The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.

Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.

Effects of sclerostin antibody on healing of a non-critical size femoral bone defect.

Sclerostin is a glycoprotein secreted by osteocytes and inhibits osteoblastogenesis via inhibition of Wnt signaling. We hypothesized that sclerostin antibody (Scl-AbIII) would accelerate the healing of a murine femoral non-critical size bone defect model. A unilateral and unicortical 0.8 mm-sized drill hole was made in the proximal femoral shaft of adult female nude mice. One group of mice received subcutaneous injections of Scl-AbIII and a second group received vehicle only. Reporter MC3T3 osteoprogenitor cells were injected via the tail vein 3 days after surgery to monitor systemic trafficking of exogenous osteoprogenitors. Bioluminescence imaging (BLI), microcomputed tomography (microCT), micropositron emission tomography (microPET) and histological analysis were used to compare the bone healing responses to Scl-AbIII treatment. Bone mineral density (BMD) significantly increased at the defect site after week 1, and was significantly higher in the treatment compared with the control group at all time points. This finding was also confirmed on histological analysis by increased deposition of new woven bone. MicroPET scanning showed a trend for greater activity in the control group at day 21 compared with the Scl-AbIII group, indicating early bone maturation following treatment with Scl-AbIII. Whereas the BLI signals derived from the injected osteoprogenitor cells showed no differences between vehicle and Scl-AbIII treated groups, systemic migration of MC3T3 cells to the bone defect was clearly identified in both groups using immunohistochemistry. Systemic administration of Scl-AbIII resulted in earlier healing and maturation of a non-critical size bone defect. These findings underscore the potential use of Scl-AbIII for treatment of complicated fractures, non-unions, and other clinical scenarios.

Sclerostin expression is induced by BMPs in human Saos-2 osteosarcoma cells but not via direct effects on the sclerostin gene promoter or ECR5 element.

Sclerostin is a secreted inhibitor of Wnt signaling and plays an essential role in the regulation of bone mass. The expression of sclerostin is largely restricted to osteocytes although its mode of transcriptional regulation is not well understood. We observed regulated expression of sclerostin mRNA and protein that was directly correlated with the mineralization response in cultured human Saos-2 osteosarcoma cells and rat primary calvarial cells. Sclerostin mRNA and protein levels were increased following treatment of cells with BMP2, BMP4 and BMP7. Analysis of deletion mutants from the -7.4 kb upstream region of the human sclerostin promoter did not reveal any specific regions that were responsive to BMPs, Wnt3a, PTH, TGFß1 or Activin A in Saos-2 cells. The downstream ECR5 element did not show enhancer activity in Saos-2 cells and also was not affected when Saos-2 cells were treated with BMPs or PTH. Genome-wide microarray analysis of Saos-2 cells treated with BMP2 showed significant changes in expression of several transcription factors with putative consensus DNA binding sites in the region of the sclerostin promoter. However, whereas most factors tested showed either a range of inhibitory activity (DLX family, MSX2, HEY1, SMAD6/7) or lack of activity on the sclerostin promoter including SMAD9, only MEF2B showed a positive effect on both the promoter and ECR5 element. These results suggest that the dramatic induction of sclerostin gene expression by BMPs in Saos-2 cells occurs indirectly and is associated with late stage differentiation of osteoblasts and the mineralization process.

Dickkopf-1 regulates bone formation in young growing rodents and upon traumatic injury.

The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.

New variants in the Enpp1 and Ptpn6 genes cause low BMD, crystal-related arthropathy, and vascular calcification.

A large genome-wide, recessive, N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen was performed on a mixed C57BL/6J and C3H.SW-H2/SnJ mouse background to identify genes regulating bone mass. Approximately 6500 male and female G(3) hybrid mice were phenotyped at 8 and 10 wk of age by DXA analysis for evidence of changes in unadjusted or body weight-adjusted BMD or BMC. Phenodeviant lines were identified based on statistical criteria that included a false discovery rate (FDR) <20% and Z-score >2.8. Genome-wide mapping scans were initiated on 22 lines, with evidence of high or low BMD or BMC that deviated by approximately -30% to +50% from the means. Several lines were discontinued as showing lack of heritability, but two heritable lines were identified with narrow chromosomal regions that allowed sequencing of potential mutant candidate genes. Novel mutations were identified in the Enpp1 (C397S) gene on chromosome 10 (line 4482) and the Ptpn6 (I482F) gene on chromosome 6 (line 4489) that were both associated with low bone mass. In addition, the phenotype of the Enpp1 mice showed a striking joint disease and calcification of blood vessels including the aorta, myocardium, and renal arteries and capillaries. These results support a role for the Enpp1 gene in the pathogenesis associated with mineralization of articular cartilage and vascular calcification. This work confirms the utility of the chemical mutagenesis approach for identification of potential disease genes and confirms the role of Enpp1 and Ptpn6 in regulating mineralization and skeletal bone mass.

Identification of three loci affecting HDL-cholesterol levels in a screen for chemically induced recessive mutations in mice.

We conducted a genome-wide screen using the mutagen N-ethyl-N-nitrosourea to identify recessive mutations in genes that lead to altered lipid traits in mice. We screened 7,546 G3 mice that were of mixed C57BL/6J (B6) x C3.SW-H2(b)/SnJ (C3) genomes and identified three pedigrees with differences in plasma HDL-cholesterol. Genome scan analyses mapped three distinct loci to chromosomes 3, 4, and 7. An S1748L missense mutation was identified in ABCA1 in one pedigree with undetectable levels of HDL-cholesterol and resulted in reduced protein levels. This phenotype was completely penetrant, semi-dominant, and cosegregated with high plasma triglycerides. Mice in a second pedigree had very high levels of plasma total cholesterol and HDL-cholesterol (up to 800 mg/dl total cholesterol). Despite a high degree of phenotype lability and reduced penetrance, an I68N missense mutation was identified in the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). Finally, a second high HDL-cholesterol pedigree of mice, again with a highly labile phenotype and reduced penetrance, was mapped to a 7 Mb locus on chromosome 3. These results illustrate the use of a hybrid background for simultaneous screening and mapping of mutagenized pedigrees of mice and identification of three novel alleles of HDL-cholesterol phenotypes.

Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone.

A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/beta-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/beta-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/beta-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3beta inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3beta inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/beta-catenin signaling is a normal physiological response to load and that activation of the Wnt/beta-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.

Smooth muscle-specific expression of SV40 large TAg induces SMC proliferation causing adaptive arterial remodeling.

To study the effects of enhanced smooth muscle cell (SMC) proliferation on arterial vessel geometry in the absence of vessel trauma, we developed a transgenic mouse model expressing SV40 large T antigen under control of the 2.3-kb smooth muscle-myosin heavy chain promoter. Transgenic mice studied at ages from 3 to 13 wk showed a 3.2-fold increase in arterial wall SMC density, with 28% of SMC exhibiting proliferative cell nuclear antigen staining, confirming enhanced SMC proliferation, which was accompanied by two- to threefold increases in arterial wall areas (P < 0.05). Remarkably, despite increased vessel wall mass, the lumen area was not compromised, but rather was increased. A tightly conserved linear relationship was found between arterial circumference and wall thickness with slopes of 0.036 for both transgenics (r = 0.93, P < 0.01) and controls (r = 0.77, P < 0.01), suggesting the hypothesis that the conservation of wall stress functions as a primary determinant of adaptive arterial remodeling. This establishes a new model of adaptive vessel remodeling occurring in response to a proliferative input in the absence of mechanical injury or primary flow perturbation.