Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is ...remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.
Cells that form bone (osteoblasts) express both ephrinB2 and EphB4, and previous work has shown that pharmacological inhibition of the eph‐rinB2/EphB4 interaction impairs osteoblast differentiation ...in vitro and in vivo. The purpose of this study was to determine the role of ephrinB2 signaling in the osteoblast lineage in the process of bone formation. Cultured osteoblasts from mice with osteoblast‐specific ablation of ephrinB2 showed delayed expression of osteoblast differentiation markers, a finding that was reproduced by ephrinB2, but not EphB4, RNA interference. Microcomputed tomography, histomorphometry, and mechanical testing of the mice lacking ephrinB2 in osteoblasts revealed a 2‐fold delay in bone mineralization, a significant reduction in bone stiffness, and a 50% reduction in osteoblast differentiation induced by anabolic parathyroid hormone (PTH) treatment, compared to littermate sex‐ and age‐matched controls. These defects were associated with significantly lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis, indicated by TUNEL staining and transmission electron microscopy of bone samples, and a 2‐fold increase in annexin V staining and 7‐fold increase in caspase 8 activation in cultured ephrinB2 deficient osteoblasts. We conclude that osteoblast differentiation and bone strength are maintained by antiapoptotic actions of ephrinB2 signaling within the osteoblast lineage.—Tonna, S., Takyar, F. M., Vrahnas, C., Crimeen‐Irwin, B., Ho, P. W. M., Poulton, I. J., Brennan, H. J., McGregor, N. E., Allan, E. H., Nguyen, H., Forwood, M. R., Tatarczuch, L., Mackie, E. J., Martin, T. J., Sims, N. A., EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis. FASEB J. 28, 4482–4496 (2014). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Herpesviruses have been reported in several marsupial species, but molecular classification has been limited to four herpesviruses in macropodids, a gammaherpesvirus in two antechinus species ...(Antechinus flavipes and Antechinus agilis), a gammaherpesvirus in a potoroid, the eastern bettong (Bettongia gaimardi) and two gammaherpesviruses in koalas (Phascolarctos cinereus). In this study we examined a range of Australian marsupials for the presence of herpesviruses using molecular and serological techniques, and also assessed risk factors associated with herpesvirus infection. Our study population included 99 koalas (Phascolarctos cinereus), 96 eastern grey kangaroos (Macropus giganteus), 50 Tasmanian devils (Sarcophilus harrisii) and 33 common wombats (Vombatus ursinius). In total, six novel herpesviruses (one alphaherpesvirus and five gammaherpesviruses) were identified in various host species. The overall prevalence of detection of herpesvirus DNA in our study population was 27.2% (95% confidence interval (CI) of 22.6-32.2%), but this varied between species and reached as high as 45.4% (95% CI 28.1-63.7%) in common wombats. Serum antibodies to two closely related macropodid herpesviruses (macropodid herpesvirus 1 and 2) were detected in 44.3% (95% CI 33.1-55.9%) of animals tested. This also varied between species and was as high as 92% (95% CI 74.0-99.0%) in eastern grey kangaroos. A number of epidemiological variables were identified as positive predictors for the presence of herpesvirus DNA in the marsupial samples evaluated. The most striking association was observed in koalas, where the presence of Chlamydia pecorum DNA was strongly associated with the presence of herpesvirus DNA (Odds Ratio = 60, 95% CI 12.1-297.8). Our results demonstrate the common presence of herpesviruses in Australian marsupials and provide directions for future research.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in ...arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Metaphyseal chondrodysplasia, Schmid type (MCDS) is characterized by mild short stature and growth plate hypertrophic zone expansion, and caused by collagen X mutations. We recently demonstrated the ...central importance of ER stress in the pathology of MCDS by recapitulating the disease phenotype by expressing misfolding forms of collagen X (Schmid) or thyroglobulin (Cog) in the hypertrophic zone. Here we characterize the Schmid and Cog ER stress signaling networks by transcriptional profiling of microdissected mutant and wildtype hypertrophic zones. Both models displayed similar unfolded protein responses (UPRs), involving activation of canonical ER stress sensors and upregulation of their downstream targets, including molecular chaperones, foldases, and ER-associated degradation machinery. Also upregulated were the emerging UPR regulators Wfs1 and Syvn1, recently identified UPR components including Armet and Creld2, and genes not previously implicated in ER stress such as Steap1 and Fgf21. Despite upregulation of the Chop/Cebpb pathway, apoptosis was not increased in mutant hypertrophic