Nf1 Haploinsufficiency Alters Myeloid Lineage Commitment and Function, Leading to Deranged Skeletal Homeostasis

Authors

Steven D. Rhodes, Hao Yang, Ruizhi Dong, Keshav Menon, Yongzheng He, Zhaomin Li, Shi Chen, Karl W. Staser, Li Jiang, Xiaohua Wu,Xianlin Yang, Xianghong Peng, Khalid S. Mohammad, Theresa A. Guise,Mingjiang Xu and Feng-Chun Yang

Abstract

While nullizygous loss of NF1 leads to myeloid malignancies, haploinsufficient loss of NF1 (Nf1) has been shown to contribute to osteopenia and osteoporosis which occurs in approximately 50 percent of neurofibromatosis type 1 (NF1) patients. Bone marrow mononuclear cells of haploinsufficient NF1 patients and Nf1+/− mice exhibit increased osteoclastogenesis and accelerated bone turnover, however the culprit hematopoietic lineages responsible for perpetuating these osteolytic manifestations have yet to be elucidated. Here we demonstrate that conditional inactivation of a single Nf1 allele within the myeloid progenitor cell population (Nf1-LysM) is necessary and sufficient to promote multiple osteoclast gain-in-functions, resulting in enhanced osteoclastogenesis and accelerated osteoclast bone lytic activity in response to pro-resorptive challenge in vivo. Surprisingly, mice conditionally Nf1 heterozygous in mature, terminally differentiated osteoclasts (Nf1-Ctsk) do not exhibit any of these skeletal phenotypes, indicating a critical requirement for Nf1 haploinsufficiency at a more primitive/progenitor stage of myeloid development in perpetuating osteolytic activity. We further identified p21Ras dependent hyper-phosphorylation of Pu.1 within the nucleus of Nf1 haploinsufficent myelomoncytic osteoclast precursors, providing a novel therapeutic target for the potential treatment of NF1 associated osteolytic manifestations.

Link To Article

http://dx.doi.org/10.1002/jbmr.2538

Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation in Vivo and Osteoprogenitor Differentiation ex Vivo

Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased 3-fold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg).

Diacylglycerol Kinase ζ (DGKζ) is a Critical Regulator of Bone Homeostasis via Modulation of c-Fos Levels in Osteoclasts

Authors

Ali Zamani, Corinne Decker, Viviana Cremasco, Lindsey Hughes, Deborah V. Novack and Roberta Faccio

Abstract

Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs).

Strikingly, DGKζ-/- mice are osteoporotic due to a marked increase in OC numbers. In vitro, DGKζ-/- bone marrow macrophages (BMMs) form more numerous, larger and highly resorptive OCs. Surprisingly, while increased DAG levels do not alter RANK/RANKL osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine M-CSF. We find that M-CSF is responsible for increased DGKζ-/- OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ-/- cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2+/− BMMs, which have reduced DAG levels and form fewer OCs due to impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2+/− mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression.

Link To Article

http://dx.doi.org/10.1002/jbmr.2533

Deletion of Filamin A in Monocytes Protects Cortical and Trabecular Bone from Post-menopausal Changes in Bone Microarchitecture

Authors

S. Goldberg, J. Glogauer, M. D. Grynpas, M. Glogauer

Abstract

The objective of the study was to determine the in vivo role of Filamin A (FLNA) in osteoclast generation and function, through the assessment of trabecular bone morphology, bone turnover, and the resulting changes in mechanical properties of the skeleton in mice with targeted deletion of FLNA in pre-osteoclasts. Using a conditional targeted knockdown of FLNA in osteoclasts, we assessed bone characteristics in vivo including micro-computed tomography (micro-ct), histomorphometric analyses, and bone mechanical properties. These parameters were assessed in female mice at 5 months of age, in an aging protocol (comparing 5-month-old and 11-month-old mice) and an osteoporosis protocol [ovariectomized (OVX) at 5 months of age and then sacrificed at 6 and 11 months of age]. In vivo bone densitometry, mechanical and histomorphometric analyses revealed a mild osteoporotic phenotype in the FLNA-null 5-month and aging groups. The WT and FLNA-KO bones did not appear to age differently. However, the volumetric bone mineral density decrease associated with OVX in WT is absent in FLNA-KO-OVX groups. The skeleton in the FLNA-KO-OVX group does not differ from the FLNA-KO group both in mechanical and structural properties as shown by mechanical testing of femora and vertebrae and histomorphometry of vertebrae. Additionally, FLNA-KO femora are tougher and more ductile than WT femora. The result of this study indicates that while FLNA-KO bones are weaker than WT bones, they do not age differently and are protected from estrogen-mediated post-menopausal osteoporosis.

