The Ewing's Sarcoma Fusion Protein, EWS-FLI, Binds Runx2 and Blocks Osteoblast Differentiation

Authors

Xiaodong Li,Meghan E. McGee-Lawrence, Matthew Decker, Jennifer J. Westendorf

Abstract

Ewing's sarcomas are highly aggressive round cell tumors of bone and soft tissues that afflict children and young adults. The majority of these tumors harbor the t(11;22) translocation and express the fusion protein EWS-FLI. Modern molecular profiling experiments indicate that Ewing's tumors originate from mesenchymal precursors in young individuals. EWS-FLI alters the morphology of mesenchymal cells and prevents lineage specification; however, the molecular mechanisms for differentiation arrest are unclear. We recently showed that EWS-FLI binds Runx2, a master regulator of osteoblast differentiation. In this report, we demonstrate that FLI sequences within EWS-FLI are responsible for interactions with Runx2. EWS-FLI blocks the expression of osteoblastic genes in a multipotent progenitor cell line that requires Runx2 to integrate bone morphogenic protein (Bmp)2 signaling while increasing proliferation and altering cell morphology. These results demonstrate that EWS-FLI blocks the ability of Runx2 to induce osteoblast specification of a mesenchymal progenitor cell. Disrupting interactions between Runx2 and EWS-FLI1 may promote differentiation of the tumor cell

Link to Article

http://dx.doi.org/10.1002/jcb.22782

Vitamin D Deficiency Promotes Growth of MCF-7 Human Breast Cancer in a Rodent Model of Osteosclerotic Bone Metastasis

Authors

Li Laine Ooia, Yu Zhenga, Hong Zhoua, Trupti Trivedia, Arthur D. Conigraveb, Markus J. Seibela, and Colin R. Dunstan

Abstract

Breast cancer metastases to bone are common in advanced stage disease. We have recently demonstrated that vitamin D deficiency enhances breast cancer growth in an osteolytic mouse model of breast cancer metastasis. In this study, we examined the effects of vitamin D deficiency on tumor growth in an osteosclerotic model of intra-skeletal breast cancer in mice.
Methods

The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on proliferation and apoptosis of MCF-7 breast cancer cells, and changes in the expression of genes within the vitamin D metabolic pathway (VDR, 1α- and 24-hydroxylase) were examined in vitro. MCF-7 breast cancer cells were injected intra-tibially into vitamin D deficient and vitamin D sufficient mice co-treated with and without osteoprotegerin (OPG). The development of tumor-related lesions was monitored via serial X-ray analysis. Tumor burden and indices of proliferation and apoptosis were determined by histology along with markers of bone turnover and serum intact PTH levels.
Results

In vitro, MCF-7 cells expressed critical genes for vitamin D signalling and metabolism. Treatment with 1,25(OH)2D3 inhibited cell growth and proliferation, and increased apoptosis. In vivo, osteosclerotic lesions developed faster and were larger at endpoint in the tibiae of vitamin D deficient mice compared to vitamin D sufficient mice (1.49 ± 0.08 mm2 versus 1.68 ± 0.15 mm2, p < 0.05). Tumor area was increased by 55.8% in vitamin D deficient mice (0.81 ± 0.13 mm2 versus 0.52 ± 0.11 mm2 in vitamin D sufficient mice). OPG treatment inhibited bone turnover and caused an increase in PTH levels, while tumor burden was reduced by 90.4% in vitamin D sufficient mice and by 92.6% in vitamin D deficient mice. Tumor mitotic activity was increased in the tibiae of vitamin D deficient mice and apoptosis was decreased, consistent with faster growth.
Conclusion

Vitamin D deficiency enhances both the growth of tumors and the tumor-induced osteosclerotic changes in the tibiae of mice following intratibial implantation of MCF-7 cells. Enhancement of tumor growth appears dependent on increased bone resorption rather than increased bone formation induced by these tumors.

Link to Article

http://dx.doi.org/10.1016/j.bone.2010.07.012

Modification of Osteoarthritis in the Guinea Pig with Pulsed Low-Intensity Ultrasound Treatment

Authors

Gurkan, I. and Ranganathan, A. and Yang, X. and Horton Jr, W.E. and Todman, M. and Huckle, J. and Pleshko, N. and Spencer, R.G.

Abstract

Objective: The Hartley guinea pig develops articular cartilage degeneration similar to that seen in idiopathic human osteoarthritis (OA). We investigated whether the application of pulsed low-intensity ultrasound (PLIUS) to the Hartley guinea pig joint would prevent or attenuate the progression of this degenerative process.

Methods: Treatment of male Hartley guinea pigs was initiated at the onset of degeneration (8 weeks of age) to assess the ability of PLIUS to prevent OA, or at a later age (12 months) to assess the degree to which PLIUS acted to attenuate the progression of established disease. PLIUS (30mW/cm2) was applied to stifle joints for 20min/day over periods ranging from 3 to 10 months, with contralateral limbs serving as controls. Joint cartilage histology was graded according to a modified Mankin scale to evaluate treatment effect. Immunohistochemical staining for interleukin-1 receptor antagonist (IL-1ra), matrix metalloproteinase (MMP)-3, MMP-13, and transforming growth factor (TGF)-β1 was performed on the cartilage to evaluate patterns of expression of these proteins.

