Abstract
Neurofibromatosis Type I (NF1) syndrome is characterized by neurofibromas andneural tumors but is also associated with skeletal abnormalities. The cellularpathophysiology of skeletal abnormalities in NF1 is not understood. Theseabnormalities result from constitutive active RAS and its downstream effectors, RASERKpathway, due to mutation of NF1 gene which converts active RAS-GTP intoinactive RAS-GDP. In osteoblast cells, RAS-ERK pathway is involved in cellproliferation and differentiation and is also involved in mechanical signals transduction. In this study, we propose that Nf1 mutation in osteoblast cells will affect the responseto mechanical stimulation through the RAS pathway. The Flexcell tension system wasused to mechanically stimulate calvarial osteoblast precursor from conditional knockoutmice, Nf1(ob-/-), and wild type calvarial osteoblast precursor cells, (WT. Theprotocol of cyclic mechanical strain was 2% to 4% elongation at 0.16 Hz (10 cycles perminute) for 24h. Mechanically stimulated cells showed lower expression levels of theosteoblast marker gene, RUNX2, measured at 4h and 8h post-stretch. Mineralizedmatrix deposition, assessed by Alizarin red staining, was decreased in Nf1(ob-/-)compared to (WT) cells following mechanical stimulation. the Nf1(ob-/-) and WTosteoblast precursor cells were then treated with RAS inhibitor (FTI-277), for 4h and8h. RUNX2 expression level was increased in Nf1(ob-/-) cells compared to non-treatedcells. However, the opposite result was seen in (WT) cells. The FTI-277 treatmentresulted in lower RUNX2 expression level and lower mineralized matrix deposition. This response of (WT) cells was normal. However, the Nf1(ob-/-) response showedthat 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. Ourresults indicate that, the hyper-active RAS in NF1 mutant osteoblast will result in cellsbeing stuck in proliferative state and unable to differentiate.
Highlights
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by loss of function mutations in the NF1 gene with an incidence of approximately 1 in 3000, making it one of the most common genetic disorders [1,2]
Knowing that neurofibromin is expressed in bone cells and acting on RAS signaling pathway and that bone cells can adapt to mechanical stimulation through activation of the RAS signaling pathway, we hypothesize that the response of NF1 mutant osteoblast to mechanical stress is defective which contributes to the skeletal tissue abnormalities in NF1 patients
This result supports the previous finding of the lack of NF1 in osteoblast resulting in reduced bone mineral density (BMD) and reduced mechanical properties [40]
Summary
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by loss of function mutations in the NF1 gene with an incidence of approximately 1 in 3000, making it one of the most common genetic disorders [1,2]. Knowing that neurofibromin is expressed in bone cells and acting on RAS signaling pathway and that bone cells can adapt to mechanical stimulation through activation of the RAS signaling pathway, we hypothesize that the response of NF1 mutant osteoblast to mechanical stress is defective which contributes to the skeletal tissue abnormalities in NF1 patients. We conduct multiple experiments to determine how NF1 mutant osteoblast cells respond to mechanical stress in terms of bone formation and differentiation.
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