Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

Authors

Naobumi Hosogane, Zhiping Huang, Bernard A. Rawlins, Xia Liu, Oheneba Boachie-Adjei, Adele L. Boskey, and Wei Zhu

Abstract

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations.

Link to Article

http://dx.doi.org/10.1016/j.biocel.2010.03.020

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model

Authors

Maxim Moreau, Pascale Rialland, Jean-Pierre Pelletier, Johanne Martel-Pelletier, Daniel Lajeunesse, Christelle Boileau, Judith Caron, Diane Frank, Bertrand Lussier, Jerome RE del Castillo, Guy Beauchamp, Dominique Gauvin, Thierry Bertaim, Dominique Thibaud and Eric Troncy

Abstract

The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA). Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving 4 TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for 8 weeks. At baseline, week 4 and week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (MMP-1, MMP-13, and ADAMTS5) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE2 and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an alpha-threshold of 0.05. A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE2 (P = 0.02), nitrites/ nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone. Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.

Link to Article

http://dx.doi.org/10.1186/ar3373

Osteoclast-specific inactivation of the integrin-linked kinase (ILK) inhibits bone resorption

Authors

Tanya Dossa, Alice Arabian, Jolene J. Windle, Shoukat Dedhar, Steven L. Teitelbaum, F. Patrick Ross, G. David Roodman, René St-Arnaud

Abstract

Bone resorption requires the adhesion of osteoclasts to extracellular matrix (ECM) components, a process mediated by the αvβ3 integrin. Following engagement with the ECM, integrin receptors signal via multiple downstream effectors, including the integrin-linked kinase (ILK). In order to characterize the physiological role of ILK in bone resorption, we generated mice with an osteoclast-specific Ilk gene ablation by mating mice with a floxed Ilk allele with TRAP-Cre transgenic mice. The TRAP-Cre mice specifically excised floxed alleles in osteoclasts, as revealed by crossing them with the ROSA26R reporter strain. Osteoclast-specific Ilk mutant mice appeared phenotypically normal, but histomorphometric analysis of the proximal tibia revealed an increase in bone volume and trabecular thickness. Osteoclast-specific Ilk ablation was associated with an increase in osteoclastogenesis both in vitro and in vivo. However, the mutant osteoclasts displayed a decrease in resorption activity as assessed by reduced pit formation on dentin slices in vitro and decreased serum concentrations of the C-terminal telopeptide of collagen in vivo. Interestingly, compound heterozygous mice in which one allele of Ilk and one allele of the β3 integrin gene were inactivated (ILK+/−; β) also had increased trabecular thickness, confirming that β3 integrin and Ilk form part of the same genetic cascade. Our results show that ILK is important for the function, but not the differentiation, of osteoclasts.

Link to Article

http://dx.doi.org/10.1002/jcb.22609

Microfibril-associated Glycoprotein-1, an Extracellular Matrix Regulator of Bone Remodeling

Authors

Clarissa S. Craft, Wei Zou, Marcus Watkins, Susan Grimston, Michael D. Brodt, Thomas J. Broekelmann, Justin S. Weinbaum, Steven L. Teitelbaum, Richard A. Pierce, Roberto Civitelli, Matthew J. Silva and Robert P. Mecham

Abstract

MAGP1 is an extracellular matrix protein that, in vertebrates, is a ubiquitous component of fibrillin-rich microfibrils. We previously reported that aged MAGP1-deficient mice (MAGP1Δ) develop lesions that are the consequence of spontaneous bone fracture. We now present a more defined bone phenotype found in MAGP1Δ mice. A longitudinal DEXA study demonstrated age-associated osteopenia in MAGP1Δ animals and μCT confirmed reduced bone mineral density in the trabecular and cortical bone. Further, MAGP1Δ mice have significantly less trabecular bone, the trabecular microarchitecture is more fragmented, and the diaphyseal cross-sectional area is significantly reduced. The remodeling defect seen in MAGP1Δ mice is likely not due to an osteoblast defect, because MAGP1Δ bone marrow stromal cells undergo osteoblastogenesis and form mineralized nodules. In vivo, MAGP1Δ mice exhibit normal osteoblast number, mineralized bone surface, and bone formation rate. Instead, our findings suggest increased bone resorption is responsible for the osteopenia. The number of osteoclasts derived from MAGP1Δ bone marrow macrophage cells is increased relative to the wild type, and osteoclast differentiation markers are expressed at earlier time points in MAGP1Δ cells. In vivo, MAGP1Δ mice have more osteoclasts lining the bone surface. RANKL (receptor activator of NF-κB ligand) expression is significantly higher in MAGP1Δ bone, and likely contributes to enhanced osteoclastogenesis. However, bone marrow macrophage cells from MAGP1Δ mice show a higher propensity than do wild-type cells to differentiate to osteoclasts in response to RANKL, suggesting that they are also primed to respond to osteoclast-promoting signals. Together, our findings suggest that MAGP1 is a regulator of bone remodeling, and its absence results in osteopenia associated with an increase in osteoclast number.