Results: PLIUS did not fully prevent cartilage degeneration in the prevention groups, but diminished the severity of the disease, with the treated joints showing markedly decreased surface irregularities and a much smaller degree of loss of matrix staining as compared to controls. PLIUS also attenuated disease progression in the groups with established disease, although to a somewhat lesser extent as compared to the prevention groups. Immunohistochemical staining demonstrated a markedly decreased degree of TGF-β1 production in the PLIUS-treated joints. This indicates less active endogenous repair, consistent with the marked reduction in cartilage degradation.

Conclusions: PLIUS exhibits the ability to attenuate the progression of cartilage degeneration in an animal model of idiopathic human OA. The effect was greater in the treatment of early, rather than established, degeneration.

Link to Article

http://linkinghub.elsevier.com/retrieve/pii/S1063458410000336

Effect of HIP/ribosomal protein L29 deficiency on mineral properties of murine bones and teeth

Authors

Sloofman, L.G. and Verdelis, K. and Spevak, L. and Zayzafoon, M. and Yamauchi, M. and Opdenaker, L.M. and Farach-Carson, M.C. and Boskey, A.L. and Kirn-Safran, C.B.

Abstract

Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues.

Link to Article

http://linkinghub.elsevier.com/retrieve/pii/S8756328210005296

Aged mice have enhanced endocortical response and normal periosteal response compared to young-adult mice following 1 week of axial tibial compression

Authors

Brodt, M.D. and Silva, M.J.

Abstract

With aging, the skeleton may lose its ability to respond to positive mechanical stimuli. We hypothesized that aged mice are less responsive to loading compared to young-adult mice. We subjected aged (22 mo) and young-adult (7 mo) BALB/c male mice to daily bouts of axial tibial compression for one week, and evaluated cortical and trabecular responses using microCT and dynamic histomorphometry. The right legs of 95 mice were loaded for 60 rest-inserted cycles/day to 8, 10 or 12 N peak force (generating mid-diaphyseal strains of 900-1900 µ endocortically and 1400-3100 µ periosteally). At the mid-diaphysis, mice from both age groups showed a strong anabolic response on the endocortex (Ec) and periosteum (Ps) (Ec.MS/BS and Ps.MS/BS: loaded [right] vs. control [left], p < 0.05). Generally, bone formation increased with increasing peak force. At the endocortical surface, contrary to our hypothesis, aged mice had a significantly greater response to loading than young-adult mice (Ec.MS/BS and Ec.BFR/BS: 22-mo vs. 7-mo, p < 0.001). Responses at the periosteal surface did not differ between age groups (p < 0.05). The loading-induced increase in bone formation resulted in increased cortical area in both age groups (loaded vs. control, p < 0.05). In contrast to the strong cortical response, loading only weakly stimulated trabecular bone formation. Serial (in vivo) microCT at the proximal metaphysis revealed that loading caused a loss of trabecular bone in 7-mo mice whereas it appeared to prevent bone loss in 22-mo mice. In summary, one week of daily tibial compression stimulated a robust endocortical and periosteal bone formation response at the mid-diaphysis in both young-adult and aged male BALB/c mice. We conclude that aging does not limit the short-term anabolic response of cortical bone to mechanical stimulation in our animal model.

Link to Article

http://www3.interscience.wiley.com/journal/123333914/abstract

Nuclear alphaNAC influences bone matrix mineralization and osteoblast maturation in vivo

Authors

Meury, T. and Akhouayri, O. and Jafarov, T. and Mandic, V. and St-Arnaud, R.

Abstract

Nascent-polypeptide-associated complex and coactivator alpha (NAC) is a protein shuttling between the nucleus and the cytoplasm. Upon phosphorylation at residue serine 43 by integrin-linked kinase, NAC is translocated to the nuclei of osteoblasts, where it acts as an AP-1 coactivator to increase osteocalcin gene transcription. To determine the physiological role of nuclear NAC, we engineered a knock-in mouse model with a serine-to-alanine mutation at position 43 (S43A). The S43A mutation resulted in a decrease in the amount of nuclear NAC with reduced osteocalcin gene promoter occupancy, leading to a significant decrease in osteocalcin gene transcription. The S43A mutant bones also expressed decreased levels of 1(I) collagen mRNA and as a consequence had significantly less osteoid tissue. Transient transfection assays and chromatin immunoprecipitation confirmed the 1(I) collagen gene as a novel NAC target. The reduced quantity of bone matrix in S43A mutant bones was mineralized faster, as demonstrated by the significantly reduced mineralization lag time, producing a lower volume of immature, woven-type bone characterized by poor lamellation and an increase in the number of osteocytes. Accordingly, the expression of the osteocyte differentiation marker genes DMP-1 (dentin matrix protein 1), E11, and SOST (sclerostin) was increased. The accelerated mineralization phenotype was cell autonomous, as osteoblasts isolated from the calvaria of S43A mutant mice mineralized their matrix faster than did wild-type cells. Thus, inhibition of NAC nuclear translocation results in an osteopenic phenotype caused by reduced expression of osteocalcin and type I collagen, accelerated mineralization, and immature woven-bone formation.

Link to Article

http://mcb.asm.org/cgi/content/abstract/30/1/43