Hyperactive Ras/MAPK signaling is critical for tibial nonunion fracture in neurofibromin-deficient mice

Abstract

Neurofibromatosis type 1 (NF1) is a common genetic disorder affecting 1 in 3500 individuals. Patients with NF1 are predisposed to debilitating skeletal manifestations, including osteopenia/osteoporosis and long bone pseudarthrosis (nonunion fracture). Hyperactivation of the Ras/mitogen-activated protein kinase (MAPK) pathway in NF1 is known to underlie aberrant proliferation and differentiation in cell lineages, including osteoclast progenitors and mesenchymal stem cells (MSCs) also known as osteoblast progenitors (pro-OBLs). Our current study demonstrates the hyper Ras/MAPK as a critical pathway underlying the pathogenesis of NF1-associated fracture repair deficits. Nf1-deficient pro-OBLs exhibit Ras/MAPK hyperactivation. Introduction of the NF1 GTPase activating-related domain (NF1 GAP-related domain) in vitro is sufficient to rescue hyper Ras activity and enhance osteoblast (OBL) differentiation in Nf1(-/-) pro-OBLs and NF1 human (h) MSCs cultured from NF1 patients with skeletal abnormalities, including pseudarthrosis or scoliosis. Pharmacologic inhibition of mitogen-activated protein kinase kinase (MEK) signaling with PD98059 partially rescues aberrant Erk activation while enhancing OBL differentiation and expression of OBL markers, osterix and osteocalcin, in Nf1-deficient murine pro-OBLs. Similarly, MEK inhibition enhances OBL differentiation of hMSCs. In addition, PD98059 rescues aberrant osteoclast maturation in Nf1 haploinsufficient bone marrow mononuclear cells (BMMNCs). Importantly, MEK inhibitor significantly improves fracture healing in an NF1 murine model, Col2.3Cre;Nf1(flox/-). Collectively, these data indicate the Ras/MAPK cascade as a critical pathway in the pathogenesis of bone loss and pseudarthrosis related to NF1 mutations. These studies provide evidence for targeting the MAPK pathway to improve bone mass and treat pseudarthrosis in NF1.

Figure 1.

MAPK hyperactivation in Col2.3Cre⁺;Nf1^flox/− tibiae. (A) Representative histological sections from the tibia fracture site of WT, Nf1^+/−, and Col2.3Cre; Nf1^flox/− mice are shown. Red arrows indicate cells that are p44/42 positive (brown). (B) Quantitative measurement of p44/42-positive cells (p44/42-positive cells/total cells) is shown. Data are represented as mean ± SEM from three individual experiments (*P < 0.05 for WT versus Nf1^+/−mice, **P < 0.01 for Nf1^+/− versus Col2.3Cre; Nf1^flox/− mice, ***P < 0.001 for WT versus Col2.3Cre; Nf1^flox/− mice).

Figure 2.

PD98059 inhibits hyperactive Ras/MAPK signaling in Nf1^−/− pro-OBLs. (A) Ras activity in WT, Nf1^+/−, Nf1^−/− pro-OBLs was measured at indicated time following stimulation with PDGF (20 ng/ml). A representative immunoblot is shown. (B) Phosphorylation of Erk1/2 (p44/42) was measured in WT, Nf1^+/−, Nf1^−/− pro-OBLs in response to PDGF stimulation in the presence or absence of PD98059 (100 nm). Data represent one of the four independent results.

Figure 3.

Expression of NF1 GRD promotes OBL differentiation in Nf1^−/− pro-OBL cultures. (A) OBL differentiation was evaluated by ALP staining after 5 days of culture in OBL differentiation medium using WT and Nf1^−/− pro-OBLs transduced with MSCV-pac control or MSCV-NF1 GRD-pac. Representative high power fields (20×) of OBL cultures were used to quantify OBL differentiation by measuring ALP expression relative to WT controls (**P < 0.001 for NF1 GRD Nf1^−/− pro-OBLs versus WT MSCV-pac, and MSCV-pac Nf1^−/− pro-OBLs, ***P < 0.001 for MSCV-pac Nf1^−/− pro-OBLs versus MSCV-WT, and NF1 GRD Nf1^−/− pro-OBLs). (B) Ras activity in Nf1^−/− pro-OBLs transduced with either MSCV-pac or NF1 GRD was measured at indicated times following PDGF (20 ng/ml) stimulation.

Figure 4.

