameters for the transfection of large plasmids and a novel strategy based on promoting bone formation for prevention and therapy of bone disorders.

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

“…Recently, canonical biochemical components such as the Wnt signaling pathway and β-catenin were reported to present mechanical sensitivity [49]. Previous studies showed that Wnt/β-catenin signaling is a key signaling pathway in osteoblast differentiation and bone formation [33,50,51]. We also found that knockdown of β-catenin inhibited osteoblast differentiation in MC3T3-E1 cells (Figure 5c,d).…”

“…Therefore, the mechanism of irisin in osteoblast differentiation and bone formation under microgravity warrants an investigation. 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].…”

Silencing of lncRNA AK045490 Promotes Osteoblast Differentiation and Bone Formation via β-Catenin/TCF1/Runx2 Signaling Axis

“…It has been widely recognized that β-catenin/TCF signaling plays a key role in regulating osteoblast differentiation by targeting Runx2 [9]. Moreover, our previous study demonstrated that MACF1 play a positive regulatory role in osteoblast differentiation and bone formation by promoting nuclear translocation of β-catenin/TCF1/Runx2 signaling axis [20,21]. RUNX2, TCF1, and LEF1 were master transcription factors regulating osteoblast differentiation [22,23].…”

Mesenchymal MACF1 Facilitates SMAD7 Nuclear Translocation to Drive Bone Formation

“…Construction of the MACF1 knockdown MC3T3-E1 cell line (KD), and transfection of pKH3S-ACF plasmid into MACF1 KD cells were reported previously [20,21]. Cells 2020, 9, 616 4 of 20 Growth medium (10% FBS + 1% pen-strep + 1% L-glutamine in α-MEM) were used for subculture, and osteo-induction medium (10% FBS + 1% pen-strep + 1% L-glutamine + 0.1 µM dexamethasone + 50 µg/mL ascorbic acid + 10 mM beta-glycerophosphate in α-MEM) were used for osteo-induction.…”