The Function Of Nf1+/-Osteoclast And The Relationship With Osteopontin

Neurofibromatosis type 1 (NF1) is a common, autosomal-dominant disorder caused by mutations in the NF1 tumor suppressor gene. Individuals with NF1 have a high incidence of both malignant as well as non-malignant complications. Many laboratories are pursuing concentrated efforts to understand the pathogenesis of the peripheral and central nervous system tumors that affect a wide range of NF1 patients. Despite NF1 being considered a neurocristopathy, NF1 functions as a GTPase in mesothelial derived tissues including blood cells, fibroblasts and osteoprogenitor cells that are relevant to skeletal development. However, less effort to date has been focused on understanding the molecular mechanisms underlying the pathogenesis of many of the non-malignant manifestations of NF1, particularly the skeletal manifestations that cumulatively affect up to 50% of all NF1 patients. Recent clinical studies have found that individuals with NF1 are at significant risk for both generalized and focal skeletal abnormalities. Several groups have reported that NF1 have reduced including a controlled trial using WHO criteria for osteoporosis and osteopenia, have provided evidence that NF1 patients have a significantly higher incidence of osteoporosis and osteopenia.Osteoclasts play a central role in the regulation of bone mass. Osteoclasts are terminally differentiated multinucleated giant cells with a principal bone resorptive activity. Bone resorption appears to proceed by the intricate coordination of the processes of attachment to bone, polarized secretion of acid and proteases, and active motility of osteoclasts along the bone surface. As osteoclasts crawl over bone surfaces they require rapid attachment and release from the extracellular matrix. The adhesion structures, podosomes, are uniquely designed for this purpose. After osteoclasts attach to the bone surface, podosomes develop highly polarized cytoplasmic organization in their ruffled borders. Recently, we reported a gain-of–function in the osteoclast bone resorption derived from Nf1+/- mice.Osteopontin (OPN) has been shown to be multifunctional, with activities in cell migration, cell survival, inhibition of calcification, regulation of immune cell function, and tumor cell metastasis. Increased levels of OPN protein have been linked to many disease states including atherosclerosis, transformation, tumor metastasis and renal diseases. Osteopontin (OPN) has also been reported involved in bone resorption, angiogenesis, wound healing, and tissue injuries following binding with type I collagen , fibronectin , and osteocalcin. OPN is a secreted, non-collagenous, sialic acid-rich phosphog- lycoprotein and acts both as chemokine and cytokine . Recently, OPN has been shown interacts with several integrins and CD44 variants which causes cell adhesion, migration, extracellular matrix invasion, and cell proliferation. In bone, it is predominantly expressed in osteoblasts and in terminally differentiated chondrocytes at the chondroosseous border. OPN-deficient (OPN-/-) mice are resistant to ovariectomy-induced bone resorption. Yu et al. have shown that haploinsufficiency of Nf1 enhances osteopontin expression in bone marrow-inducible osteoprogenitors and cortical bone. Kolanczyk et al. reported a significant upregulation of osteopontin in chondrocytes derived from a conditional Nf1 knockout mice, Nf1Prx1 mice. This upregulation of OPN expression may regulated by Ras. These studies imply an association between OPN and gain in functions in Nf1 haploinsufficient osteoclasts. In the present study, we proposed to investigate the role of OPN in regulating multiple cellular functions of Nf1+/- osteoclasts and related signaling axis. Our study indicates that Nf1 haploinsufficient osteoclasts are hypersensitive to OPN, which leads to increased osteoclast migration, adhesion, and bone resorption. Further, hyper phosphorylation of Ras/MAPK pathway is responsible for the gain in the osteoclast functions associated with OPN. Target of OPN may be a new treatment for the osseous manifestations in NF1 patients. PART 1Osteoclast hyperfunction in neurofibromatosis type 1 murine modelObjective: Investigate cellular functions of osteoclasts derived from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice,and analyze the mechanism of the osteoprosis in Neurofibromatosis type 1.Method: Stain histological sections of tibias from Nf1+/- and Nf1+/+ mice with H&E or tartrate-resistant acid phosphatase (TRAP) activity, and measure the osteoclasts contained in metaphysis; Culture the low density bone marrow cells from Nf1+/- and Nf1+/+ mice with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), examine the frequency of osteoclast progenitor, and evaluate multiple functions, including migration, adhesion, and bone absorption.Result: Nf1+/- mice contain numerous enlarged multinucleated osteoclasts in vivo than Nf1-/- mice.