Robert P. Marshall

Impaired gastric acidification negatively affects calcium homeostasis and bone mass.

Activation of osteoclasts and their acidification-dependent resorption of bone is thought to maintain proper serum calcium levels. Here we show that osteoclast dysfunction alone does not generally affect calcium homeostasis. Indeed, mice deficient in Src, encoding a tyrosine kinase critical for osteoclast activity, show signs of osteopetrosis, but without hypocalcemia or defects in bone mineralization. Mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells, have the expected defects in gastric acidification but also secondary hyperparathyroidism and osteoporosis and modest hypocalcemia. These results suggest that alterations in calcium homeostasis can be driven by defects in gastric acidification, especially given that calcium gluconate supplementation fully rescues the phenotype of the Cckbr-mutant mice. Finally, mice deficient in Tcirg1, encoding a subunit of the vacuolar proton pump specifically expressed in both osteoclasts and parietal cells, show hypocalcemia and osteopetrorickets. Although neither Src- nor Cckbr-deficient mice have this latter phenotype, the combined deficiency of both genes results in osteopetrorickets. Thus, we find that osteopetrosis and osteopetrorickets are distinct phenotypes, depending on the site or sites of defective acidification (pages 610–612).

Control of bone formation by the serpentine receptor Frizzled-9.

Fzd9, induced upon osteoblast differentiation, is required for bone matrix mineralization in primary osteoblasts.

Micro-structural basis for particular vulnerability of the superolateral neck trabecular bone in the postmenopausal women with hip fractures

In this study we analyzed the trabecular bone micro-architecture in the inferomedial and superolateral subregions of the femoral neck in a group with hip fractures and a control group of elderly women, with aim to clarify the micro-structural basis of bone fragility. Proximal femora from 29 Caucasian female cadavers were collected at Institute of Forensic Medicine in Belgrade (15 women with hip fracture: age 79.5±8.5 yrs.; and 14 women without hip fractures: age 74.1±9.3 yrs.). The femoral neck section was scanned in dry conditions using a micro-computed tomography (Scanco μCT 40), at 70 kV, 114 μA, 300 ms integration time, 36 μm resolution, isotropic, 1024×1024 pixels per slice, automatically evaluating trabecular micro-architecture using the built-in program of the micro-CT with direct 3D morphometry. The samples were foam padded to avoid any movement artifacts during scanning. Analysis of the neck section in the fracture group compared to the control cases demonstrated significantly lower bone volume fraction (mean: 6.3% vs. 11.2%, p=0.002), lower connectivity density (0.33/mm(3) vs. 0.74/mm(3), p=0.019) and higher trabecular separation (0.87 mm vs. 0.83 mm, p=0.030). Division into the superolateral and inferomedial regions of interest revealed that the superolateral neck displayed even more differences in micro-architectural properties between the fracture and non-fracture groups. Namely, while in the inferomedial neck only bone volume fraction and degree of anisotropy displayed significant inter-group variability (lower BV/TV with higher degree of anisotropy in the fracture group), in the superolateral neck almost all parameters were different between the fracture cases and the controls, where the fracture group showed a lower trabecular bone volume fraction (3.6% vs. 8.2%, p=0.001), lower connectivity (0.21 vs. 0.63/mm(3), p=0.008), more rod like trabecular structure (SMI: 2.94 vs. 2.62, p=0.049), higher separation and the thinned trabeculae (Tb.Sp: 0.89 vs. 0.85 mm, p=0.013; Tb.Th: 0.17 vs. 0.20 mm, p=0.05). In addition, after adjusting for the effects of BV/TV, the majority of differences disappeared, demonstrating that the bone loss manifests itself via the changes in micro-architectural parameters: trabecular thinning, rising the spacing between individual trabeculae, reducing trabecular connectivity and accentuating trabecular perforations leading to predominance of rod-like trabecular elements. Preferential impairment of the superolateral neck trabecular structure and organization in women with hip fracture reveals the region-dependent micro-structural basis of bone fragility in elderly women.

Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.

