The T-lymphocyte secreted pro-inflammatory cytokine, interleukin-17F (IL-17F), was found to be a key mediator in the cellular response of the immune system in the early phase of fracture repair but its intracellular signaling processes are currently not known in osteoblasts. The objective of this study was to identify the signaling proteins and crucial gene targets involved in osteoblast activation via IL-17F. It was hypothesised that IL-17F stimulated osteoblast maturation through a novel GSK3beta / beta-catenin independent pathway. Mouse pre-osteoblast cell line (MC3T3-E1) was used for IL-17F or Wnt3a treatment. Desired proteins were detected using western blot analysis (antibodies: Phospho-GSK-3beta (Tyr 216), Phospho-GSK-3beta (Ser9), Runx2/cbfa1, TRAF6, Act1, p-ERK2, p-JNK and p-MAPK, C/EBP-beta and & delta). Gene-specific siRNAs of mouse IL-17Ra, IL-17Rc and a non-targeting siRNA (control) were utilised. MC3T3-E1 were transfected with IL-17Ra, IL-17Rc or Negative Control and treated with IL-17F. Chromatin Immunoprecipitation (ChIP-qPCR) was used to evaluate the mouse Runx2 P1 promoter region. IL-17F increased expression of Col1, BSP, Runx2/cbfa1 and osteocalcin in MC3T3-E1 cells. Western blot analysis confirmed expression of known Wnt signaling proteins TRAF6, Act1, p-ERK2, p-JNK and p-MAPK in both IL-17F and Wnt3a treated cultures, including up-regulation of Runx2/cbfa1, a key transcription factor associated with osteoblast differentiation. IL-17F up-regulation of Runx2/cbfa1 appears independent of the Wnt/beta-catenin pathway as phosphorylated GSK-3beta at the Ser9 site was not detected with IL-17F treatment. Despite this, IL-17F treatment still increased expression of Runx2/cbfa1 downstream, lending evidence for a GSK3beta/beta-catenin independent manner of IL-17F stimulated osteogenesis. While IL-17F and Wnt3a both induced expression of C/EBP-delta, only IL-17F treatment induced expression of C/EBP-beta, an upstream transcription factor of Runx2/cbfa1. Further, siRNA knock down of the IL-17 receptors directly decreased Act1, C/EBP-beta and Runx2/cfba1 expression. By ChIP analysis, IL-17F was shown to upregulate C/EBP-beta expression and stimulated its binding to the P1 Promoter of the Runx2/cbfa1 gene. The C/EBP-beta transcription factor was shown to be a key regulator of early osteogenesis. C/EBP-beta up-regulates Runx2/cbfa1 expression by directly binding to the Runx2/cbfa1 P1 promoter in osteoblasts. C/EBP-beta was activated in the osteoblast by IL-17F but not by Wnt3a adding further support to a novel GSK3beta/beta-catenin independent pathway. Our data shows that IL-17F, a cytokine secreted by T-lymphocytes, stimulates osteoblast maturation through a novel GSK3beta/beta-catenin independent pathway and reveals a crucial interaction between C/EBP-beta and the Runx2/cbfa1 P1 promoter not previously been shown in osteogenesis signaling further

Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1-KO) exhibit progressive ectopic calcification of spinal tissues, including the annulus fibrosus (AF) of intervertebral discs (J Bone Miner Res 28:1135–49, 2013, Bone 90:37–49, 2016). Our purpose was twofold: (1) to compare ectopic calcifications in ENT1-KO mice with those in human DISH, and (2) to investigate the molecular pathways underlying pathological calcification in ENT1-KO mice. Studies were performed with age-matched wild-type (WT) and ENT1-KO mice, as well as human cadaveric vertebral columns meeting radiographic criteria for DISH. Mouse and human specimens were scanned using high-resolution, micro-computed tomography (micro-CT). As well, some samples were decalcified and processed for histological assessment. Calcified lesions in selected specimens were examined using energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). To investigate molecular changes associated with ectopic calcification, we isolated AF tissue from thoracic intervertebral discs of WT and ENT1-KO mice. Tissues were then subjected to transcriptomic and proteomic analyses. Micro-CT of ENT1-KO mice revealed ectopic calcification of spinal tissues, first appearing in the cervical-thoracic region and extending caudally with advancing age. Histological examination of calcified lesions in mice revealed accumulations of amorphous, eosinophilic, acellular material in paraspinal ligaments and entheses, intervertebral discs, mandibular symphysis, and sternocostal articulations. There was no evidence of inflammation associated with these lesions. EDX of calcified lesions revealed a high content of calcium and phosphorus in a molar ratio of ∼1.6, with hydroxyapatite detected by micro-XRD. Ten human cadaveric spines (three females and seven males, mean age 81 years) that met radiographic criteria for DISH were analysed in detail by micro-CT. Remarkable heterogeneity in the density and morphology of ectopic calcifications was observed. Analyses of calcifications by EDX and XRD again yielded a calcium/phosphorus ratio of ∼1.6 and a crystalline diffraction pattern matching hydroxyapatite. Histological examination of human lesions revealed regions of mature ossification and other areas of irregular amorphous calcification that resembled lesions in ENT1-KO mice. Microarray analysis of AF tissue from WT and ENT1-KO mice showed extensive dysregulation of transcription in affected tissues. Cell cycle-associated transcripts were the most affected, including the E2f family of transcription factors and proliferating cell nuclear antigen. In addition, expression of genes involved in the regulation of mineralization and bone development were dysregulated. Proteomic analyses confirmed transcriptomic changes and revealed alterations in known modulators of biomineralization such as matrix Gla-protein. Many of the characteristics of ectopic calcification in ENT1-KO mice resemble those of DISH in humans. Human lesions were found to be heterogeneous with regions of pathological ossification and amorphous calcification, the latter resembling lesions in the mouse model. Our studies of the molecular events associated with ectopic calcification in ENT1-KO mice may provide insights into the pathogenesis of DISH in humans. ENT1-KO mice may also be useful for evaluating therapeutics for the prevention of ectopic calcification in DISH and related disorders

Background. Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs). A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto(tm) (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast proliferation, function, matrix mineralisation and gene expression compared to enoxaparin, a commonly used LMWH. Methods. Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 μg/ml) or enoxaparin (0.1, 1.0 and 10 international units/ml). The effect of each drug on osteoblast function and matrix mineralisation was evaluated by measuring alkaline phosphatase activity and calcium deposition, respectively. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR). Results. Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast proliferation. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity, with a moderate reduction in calcium deposition also observed. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. Conclusion. These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function. The increased duration of recommended Rivaroxaban therapy (2 and 5 weeks) post-arthroplasty compared to Enoxaparin therapy (average one week) may have a more pronounced effect on bone homeostasis

Purpose. Limb regeneration as it occurs in amphibians has two basic requirements: a source of multipotent cells capable of generating various tissues, and reorganization of those cells to form the one and only pattern of tissue appropriate to restore the missing parts. In the current biomedical world, there is much work dedicated to tissue engineering and to the differentiation of stem cells into various mature cell types. Neither of these approaches however, will by themselves succeed in regenerating a complex structure such as a limb. In our lab, we decided to focus on the pattern organization side of the equation by testing the potential of mammalian limb bud tissue to change its positional identity, and to manipulate that potential. Method. We used mouse embryos for our mammalian model. Small groups of cells were transplanted from one region of the limb bud into another, and the resulting effect on the positional identity of those cells was assessed using molecular markers of the upper arm, forearm and hand. We knocked out a genetic regulator of cell fate named Ezh2 specifically in the limb bud to test its role in committing cells to a given positional identity along the proximodistal limb axis. Results. We found that, similar to what one would expect for regenerating amphibians, mouse cells in the limb bud can alter their identity from that destined to make a hand to one poised to contribute to an upper arm, and vice versa. However, the cells ability to reset their identity is gradually lost during early development. In Ezh2 mutant mice, this cellular plasticity is prolonged, indicating that Ezh2 regulates commitment to a given fate. Ezh2 functions epigenetically by altering access of transcription factors to DNA. Conclusion. There is inherent plasticity in the ability of early mammalian limb bud cells with regard to positional identity. This plasticity can be prolonged by the alteration of a single gene. Many genes related to Ezh2 are functional in regulating cell fate. Manipulation of this family of genes to facilitate pattern reorganisation among a source of stem cells capable of making every relevant tissue type is one potential approach to engineering organised tissue in mammals