zones. Ultrastructural analysis of mutant growth plates revealed ER stress and disrupted chondrocyte maturation throughout mutant hypertrophic zones. This disruption was defined by profiling the expression of wildtype growth plate zone gene signatures in the mutant hypertrophic zones. Hypertrophic zone gene upregulation and proliferative zone gene downregulation were both inhibited in Schmid hypertrophic zones, resulting in the persistence of a proliferative chondrocyte-like expression profile in ER-stressed Schmid chondrocytes. Our findings provide a transcriptional map of two chondrocyte UPR gene networks in vivo, and define the consequences of UPR activation for the adaptation, differentiation, and survival of chondrocytes experiencing ER stress during hypertrophy. Thus they provide important insights into ER stress signaling and its impact on cartilage pathophysiology.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The tissue destruction seen in chronic periodontitis is commonly accepted to involve extensive upregulation of the host inflammatory response. Protease-activated receptor 2 (PAR-2)-null mice infected ...with Porphyromonas gingivalis did not display periodontal bone resorption in contrast to wild-type-infected and PAR-1-null-infected mice. Histological examination of tissues confirmed the lowered bone resorption in PAR-2-null mice and identified a substantial decrease in mast cells infiltrating the periodontal tissues of these mice. T cells from P. gingivalis-infected or immunized PAR-2-null mice proliferated less in response to antigen than those from wild-type animals. CD90 (Thy1.2) expression on CD4⁺ and CD8⁺ T-cell-receptor β (TCRβ) T cells was significantly (P < 0.001) decreased in antigen-immunized PAR-2-null mice compared to sham-immunized PAR-2-null mice; this was not observed in wild-type controls. T cells from infected or antigen-immunized PAR-2-null mice had a significantly different Th1/inflammatory cytokine profile from wild-type cells: in particular, gamma interferon, interleukins (interleukin-2, -3, and -17), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha demonstrated lower expression than wild-type controls. The absence of PAR-2 therefore appears to substantially decrease T-cell activation and the Th1/inflammatory response. Regulation of such proinflammatory mechanisms in T cells and mast cells by PAR-2 suggests a pivotal role in the pathogenesis of the disease.
PAR‐2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR‐2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow ...cultures and may protect bone from uncontrolled resorption.
Introduction: Protease‐activated receptor‐2 (PAR‐2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR‐2 is also activated by a peptide (RAP) that corresponds to the “tethered ligand” created by cleavage of the receptor's extracellular domain. The effect of activating PAR‐2 on osteoclast differentiation was investigated.
Materials and Methods: Mouse bone marrow cultures have been used to investigate the effect of PAR‐2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 1,25(OH)2D3, and interleukin‐11 (IL‐11). Expression of PAR‐2 by mouse bone marrow, mouse bone marrow stromal cell‐enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT‐PCR.
Results: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL‐11. Semiquantitative RT‐PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL‐11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL‐11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR‐2 led to reduced expression of prostaglandin G/H synthase‐2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL‐11. RAP inhibited PTH‐ or 1,25(OH)2D3‐induced expression of IL‐6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL‐treated RAW264.7 cells.
Conclusion: These observations indicate that PAR‐2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL‐11. Therefore, the role of PAR‐2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
MalF has been shown to be required for virulence in the important avian pathogen
To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite ...profiles of a mutant with a transposon inserted in
(MalF-deficient ST mutant 04-1; Δ
) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the Δ
mutant, compared to the wild type. Stable isotope labeling using U-
Cglycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into
and that, in the absence of MalF, the transcription of
, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the Δ
mutant is due to perturbed glycerol uptake and metabolism and that the operon including
should be reannotated as
to reflect its function in glycerol transport.
Many mycoplasmas are pathogenic and cause disease in humans and animals.
causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of
, as well as its effect on expression of selected genes, cell phenotype, and H
O
production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.
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