Neurofibromatosis Type 1 (NF1) Skeletal Lesion And Its Mechanism

Neurofibromatosis type 1 (NF1) is a common, autosomal-dominant disorder caused by mutations in the NF1 tumor suppressor gene. One in 3500 individuals worldwide is born with NF1. The NF1 gene is extremely large and complex. It spans 350 kilobases of the genome and there are 60 exons. There are three genes within the intron of 27b of the NF1 gene that are transcribed in the opposite orientation of the NF1 gene for which no function has been ascribed. The only region of NF1 for which a function has been ascribed is the GAP Related domains of NF1 which accelerate the hydrolysis of Ras thousands of fold.The NF1 gene encodes for the protein neurofibromin, and is expressed in a broad range of cell and tissue types. Neurofibromin negatively regulates the activity of an intracellular signaling molecule p21ras (Ras) by functioning as a GTPase activating protein (Ras-GAP). The Ras-GAP function of neurofibromin has been linked to a variety of the clinical symptoms associated with NF1. Ras is on the upstream of any singal pathway. Haploinsufficiency or a complete deficiency in NF1 results in a dose-dependent elevation in Ras activity, which in turn can activate a variety of signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway, and the phosphatidylinositol-3-phosphate kinase (PI-3K) pathway. These pathways have the capacity to affect cellular proliferation and differentiation in a cell type specific manner.NF1 is involved nervous, vascular, and skeletal system compoments and it always occur in children or adolescent. Fifty percent of NF1 patients have osseous manifestations including a high incidence of osteoporosis. Recently, in USA and Euroupe, 4 labs reported NF1 patient show osteoporosis and osteopenia. Some NF1 patients suffer scoliosis, its prevalence is 10-70%. Spine abnormal manifestation involves kyphoscoliosis scoliosis and cervical twist. Additionally, pseudarthrosis is another common manifestation in NF1 patient. When the child is 1 year old, his tibia presents bow, and about 2 year old Fracture occured, the Fracture is difficult to heal and pseudarthrosis occurs. Pseudarthrosis make function and appearance abnormal,at last some patients have to take amputation. But until now, the mechanism underlying NF1 bone defects is not clear and no effective treatment.Bone renew continuously. Old bone resorps and new bone forms. This process is the preventive maintenance of mechanical strength by continuously replacing fatigued bone by new''fresh''bone. The bone is the main reservoir of calcium ions, and the remodeling is critical for Ca ?uxes into and from the extracellular ?uid to maintain an appropriate level of blood calcium. Bone homeostasis is maintained by balancing skeletal matrix formation and resorption. Osteoclasts are specialized cells derived from the myeloid monocyte/macrophage lineage that successively adhere to bone matrix and resorb bone, while osteoblasts generate new skeletal matrix. Imbalances in the morphogenesis and remodeling of bone are known to lead to pathological perturbations of skeletal structure and function. Osteoclasts are multinucleared cells that derive from hematopoietic progenitors in the bone marrow which also give rise to monocytes in peripheral blood, and to the various types of tissue macrophages. Osteoclasts are formed by the fusion of precursor cells. The exptriment show that M-CSF and RANKL mediate ostocalst proliferation and differentiation, which play a critical role in physiological and pathological function of osteoclast.Yang's reresults show that, in vitro, there is a decresed differentiation in osteoblast of Nf1+/- mice, and this change is directly associated with Ras activity. Karsenty found that only change of osetobalst function can not cause bone diseases. Recently, Yang reported when both abnormal in ostobalst and ostocals caused the osteoporosis, which is similar to the clinical result. NFpatient and Nf1+/-mice have Ras hyperactitive, which induced osteoclast function haperactitive. Based on these results, we presume osteoclast as a important role in NF1 skeletal lesion. The osteoclast function includes migration, adhesion and resorption, and these functions are associated with