IL-23 Is Critical for Induction of Arthritis, Osteoclast Formation, and Maintenance of Bone Mass

Authors

Iannis E. Adamopoulos, Marlowe Tessmer, Cheng-Chi Chao, Sarvesh Adda, Dan Gorman, Mary Petro, Chuan-Chu Chou, Robert H. Pierce, Wei Yao, Nancy E. Lane, Drake Laface, and Edward P. Bowman

Abstract

The role of IL-23 in the development of arthritis and bone metabolism was studied using systemic IL-23 exposure in adult mice via hydrodynamic delivery of IL-23 minicircle DNA in vivo and in mice genetically deficient in IL-23. Systemic IL-23 exposure induced chronic arthritis, severe bone loss, and myelopoiesis in the bone marrow and spleen, which resulted in increased osteoclast differentiation and systemic bone loss. The effect of IL-23 was partly dependent on CD4+ T cells, IL-17A, and TNF, but could not be reproduced by overexpression of IL-17A in vivo. A key role in the IL-23–induced arthritis was made by the expansion and activity of myeloid cells. Bone marrow macrophages derived from IL-23p19−/− mice showed a slower maturation into osteoclasts with reduced tartrate-resistant acid phosphatase-positive cells and dentine resorption capacity in in vitro osteoclastogenesis assays. This correlated with fewer multinucleated osteoclast-like cells and more trabecular bone volume and number in 26-wk-old male IL-23p19−/− mice compared with control animals. Collectively, our data suggest that systemic IL-23 exposure induces the expansion of a myeloid lineage osteoclast precursor, and targeting IL-23 pathway may combat inflammation-driven bone destruction as observed in rheumatoid arthritis and other autoimmune arthritides.

Link to Article

http://dx.doi.org/10.4049/jimmunol.1190033

Postnatally induced inactivation of Osterix in osteoblasts results in the reduction of bone formation and maintenance

Authors

Wook-Young Baek, Benoit de Crombrugghe, Jung-Eun Kim

Abstract

Osterix (Osx) is a zinc-finger-containing transcription factor that is highly specific to osteoblasts in vivo. Because Osx homozygous null mutants die in the immediate perinatal period showing a complete absence of bone formation, it is impossible determine the role that Osx plays in bones that have already formed after birth. To determine whether Osx is essential for bone maintenance and homeostasis, we conditionally inactivated the Osx gene in adult bone using the Cre/loxP recombination system. In previous reports, 2.3-kb Col1a1-CreERT2 mice that expressed a Cre recombinase that is transiently inducible by 4-hydroxytamoxifen (4-OHT) were intercrossed with Rosa26R (R26R) reporter mice, which resulted in the production of Cre-expressing osteoblasts that were detected upon X-gal staining. In the present study, inducible Col1a1-CreERT2 transgenic mice and conditional Osx mice (Osxflox/+) were used to generate Osxflox/−;Col1a1-CreERT2 mice. The Osx gene in Osxflox/−;Col1a1-CreERT2 mice was inactivated in the osteoblasts of already formed bones by active Cre recombinase after the administration of 4-OHT. The bones from 4-OHT-treated Osxflox/−;Col1a1-CreERT2 mice and oil-treated control mice were analyzed by radiography, histology, and histomorphometry. Even though no significant difference was observed in the radiographic images of the whole mouse skeletons, the mineralized trabecular bone volume and number in lumbar vertebrae were remarkably reduced in 4-OHT-treated Osxflox/−;Col1a1-CreERT2 mice. In addition, the rate of bone formation and area of mineralized surface were also reduced in 4-OHT-treated Osxflox/−;Col1a1-CreERT2 mice. Osx inactivation in already formed bones during the postnatal period caused a functional defect in osteoblasts that was followed by a reduction of bone formation, even though there were no apparent differences in osteoblast proliferation and osteoclast formation. Taken together, these results indicate that Osx is required to maintain osteoblast function following adult bone maintenance.

Link to Article

http://dx.doi.org/10.1016/j.bone.2009.12.007

Regulation of postnatal trabecular bone formation by the osteoblast endothelin A receptor

Authors

Gregory A. Clines, Khalid S. Mohammad, Jessica M. Grunda, Katrina L. Clines, Maria Niewolna, C. Ryan McKenna, Christopher R. McKibbin, Masashi Yanagisawa, Larry J. Suva, John M. Chirgwin, Theresa A. Guise

Abstract

Endothelin-1 (ET-1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET-1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis had not been defined. ETAR was inactivated in osteoblasts by crossing ETAR-floxed and osteocalcin-Cre mice. Histomorphometric analyses were performed on 4, 8 and 12 week-old osteoblast-targeted ETAR knockout (KO) and wild type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO vs. WT mice in both males and females. Bone formation rate, osteoblast density and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks, to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition compared to WT. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared to WT. In vitro, ET-1 increased osteoblast proliferation, survival and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET-1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone.

Link to Article

http://dx.doi.org/10.1002/jbmr.450

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