Mechanical Signaling in NF1 Osteoblast Cells

Abstract

Neurofibromatosis Type I (NF1) syndrome is characterized by neurofibromas and neural tumors but is also associated with skeletal abnormalities. The cellular pathophysiology of skeletal abnormalities in NF1 is not understood. These abnormalities result from constitutive active RAS and its downstream effectors, RASERK pathway, due to mutation of NF1 gene which converts active RAS-GTP into inactive RAS-GDP. In osteoblast cells, RAS-ERK pathway is involved in cell proliferation and differentiation and is also involved in mechanical signals transduction.In this study, we propose that Nf1 mutation in osteoblast cells will affect the response to mechanical stimulation through the RAS pathway. The Flexcell tension system was used to mechanically stimulate calvarial osteoblast precursor from conditional knockout mice, Nf1(ob-/-), and wild type calvarial osteoblast precursor cells, (WT. The protocol of cyclic mechanical strain was 2% to 4% elongation at 0.16 Hz (10 cycles per minute) for 24 h. Mechanically stimulated cells showed lower expression levels of the osteoblast marker gene, RUNX2, measured at 4 h and 8 h post-stretch. Mineralized matrix deposition, assessed by Alizarin red staining, was decreased in Nf1(ob-/-) compared to (WT) cells following mechanical stimulation. the Nf1(ob-/-) and WT osteoblast precursor cells were then treated with RAS inhibitor (FTI-277), for 4 h and 8 h. RUNX2 expression level was increased in Nf1(ob-/-) cells compared to non-treated cells. However, the opposite result was seen in (WT) cells. The FTI-277 treatment resulted in lower RUNX2 expression level and lower mineralized matrix deposition.This response of (WT) cells was normal. However, the Nf1(ob-/-) response showed that these cells although they have hyper-active RAS, but when it is exposed to stress, it loses its ability to express osteoblast markers or lay down mineralized matrix. Our results indicate that, the hyperactive RAS in NF1 mutant osteoblast will result in cells being stuck in proliferative state and unable to differentiate.

The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture

“…4 Moreover, the NF1 gene expression is also found in adult osteoblasts and osteoclasts, as well as hypertrophic chondrocytes, which presents during endochondral ossification. 5 The involvement of the NF1 gene and skeletal development provides a potential therapeutic target to modulate bone fracture healing.…”

“…The neurofibromin has been shown to be essential in osteoblast functioning; therefore, mutation in the NF1 gene results in skeletal abnormalities such as scoliosis, unilateral growth and congenital pseudoarthroses of long bones 4 . Moreover, the NF1 gene expression is also found in adult osteoblasts and osteoclasts, as well as hypertrophic chondrocytes, which presents during endochondral ossification 5 . The involvement of the NF1 gene and skeletal development provides a potential therapeutic target to modulate bone fracture healing.…”