the skeletal lesion. Another study showed that acin formation influences osteoclast migration, adhesion and resorption. Acin compose cytoskeletal. Cytoskeletal formation, function are controled by Rho gene.Rho family includes Rac, Cdc42, and Rho. In general, Rho gene controls motion, adhesion, and deformation in mammal. Furthermore, Rho gene controls the signal pathway of cell cycle, expression, survival, and aptosis. Rac is regard as the core in Rho family. it have three isofome: Rac1, Rac2 and Rac3. Rac1 express in general tissues, Rac2 in haemopoiesis and Rac3 in nervous system. Yang found that Rac-GTPase control not only affects cell appearance and function, but also plays a important role in adjusting mast cell migration, adhesion and degranulation. Mast cell and osteoclast are both derived from monohaemopoietic stem cell. So, we think that ostocalst gain-in-function is associated with Rac. This study focus on the osteoclast function of NF1patient and Nf1+/- mice, the functionof Rac1, and exploring a effective treatment of NF1.This study is composed of three parts:Part one Nf1+/- mice osteoclast assayObject: to investigate Nf1+/- mice osteoclast formation and function in Nf1+/- mice.Method: the marrow mononuclear cells (BMMNCs) were taken from 3 paires gene WT and Nf1+/- mice for osteoclast colony formation, osteoclast formation, proliferation, migration, bone resorption and Actin ring formation. In the presence of M-CSF and RANKL, BMMCs was induced to osteoclast. Each experiment conducted on more than three independent occasions. Student's t-test was used to evaluate statistical difference. P-values less than 0.05 were considered significant.Result: Nf1+/–mice showed doubled osteoclast and osteoclast progenitor number and have a significant increase in migration, proliferation, actin ring formation , bone resorption of the Nf1+/– osteoclast compared with wild type. Conclusion: Nf1+/–mice has a increased number and function of osteoclast.Part two The mechanism of the osteoclast gains-in-function in Nf1+/- mice Object: to investigate mechanism in increased Nf1+/- mice osteoclast formation and functionMethod: Rac1 conditional (Rac1?ox/?ox) and null alleles were generated by Dr Kwiatkowski as previously reported. The conditional Rac1 allele contains loxP sites ?anking exon 1; upon cre-mediated excision this allele generates a null allele. Rac1 conditional knockout mice were crossed with MxCre-transgenic C57BL/6 mice. Animals received three intraperitoneal injections at 3 months of age with 300μg poly I:-poly C (poly I:C) diluted in sterile phosphate-buffered saline (PBS). Mice were sacrificed for bone marrow cell harvest 48 h after the last injection. Take comparable 3 paires gene WT, Nf1+/-, Rac1-/-and Nf1+/-;Rac1-/-mice, take the bone marrow mononuclear cells (BMMNCs). To examine the Rac1-GTPase and Rac2 GTPase activity, Rac activation was determined using a PAK pulldown assay. Then to assay osteoclast colony formation, osteoclast formation, proliferation, migration, bone resorption and Actin ring formation. In the present of M-CSF and RANKL, BMMCs induce to osteoclasts, we utilized clonogenic assays to measure colony-forming-unit-macrophage (CFU-M) and osteoclast progenitors following tartrate resistant acid phosphatase (TRAP) staining (TRAP+ CFU-M) from freshly isolated bone marrow cells, Through Thymidine incorporation assay proliferation. Utilize Transwell assay migration and through dentine resoption assay to test bone resoption. Utilize FITC-phyloitine and confocol scan observe Actin ring formation. Erk phosphorylation and Akt phosphorylation were determined by western blot using phosphospecific antibody. Osteoclasts were deprived of serum and growth factors for 9h followed by stimulation with 30 ng/ml M-CSF for various amounts of time. Densitometry of individual bands was conducted using NIH Image software. To validate our biochemical results, PD98059, a pharmacological inhibitor of Mek within the Ras-Erk signaling pathway was utilized in the osteoclast culture and the effects on f-actin organization were examined. Each experiment conducted on more than three independent occasions. Student's t-test and ANOVA were used to evaluate statistical difference. P-values less than 0.05 were