(TRAP+ area/total area Nf1+/+, 0.88%±0.0014; Nf1+/-, 2.33%±0.0013,P<0.01 ) , The number of Nf1+/- osteoclast formated from 1×105 low dencity bone marrow cells are higher than that of Nf1+/+ with significant difference. (Nf1+/+,41.75±13.14 ; Nf1+/-, 61.17±18.17, P<0.01). The mutiple functions of osteoclasts derived from Nf1 heterozygote are higher than those of wild type with significant difference(adhesion Nf1+/+,53±11.08; Nf1+/-,108±11.67, P<0.01; migration Nf1+/+,88.33±12.40; Nf1+/-,239.83±67.77, P<0.01; pit formation Nf1+/+, 18.37%±0.0367; Nf1+/-,40.44%±0.1052, P<0.01).Conclusion: The hyperfuctions of osteoclast in Nf1+/- mice may be responsible for the pathogenesis of NF1 osteoprosis.PART 2 OSTEOPONTIN PREFERENTIALLY PROMOTES MULTIPLE FUNCTIONS OF MURINE Nf1+/- OSTEOCLASTObjective: Test the osteoclast function of neurofibromatosis type 1 mice with exogenous osteopontin, analysis the mechanism of the bone change in neurofibromatosis type 1.Method: We culture the low density bone marrow cells from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice ( 4~6weeks old) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand( RANKL), with or without exogenous osteopontin,The function of osteoclast and osteoclast progenitor in formation, migration, adhesion, and bone absorption are tested. Osteoclast formation(Trap+cells Nf1+/+ without OPN 39.25±11.9with OPN 47±2.34;Nf1+/- without OPN 51.5±5.90 with OPN 70.75±0.81),adhesion(adhesiion cellsNf1+/+ without OPN 52.20±11.79, with OPN,69.80±7.65 ; Nf1+/- without OPN 80.40±13.61, with OPN 162.50±10.52), migration(migration cells Nf1+/+ without OPN 87.20±5.17, with OPN 238.17±28.20;Nf1+/- without OPN 223.17±29.02 ,with OPN 582.13±51.51),pit formation(area of bone absorptionNf1+/+ without OPN 4.18%±0.0011 with OPN 17.08%±0.018;Nf1+/- without OPN 9.11%±0.008, with OPN 72.14%±0.083)the difference is significant.Result: Given exogenous osteopontin, the osteoclast functions of Nf1 heterozygote are higher than wild type. The difference is significant.Conclusion: The hyperfunction of osteoclast in Nf1 heterozygote are related with osteopontin, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1.PART 3 AUTOCRINE OSTEOPONTIN PROMOTES MULTIPLE FUNCTIONS OF MURINE Nf1+/- OSTEOCLASTObjective: Test the osteoclast function of neurofibromatosis type 1 mice with exogenous osteopontin antibody, analysis the mechanism of the bone change in neurofibromatosis type 1. Method: Measure the OPN concentration in osteoclast culture superenant with ELISA. culture the low density bone marrow cells from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice ( 4~6weeks old ) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand( RANKL), with or without exogenous Anti-osteopontin,The function of osteoclast and osteoclast progenitor in formation, migration, adhesion, and bone absorption are tested.Result: The concentration of OPN in osteoclast culture media (Nf1+/+ 3days,54.60±6.72ng/ml ; 6day , 80.04±11.11ng/ml ; Nf1+/-3days ,71.34±9.18ng/ml, 6days,120.73±3.4ng/ml p<0.05) the difference is significant. Osteoclast formation (Trap+cells Nf1+/+ without anti-OPN antibody42.0±6.08with anti-OPN antibody ; Nf1+/- without anti-OPN antibody72.33±5.75with anti-OPN antibody 41.67±11.86), adhesion(adhesiion cellsNf1+/+ without anti-OPN antibody 39.25±11.9 with anti-OPN antibody, 27±2.34 ; Nf1+/- without anti-OPN antibody 51.5±5.90, with anti-OPN antibody 10.75±0.81,), migration(migration cellsNf1+/+ without anti-OPN antibody150.20±35.28, with anti-OPN antibody 114.8±24.05;Nf1+/- without anti-OPN antibody241.60±37.10 ,with anti-OPN antibody111.80±21.13),pit formation ( area of bone absorptionNf1+/+ without anti-OPN antibody4.18%±0.0011 with anti-OPN antibody2.08%±0.0041 ; Nf1+/- without anti-OPN antibody19.11%±0.046, with anti-OPN antibody1.81%±0.002).the difference are significant.Conclusion: The hyperfunction of osteoclast in Nf1 heterozygote are related with autocrine osteopontin, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1. PART 4 Paracrine secretion of osteopontin by Nf1+/- osteoblasts promote osteoclast activation in a neurofibromatosis type I murine modelObjective: Test the osteoclast function of neurofibromatosis type 1 mice with osteoblast conditioned media, analysis the relationship of osteoclast hyperfunction with the paracrine of osteopontin by haploinsufficient loss of Nf1 osteoblast.Method: Measure the OPN concentration in osteoblast conditioned media with ELISA. culture the low density bone marrow cells from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice (age at 4~6weeks ) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand(RANKL), with or without OBCM and exogenous Anti-osteopontin antibody,The function of osteoclast and osteoclast progenitor in formation, migration, adhesion, and bone absorption are tested.Result: The concentration of OPN in osteoblast conditioned media ((Nf1+/+141.09±11.31ng/ml;Nf1+/-241.08±18.63ng/ml,p<0.01) the difference is significant. Conditioned media from Nf1+/- osteoblasts promotes the Nf1+/- osteoclast formation. Neutralizing antibody for OPN inhibited bioactivity of Nf1+/- OBCM. the difference are significant.Conclusion: The hyperfunction of osteoclast in Nf1 heterozygote are related with paratocrine osteopontin by osteoblast, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1.