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

Streptozotocin-induced diabetes in rats diminishes the size of the osteoprogenitor pool in bone marrow

Authors

E. Weinberg, T. Maymon, O. Moses, M. Weinre

Abstract

Aims

Bone formation is reduced in animals and humans with type 1 diabetes, leading to lower bone mass and inferior osseous healing. Since bone formation greatly depends on the recruitment of osteoblasts from their bone marrow precursors, we tested whether experimental type 1 diabetes in rats diminishes the number of bone marrow osteoprogenitors.

Methods

Diabetes was induced by 65 mg/kg streptozotocin and after 4 weeks, femoral bone marrow cells were extracted and cultured. Tibia and femur were frozen for further analysis.

Results

The size of the osteoprogenitor pool in bone marrow of diabetic rats was significantly reduced, as evidenced by 1) lower (∼35%) fraction of adherent stromal cells (at 24 h of culture); 2) lower (20-25%) alkaline phosphatase activity at 10 days of culture; and 3) lower (∼40%) mineralized nodule formation at 21 days of culture. Administration of insulin to hyperglycemic rats normalized glycemia and abrogated most of the decline in ex-vivo mineralized nodule formation. Apoptotic cells in tibial bone marrow were more numerous in hyperglycemic rats. Also, the levels of malondialdehyde (indicator of oxidative stress) were significantly elevated in bone marrow of diabetic animals.

Conclusions

Experimental type 1 diabetes diminishes the osteoprogenitor population in bone marrow, possibly due to increased apoptosis via Oxidative Stress. Reduced number of osteoprogenitors is likely to impair osteoblastogenesis, bone formation, and bone healing in diabetic animals.

Link to Article

http://dx.doi.org/10.1016/j.diabres.2013.11.015

Osteoblast CFTR Inactivation Reduces Differentiation and Osteoprotegerin Expression in a Mouse Model of Cystic Fibrosis-Related Bone Disease

Authors

Michael S. Stalvey, Katrina L. Clines, Viktoria Havasi, Christopher R. McKibbin, Lauren K. Dunn, W. Joon Chung, Gregory A. Clines

Abstract

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.

Link To Article

http://dx.doi.org/10.1371/journal.pone.0080098