considered significant.Result: our data showed that the Rho-GTPase Rac1 is a crucial Ras-mediated effector in Nf1 haploinsufficient (Nf1+/-) osteoclasts. Nf1+/- mice were intercrossed with conditional Rac1?ox/?oxMxcre+ (Rac1-/-) mice to generate Nf1+/-; Rac1-/- mice. Increased Rac1 activation was seen in Nf1+/- osteoclasts as compared to that in control cells, Genetic disruption of Rac1 restored the pathological increase in osteoclast progenitor cells in Nf1+/- mice and was sufficient to correct the increased Nf1+/- osteoclast function and osteoclast belt formation, an Actin structure observed in mature osteoclasts critical for bone resorption and lytic activity. The increased Akt and Erk phosphorylation correlates with the gain in proliferation, migration, and bone resorption observed in Nf1+/- osteoclasts. Hyperactivation of Akt and Erk phosphorylation in Nf1+/- osteoclast progenitors is normalized by deletion of Rac1. Upon culture with PD98059 (50 mM), the size and the number of osteoclasts observed in the Nf1+/- cultures were reduced and identical to that observed in the WT cultures.Conclusion: these data demonstrate that Rac1 critically contributes to increased osteoclast function induced by haploinsufficiency of Nf1. Genetic disruption of Rac1 restored the pathological increase in osteoclast progenitor cells in Nf1+/- mice and was sufficient to correct the increased Nf1+/- osteoclast function and osteoclast belt formation. Rac1-Erk/ Rac1-Akt pathway is the increased osteoclast function signal pathway. Hyperactivation of Erk and Akt in Nf1+/- osteoclast progenitors is normalized by deletion of Rac1 and inhibition of the Ras-Erk pathway corrects the osteoclast formation observed in Nf1+/- osteoclasts. This implicated Rac1 as a rational therapeutic target for osteoporosis. Part three NF1 patient osteoclast assayObject: to investigate NF1 patient osteoclast formation and function in NF1 patient and its mechanismMethod: According to WHO NF1 diognosis criteria, harvest 32 pairs of compared normal and NF1 person, take peripheral blood 10ml each person. Get mononuclear cells from peripheral blood (PBMNCs) after centrifugalization. Osteoclast formation and function assay, includes osteoclast formation, migration, adhension, resorption and actin ring formation. In the presence of M-CSF and RANKL, PBMNCs was induced to osteoclast. We utilized osteoclast progenitors following tartrate resistant acid phosphatase (TRAP) staining from freshly isolated PBMNCs. 24-Transwell assay was used for migration and dentine resoption assay to test bone resoption. We used FITC-phyloitine and confocol scan observe Actin ring formation. Osteoclasts were deprived of serum and growth factors for 9 hours and stimulated with 30ng/ml M-CSF. Akt and Erk phosphorylation were determined by Western blot using phospho-specific antibody for Akt and Erk. Densitometry of individual bands was conducted using NIH Image software. To examine the Rac1-GTPase activity, a PAK pulldown assay was performed.Each experiment conducted on more than three independent occasions. Student's t-test was used to evaluate statistical difference. P-values less than 0.05 were considered significant.Result: The individuals with NF1 had increased osteoclast formation following M-CSF and RANKL stimulation as compared to control individuals. In addition, osteoclasts of individuals with NF1 had increased migration, adhension and bone resorption. The gain in bone absorption is correlated with the elevated Actin ring formation and Rac1-GTP activity. Furthermore, NF1 osteoclasts had elevated phosphorylation of Erk and Akt, consistent with elevated cellular functions.Conclusion: NF1 patient showed osteoclast formation, migration, adhension, bone resorption, actin ring formation have significant inceased compared with health person. The increased Akt and Erk phosphorylation correlates with the gain in formation, migration, actin ring formation and bone resorption observed in NF1 osteoclasts. In addition, increased Rac1 activation was also seen in NF1 osteoclasts as compared to that in health person, consistent with hypermotility and increased actin ring formation in NF1 osteoclasts. Rac1-Erk/ Rac1-Akt pathway is the increased osteoclast function signal pathway.