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

Overexpression of secreted frizzled-related protein 1 inhibits bone formation and attenuates parathyroid hormone bone anabolic effects

Authors

Yao, W. and Cheng, Z. and Shahnazari, M. and Dai, W. and Johnson, M.L. and Lane, N.E.

Abstract

Secreted frizzled-related protein 1 (sFRP1) is an antagonist of Wnt signaling, an important pathway in maintaining bone homeostasis. In this study we evaluated the skeletal phenotype of mice overexpressing sFRP1 (sFRP1 Tg) and the interaction of parathyroid hormone (PTH) treatment and sFRP1 (over)expression. Bone mass and microarchitecture were measured by micro-computed tomography (µCT). Osteoblastic and osteoclastic cell maturation and function were assessed in primary bone marrow cell cultures. Bone turnover was assessed by biochemical markers and dynamic bone histomorphometry. Real-time PCR was used to monitor the expression of several genes that regulate osteoblast maturation and function in whole bone. We found that trabecular bone mass measurements in distal femurs and lumbar vertebral bodies were 22% and 51% lower in female and 9% and 33% lower in male sFRP1 Tg mice, respectively, compared with wild-type (WT) controls at 3 months of age. Genes associated with osteoblast maturation and function, serum bone formation markers, and surface based bone formation were significantly decreased in sFRP1 Tg mice of both sexes. Bone resorption was similar between sFRP1 Tg and WT females and was higher in sFRP1 Tg male mice. Treatment with hPTH(1-34) (40 µg/kg/d) for 2 weeks increased trabecular bone volume in WT mice (females: +30% to 50%; males: +35% to 150%) compared with sFRP1 Tg mice (females: +5%; males: +18% to 54%). Percentage increases in bone formation also were lower in PTH-treated sFRP1 Tg mice compared with PTH-treated WT mice. In conclusion, overexpression of sFRP1 inhibited bone formation as well as attenuated PTH anabolic action on bone. The gender differences in the bone phenotype of the sFRP1 Tg animal warrants further investigation.

Link to Article

http://www3.interscience.wiley.com/journal/123210016/abstract

Ex Vivo Transfer of the Hoxc-8-interacting Domain of Smad1 by a Tropism-modified Adenoviral Vector Results in Efficient Bone Formation in a Rabbit Model of Spinal Fusion

Authors

Douglas, J.T. and Rivera, A.A. and Lyons, G.R. and Lott, P.F. and Wang, D. and Zayzafoon, M. and Siegal, G.P. and Cao, X. and Theiss, S.M.

Abstract

Study Design: Ex vivo gene transfer for spinal fusion.

Objective: This study aimed to evaluate ex vivo transfer of the nuclear-localized Hoxc-8-interacting domain of Smad1 (termed Smad1C) to rabbit bone marrow stromal cells (BMSCs) by a tropism-modified human adenovirus serotype 5 (Ad5) vector as a novel therapeutic approach for spinal fusion.

Summary of Background Data: Novel approaches are needed to improve the success of bone union after spinal fusion. One such approach is the ex vivo transfer of a gene encoding an osteoinductive factor to BMSCs which are subsequently reimplanted into the host. We have previously shown that heterologous expression of the Hoxc-8-interacting domain of Smad1 in the nuclei of osteoblast precursor cells is able to stimulate the expression of genes related to osteoblast differentiation and induce osteogenesis in vivo. Gene delivery vehicles based on human Ad5 are well suited for gene transfer for spinal fusion because they can mediate high-level, short-term gene expression. However, Ad5-based vectors with native tropism poorly transduce BMSCs, necessitating the use of vectors with modified tropism to achieve efficient gene transfer.

Methods: The gene encoding Smad1C was transferred to rabbit BMSCs by an Ad5 vector with native tropism or a vector retargeted to αv integrins, which are abundantly expressed on rabbit BMSCs. Transduced BMSCs were maintained in osteoblastic differentiation medium for 30 days. Alkaline phosphatase activity was determined and cells stained for calcium deposition. As positive controls for osteogenesis, we used Ad5 vectors expressing bone morphogenetic protein 2. As negative controls, BMSCs were mock-transduced or transduced with an Ad5 vector expressing β-galactosidase. In an immunocompetent rabbit model of spinal fusion, transduced BMSCs were coated onto absorbable gelatin sponge and implanted between decorticated transverse processes L6 and L7 of 8-week-old female New Zealand white rabbits. Animals were killed 4 weeks after implantation of the sponges, the fusion masses harvested and the area of new bone quantified using image analysis software.

Results: The Smad1C-expressing tropism-modified Ad5 vector mediated a significantly higher level of alkaline phosphatase activity and calcium deposition in transduced rabbit BMSCs than all other vectors. The rabbit BMSCs transduced ex vivo with the Smad1C-expressing tropism-modified Ad5 vector mediated a greater amount of new bone formation than BMSCs transduced with any other vector.

Conclusions: Delivery of the Smad1C gene construct to BMSCs by an αv integrin-targeted Ad5 vector shows promise for spinal fusion and other applications requiring the formation of new bone in vivo.

Link to Article

http://journals.lww.com/jspinaldisorders/Abstract/2010/02000/Ex_Vivo_Transfer_of_the_Hoxc_8_interacting_Domain.13.aspx

Multiple roles for CCR2 during fracture healing

Authors

Xing, Z. and Lu, C. and Hu, D. and Yu, Y. and Wang, X. and Colnot, C. and Nakamura, M. and Wu, Y. and Miclau, T. and Marcucio, R.S.

Abstract

Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2–/– mice compared with wild-type controls at 3 days injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after in mutant mice. At day 14, Ccr2–/– mice had less bone in their calluses. At day 21, Ccr2–/– mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2–/– osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2–/– mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.

Link to Article

http://dmm.biologists.org/content/early/2010/03/25/dmm.003186.full.pdf