Link To Article

http://dx.doi.org/10.1007/s00223-015-9994-4

Influence of age and gender on microarchitecture and bone remodeling in subchondral bone of the osteoarthritic femoral head

Authors

Guangyi Li, Qiujian Zheng, Euphemie Landao-Bassonga, Tak S. Cheng, Nathan J. Pavlos, Yuanchen Ma, Changqing Zhang, Ming H. Zheng

Abstract

Introduction Age and gender have been reported to have a remarkable impact on bone homeostasis. However, subchondral bone, which plays a pivotal role in the initiation and progression of OA, has been poorly investigated in this area. This study was to investigate age- and gender- related changes of microarchitecture and bone remodeling in subchondral bone in OA.

Methods Subchondral trabecular bone (STB) and deeper trabecular bone (DTB) specimens were extracted in the load-bearing region of femoral heads from 110 patients with OA. Micro-CT and histomorphometry were performed to analyze microarchitectural and bone remodeling changes of all specimens.

Results Compared to DTB, STB showed more sclerotic microarchitecture, more active bone remodeling and higher frequency of bone cysts. There were no gender differences for both microarchitecture and bone remodeling in STB. However, gender differences were found in DTB, with thinner Tb.Th, higher Tb.N, higher OS/BV and ES/BV in males. In both STB and DTB, no correlation between microarchitecture and age was found in both genders. However, bone remodeling of STB increased significantly with age in males, while bone remodeling of DTB increased significantly with age in females. No age or gender preference was found in subchondral bone cysts (SBCs) frequency. The cyst volume fraction was correlated with neither age nor gender.

Conclusions There were distinct differences in microarchitecture and bone remodeling between STB and DTB, which may be due to the distinct biomechanical and biochemical functions of these two bone structures in maintaining joint homeostasis. OA changed the normal age- and gender-dependence of bone homeostasis in joints, in a site-specific manner.

Link To Article

http://dx.doi.org/10.1016/j.bone.2015.04.019

Sclerostin antibody treatment causes greater alveolar crest height and bone mass in an ovariectomized rat model of localized periodontitis

Authors

Hui Chen, Xinchen Xu, Min Liu, Wen Zhang, Hua-zhu Ke, An Qin, Tingting Tang, Eryi Lu

Abstract

Introduction Periodontitis and osteoporosis are bone destructive diseases with a high prevalence in the adult population. The concomitant presence of osteoporosis may be a risk factor of progression of periodontal destruction. We studied the effect of sclerostin-neutralizing monoclonal antibody (Scl-Ab) on alveolar bone endpoints in an ovariectomized (OVX) rat model of induced experimental periodontitis.

Methods Sixty female, 4-month-old Sprague–Dawley rats underwent sham operation or bilateral OVX and were left untreated for 2 months. Experimental periodontitis (ligature) was established by placing silk sutures subgingival to the right maxillary first and second molar teeth for 4 weeks, and feeding the rats food and high-sugar drinking water during this period. Thereafter, ligatures were removed and 25 mg/kg vehicle or Scl-Ab was administered subcutaneously twice weekly for 6 weeks. Rats were randomized into four groups: (1) Control (Sham + Vehicle), (2) Sham + Ligature + Vehicle, (3) OVX + Ligature + Vehicle, and (4) OVX + Ligature + Scl-Ab. Terminal blood and right maxilla specimens were collected for analyses.

Results Group 3 rats showed lower bone volume fraction (BVF) of alveolar bone with higher bone resorption and lower bone formation than Group 2 rats. Group 4 rats had higher alveolar crest height, as assessed by linear distance of cementoenamel junction to the alveolar bone crest and greater alveolar bone mass using Micro CT, than Group 3 rats. Significantly higher values of mineral apposition rate (MAR) and mineralizing surface/bone surface (MS/BS) were also observed in Group 4 rats by analyzing polychrome sequential labeling data. Increased serum osteocalcin and osteoprotegerin, and deceased serum tartrate-resistant acid phosphatase and CTx-1 illustrate the ability of Scl-Ab to increase alveolar bone mass by enhancing bone formation and decreasing bone resorption in an animal model of estrogen deficiency osteopenia plus periodontitis.

Conclusion Scl-Ab could be a potential bone anabolic agent for improving alveolar crest height and higher alveolar bone mass in conditions where alveolar bone loss in periodontitis is compounded by estrogen deficiency osteopenia.

Link To Article

http://dx.doi.org/10.1016/j.bone.2015.04.002