Cartilage-Specific Overexpression of ERRγ Results in Chondrodysplasia and Reduced Chondrocyte Proliferation

Authors

Marco Cardelli, Ralph A. Zirngibl, Jonathan F. Boetto, Kristen P. McKenzie, Tammy-Claire Troy, Kursad Turksen, Jane E. Aubin

Abstract

While the role of estrogen receptor-related receptor alpha (ERRα) in chondrogenesis has been investigated, the involvement of ERR gamma (ERRγ) has not been determined. To assess the effect of increased ERRγ activity on cartilage development in vivo, we generated two transgenic (Tg) lines overexpressing ERRγ2 via a chondrocyte-specific promoter; the two lines exhibited ~3 and ~5 fold increased ERRγ2 protein expression respectively in E14.5 Tg versus wild type (WT) limbs. On postnatal day seven (P7), we observed a 4–10% reduction in the size of the craniofacial, axial and appendicular skeletons in Tg versus WT mice. The reduction in bone length was already present at birth and did not appear to involve bones that are derived via intramembranous bone formation as the bones of the calvaria, clavicle, and the mandible developed normally. Histological analysis of P7 growth plates revealed a reduction in the length of the Tg versus WT growth plate, the majority of which was attributable to a reduced proliferative zone. The reduced proliferative zone paralleled a decrease in the number of Ki67-positive proliferating cells, with no significant change in apoptosis, and was accompanied by large cell-free swaths of cartilage matrix, which extended through multiple zones of the growth plate. Using a bioinformatics approach, we identified known chondrogenesis-associated genes with at least one predicted ERR binding site in their proximal promoters, as well as cell cycle regulators known to be regulated by ERRγ. Of the genes identified, Col2al, Agg, Pth1r, and Cdkn1b (p27) were significantly upregulated, suggesting that ERRγ2 negatively regulates chondrocyte proliferation and positively regulates matrix synthesis to coordinate growth plate height and organization.

Link To Article

http://dx.doi.org/10.1371/journal.pone.0081511

Deleterious effects of osteoarthritis on the structure and function of the meniscal enthesis

Authors

Adam C. Abraham, Hannah M. Pauly, Tammy L. Haut Donahue

Abstract

Objective

The ability of menisci to prevent osteoarthritis (OA) is dependent on the integrity of the complex meniscal entheses, the attachments of the menisci to the underlying subchondral bone. The goal of this study was to determine mechanical and structural changes in meniscal entheses after the onset of osteoarthritis.

Design

Healthy and osteoarthritic meniscal entheses were evaluated for changes in histomorphological characteristics, mineralization, and mechanical properties. GAG and calcium in the insertion were evaluated with histological staining techniques. The extent of calcium deposition was assessed and tidemark integrity was quantified. Changes in the mineralized zone of the insertion was examined using micro-computed tomography to determine bone mineral density, cortical zone thickness, and mineralization gradient. Mechanical properties of the entheses were measured using nanoindentation techniques to obtain material properties based on viscoelastic analysis.

Results

GAG thickness in the calcified fibrocartilage zone and calcium content were significantly greater in osteoarthritic anterior meniscal entheses. Tidemark integrity was significantly decreased in OA tissue, particularly in the medial anterior enthesis. The mineralized zone of osteoarthritic meniscal entheses was significantly thicker than in healthy entheses and showed decreased bone mineral density. Fitting of mineralization data to a sigmoidal Gompertz function revealed a lower rate of increase in mineralization in osteoarthritic tissue. Analysis of viscoelastic mechanical properties revealed increased compliance in osteoarthritic tissue.

Conclusions

These data suggest that significant changes occur at meniscal enthesis sites with the onset of osteoarthritis. Mechanical and structural changes in meniscal entheses may contribute to meniscal extrusion, which has been show to increase the progression of OA.

Link To Article

http://dx.doi.org/10.1016/j.joca.2013.11.013

Palmitic Acid and DGAT1 Deficiency Enhance Osteoclastogenesis while Oleic Acid-Induced Triglyceride Formation Prevents it

Authors

Zoi Drosatos-Tampakaki, Konstantinos Drosatos, Yasemin Siegelin, Shan Gong, Salmiyeh Khan, Thomas Van Dyke, Ira J. Goldberg, P. Christian Schulze, Ulrike Schulze-Späte

Abstract

Both obesity and diabetes mellitus are associated with alterations in lipid metabolism as well as a change in bone homeostasis and osteoclastogenesis. We hypothesized that increased fatty acid levels affect bone health by altering precursor cell differentiation and osteoclast activation. Here we show that palmitic acid (PA, 16:0) enhances RANKL-stimulated osteoclastogenesis and is sufficient to induce osteoclast differentiation even in the absence of RANKL. TNFα expression is crucial for PA-induced osteoclastogenesis as shown by increased TNFα mRNA levels in PA-treated cells and abrogation of PA-stimulated osteoclastogenesis by TNFα neutralizing antibodies. In contrast, oleic acid (OA, 18:1) does not enhance osteoclast differentiation, leads to increased intracellular triglyceride accumulation and inhibits PA-induced osteoclastogenesis. Adenovirus-mediated expression of diacylglycerol acyl transferase (DGAT) 1, a gene involved in triglyceride synthesis, also inhibits PA-induced osteoclastogenesis, suggesting a protective role of DGAT1 for bone health. Accordingly, Dgat1 knockout mice have larger bone marrow-derived osteoclasts and decreased bone mass indices. In line with these findings, mice on a high fat PA-enriched diet have a greater reduction in bone mass and structure than mice on a high fat OA-enriched diet. Thus, we propose that TNFα mediates saturated fatty acid-induced osteoclastogenesis that can be prevented by DGAT activation or supplementation with OA.