Decreased bone formation and increased osteoclastogenesis cause bone loss in mucolipidosis II

Mucolipidosis type II (MLII) is a severe multi‐systemic genetic disorder caused by missorting of lysosomal proteins and the subsequent lysosomal storage of undegraded macromolecules. Although affected children develop disabling skeletal abnormalities, their pathogenesis is not understood. Here we report that MLII knock‐in mice, recapitulating the human storage disease, are runted with accompanying growth plate widening, low trabecular bone mass and cortical porosity. Intralysosomal deficiency of numerous acid hydrolases results in accumulation of storage material in chondrocytes and osteoblasts, and impaired bone formation. In osteoclasts, no morphological or functional abnormalities are detected whereas osteoclastogenesis is dramatically increased in MLII mice. The high number of osteoclasts in MLII is associated with enhanced osteoblastic expression of the pro‐osteoclastogenic cytokine interleukin‐6, and pharmacological inhibition of bone resorption prevented the osteoporotic phenotype of MLII mice. Our findings show that progressive bone loss in MLII is due to the presence of dysfunctional osteoblasts combined with excessive osteoclastogenesis. They further underscore the importance of a deep skeletal phenotyping approach for other lysosomal diseases in which bone loss is a prominent feature.

Osteopetrosis, osteopetrorickets and hypophosphatemic rickets differentially affect dentin and enamel mineralization

Osteopetrosis (OP) is an inherited disorder of defective bone resorption, which can be accompanied by impaired skeletal mineralization, a phenotype termed osteopetrorickets (OPR). Since individuals with dysfunctional osteoclasts often develop osteomyelitis of the jaw, we have analyzed, if dentin and enamel mineralization are differentially affected in OP and OPR. Therefore, we have applied non-decalcified histology and quantitative backscattered electron imaging (qBEI) to compare the dental phenotypes of Src(-/-), oc/oc and Hyp(-/0) mice, which serve as models for OP, OPR and hypophosphatemic rickets, respectively. While both, Src(-/-) and oc/oc mice, were characterized by defects of molar root formation, only oc/oc mice displayed a severe defect of dentin mineralization, similar to Hyp(-/0) mice. Most importantly, while enamel thickness was not affected in either mouse model, the calcium content within the enamel phase was significantly reduced in oc/oc, but not in Src(-/-) or Hyp(-/0) mice. Taken together, these data demonstrate that dentin and enamel mineralization are differentially affected in Src(-/-) and oc/oc mice. Moreover, since defects of dental mineralization may trigger premature tooth decay and thereby osteomyelitis of the jaw, they further underscore the importance of discriminating between OP and OPR in the respective individuals.

Human apolipoprotein E isoforms differentially affect bone mass and turnover in vivo.

The primary role of apolipoprotein E (apoE) is to mediate the cellular uptake of lipoproteins. However, a new role for apoE as a regulator of bone metabolism in mice has recently been established. In contrast to mice, the human APOE gene is characterized by three common isoforms APOE ε2, ε3, and ε4 that result in different metabolic properties of the apoE isoforms, but it remains controversial whether the APOE polymorphism influences bone traits in humans. To clarify this, we investigated bone phenotypes of apoE knock‐in (k.i.) mice, which express one human isoform each (apoE2 k.i., apoE3 k.i., apoE4 k.i.) in place of the mouse apoE. Analysis of 12‐week‐old female k.i. mice revealed increased levels of biochemical bone formation and resorption markers in apoE2 k.i. animals as compared to apoE3 k.i. and apoE4 k.i., with a reduced osteoprotegerin (OPG)/receptor activator of NF‐κB ligand (RANKL) ratio in apoE2 k.i., indicating increased turnover with prevailing resorption in apoE2 k.i. Accordingly, histomorphometric and micro–computed tomography (µCT) analyses demonstrated significantly lower trabecular bone mass in apoE2 than in apoE3 and apoE4 k.i. animals, which was reflected by a significant reduction of lumbar vertebrae maximum force resistance. Unlike trabecular bone, femoral cortical thickness, and stability was not differentially affected by the apoE isoforms. To extend these observations to the human situation, plasma from middle‐aged healthy men homozygous for ε2/ε2, ε3/ε3, and ε4/ε4 (n = 21, n = 80, n = 55, respectively) was analyzed with regard to bone turnover markers. In analogy to apoE2 k.i. mice, a lower OPG/RANKL ratio was observed in the serum of ε2/ε2 carriers as compared to ε3/ε3 and ε4/ε4 individuals (p = 0.02 for ε2/ε2 versus ε4/ε4). In conclusion, the current data strongly underline the general importance of apoE as a regulator of bone metabolism and identifies the APOE ε2 allele as a potential genetic risk factor for low trabecular bone mass and vertebral fractures in humans.

