Netrin-1 is a critical autocrine/paracrine factor for osteoclast differentiation

Authors

Aránzazu Mediero, Bhama Ramkhelawon, Miguel Perez-Aso, Kathryn J. Moore and Bruce N. Cronstein

Abstract

Bone metabolism is a vital process that involves resoption by osteoclasts and formation by osteoblasts which is closely regulated by immune cells. The neuronal guidance protein Netrin-1 regulates immune cell migration and inflammatory reactions, but its role in bone metabolism is unknown. During osteoclast differentiation osteoclast precursors increase expression of Netrin-1 and its receptor Unc5b. Netrin-1 binds, in an autocrine and paracrine manner, to Unc5b to promote osteoclast differentiation in vitro and absence of Netrin-1 or antibody-mediated blockade of Netrin-1 or Unc5b prevents osteoclast differentiation of both murine and human precursors. We confirmed the functional relationship of Netrin-1 in osteoclast differentiation in vivo using Netrin-1-deficient (Ntn1-/-) or wildtype (WT) bone marrow transplanted mice. Notably, Ntn1-/- chimeras have markedly diminished osteoclasts, as well as increased cortical and trabecular bone density and volume compared to WT mice. Mechanistic studies revealed that Netrin-1 regulates osteoclast differentiation by altering cytoskeletal assembly. Netrin-1 increases LARG (regulator of Rho-GEF subfamily) and RGMa (repulsive guidance molecule) association with Unc5b, which increases expression and activation of cytoskeletal regulators RhoA and focal adhesion kinase (FAK). Netrin-1 and its receptor unc5b likely play a role in fusion of osteoclast precursors since Netrin-1 and DC-STAMP are tightly linked. These results identify Netrin-1 as a key regulator of osteoclast differentiation that may be a new target for bone therapies.

Link To Article

http://dx.doi.org/10.1002/jbmr.2421

Treatment with eldecalcitol positively affects mineralization, microdamage, and collagen crosslinks in primate bone

Authors

Mitsuru Saito, Marc D. Grynpas, David B. Burr, Matthew R. Allen, Susan Y. Smith, Nancy Doyle, Norio Amizuka, Tomoka Hasegawa, Yoshikuni Kida, Keishi Marumo, Hitoshi Saito

Abstract

Eldecalcitol (ELD), an active form of vitamin D analog approved for the treatment of osteoporosis in Japan, increases lumbar spine bone mineral density (BMD), suppresses bone turnover markers, and reduces fracture risk in patients with osteoporosis. We have previously reported that treatment with ELD for 6 months improved the mechanical properties of the lumbar spine in ovariectomized (OVX) cynomolgus monkeys. ELD treatment increased lumbar BMD, suppressed bone turnover markers, and reduced histomorphometric parameters of both bone formation and resorption in vertebral trabecular bone. In this study, we elucidated the effects of ELD on bone quality (namely, mineralization, microarchitecture, microdamage, and bone collagen crosslinks) in OVX cynomolgus monkeys in comparison with OVX-vehicle control monkeys. Density fractionation of bone powder prepared from lumbar vertebrae revealed that ELD treatment shifted the distribution profile of bone mineralization to a higher density, and backscattered electron microscopic imaging showed improved trabecular bone connectivity in the ELD-treated groups. Higher doses of ELD more significantly reduced the amount of microdamage compared to OVX-vehicle controls. The fractionated bone powder samples were divided according to their density, and analyzed for collagen crosslinks. Enzymatic crosslinks were higher in both the high-density (≥2.0 mg/mL) and low-density (<2.0 mg/mL) fractions from the ELD-treated groups than in the corresponding fractions in the OVX-vehicle control groups. On the other hand, non-enzymatic crosslinks were lower in both the high- and low-density fractions. These observations indicated that ELD treatment stimulated the enzymatic reaction of collagen crosslinks and bone mineralization, but prevented non-enzymatic reaction of collagen crosslinks and accumulation of bone microdamage. Bone anti-resorptive agents such as bisphosphonates slow down bone remodeling so that bone mineralization, bone microdamage, and non-enzymatic collagen crosslinks all increase. Bone anabolic agents such as parathyroid hormone decrease bone mineralization and bone microdamage by stimulating bone remodeling. ELD did not fit into either category. Histological analysis indicated that the ELD treatment strongly suppressed bone resorption by reducing the number of osteoclasts, while also stimulating focal bone formation without prior bone resorption (bone minimodeling). These bidirectional activities of ELD may account for its unique effects on bone quality.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.11.025