Link To Article

http://dx.doi.org/10.1002/jbmr.2150

Modulation of endochondral ossification by MEK inhibitors PD0325901 and AZD6244 (Selumetinib)

Authors

J. El-Hossa, M. Kolind, M.T. Jackson, N. Deo, K. Mikuleca, M.M. McDonald, C.B. Little, D.G. Little, A. Schindeler

Abstract

MEK inhibitors (MEKi) PD0325901 and AZD6244 (Selumetinib) are drugs currently under clinical investigation for cancer treatment, however the Ras–MAPK pathway is also an important mediator of normal bone cell differentiation and function. In this study we examined the effects of these compounds on endochondral processes using both in vitro and in vivo models. Treatment with PD0325901 or AZD6244 significantly increased Runx2 and Alkaline phosphate gene expression in calvarial osteoblasts and decreased TRAP + cells in induced osteoclast cultures. To test the effects of these drugs on bone healing, C57/Bl6 mice underwent a closed tibial fracture and were treated with PD0325901 or AZD6244 at 10 mg/kg/day. Animals were culled at day 10 and at day 21 post-fracture for analysis of the fracture callus and the femoral growth plate in the contralateral leg. MEKi treatment markedly increased cartilage volume in the soft callus at day 10 post-fracture (+ 60% PD0325901, + 20% AZD6244) and continued treatment led to a delay in cartilage remodeling. At the growth plate, we observed an increase in the height of the hypertrophic zone relative to the proliferative zone of + 78% in PD0325901 treated mice. Osteoclast surface was significantly decreased both at the terminal end of the growth plate and within the fracture calluses of MEKi treated animals. The mechanistic effects of MEKi on genes encoding cartilage matrix proteins and catabolic enzymes were examined in articular chondrocyte cultures. PD0325901 or AZD6244 led to increased matrix protein expression (Col2a1 and Agc1) and decreased expression of catabolic factors (Mmp13 and Adamts-5). Taken together, these data support the hypothesis that MEKi treatment can impact chondrocyte hypertrophy, matrix resorption, and fracture healing. These compounds can also affect bone architecture by expanding the hypertrophic zone of the growth plate and reducing osteoclast surface systemically.

Link To Article

http://dx.doi.org/10.1016/j.bone.2013.11.013

Increased Col10a1 expression is not causative for the phenotype of Phex-deficient Hyp mice

Authors

Timur Yorgan, Carsten Rendenbach, Anke Jeschke, Michael Amling, Kathryn S.E. Cheah,Thorsten Schinke

Abstract

X-linked hypophosphatemic rickets (XLHR) is a severe disorder of phosphate homeostasis and skeletal mineralization caused by mutations of PHEX, encoding a bone-specific endopeptidase. Phex-deficient Hyp mice have been extensively studied to understand the molecular bases of XLHR, and here it was found that Fgf23, encoding a major phosphaturic hormone, was transcriptionally activated in bone-forming osteoblasts. We and others could additionally show that Col10a1 expression is increased in Hyp osteoblasts and bones, thereby raising the possibility that ectopic production of type X collagen could contribute to the impaired mineralization of the Hyp bone matrix. Here we show that an additional deficiency of the Col10a1 gene does not overtly affect the skeletal phenotype of Hyp mice. More specifically, Col10a1-deficient Hyp mice displayed severe disturbances of skeletal growth, bone mass acquisition and bone matrix mineralization, and they were essentially indistinguishable from Hyp littermates. This was confirmed by non-decalcified histology and bone-specific histomorphometry quantifying all relevant parameters of growth plate maturation, trabecular bone architecture and osteoid accumulation. Taken together, our results show that increased Col10a1 expression in Phex-deficient osteoblasts is not a major cause of the XLHR phenotype, which was an important issue to address based on the previous findings.

Link To Article

http://dx.doi.org/10.1016/j.bbrc.2013.11.033

Radiation‑induced cytochrome c release and the neuroprotective effects of the pan-caspase inhibitor z‑VAD‑fmk in the hypoglossal nucleus

Authors

Jianguo Li, Yan Wang, Liqing Du, Chang Xu, Jia Cao, Qin Wang, Qiang Liu, Feiyue Fan

Abstract

Numerous studies have demonstrated that neuronal cell death occurs via extrinsic (death receptors) and intrinsic (mitochondria) pathways. Radiation induces caspase activation fundamentally via the mitochondrial pathway. To investigate the role of caspase, a cell permeable pan-caspase inhibitor, z-VAD-fmk [N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone], was used to investigate the effects of caspase blockade in vivo following irradiation. Adult male Sprague‑Dawley rats (weight, 250‑300 g) underwent irradiation at room temperature with a 4‑Gy dose of radiation. Since z-VAD-fmk does not penetrate the blood‑brain barrier, it was applied intracerebroventricularly via a bolus injection (0.2 µg/h for 1 h). Terminal deoxynucleotidyl transferase dUTP nick end‑labeling (TUNEL) demonstrated that z-VAD‑fmk reduced the numbers of TUNEL-positive cells within the hypoglossal nucleus, suggesting that intervention in the caspase cascade following radiation may have therapeutic applications. The caspase inhibitor z-VAD-fmk reduced the expression and activation of caspase-3, caspase-8 and caspase‑9 in the irradiated rats, indicating that caspase may be a potential therapeutic target in the treatment of brain radiation injury. Treatment with z-VAD-fmk also reduced the appearance of cytochrome c within the cytosolic fraction following radiation. The hypoglossal nucleus may be used as a model of radiation‑induced injury in the central nervous system, providing visual information and displaying apoptotic nuclear morphology.

Link To Article

http://dx.doi.org/10.3892/etm.2013.1419