Pharmacologic MEK inhibition improves OBL differentiation and enhances mRNA expression of OBL differentiation markers. (A) WT, Nf1^+/− and Nf1^−/− pro-OBLs were cultured in OBL differentiating medium in the presence or absence of PD98059 (100 nm). OBL differentiation was measured by quantifying ALP expression from representative photomicrographs (20×). Data reflect three independent experiments (*P < 0.05 for Nf1^−/− pro-OBLs treated with PD98059 versus WT pro-OBLs, **P < 0.01 for Nf1^+/− pro-OBLs treated with vehicle versus PD98059 treated Nf1^+/− pro-OBLs and WT pro-OBLs, ***P < 0.001 for Nf1^−/− pro-OBLs treated with vehicle versus PD98059). (B) Osterix mRNA expression in untreated versus PD98059 (100 nm) treated WT and Nf1^−/− pro-OBLs was determined by a real-time PCR. Osterix expression, relative to untreated WT, was quantified in three individual experiments (***P < 0.001 for untreated WT versus untreated Nf1^−/− pro-OBLs and untreated Nf1^−/− pro-OBLs versus PD98059 treated Nf1^−/− pro-OBLs). (C) Osteocalcin mRNA expression in untreated versus PD98059 treated WT and Nf1^−/− pro-OBLs was determined by a real-time PCR in five individual experiments. Osteocalcin expression was measured relative to untreated WT (***P < 0.001 for untreated WT versus untreated Nf1^−/− pro-OBLs and untreated Nf1^−/− pro-OBLs versus PD98059 treated Nf1^−/− pro-OBLs).

Figure 5.

Inhibition of MAPK pathway attenuates frequency of osteoclast progenitors and osteoclast maturation of Nf1^+/− BMMNCs. (A) CFU-M formation was measured in WT and Nf1^+/−BMMNCs following M-CSF (30 ng/ml) stimulation in the presence of escalating dose of PD98059. Data represent the mean ± SEM of three individual experiments (**P < 0.01 for untreated and PD98059 (100 nm) treated WT versus untreated and PD98059 (100 nm) treated Nf1^+/−CFU-M, *P < 0.05 for PD98059 treated (250 nM, 500 nM) WT versus PD98059-treated (250, 500 nm) Nf1^+/−CFU-M. (B) WT and Nf1^+/−BMMNCs were cultured for 7 days and stained with TRACP to identify mature osteoclasts. (a) Data represent the mean ± SEM of % TRACP-positive area in response to varying concentrations of PD98059 of three independent experiments (**P < 0.01 for untreated WT versus untreated Nf1^+/−osteoclasts). (b) Representative photographs (20×) of untreated and PD98059 (100 nM) treated WT and Nf1^+/−osteoclasts are shown.

Figure 6.

MEK inhibition promotes tibial fracture-healing in Col2.3Cre; Nf1^flox/−mice in vivo. Tibial fractures were generated for WT and Col2.3Cre;Nf1^flox/− mice. Osmotic pumps containing either vehicle control (PBS) or PD98059 were implanted subcutaneously in the contralateral dorsal flank of Col2.3Cre;Nf1^flox/− mice. WT mice received vehicle control (PBS) implants. (A) Showing are representative radiographs acquired at 0, 14, 21, 28 days post-fracture in WT mice with PBS vehicle and Col2.3Cre;Nf1^flox/− mice with or without PD98059 (10 mg/kg/day). (B, a) Representative photomicrographs (20×) of MacNeal's/Von Kossa counter stain shows accumulation of cartilage and infiltration of marrow cells filling the fracture area in the control pump group. (B, b) Representative photomicrographs (20×) of TRACP stains are shown. Red area represents TRACP-positive-staining osteoclasts. (C) TRACP-positive osteoclasts to the bone surface ratio (Oc.N/BS) was enumerated as shown (***P < 0.01). (D) The ratio of bone volume/tissue volume was quantified to calculate percent tissue mineralization (***P < 0.01). (E) The number of OBLs per bone surface (Ob.N/BS) was quantified as shown (**P < 0.01).). Data represent mean ± SEM from five mice per treatment group.

Figure 7.

Inhibition of Ras/MAPK signaling enhances OBL differentiation in NF1 hMSCs. (A) Reintroduction of the NF1 GRD into NF1 patient hMSCs restores OBL differentiation. ALP expression, relative to WT control, is shown (***P < 0.001 for MSCV-pac control hMSCs versus MSCV-pac NF1 hMSCs, **P < 0.01 for MSCV-pac NF1 hMSCs versus NF1 GRD NF1 hMSCs,). (B) Pharmacologic MEK inhibition (100 nm PD98059) enhances OBL differentiation in NF1 patient hMSCs (*P < 0.05 for vehicle-treated NF1 hMSCs versus control hMSCs, and NF1 hMSCs vehicle versus PD98059 -reated NF1 hMSCs. Data are representative of three NF1 and two control samples.