Received Date: September 10, 2017; Accepted Date: September 14, 2017; Published Date: September 18, 2017
Citation: Bamaga I, McHugh KP (2017) Mechanical Signaling in NF1 Osteoblast Cell. J Den Craniofac Res Vol.2 No.2:13. doi: 10.21767/2576-392X.100013
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.
Neurofibromatosis; Osteoblasts; Bone; Mechanical signaling; Craniofacial
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]. NF1 syndrome is primarily characterized by subcutaneous neurofibromas and neural tumors. In addition, NF1 is associated with several skeletal abnormalities including, scoliosis, tibial bowing and sphenoid wing dysplasia [3,4]. Unfortunately, the cellular pathophysiology of the NF1 skeletal dysplasia is still not fully understood .The main functional domain of NF1 gene is known to be located between exon 27 and 34, known as RAS-GAP domain which gives the NF1 gene it’s tumor suppressor property [6,7].The NF1 gene encodes neurofibromin, a RAS GTPase-activating protein (GAP) that promotes the conversion of an active RAS-GTP-bound form to an inactive RAS-GDP form and functions to negatively regulate the activity of RAS effectors, including the RAF–MEK–ERK signaling pathway [8,9]. Thus, NF1 mutations results in activation of canonical mitogen-activated protein kinase (MAPK) signaling [10,11]. Of relevance to skeletal development, NF1 expression has been reported in hypertrophic chondrocytes, which are an important intermediate step during endochondral ossification, and also in adult osteoblast and osteoclasts [12,13] either of which might explain the skeletal involvement in NF1. It is established that skeletal tissue can sense mechanical loading which induces bone remodeling activity, resulting in structural changes through different cellular pathways. Several studies have shown that, RAS-MAPK-ERK pathway is the main contributor in mechanical signaling response in osteoblast cells [14-17]. The role of MAPK signaling components have been shown to favor osteoblastic cell proliferation and differentiation. In particular, ERK1/2 is involved in cell proliferation, differentiation and the survival of several cell types, including osteoblasts [18,19]. ERK1/2 signals can promote the proliferation and anabolism of osteoblasts in order to facilitate bone turnover, thereby contributing to the homeostasis of bone tissue [20,21].
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.
Here we conduct multiple experiments to determine how NF1 mutant osteoblast cells respond to mechanical stress in terms of bone formation and differentiation. We tested if antagonizing the hyper-active RAS in NF1 osteoblast cells would reverse the effect of the mutation.In the mouse model employed (herein called NF1(ob-/-) mice), ablation of NF1 occurs at the pre-osteoblast stage and is restricted to bone forming cells [22-24].The global Nf1 knock-out mouse models, such as Nf1(-/-) is prenatal lethal due to cardiomyopathies and the Nf1 heterozygotic mouse Nf1(-/+) mice does not develop any skeletal abnormalities. Nf1(ob-/-) mice allows us to study the effect of Nf1 mutation in osteoblast. In this model, the Nf1 gene has been knocked-out using the Cre-LoxP system under the control of 2.3kb collagen1-alpha1 promoter (Col11), which is primarily expressed in osteoblast [23,25]. Our current view of the NF1 bone pathology, based on the analysis of osteoblast function, whether the imbalance of bone homeostasis and improper response of osteoblast cell to mechanical stress cause the NF1 skeletal manifestations [26-35].
The NF1 dystrophic skeletal pathologies, including the progressive sphenoid wing dysplasia (SWD), are associated with functional disability and changes in bone shapefor which treatment or prevention is not available [1,36,37]. This highlights the dearth of knowledge related to NF1 pathophysiology in bone. We conducted this study to understand how ablation of NF1 in osteoblasts will affect their function in response to mechanical stress. Several Nf1 mouse models have been developed to study cancer and bone abnormalities . Initially, studies focused on generation of mice with a targeted mutation in Nf1 gene. Then the next generation models focused on exon-specific knock-out mice. However recently tissue-specific Nf1 gene knock-out has shown the function of neurofibromin in specific cell types. Here we isolated osteoblasts from mice in which Nf1 gene has been deleted using 2.3 collagen1-alpha1 promoter (Col1), which is expressed mainly in osteoblasts [23,25]. Unlike ablation of Nf1 in osteo-chondroprogenitor, these cells allowed us to study the effect on osteoblast function. In regard of RAS-GAP and Nf1, constitutively active RAS and its downstream kinase ERK1/2, are thought to underlie NF1 skeletal manifestations [23,24,39]. In the experiments presented here, osteoblast cells from Nf1(ob-/-) produce little mineralized matrix when growing in an osteoinductive medium (OIM) compared to NF1(WT) cells.