Enhanced trabecular micro-architecture of the femoral neck in hip osteoarthritis vs. healthy controls: a micro-computer tomography study in postmenopausal women

PurposeA controversial relationship between osteoarthritis (OA) and bone fragility has been attracting considerable attention. However, despite interest in the effects of OA on femoral neck fracture risk and numerous studies analysing the changes in the arthritic femoral head, there is insufficient data about femoral neck 3D bone micro-architecture in individuals with hip osteoarthritis. We compared trabecular micro-architecture of the femoral neck between postmenopausal women with coxarthrosis and controls to explore whether coxarthrosis may indicate reduced bone fragility from the trabecular micro-architectural perspective.MethodsThe study sample included nine women with hip osteoarthritis and 13 age-matched controls. The femoral neck sections were scanned using micro-computed tomography, evaluating the cancellous bone from the superolateral and inferomedial neck subregions.ResultsOsteoarthritic subjects demonstrated a general trend of improved trabecular micro-architecture in both analysed subregions when compared with age-matched controls. In particular, several architectural properties that are important predictors of cancellous bone strength showed significantly better values in the OA group, even after adjusting for bone volume fraction. Namely, the OA group expressed higher trabecular connectivity (p = 0.008), lower SMI indicating more plate-like structure (p = 0.005), and reduced anisotropy (p = 0.006) particularly in the inferomedial neck. Osteoarthritic cases also trended towards higher BV/TV, particularly in the superolateral neck. All micro-architectural parameters displayed significant regional heterogeneity (p ≤ 0.01), with the inferomedial neck region showing more favourable values than the superolateral region.ConclusionsEnhanced trabecular micro-architecture of the femoral neck in postmenopausal osteoarthritic subjects suggests reduced cancellous bone fragility in comparison with their age-matched healthy controls.

Increased trabecular bone formation in mice lacking the growth factor midkine

Midkine (Mdk) and pleiotrophin (Ptn) comprise a family of heparin‐binding growth factors known primarily for their effects on neuronal cells. Since transgenic mice overexpressing Ptn have been reported to display increased bone density, we have previously analyzed Ptn‐deficient mice but failed to detect any abnormality of skeletal development and remodeling. Together with the finding that Mdk expression increases in the course of primary osteoblast differentiation, we reasoned that Mdk, rather than Ptn, could play a physiologic role in bone formation. Here, we show that Mdk‐deficient mice display an increased trabecular bone volume at 12 and 18 months of age, accompanied by cortical porosity. Histomorphometric quantification demonstrated an increased bone‐formation rate compared with wild‐type littermates, whereas bone resorption was differentially affected in trabecular and cortical bone of Mdk‐deficient mice. To understand the effect of Mdk on bone formation at the molecular level, we performed a genome‐wide expression analysis of primary osteoblasts and identified Ank and Enpp1 as Mdk‐induced genes whose decreased expression in Mdk‐deficient osteoblasts may explain, at least in part, the observed skeletal phenotype. Finally, we performed ovariectomy and observed bone loss only in wild‐type but not in Mdk‐deficient animals. Taken together, our data demonstrate that Mdk deficiency, at least in mice, results in an increased trabecular bone formation, thereby raising the possibility that Mdk‐specific antagonists might prove beneficial in osteoporosis therapy.

Short-term activation of liver X receptors inhibits osteoblasts but long-term activation does not have an impact on murine bone in vivo

Liver X receptors (LXRs) are nuclear receptors that play a crucial role in the transcriptional control of lipid metabolism. Pharmacological LXR activation is an attractive concept for the treatment of atherosclerosis. Genetic LXR deficiency in mice has been shown to have an effect on bone turnover and structure and LXR activation is known to influence the osteogenic differentiation of bone marrow stromal cells. Therefore, therapeutic pharmacological LXR activation may have relevant effects on bone. Here, using two synthetic LXR ligands, T0901317 and GW3965, we investigated the effect of LXR activation on murine osteoblasts and the influence of long-term LXR activation on bone in vivo in mice. Short term (48 h) in vitro treatment of primary murine osteoblasts with T0901317 resulted in a dose-dependent decrease of osteocalcin and alkaline phosphatase mRNA and protein. In vivo, a 6-day treatment of C57BL/6J mice with T0901317 led to a 40% reduction of serum osteocalcin concentrations. Long-term (12-week) oral administration of T0901317 or GW3965 influenced the expression of established LXR target genes in liver and intestine, but did not alter trabecular and cortical bone structure or bone turnover as determined by total skeleton radiography, histomorphometric analysis of lumbar vertebral trabecular bone, micro CT analysis of femur cortical bone and biochemical determination of bone formation and resorption markers. We conclude that short-term pharmacological LXR activation has the potential to profoundly influence osteoblast function, but that long-term LXR activation in vivo has no adverse effects on the murine skeleton.