Purpose. The data regarding the effects of noggin on bone morphogenetic protein (BMP)-induced osteogenesis of mesenchymal stem cells (MSCs) are controversial. Most studies performed in rodent cells/models indicated that noggin was a negative regulator of BMP-2-induced osteogenesis; however, one study conducted with human MSCs in culture showed that the addition of noggin induced osteogenesis in vitro. To clear the controversy, we designed this study to evaluate the effects of knocking down noggin gene expression on BMP-2-induced osteogenesis of human bone marrow-derived primary MSCs in vitro. Method. MSCs were isolated from human tibial bone marrow by density gradient centrifugation. Two noggin small interfering RNAs (siRNAs) were used in this study to knockdown noggin gene expression. There were four study groups: MSCs with no transfection of siRNA (named as NT group), MSCs transfected with non-targeting negative control siRNA (named as control group), MSCs transfected with noggin siRNA1 (named as NOGsi1 group), and MSCs transfected with noggin siRNA2 (named as NOGsi2 group). After transfection, MSCs were induced to undergo osteogenic differentiation by incubating in basal medium containing 0.1 μg/ml BMP-2 for 35 days. The expression levels of osteoblastic marker genes were measured by real-time quantitative PCR on day 14. Also assessed was alkaline phosphatase (ALP) activity by a colorimetric kinetic assay and Fast Blue B staining on day 14. Calcium deposition was determined by the calcium assay on day 35. Results. The expression levels of integrin binding sialoprotein (IBSP) and osteocalcin (OC) were significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT and control groups (all p<0.038). Although the expression level of runt-related transcription factor 2 (RUNX2) was also reduced in NOGsi1 and NOGsi2 groups compared with NT and control groups, it did not reach statistical significance. ALP activity was significantly lower in NOGsi1 and NOGsi2 groups than that of NT group (both p<0.024). The same pattern was also observed in ALP Fast Blue B staining. Calcium deposition was also significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT group (both p<=0.048). Conclusion. Noggin suppression by siRNA inhibits BMP-2-induced osteogenesis of human bone marrow-derived MSCs. Our results, contrary to the extensive studies conducted in rodent cells/models, corroborated with the previous study that the addition of noggin in the cell culture increased osteogenesis of human MSCs. This suggests that the effects of noggin on BMP-2-induced osteogenesis of MSCs might be species-specific

Objectives

Salubrinal is a synthetic agent that elevates phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) and alleviates stress to the endoplasmic reticulum. Previously, we reported that in chondrocytes, Salubrinal attenuates expression and activity of matrix metalloproteinase 13 (MMP13) through downregulating nuclear factor kappa B (NFκB) signalling. We herein examine whether Salubrinal prevents the degradation of articular cartilage in a mouse model of osteoarthritis (OA).

Construction of a functional skeleton is accomplished through co-ordination of the developmental processes of chondrogenesis, osteogenesis, and synovial joint formation. Infants whose movement in utero is reduced or restricted and who subsequently suffer from joint dysplasia (including joint contractures) and thin hypo-mineralised bones, demonstrate that embryonic movement is crucial for appropriate skeletogenesis. This has been confirmed in mouse, chick, and zebrafish animal models, where reduced or eliminated movement consistently yields similar malformations and which provide the possibility of experimentation to uncover the precise disturbances and the mechanisms by which movement impacts molecular regulation. Molecular genetic studies have shown the important roles played by cell communication signalling pathways, namely Wnt, Hedgehog, and transforming growth factor-beta/bone morphogenetic protein. These pathways regulate cell behaviours such as proliferation and differentiation to control maturation of the skeletal elements, and are affected when movement is altered. Cell contacts to the extra-cellular matrix as well as the cytoskeleton offer a means of mechanotransduction which could integrate mechanical cues with genetic regulation. Indeed, expression of cytoskeletal genes has been shown to be affected by immobilisation. In addition to furthering our understanding of a fundamental aspect of cell control and differentiation during development, research in this area is applicable to the engineering of stable skeletal tissues from stem cells, which relies on an understanding of developmental mechanisms including genetic and physical criteria. A deeper understanding of how movement affects skeletogenesis therefore has broader implications for regenerative therapeutics for injury or disease, as well as for optimisation of physical therapy regimes for individuals affected by skeletal abnormalities.

Cite this article: Bone Joint Res 2015;4:105–116