Hyperactive Transforming Growth Factor-β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

Abstract

Dysregulated transforming growth factor beta (TGF-b) signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGFb1 signaling pivotally underpins osseous defects in Nf1 flox/À ;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-b1 levels are fivefold to sixfold increased both in Nf1 flox/À ;Col2.3Cre mice and in a cohort of NF1 patients. Nf1-deficient osteoblasts, the principal source of TGF-b1 in bone, overexpress TGF-b1 in a gene dosage-dependent fashion. Moreover, Nf1-deficient osteoblasts and osteoclasts are hyperresponsive to TGF-b1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras-dependent hyperactivation of the canonical TGF-b1-Smad pathway. Reexpression of the human, fulllength neurofibromin guanosine triphosphatase (GTPase)-activating protein (GAP)-related domain (NF1 GRD) in primary Nf1-deficient osteoblast progenitors, attenuated TGF-b1 expression levels and reduced Smad phosphorylation in response to TGF-b1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF-b receptor 1 (TbRI) kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1 flox/À ;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-b1 signaling in the pathogenesis of NF1-associated osteoporosis and pseudarthrosis, thus implicating the TGF-b signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies.

Bone marrow mesenchymal stem cells and TGF-β signaling in bone remodeling

“…Hyperactive TGF-β1 signaling has been implicated as the primary factor underlying the pathophysiology of the osseous defects in Nf1 fl/-Col2.3 Cre mice, a model of NF1 that closely recapitulates the skeletal abnormalities found in human disease (85). The exact mechanisms mediating mutant neurofibromin-associated enhancement of TGF-β production and signaling remain unknown.…”

Role of endoplasmic reticulum stress in disuse osteoporosis

“…Micro-computed tomography (µCT) was performed using a ScancovivaCT 40 (Scanco Medical AG, Bassersdorf, Switzerland), a high-resolution desk-top system as previously reported [24]. Briefly, the excised left distal femora were scanned using an 8 X-ray source set at 60 kV with 6-μm pixel size.…”

“…On day 14, cells were stained using a HEMA-3 quick staining kit (Fisher Scientific, Waltham, MA, USA). The number of CFU-F colonies with more than 50 cells was counted, and the clusters of cells that did not present fibroblast-like morphology were excluded [24,33,34].…”

Osteoclasts—Key Players in Skeletal Health and Disease

“…In diseases such as Marfan's syndrome, mutations in matrix proteins reduce the sequestration of TGF-β and increase free TGF-β levels, causing osteoblast lineage cells to secrete more RANKL, providing another mechanism for osteolytic effects of this factor (241–243). Aberrant activation of TGF-β may also play a role in the bone abnormalities in neurofibromatosis (244). …”