Osteoblast extracellular Ca2+-sensing receptor regulates bone development, mineralization and turnover

Authors

Melita M. Dvorak-Ewell, Tsui-Hua Chen, Nathan Liang, Caitlin Garvey, Betty Liu, Chialing Tu, Wenhan Chang, Daniel D. Bikle, Dolores M. Shoback

Abstract

The extracellular Ca2+-sensing receptor (CaR), a G protein-coupled receptor responsible for maintenance of calcium homeostasis, is implicated in regulation of skeletal metabolism. To discern the role of the osteoblast CaR in regulation of bone development and remodeling, we generated mice in which the CaR is excised in a broad population of osteoblasts expressing the 3.6 kb a1(I) collagen promoter. Conditional knockouts had abnormal skeletal histology at birth and developed progressively reduced mineralization secondary to retarded osteoblast differentiation, evident by significantly reduced numbers of osteoblasts and decreased expression of collagen I, osteocalcin and sclerostin mRNAs. Elevated expression of ankylosis protein, ectonucleotide pyrophosphatase/phosphodiesterase 1, and osteopontin mRNAs in the conditional knockout indicate altered regulation of genes important in mineralization. Knockout of the osteoblast CaR also resulted in increased expression of the receptor activator of nuclear factor kappa B ligand (RANK-L), the major stimulator of osteoclast differentiation and function, consistent with elevated osteoclast numbers in vivo. Osteoblasts from the conditional knockouts exhibited delayed differentiation, reduced mineralizing capacity, altered expression of regulators of mineralization and increased ability to promote osteoclastogenesis in co-culture experiments. We conclude that CaR signaling in a broad population of osteoblasts is essential for bone development and remodeling and plays an important role in the regulation of differentiation and expression of regulators of bone resorption and mineralization.

Link to Article

http://dx.doi.org/10.1002/jbmr.520

Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Authors

Tracy Brennan, Naga Suresh Adapala, Mary F. Barbe, Vanessa Yingling, and Archana Sanjay

Abstract

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (CblYF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation.

Link to Article

http://dx.doi.org/10.1007/s00223-011-9531-z

Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

Authors

Jason W. Ashley, Zhenqi Shi, Haibo Zhao, Xingsheng Li, Robert A. Kesterson, Xu Feng

Abstract

CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68−/− osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68−/− osteoclasts.

Link to Article

http://dx.doi.org/10.1371/journal.pone.0025838

Trabecular bone histomorphometry in humans with type 1 diabetes Mellitus

Authors

Laura A.G. Armas, Mohammed P. Akhter, Andjela Drincic, Robert R. Recker

Abstract

Patients with Type 1 Diabetes Mellitus (DM) have markedly increased risk of fracture, but little is known about abnormalities in bone micro-architecture or remodeling properties that might give insight into the pathogenesis of skeletal fragility in these patients. We report here a case–control study comparing bone histomorphometric and micro-CT results from iliac biopsies in 18 otherwise healthy subjects with Type 1 Diabetes Mellitus with those from healthy age- and sex- matched non-diabetic control subjects. Five of the diabetics had histories of low-trauma fracture. Transilial bone biopsies were obtained after tetracycline labeling. The biopsy specimens were fixed, embedded, and scanned using a desktop μCT at 16 micron resolution. They were then sectioned and quantitative histomorphometry was performed as previously described by Recker et al. 1988.[1] Two sections, > 250 μm apart, were read from the central part of each biopsy. Overall there were no significant differences between diabetics and controls in histomorphometric or micro-CT measurements. However, fracturing diabetics had structural and dynamic trends different from nonfracturing diabetics by both methods of analysis. In conclusion, Type 1 Diabetes Mellitus does not result in abnormalities in bone histomorphometric or micro-CT variables in the absence of manifest complications from the diabetes. However, diabetics suffering fractures may have defects in their skeletal microarchitecture that may underlie the presence of excess skeletal fragility.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.09.055

Lamin A/C deficiency is associated with fat infiltration of muscle and bone

Authors

Jessica Tong, Wei Li, Christopher Vidal, Li Sze Yeo, Diane Fatkin, Gustavo Duque

Abstract

Sarcopenia and osteopenia are two common components of the frailty syndrome that may share a common underlying mechanism. Since frailty has been associated with increased fat infiltration in muscle and bone, we hypothesized that lamin A/C, a protein of the nuclear envelope that regulates adipose differentiation, could be associated with the pathophysiology of both osteo and sarcopenia in the frailty syndrome. Four-week-old lamin A/C null (Lmna−/−), heterozygous (Lmna+/−) and wild type (WT) mice were sacrificed and their mid-thigh analyzed for fat infiltration using invasive (histology) and non-invasive (μCT) methods. Lmna−/− mice showed a significant increase in inter- (∼4-fold) and intra-myofiber (∼2.5-fold) fat and marrow fat infiltration (∼40-fold), with a significant decrease in muscle volume (−42.8%) and bone volume (−21.8%), as compared with WT controls. Furthermore, fat infiltration happened concomitantly with a significant decline in muscle and bone strength in Lmna−/− mice. From a mechanistic approach, high levels of pro-adipogenic factors PPARγ and C/EBPα were associated with a reduction in myogenic and osteogenic factors from the Wnt-10b/β-catenin signalling pathway in Lmna−/− mice. In conclusion, lamin A/C could constitute the determinant factor in the pathogenesis and potential treatment of both sarcopenia and osteopenia, which are commonly observed in the frailty syndrome.

Link to Article

http://dx.doi.org/10.1016/j.mad.2011.09.004

Nanostructured implant surface effect on osteoblast gene expression and bone-to-implant contact in vivo

Authors

Gustavo Mendonça, Daniela Baccelli Silveira Mendonça, Luis Gustavo Pagotto Simões, André Luis Araújo, Edson Roberto Leite, Alexsander Luiz Golin, Francisco J.L. Aragão, Lyndon F. Cooper

Abstract

The aim of this study was to investigate the response of nanostructured implant surfaces at the level of osteoblast differentiation and its effects in bone-to-implant contact (BIC) and removal-torque values (RTV). CpTi grade IV implants (1.6 × 4.0 mm) were machined or machined and subsequently coated with an oxide solution. The surfaces were divided into: machined (M), titania-anatase (An), titania-rutile (Ru), and zirconia (Zr). Surfaces were examined by scanning electron microscopy, atomic force microscopy, and by X-ray microanalysis. Implants were inserted in rat tibia and harvested from 0 to 21 days for measurement of Alkaline Phosphatase, Bone Sialoprotein, Osteocalcin, Osteopontin, and RUNX-2 mRNA levels by real time PCR; from 0 to 56 days for RTV; and from 0 to 56 days for BIC. The roughness parameter (Sa) was compared by one-way ANOVA followed by Tukey Test. Comparison of Torque removal values and histomorphometric measurements on implants in vivo was performed by Kruskal–Wallis test and the significance level for all statistical analyses was set at p ≤ 0.05. mRNA levels on all nanostructured surfaces were increased compared to M. At 56 days, the mean RTV in Ncm was 11.6 ± 2.5, 11.3 ± 2.4, 11.1 ± 3.5, 9.7 ± 1.4 for An, Ru, Zr, and M, respectively. Higher BIC (%) was measured for all the nanostructured surfaces versus M at 21 and 56 days (p < 0.05). Nanostructured topographic features composed of TiO2 or ZrO2 applied to machined cpTi implant promoted greater mesenchymal stem cell commitment to the osteoblast phenotype and associated increased BIC and physical association with bone.

Link to Article

http://dx.doi.org/10.1016/j.msec.2011.08.021