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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.

SUBMITTER: Sharma R

PROVIDER: S-EPMC3820137 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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

Sharma Richa R Wu Xiaohua X Rhodes Steven D SD Chen Shi S He Yongzheng Y Yuan Jin J Li Jiliang J Yang Xianlin X Li Xiaohong X Jiang Li L Kim Edward T ET Stevenson David A DA Viskochil David D Xu Mingjiang M Yang Feng-Chun FC

Human molecular genetics 20130717 23

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 ost ...[more]

PMID: 23863460

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Project description:Osteoblasts are implicated in the building of the vertebrate skeleton. The current study aimed to investigate the role of microRNA-495 (miR-495) in the osteoblasts of mice with tibial fractures and the underlying mechanism involving in aquaporin-1 (AQP1) and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Initially, a microarray-based analysis was performed to screen the differentially expressed genes and miRNAs associated with tibial fracture. Following the establishment of a tibial fracture mouse model, the positive rate of the AQP1 protein in the fracture tissue was detected by immunohistochemistry (IHC). Next, to verify the binding site between miR-495 on AQP1, bioinformatics data were employed in addition to the application of a dual-luciferase reporter gene assay. The osteoblast cell line MC3T3-E1 was treated with miR-495 mimic, miR-495 inhibitor and Anisomycin to explore the potent effects of miR-495 on proliferation and differentiation of osteoblasts in mice with tibial fracture. The expression of miR-495, AQP1, p38 MAPK, PCNA, Cyclin D1, OCN, and OPN was subsequently evaluated by RT-qPCR and Western blot analysis. Cell viability, the number of calcium nodules and alkaline phosphatase (ALP) activity were detected by MTT assay, alizarin red staining, and ALP activity assay, respectively. Our results revealed that miR-495 was down-regulated while AQP1 was up-regulated in the mice with tibial fractures. AQP1 was verified as a target gene of miR-495. When the cells were treated with overexpressed miR-495 or activated p38 MAPK signaling pathway, elevated expression of PCNA, Cyclin, D1, OCN, and OPN along with an increased amount of calcium nodules, higher cell viability, and enhanced ALP activity was detected, while the expression of AQP1 was reduced. Collectively, the key findings of the present study support the notion that overexpressed miR-495 may activate the p38 MAPK signaling pathway to inhibit AQP1 and to promote the proliferation and differentiation of osteoblasts in mice with tibial fracture.

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

Publications

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

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