Microdamage generation by tapered and cylindrical mini-screw implants after pilot drilling

Authors

Emily Taing-Watson, Thomas R. Katona, Kelton T. Stewart, Ahmed Ghoneima, Gabriel T. M. Chu, Hee-Moon Kyung, and Sean S. Liu

Abstract

Objective: To investigate the relationship between mini-screw implant (MSI) diameter (1.6 vs 2.0 mm) and shape (tapered vs cylindrical) and the amount of microdamage generated during insertion.

Materials and Methods: Thirty-six cylindrical and 36 tapered MSIs, 6 mm long, were used in this study. Half of each shape was 1.6 mm in diameter, while the other half was 2.0 mm. After pilot drilling, four and five MSIs were inserted, respectively, into fresh cadaveric maxillae and mandibles of dogs. Bone blocks containing the MSIs were sectioned and ground parallel to the MSI axis. Epifluorescent microscopy was used to measure overall cortical thickness, crack length, and crack number adjacent to the MSI. Crack density and total microdamage burden per surface length were calculated. Three-way analysis of variance (ANOVA) was used to test the effects of jaw, and MSI shape and diameter. Pairwise comparisons were made to control the overall significance level at 5%.

Results: The larger (2.0 vs 1.6 mm) cylindrical MSIs increased the numbers, lengths, and densities of microcracks, and the total microdamage burden. The same diameter cylindrical and tapered MSIs generated a similar number of cracks and crack lengths. More total microdamage burden was created by the 2.0-mm cylindrical than the 2.0-mm tapered MSIs. Although higher crack densities were produced by the insertion of 1.6-mm tapered MSIs, there was no difference in total microdamage burden induced by 1.6-mm tapered and 1.6-mm cylindrical MSIs.

Conclusions: Pilot drilling is effective in reducing microdamage during insertion of tapered MSIs. To prevent excessive microdamage, large diameter and cylindrical MSIs should be avoided.

Link To Article

http://dx.doi.org/10.2319/062314-452.1

Suppression of Adult Neurogenesis Increases the Acute Effects of Kainic Acid

Authors

Sloka S. Iyengar, John J. LaFrancois, Daniel Friedman, Liam J. Drew, Christine A. Denny, Nesha S. Burghardt, Melody V. Wu, Jenny Hsieh, René Hen, Helen E. Scharfman

Abstract

Adult neurogenesis, the generation of new neurons in the adult brain, occurs in the hippocampal dentate gyrus (DG) and the olfactory bulb (OB) of all mammals, but the functions of these new neurons are not entirely clear. Originally, adult-born neurons were considered to have excitatory effects on the DG network, but recent studies suggest a net inhibitory effect. Therefore, we hypothesized that selective removal of newborn neurons would lead to increased susceptibility to the effects of a convulsant. This hypothesis was tested by evaluating the response to the chemoconvulsant kainic acid (KA) in mice with reduced adult neurogenesis, produced either by focal X-irradiation of the DG, or by pharmacogenetic deletion of dividing radial glial precursors. In the first 4 hrs after KA administration, when mice have the most robust seizures, mice with reduced adult neurogenesis had more severe convulsive seizures, exhibited either as a decreased latency to the first convulsive seizure, greater number of convulsive seizures, or longer convulsive seizures. Nonconvulsive seizures did not appear to change or they decreased. Four-21 hrs after KA injection, mice with reduced adult neurogenesis showed more interictal spikes (IIS) and delayed seizures than controls. Effects were greater when the anticonvulsant ethosuximide was injected 30 min prior to KA administration; ethosuximide allows forebrain seizure activity to be more easily examined in mice by suppressing seizures dominated by the brainstem. These data support the hypothesis that reduction of adult-born neurons increases the susceptibility of the brain to effects of KA.