Link to Article

http://dx.doi.org/10.1074/jbc.M110.113019

Adenosine A1 receptors regulate bone resorption in mice...

Title

Adenosine A1 receptors regulate bone resorption in mice: Adenosine A1 receptor blockade or deletion increases bone density and prevents ovariectomy-induced bone loss in adenosine A1 receptor–knockout mice

Authors

Firas M. Kara, Stephen B. Doty, Adele Boskey, Steven Goldring, Mone Zaidi, Bertil B. Fredholm, Bruce N. Cronstein

Abstract

Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and developing new treatments, we studied the effect of adenosine A1 receptor blockade or deletion on bone density. The bone mineral density (BMD) in adenosine A1 receptor–knockout (A1R-knockout) mice was analyzed by dual x-ray absorptiometry (DXA) scanning, and the trabecular and cortical bone volume was determined by microfocal computed tomography (micro-CT). The mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analyzed by DXA scanning and micro-CT. A histologic examination of bones obtained from A1R-knockout and wild-type mice was carried out. Visualization of osteoblast function (bone formation) after tetracycline double-labeling was performed by fluorescence microscopy. Micro-CT analysis of bones from A1R-knockout mice showed significantly increased bone volume. Electron microscopy of bones from A1R-knockout mice showed the absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistologic analysis demonstrated that although osteoclasts were present in the A1R-knockout mice, they were smaller and often not associated with bone. No morphologic changes in osteoblasts were observed, and bone-labeling studies revealed no change in the bone formation rates in A1R-knockout mice. These results suggest that the adenosine A1 receptor may be a useful target in treating diseases characterized by excessive bone turnover, such as osteoporosis and prosthetic joint loosening.

Link to Article

http://dx.doi.org/10.1002/art.27219

Bone loss caused by iron overload in a murine model: importance of oxidative stress

Authors

Jaime Tsay, Zheiwei Yang, F. Patrick Ross, Susanna Cunningham-Rundles, Hong Lin, Rhima Coleman, Philipp Mayer-Kuckuk, Stephen B. Doty, Robert W. Grady, Patricia J. Giardina, Adele L. Boskey, and Maria G. Vogiatzi

Abstract

Osteoporosis is a frequent problem in disorders characterized by iron overload, such as the thalassemias and hereditary hemochromatosis. The exact role of iron in the development of osteoporosis in these disorders is not established. To define the effect of iron excess in bone, we generated an iron-overloaded mouse by injecting iron dextran at 2 doses into C57/BL6 mice for 2 months. Compared with the placebo group, iron-overloaded mice exhibited dose-dependent increased tissue iron content, changes in bone composition, and trabecular and cortical thinning of bone accompanied by increased bone resorption. Iron-overloaded mice had increased reactive oxygen species and elevated serum tumor necrosis factor-α and interleukin-6 concentrations that correlated with severity of iron overload. Treatment of iron-overloaded mice with the antioxidant N-acetyl-L-cysteine prevented the development of trabecular but not cortical bone abnormalities. This is the first study to demonstrate that iron overload in mice results in increased bone resorption and oxidative stress, leading to changes in bone microarchitecture and material properties and thus bone loss.

Link to Article

http://dx.doi.org/10.1182/blood-2009-12-260083