Combined MEK Inhibition and BMP2 Treatment Promotes Osteoblast Differentiation and Bone Healing in Nf1Osx−/− Mice

Abstract

Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase-activating protein neurofibromin. Non-bone union following fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hematopoietic cells to TGFβ. However, clinically relevant pharmacological approaches to augment bone union in these patients remain limited. In this study, we report the generation of a novel conditional mutant mouse line used to model NF1 pseudoarthrosis, in which Nf1 can be ablated in an inducible fashion in osteoprogenitors of post-natal mice, thus circumventing the dwarfism associated with previous mouse models where Nf1 is ablated in embryonic mesenchymal cell lineages. An ex vivo-based cell culture approach based on the use of Nf1flox/flox bone marrow stromal cells showed that loss of Nf1 impairs osteoprogenitor cell differentiation in a cell-autonomous manner, independent of developmental growth plate-derived or paracrine/hormonal influences. In addition, in vitro gene expression and differentiation assays indicated that chronic ERK activation in Nf1-deficient osteoprogenitors blunts the pro-osteogenic property of BMP2, based on the observation that only combination treatment with BMP2 and MEK inhibition promoted the differentiation of Nf1-deficient osteoprogenitors. The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1osx−/− mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol (PEG)-based delivery method. Collectively, these results provide novel evidence for a cell-autonomous role of neurofibromin in osteoprogenitor cells and insights about a novel targeted approach for the treatment of NF1 pseudoarthrosis.

Ca2+/Mg2+ homeostasis-related TRPM7 channel mediates chondrocyte hypertrophy via regulation of the PI3K-Akt signaling pathway

“…Conversely, levels of type II collagen and aggrecan remained unaffected by TRPM7 silencing during ATDC5 cell proliferation, suggesting that TRPM7 expression, although essential for chondrocyte hypertrophy, may not be critical for chondrocyte proliferation. In addition, osteoblast cells arise from osteoprogenitor cells located in the bone marrow, and their proliferation and differentiation has been implicated in bone formation and remodeling (20)(21)(22). It has been reported that intracellular Ca 2+ or Mg 2+ homeostasis, maintained through TRPM7 expression, was important for osteoblast proliferation, migration and differentiation (12).…”

The mechanism of a chitosan-collagen composite film used as biomaterial support for MC3T3-E1 cell differentiation

“…Multiple signals, such as growth/hormone factor stimulation and matrix-integrin binding, activate the Erk pathway in bone. Activated Erk promotes calvarial osteoblast differentiation through the downstream transcription factor Runx2 at Ser 301 and Ser 319 residues, and the mRNA levels of specific osteoblast differentiation-related marker genes are increased123456.…”

RASopathies: unraveling mechanisms with animal models

“…However, because Nf1 −/− mice are embryonic lethal, follow-up studies focused on creating many tissue-specific biallelic deletions of Nf1 . These models recapitulate several phenotypes observed in humans, including bowing of the tibia, impaired bone strength and short stature ( de la Croix Ndong et al, 2014 , 2015 ; El Khassawna et al, 2012 ; Kolanczyk et al, 2007 ; Kossler et al, 2011 ; Rhodes et al, 2013 ; Schindeler et al, 2008 ; Wang et al, 2011 ; Zhang et al, 2011 ). Some of these phenotypes might be caused by increased levels of pyrophosphate, a strong inhibitor of bone mineralization.…”