This result supports the previous finding of the lack of NF1 in osteoblast resulting in reduced bone mineral density (BMD) and reduced mechanical properties . Blockade of RAS protein post-translation modification is shown to prevent its targeting to the cell membrane and abrogation of its downstream effects. FTI-277 is an anti-RAS treatment which prevents RAS isoprenylation [35,41]. We hypothesize that if we inhibit hyper-active RAS using FTI-277, then the NF1 mutant osteoblast cell should be able to differentiate normally. We find that, when treated with 5µM of FTI-277 in OIM culture medium Nf1(ob-/-) cells were able to produce more mineralized matrix. However, the Nf1(WT) osteoblast cells treated with 5µM of FTI-277 produced less mineralized matrix when compared to untreated cells. This indicates that the level of RAS signaling is critical not only for osteoblast proliferation and differentiation but for normal function and matrix deposition. This is confirmed by the level of RUNX2 expression, which is known to be phosphorylated by ERK1/2 during matrix deposition [42,43].
Since we know that RAS-ERK pathway is involved in mechanical signal transduction [28,44]. We asked what is the role of NF1 in osteoblast during mechanical stress.Although different systems have been used to strain osteoblast cells, it is very difficult to compare in-vitro applied strains with those applied in-vivo because the characteristics of the strains are different, i.e. the 3D configuration and presence of interstitial fluid in-vivo. The Flexcell system has been a good platform to study the effect of mechanostimulation in many different contexts [45,46]. This system applies an equibiaxial strain to the cells using a flexible silicone bottom plate connected to a computer-controlled vacuum device. In the literature a wide variety of parameters have been used (i.e. the magnitude of stretch, the frequency of stretch and the duration of stretch, to mechanically stimulate different cell types). In terms of bone cells, there is a general concordance that the frequency is more important than the magnitude of stretch applied and 2% to 4% elongation is found to be enough to initiate osteoblast cellular response. At higher magnitudes (i.e. 10% to 12%) it is reported to be lethal to the cell [20,45]. In this study, we used the Flexcell system to apply a mechanical strain to Nf1(ob-/-) and Nf1(WT) at (10 cycles per minute (0.16 Hz) for 24h and 3% elongation). We found, in terms of protein expression that RUNX2 showed lower expression level in Nf1(ob-/-) upon mechanical stretching compared to non-stretched control cells. However, the opposite was found in Nf1(WT) cells with RUNX2 expression level increased with stretching in Nf1(WT) cells. Also we tested the downstream effects of stretching on mineralized matrix formation. It has been shown that mechanical stimulation leads to increased matrix deposition [32,47].
Our results showed, Nf1(ob-/-) cells upon mechanical stimulation were unable to responded normally and unable to form more matrix compared to Nf1(WT). Nf1(ob-/-) cells deposit less matrix when mechanically stimulated compared to non-stressed Nf1(ob-/-) cells. This potentially explains why NF1 patients have defective bone healing process and also why the skeletal lesions show a progressive nature. Bone tissue is continually being remodeled depending on the mechanical environment , therefore the defective mechanical transduction signals NF1 osteoblast cells produced upon mechanical stress are responsible for the abnormal response to stress.Coupling between bone formation and resorption and involvement of osteoclasts in this process (in vivo) may direct future studies to determine the response of osteoclast cells in NF1 to mechanical stimulation as this might bridge the gap and help to understand the cellular events leading to skeletal abnormalities in NF1 patients .
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