Link To Article

http://dx.doi.org/10.1016/j.expneurol.2014.11.009

Tgfbi Deficiency Leads to a Reduction in Skeletal Size and Degradation of the Bone Matrix

Authors

Jung-Mi Lee, Eun-Hye Lee, In-San Kim, Jung-Eun Kim

Abstract

Transforming growth factor-β-induced gene product-h3 (TGFBI/BIGH3) is an extracellular matrix protein expressed in a wide variety of tissues. TGFBI binds to type I, II, and IV collagens, as well as to biglycan and decorin and plays important roles in cell-to-cell, cell-to-collagen, and cell-to-matrix interactions. Furthermore, TGFBI is involved in cell growth and migration, tumorigenesis, wound healing, and apoptosis. To investigate whether TGFBI is involved in the maintenance of skeletal tissues, Tgfbi knockout mice were generated by crossing male and female Tgfbi heterozygous mice. Skeletal preparation showed that the skeletal size in Tgfbi knockout mice was smaller than in wild-type and heterozygous mice. However, chondrocytic cell alignment in the growth plates, bone mineral density, and bone forming rates were similar in Tgfbi knockout, wild-type, and heterozygous mice. Alterations in skeletal tissue arrangements in Tgfbi knockout mice were estimated from safranin O staining, trichrome staining, and immunohistochemistry for type II and X collagen, and matrix metalloproteinase 13 (MMP13). Cartilage matrix degradation was observed in the articular cartilage of Tgfbi knockout mice. Although the detection of type II collagen in the articular cartilage was lower in Tgfbi knockout mice than wild-type mice, the detection of MMP13 was markedly higher, indicating that Tgfbi deficiency is associated with the degradation of cartilage matrix. These results suggest that TGFBI plays an important role in maintaining skeletal tissues and the cartilage matrix in mice.

Link To Article

http://dx.doi.org/10.1007/s00223-014-9938-4

The in Vivo Effect of Prophylactic Subchondral Bone Protection of Osteoarthritic Synovial Membrane in Bone-Specific Ephb4–Overexpressing Mice

Authors

Gladys Valverde-Franco, David Hum, Koichi Matsuo, Bertrand Lussier, Jean-Pierre Pelletier, Hassan Fahmi, Mohit Kapoor, Johanne Martel-Pelletier

Abstract

Osteoarthritis (OA) is characterized by progressive joint destruction, including synovial membrane alteration. EphB4 and its ligand ephrin-B2 were found in vitro to positively affect OA subchondral bone and cartilage. In vivo in an experimental mouse model overexpressing bone-specific Ephb4 (TgEphB4), a protective effect was found on both the subchondral bone and cartilage during OA. We investigated in the TgEphB4 mouse model the in vivo effect on synovial membrane during OA. Knee OA was surgically induced by destabilization of the medial meniscus (DMM). Synovial membrane was evaluated using histology, histomorphometry, IHC, and real-time PCR. DMM-TgEphB4 mice had a significant decrease in synovial membrane thickness, vascular endothelial growth factor, and the profibrotic markers fibrin, type 1 procollagen, type 3 collagen, connective tissue growth factor, smooth muscle actin-α, cartilage oligomeric matrix protein, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 compared with DMM–wild-type (WT) mice. Moreover, factors known to modulate transforming growth factor-β signaling, transforming growth factor receptor 1/ALK1, phosphorylated Smad-1, and heat shock protein 90β were significantly decreased in DMM-TgEphB4 compared with DMM-WT mice. Ephb4 overexpression also exhibited a protective effect on synovial membrane thickness of aged (24-month-old) mice. Overexpression of bone-specific Ephb4 clearly demonstrated prevention of the development and/or progression of fibrosis in OA synovial membrane, reinforcing the hypothesis that protecting the subchondral bone prophylactically and during OA reduces the pathologic changes in other articular tissues.

Link To Article

http://dx.doi.org/10.1016/j.ajpath.2014.10.004