Autophagy is involved in neurofibromatosis type I gene‑modulated osteogenic differentiation in human bone mesenchymal stem cells

Abstract

Neurofibromatosis type I (NF1) is an autosomal dominant genetic disease that is caused by mutations in the NF1 gene. Various studies have previously demonstrated that the mTOR complex 1 signaling pathway is essential for the NF1-modulated osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Additionally, the mTOR signaling pathway plays a notable role in autophagy. The present study hypothesized that NF1 could modulate the osteogenic differentiation of BMSCs by regulating the autophagic activities of BMSCs. In the present study, human BMSCs were cultured in an osteogenic induction medium. The expression of the NF1 gene was either knocked down or overexpressed by transfection with a specific small interfering RNA (siRNA) targeting NF1 or the pcDNA3.0 NF1-overexpression plasmid, respectively. Autophagic activities of BMSCs (Beclin-1, P62, LC3B I, and LC3B II) were determined using western blotting, electron microscopy, acridine orange (AO) staining and autophagic flux/lysosomal detection by fluorescence microscopy. In addition, the autophagy activator rapamycin (RAPA) and inhibitor 3-methyladenine (3-MA) were used to investigate the effects of autophagy on NF1-modulated osteogenic differentiation in BMSCs. Inhibiting NF1 with siRNA significantly decreased the expression levels of autophagy markers Beclin-1 and LC3B-II, in addition to osteogenic differentiation markers osterix, runt-related transcription factor 2 and alkaline phosphatase. By contrast, overexpressing NF1 with pcDNA3.0 significantly increased their levels. Transmission electron microscopy, AO staining and autophagic flux/lysosomal detection assays revealed that the extent of autophagosome formation was significantly decreased in the NF1-siRNA group but significantly increased in the NF1-pcDNA3.0 group when compared with the NC-siRNA and pcDNA3.0 groups, respectively. In addition, the activity of the PI3K/AKT/mTOR pathway [phosphorylated (p)-PI3K, p-AKT, p-mTOR and p-p70S6 kinase] was significantly upregulated in the NF1-siRNA group compared with the NC-siRNA group, and significantly inhibited in the NF1-pcDNA3.0 group, compared with the pcDNA3.0 group. The knockdown effects of NF1-siRNA on the autophagy and osteogenic differentiation of BMSCs were reversed by the autophagy activator RAPA, while the overexpression effects of NF1-pcDNA3.0 on the autophagy and osteogenic differentiation of BMSCs were reversed by the autophagy inhibitor 3-MA. In conclusion, results from the present study suggest at the involvement of autophagy in the NF1-modulated osteogenic differentiation of BMSCs. Furthermore, NF1 may partially regulate the autophagic activity of BMSCs through the PI3K/AKT/mTOR signaling pathway.

Current Aspects on the Pathophysiology of Bone Metabolic Defects during Progression of Scoliosis in Neurofibromatosis Type 1

“…Overexpression of Nf in BMSC inhibits mTORC1 signaling and thus enhances autophagy and results in new bone formation [64]. In the same line, inhibition of Nf1 in BMSC enhances mTORC1 signaling and decreases the expression of autophagy markers, such as Beclin-1 and LC3B-II, as well as bone differentiation markers, such as osterix, runt-related transcription factor 2 and ALP [62]. Moreover, in the Nf1-siRNA group the activity of the PI3K/AKT/mTOR pathway was significantly upregulated, whereas administration of the autophagy activator RAPA reserved the knockdown effects of Nf1-siRNA on the autophagy and osteogenic differentiation of BMSCs and led to elevated ALP activity and calcium deposition [62].…”

“…In NF1, the PI3K-AKT-mTOR pathway also seems to be involved in the deranged osteogenic differentiation of BMSC [61,62]. The down-regulated expression of Nf in human BMSC results in enhanced mTORC1 activity and a remarkable reduction in osteoblastic differentiation markers, such as osterix, runx2 (RUNX Family Transcription Factor 2) ALP and OCN, while overexpression of Nf1 had the opposite outcome [61].…”

“…The down-regulated expression of Nf in human BMSC results in enhanced mTORC1 activity and a remarkable reduction in osteoblastic differentiation markers, such as osterix, runx2 (RUNX Family Transcription Factor 2) ALP and OCN, while overexpression of Nf1 had the opposite outcome [61]. Some studies suggest that the osteogenic differentiation is strongly dependent on autophagy [62], with mTORC1 playing a regulatory role in this process [63,64]. Overexpression of Nf in BMSC inhibits mTORC1 signaling and thus enhances autophagy and results in new bone formation [64].…”